miR-204 inhibits angiogenesis and promotes sensitivity to cetuximab in head and neck squamous cell carcinoma cells by blocking JAK2-STAT3 signaling.

Science.gov (United States)

Wu, Qingwei; Zhao, Yingying; Wang, Peihua

2018-03-01

This study aims to investigate the roles of miR-204 in tumor angiogenesis of head and neck squamous cell carcinoma (HNSCC). Here, we found that miR-204 level was reduced in HNSCC tissues relative to that in normal adjacent tissues. Overexpression of miR-204 promoted tumor angiogenesis in HNSCC cells. Mechanistically, JAK2 was identified as a direct target of miR-204, and miR-204 overexpression blocked JAK2/STAT3 pathway. Moreover, overexpression of JAK2 attenuated the inhibition of miR-204 on tumor angiogenesis of HNSCC. Furthermore, overexpression of miR-204 enhanced sensitivity of cetuximab in HNSCC cells, this effect was attenuated by JAK2 overexpression too. Importantly, JAK2 expression was negatively correlated with miR-204 level in HNSCC tissues. Therefore, miR-204 acts as a tumor suppressor by blocking JAK2/STAT3 pathway in HNSCC cells. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Discovery and characterization of LY2784544, a small-molecule tyrosine kinase inhibitor of JAK2V617F

International Nuclear Information System (INIS)

Ma, L; Clayton, J R; Walgren, R A; Zhao, B; Evans, R J; Smith, M C; Heinz-Taheny, K M; Kreklau, E L; Bloem, L; Pitou, C; Shen, W; Strelow, J M; Halstead, C; Rempala, M E; Parthasarathy, S; Gillig, J R; Heinz, L J; Pei, H; Wang, Y; Stancato, L F; Dowless, M S; Iversen, P W; Burkholder, T P

2013-01-01

Owing to the prevalence of the JAK2V617F mutation in myeloproliferative neoplasms (MPNs), its constitutive activity, and ability to recapitulate the MPN phenotype in mouse models, JAK2V617F kinase is an attractive therapeutic target. We report the discovery and initial characterization of the orally bioavailable imidazopyridazine, LY2784544, a potent, selective and ATP-competitive inhibitor of janus kinase 2 (JAK2) tyrosine kinase. LY2784544 was discovered and characterized using a JAK2-inhibition screening assay in tandem with biochemical and cell-based assays. LY2784544 in vitro selectivity for JAK2 was found to be equal or superior to known JAK2 inhibitors. Further studies showed that LY2784544 effectively inhibited JAK2V617F-driven signaling and cell proliferation in Ba/F3 cells (IC 50 =20 and 55 nM, respectively). In comparison, LY2784544 was much less potent at inhibiting interleukin-3-stimulated wild-type JAK2-mediated signaling and cell proliferation (IC 50 =1183 and 1309 nM, respectively). In vivo, LY2784544 effectively inhibited STAT5 phosphorylation in Ba/F3-JAK2V617F-GFP (green fluorescent protein) ascitic tumor cells (TED 50 =12.7 mg/kg) and significantly reduced (P<0.05) Ba/F3-JAK2V617F-GFP tumor burden in the JAK2V617F-induced MPN model (TED 50 =13.7 mg/kg, twice daily). In contrast, LY2784544 showed no effect on erythroid progenitors, reticulocytes or platelets. These data suggest that LY2784544 has potential for development as a targeted agent against JAK2V617F and may have properties that allow suppression of JAK2V617F-induced MPN pathogenesis while minimizing effects on hematopoietic progenitor cells

JANEX-1, a JAK3 inhibitor, protects pancreatic islets from cytokine toxicity through downregulation of NF-{kappa}B activation and the JAK/STAT pathway

Energy Technology Data Exchange (ETDEWEB)

Lv, Na; Kim, Eun-Kyung; Song, Mi-Young [Department of Biochemistry, Medical School and Diabetes Research Center, Chonbuk National University, Jeonju, Jeonbuk 561-756 (Korea, Republic of); Choi, Ha-Na; Moon, Woo Sung [Department of Pathology, Medical School and Diabetes Research Center, Chonbuk National University, Jeonju, Jeonbuk 561-756 (Korea, Republic of); Park, Sung-Joo [Department of Herbology, School of Oriental Medicine, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Park, Jin-Woo [Department of Biochemistry, Medical School and Diabetes Research Center, Chonbuk National University, Jeonju, Jeonbuk 561-756 (Korea, Republic of); Kwon, Kang-Beom, E-mail: desson@wonkwang.ac.kr [Department of Physiology, School of Oriental Medicine, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Park, Byung-Hyun, E-mail: bhpark@chonbuk.ac.kr [Department of Biochemistry, Medical School and Diabetes Research Center, Chonbuk National University, Jeonju, Jeonbuk 561-756 (Korea, Republic of)

2009-07-15

JANEX-1/WHI-P131, a selective Janus kinase 3 (JAK3) inhibitor, has been shown to delay the onset of diabetes in the NOD mouse model. However, the molecular mechanism by which JANEX-1 protects pancreatic {beta}-cells is unknown. In the current study, we investigated the role of JANEX-1 on interleukin (IL)-1{beta} and interferon (IFN)-{gamma}-induced {beta}-cell damage using isolated islets. JANEX-1-pretreated islets showed resistance to cytokine toxicity, namely suppressed nitric oxide (NO) production, reduced inducible form of NO synthase (iNOS) expression, and decreased islet destruction. The molecular mechanism by which JANEX-1 inhibits iNOS expression was mediated through suppression of the nuclear factor {kappa}B (NF-{kappa}B) and JAK/signal transducer and activator of transcription (STAT) pathways. Islets treated with the cytokines downregulated the protein levels of suppressor of cytokine signaling (SOCS)-1 and SOCS-3, but pretreatment with JANEX-1 attenuated these decreases. Additionally, islets from JAK3{sup -/-} mice were more resistant to cytokine toxicity than islets from control mice. These results demonstrate that JANEX-1 protects {beta}-cells from cytokine toxicity through suppression of the NF-{kappa}B and JAK/STAT pathways and upregulation of SOCS proteins, suggesting that JANEX-1 may be used to preserve functional {beta}-cell mass.

Upregulation of miR-375 level ameliorates morphine analgesic tolerance in mouse dorsal root ganglia by inhibiting the JAK2/STAT3 pathway

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Li HQ

2017-05-01

Full Text Available Haiqin Li, Rong Tao, Jing Wang, Lingjie Xia Department of Clinical Pain, The People’s Hospital of Henan Province, Zhengzhou, People’s Republic of China Abstract: Several lines of evidence indicate that microRNAs (miRNAs modulate tolerance to the analgesic effects of morphine via regulation of pain-related genes, making dysregulation of miRNA levels a clinical target for controlling opioid tolerance. However, the precise mechanisms by which miRNAs regulate opioid tolerance are unclear. In the present study, we noted that the miR-375 level was downregulated but the expression of Janus kinase 2 (JAK2 was upregulated in mouse dorsal root ganglia (DRG following chronic morphine treatment. The miR-375 levels and JAK2 expression were correlated with the progression of morphine tolerance, and upregulation of miR-375 level could significantly hinder morphine tolerance. This was ameliorated by JAK2 knockdown. Prolonged morphine exposure induced the expression of brain-derived neurotrophic factor (BDNF in a time-dependent manner in the DRG. This was regulated by the miR-375 and JAK2–signal transducer and activator of transcription 3 (STAT3 pathway, and inhibition of this pathway decreased BDNF production, and thus, attenuated morphine tolerance. More importantly, we found that miR-375 could target JAK2 and increase BDNF expression in a JAK2/STAT3 pathway-dependent manner. Keywords: morphine tolerance, miR-375, JAK2, BDNF

Novel mechanisms to inhibit HIV reservoir seeding using Jak inhibitors.

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Christina Gavegnano

2017-12-01

Full Text Available Despite advances in the treatment of HIV infection with ART, elucidating strategies to overcome HIV persistence, including blockade of viral reservoir establishment, maintenance, and expansion, remains a challenge. T cell homeostasis is a major driver of HIV persistence. Cytokines involved in regulating homeostasis of memory T cells, the major hub of the HIV reservoir, trigger the Jak-STAT pathway. We evaluated the ability of tofacitinib and ruxolitinib, two FDA-approved Jak inhibitors, to block seeding and maintenance of the HIV reservoir in vitro. We provide direct demonstration for involvement of the Jak-STAT pathway in HIV persistence in vivo, ex vivo, and in vitro; pSTAT5 strongly correlates with increased levels of integrated viral DNA in vivo, and in vitro Jak inhibitors reduce the frequency of CD4+ T cells harboring integrated HIV DNA. We show that Jak inhibitors block viral production from infected cells, inhibit γ-C receptor cytokine (IL-15-induced viral reactivation from latent stores thereby preventing transmission of infectious particles to bystander activated T cells. These results show that dysregulation of the Jak-STAT pathway is associated with viral persistence in vivo, and that Jak inhibitors target key events downstream of γ-C cytokine (IL-2, IL-7 and IL-15 ligation to their receptors, impacting the magnitude of the HIV reservoir in all memory CD4 T cell subsets in vitro and ex vivo. Jak inhibitors represent a therapeutic modality to prevent key events of T cell activation that regulate HIV persistence and together, specific, potent blockade of these events may be integrated to future curative strategies.

Jak3- and JNK-dependent vascular endothelial growth factor expression in cutaneous T-cell lymphoma

DEFF Research Database (Denmark)

Krejsgaard, T; Vetter-Kauczok, C S; Woetmann, A

2006-01-01

with small interfering RNA has no effect, whereas curcumin, an inhibitor of both Jak3 and the JNKs, almost completely blocks the VEGF production. In conclusion, we provide evidence of VEGF production in CTCL, which is promoted by aberrant activation of Jak3 and the JNKs. Inhibition of VEGF-inducing pathways...

Dexmedetomidine reduces the neuronal apoptosis related to cardiopulmonary bypass by inhibiting activation of the JAK2–STAT3 pathway

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Chen Y

2017-09-01

Full Text Available Yanhua Chen,1,* Xu Zhang,2,* Bingdong Zhang,1 Guodong He,2 Lifang Zhou,2 Yubo Xie2 1Department of Anesthesiology, Cardiovascular Institute, 2Department of Anesthesiology, the First Affiliated Hospital of Guangxi Medical University, Nanning, China *These authors contributed equally to this work Abstract: Cardiopulmonary bypass (CPB constitutes one of the primary methodologies pertaining to cardiac surgery. However, this form of surgery can cause damage to the body. Many studies have reported that dexmedetomidine confers cerebral protection. In this study, we aimed to investigate the effect and mechanism of dexmedetomidine on neuronal apoptosis caused by CPB. Here, rats were treated with different doses of dexmedetomidine by intravenous infusion 2 hours after CPB. We observed that dexmedetomidine treatment to rats reduces the S100ß, NSE levels in plasma, and neuronal apoptosis following CPB in a dose-dependent manner. Furthermore, we observed that the beneficial effect of dexmedetomidine treatment following CPB was associated with a reduction in IL6, an inflammatory cytokine in plasma and cortex. Our results suggest that dexmedetomidine provides neuroprotective effects by inhibiting inflammation and reducing neuronal apoptosis. There was a correlation between the protective effect on the brain and the dose of dexmedetomidine. In addition, dexmedetomidine administration inhibits phosphorylation of JAK2 and STAT3 proteins in the hippocampus of rats 2 hours after CPB. Therefore, we speculate that the JAK2–STAT3 pathway plays an important role in the neuroprotective effects of dexmedetomidine following brain injury induced by CPB. Keywords: apoptosis, cardiopulmonary bypass, dexmedetomidine, neuroprotective effect, JAK2, STAT3

Cancer gene profiling in non-small cell lung cancers reveals activating mutations in JAK2 and JAK3 with therapeutic implications

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Shuyu D. Li

2017-10-01

Full Text Available Abstract Background Next-generation sequencing (NGS of cancer gene panels are widely applied to enable personalized cancer therapy and to identify novel oncogenic mutations. Methods We performed targeted NGS on 932 clinical cases of non-small-cell lung cancers (NSCLCs using the Ion AmpliSeq™ Cancer Hotspot panel v2 assay. Results Actionable mutations were identified in 65% of the cases with available targeted therapeutic options, including 26% of the patients with mutations in National Comprehensive Cancer Network (NCCN guideline genes. Most notably, we discovered JAK2 p.V617F somatic mutation, a hallmark of myeloproliferative neoplasms, in 1% (9/932 of the NSCLCs. Analysis of cancer cell line pharmacogenomic data showed that a high level of JAK2 expression in a panel of NSCLC cell lines is correlated with increased sensitivity to a selective JAK2 inhibitor. Further analysis of TCGA genomic data revealed JAK2 gain or loss due to genetic alterations in NSCLC clinical samples are associated with significantly elevated or reduced PD-L1 expression, suggesting that the activating JAK2 p.V617F mutation could confer sensitivity to both JAK inhibitors and anti-PD1 immunotherapy. We also detected JAK3 germline activating mutations in 6.7% (62/932 of the patients who may benefit from anti-PD1 treatment, in light of recent findings that JAK3 mutations upregulate PD-L1 expression. Conclusion Taken together, this study demonstrated the clinical utility of targeted NGS with a focused hotspot cancer gene panel in NSCLCs and identified activating mutations in JAK2 and JAK3 with clinical implications inferred through integrative analysis of cancer genetic, genomic, and pharmacogenomic data. The potential of JAK2 and JAK3 mutations as response markers for the targeted therapy against JAK kinases or anti-PD1 immunotherapy warrants further investigation.

Total glucosides of paeony regulates JAK2/STAT3 activation and macrophage proliferation in diabetic rat kidneys.

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Wang, Kun; Wu, Yong-Gui; Su, Jing; Zhang, Jing-Jing; Zhang, Pei; Qi, Xiang-Ming

2012-01-01

Total glucosides of paeony (TGP) is the major active constituent of Paeonia lactiflora Pall., which has shown renoprotection in experimental diabetic nephropathy. Activation of Janus kinase/signal transducers and activators of transcription (JAK/STAT) is an important mechanism by which hyperglycemia contributes to renal damage. Macrophages also play an essential role in the pathogenesis of diabetic nephropathy. Herein, we investigated the ability of TGP to modulate JAK2/STAT3 activation and macrophage proliferation in rats with streptozotocin (STZ)-induced diabetes. TGP (50, 100, and 200 mg/kg) was administered orally once a day for eight weeks. Levels of p-JAK2 and p-STAT3 were determined by Western blot analysis. Immunohistochemistry and double immunohistochemistry were used to identify p-STAT3, ED-1, PCNA/ED-1, and p-STAT3/ED-1-positive (+) cells. The elevated 24-h urinary albumin excretion rate was markedly attenuated by treatment with 50, 100, and 200 mg/kg TGP. Western blot analysis showed that the significantly increased levels of p-JAK2, p-STAT3 proteins in the kidneys of diabetic rats were significantly inhibited by 50, 100, and 200 mg/kg TGP treatment. The marked accumulation and proliferation of macrophages in diabetic kidneys were significantly inhibited by TGP treatment. ED-1+/p-STAT3+ cells were significantly increased in the kidneys from the model group but were significantly inhibited by TGP treatment. These results show that TGP significantly inhibited diabetic nephropathy progression and suggest that these protective effects are associated with the ability of TGP to inhibit the JAK2/STAT3 pathway and macrophage proliferation and action.

Involvement of JAK2 upstream of the PI 3-kinase in cell-cell adhesion regulation by gastrin

International Nuclear Information System (INIS)

Ferrand, Audrey; Kowalski-Chauvel, Aline; Bertrand, Claudine; Pradayrol, Lucien; Fourmy, Daniel; Dufresne, Marlene; Seva, Catherine

2004-01-01

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway has been implicated in cell transformation and proliferation. Besides aberrant cell proliferation, loss of cell-cell adhesion during epithelial-mesenchymal transition (EMT) is an important event which occurs during development of epithelial cancers. However, the role of JAK-dependent pathways in this process is not known. We analyzed the involvement of these pathways in the regulation of E-cadherin-dependent cell-cell adhesion by gastrin, a mitogenic factor for gastrointestinal (GI) tract. We identified JAK2/STAT3 as a new pathway in gastrin signaling. We demonstrated that JAK2 functions as an upstream mediator of the phosphatidylinositol 3 (PI 3)-kinase activity in gastrin signaling. Indeed, we observed a coprecipitation of both kinases and an inhibition of gastrin-induced PI 3-kinase activation when JAK2 activity is blocked. We also demonstrated that loss of cell-cell adhesion and the increase in cell motility induced by gastrin required the activation of JAK2 and the PI 3-kinase. Indeed, the modifications in localization of adherens junctions proteins and the migration, observed in gastrin-stimulated cells, were reversed by inhibition of both kinases. These results described the involvement of JAK2 in the modulation of cell-cell adhesion in epithelial cells. They support a possible role of JAK2 in the epithelial-mesenchymal transition which occurs during malignant development

Simultaneous inhibition of JAK and SYK kinases ameliorates chronic and destructive arthritis in mice

NARCIS (Netherlands)

Llop-Guevara, A.; Porras, M.; Cendon, C.; Ceglie, I. Di; Siracusa, F.; Madarena, F.; Rinotas, V.; Gomez, L.; Lent, P.L.E.M. van; Douni, E.; Chang, H.D.; Kamradt, T.; Roman, J.

2015-01-01

INTRODUCTION: Despite the broad spectrum of antirheumatic drugs, RA is still not well controlled in up to 30-50 % of patients. Inhibition of JAK kinases by means of the pan-JAK inhibitor tofacitinib has demonstrated to be effective even in difficult-to-treat patients. Here, we discuss whether the

Efficacy of NS-018, a potent and selective JAK2/Src inhibitor, in primary cells and mouse models of myeloproliferative neoplasms.

Science.gov (United States)

Nakaya, Y; Shide, K; Niwa, T; Homan, J; Sugahara, S; Horio, T; Kuramoto, K; Kotera, T; Shibayama, H; Hori, K; Naito, H; Shimoda, K

2011-07-01

Aberrant activation of Janus kinase 2 (JAK2) caused by somatic mutation of JAK2 (JAK2V617F) or the thrombopoietin receptor (MPLW515L) plays an essential role in the pathogenesis of myeloproliferative neoplasms (MPNs), suggesting that inhibition of aberrant JAK2 activation would have a therapeutic benefit. Our novel JAK2 inhibitor, NS-018, was highly active against JAK2 with a 50% inhibition (IC(50)) of MPLW515L mutations or the TEL-JAK2 fusion gene; IC(50)=11-120 n), but showed only minimal cytotoxicity against most other hematopoietic cell lines without a constitutively activated JAK2. Furthermore, NS-018 preferentially suppressed in vitro erythropoietin-independent endogenous colony formation from polycythemia vera patients. NS-018 also markedly reduced splenomegaly and prolonged the survival of mice inoculated with Ba/F3 cells harboring JAK2V617F. In addition, NS-018 significantly reduced leukocytosis, hepatosplenomegaly and extramedullary hematopoiesis, improved nutritional status, and prolonged survival in JAK2V617F transgenic mice. These results suggest that NS-018 will be a promising candidate for the treatment of MPNs.

Combined Targeting of JAK2 and Bcl-2/Bcl-xL to Cure Mutant JAK2-Driven Malignancies and Overcome Acquired Resistance to JAK2 Inhibitors

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Michaela Waibel

2013-11-01

Full Text Available To design rational therapies for JAK2-driven hematological malignancies, we functionally dissected the key survival pathways downstream of hyperactive JAK2. In tumors driven by mutant JAK2, Stat1, Stat3, Stat5, and the Pi3k and Mek/Erk pathways were constitutively active, and gene expression profiling of TEL-JAK2 T-ALL cells revealed the upregulation of prosurvival Bcl-2 family genes. Combining the Bcl-2/Bcl-xL inhibitor ABT-737 with JAK2 inhibitors mediated prolonged disease regressions and cures in mice bearing primary human and mouse JAK2 mutant tumors. Moreover, combined targeting of JAK2 and Bcl-2/Bcl-xL was able to circumvent and overcome acquired resistance to single-agent JAK2 inhibitor treatment. Thus, inhibiting the oncogenic JAK2 signaling network at two nodal points, at the initiating stage (JAK2 and the effector stage (Bcl-2/Bcl-xL, is highly effective and provides a clearly superior therapeutic benefit than targeting just one node. Therefore, we have defined a potentially curative treatment for hematological malignancies expressing constitutively active JAK2.

The Influence of Compound Shougong Powder on JAK2-STAT3 Signaling Pathway in Mice with Lewis Lung Cancer

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SHEN Di

2014-12-01

Full Text Available Objective: To observe the influence of Compound Shougong Powder on JAK2-STAT3 signaling pathway in mice with Lewis lung cancer. Methods: Fifty C57BL/6J mice were inoculated with Lewis lung cancer cell line according to the conventional method, 40 mice bearing cancer successfully were selected 6 d later and randomly divided into 5groups, namely negative control group, cis-platinum group, high-dose Compound Shougong Powder group, middle-dose Compound Shougong Powder group and low-dose Compound Shougong Powder group, 8 mice in each group. Negative control group was drenched with normal saline (NS. Compound Shougong Powder groups were drenched with Compound Shougong Powder, 4 mg/kg for high-dose group, 2 mg/kg for middle-dose group, 1 mg/kg for low-dose group, once per day for 14 d; cis-platinum group was orally administrated 4 mg/kg/w, intraperitoneal injection of 0.1 mL for each, once per week for 2 weeks. Mice’s responses to the treatment, activity levels, mental states and so on during the treatment were observed, tumor inhibition rate was calculated, pathomorphological changes of tumor tissues were observed under light microscope after HE staining, and the expression levels of JAK2 and STAT3 proteins were detected by Western Blot. Results: After drug administration, smooth, glossy body hair and good spirit were observed in cisplatin group and high-dose Compound Shougong Powder group; glossier body hair and less activity level in middle- and low- dose Compound Shougong Powder group, and great toxic and side effects, reduced activity level and weary spirit in negative control group. The tumor inhibition rate of cisplatin group, high-, middle- and low-dose Compound Shougong Powder group and negative control group was 57.69%, 53.53%, 48.40%, 38.46% and 38.46%, respectively. The expression levels of JAK2 and STAT3 proteins in drug groups showed decreases to different degrees, and the decreases of JAK2 were more significant. Conclusion: Compound

Aspirin down Regulates Hepcidin by Inhibiting NF-κB and IL6/JAK2/STAT3 Pathways in BV-2 Microglial Cells Treated with Lipopolysaccharide

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Wan-Ying Li

2016-12-01

Full Text Available Aspirin down regulates transferrin receptor 1 (TfR1 and up regulates ferroportin 1 (Fpn1 and ferritin expression in BV-2 microglial cells treated without lipopolysaccharides (LPS, as well as down regulates hepcidin and interleukin 6 (IL-6 in cells treated with LPS. However, the relevant mechanisms are unknown. Here, we investigate the effects of aspirin on expression of hepcidin and iron regulatory protein 1 (IRP1, phosphorylation of Janus kinase 2 (JAK2, signal transducer and activator of transcription 3 (STAT3 and P65 (nuclear factor-κB, and the production of nitric oxide (NO in BV-2 microglial cells treated with and without LPS. We demonstrated that aspirin inhibited hepcidin mRNA as well as NO production in cells treated with LPS, but not in cells without LPS, suppresses IL-6, JAK2, STAT3, and P65 (nuclear factor-κB phosphorylation and has no effect on IRP1 in cells treated with or without LPS. These findings provide evidence that aspirin down regulates hepcidin by inhibiting IL6/JAK2/STAT3 and P65 (nuclear factor-κB pathways in the cells under inflammatory conditions, and imply that an aspirin-induced reduction in TfR1 and an increase in ferritin are not associated with IRP1 and NO.

A natural product-like JAK2/STAT3 inhibitor induces apoptosis of malignant melanoma cells.

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Ke-Jia Wu

Full Text Available The JAK2/STAT3 signaling pathway plays a critical role in tumorigenesis, and has been suggested as a potential molecular target for anti-melanoma therapeutics. However, few JAK2 inhibitors were being tested for melanoma therapy. In this study, eight amentoflavone analogues were evaluated for their activity against human malignant melanoma cells. The most potent analogue, compound 1, inhibited the phosphorylation of JAK2 and STAT3 in human melanoma cells, but had no discernible effect on total JAK2 and STAT3 levels. A cellular thermal shift assay was performed to identify that JAK2 is engaged by 1 in cell lysates. Moreover, compound 1 showed higher antiproliferative activity against human melanoma A375 cells compared to a panel of cancer and normal cell lines. Compound 1 also activated caspase-3 and cleaved PARP, which are markers of apoptosis, and suppressed the anti-apoptotic Bcl-2 level. Finally, compound 1 induced apoptosis in 80% of treated melanoma cells. To our knowledge, compound 1 is the first amentoflavone-based JAK2 inhibitor to be investigated for use as an anti-melanoma agent.

Epigallocatechin-3-gallate Ameliorates Seawater Aspiration-Induced Acute Lung Injury via Regulating Inflammatory Cytokines and Inhibiting JAK/STAT1 Pathway in Rats

Science.gov (United States)

Liu, Wei; Dong, Mingqing; Bo, Liyan; Li, Congcong; Liu, Qingqing; Li, Yanyan; Ma, Lijie; Xie, Yonghong; Fu, Enqing; Mu, Deguang; Pan, Lei; Jin, Faguang; Li, Zhichao

2014-01-01

Signal transducers and activators of transcriptions 1 (STAT1) play an important role in the inflammation process of acute lung injury (ALI). Epigallocatechin-3-gallate (EGCG) exhibits a specific and strong anti-STAT1 activity. Therefore, our study is to explore whether EGCG pretreatment can ameliorate seawater aspiration-induced ALI and its possible mechanisms. We detected the arterial partial pressure of oxygen, lung wet/dry weight ratios, protein content in bronchoalveolar lavage fluid, and the histopathologic and ultrastructure staining of the lung. The levels of IL-1, TNF-α, and IL-10 and the total and the phosphorylated protein level of STAT1, JAK1, and JAK2 were assessed in vitro and in vivo. The results showed that EGCG pretreatment significantly improved hypoxemia and histopathologic changes, alleviated pulmonary edema and lung vascular leak, reduced the production of TNF-α and IL-1, and increased the production of IL-10 in seawater aspiration-induced ALI rats. EGCG also prevented the seawater aspiration-induced increase of TNF-α and IL-1 and decrease of IL-10 in NR8383 cell line. Moreover, EGCG pretreatment reduced the total and the phosphorylated protein level of STAT1 in vivo and in vitro and reduced the phosphorylated protein level of JAK1 and JAK2. The present study demonstrates that EGCG ameliorates seawater aspiration-induced ALI via regulating inflammatory cytokines and inhibiting JAK/STAT1 pathway in rats. PMID:24692852

Improved efficacy of allergen-specific immunotherapy by JAK inhibition in a murine model of allergic asthma

DEFF Research Database (Denmark)

Aguilar-Pimentel, Juan Antonio; Graessel, Anke; Alessandrini, Francesca

2017-01-01

)-induced allergic airway inflammation and allergen-specific immunotherapy was combined with the administration of Tofacitinib (TOFA, a FDA-approved JAK inhibitor) from 48 hours prior to 48 hours after therapeutic OVA-injection. The effect of TOFA on human FOXP3+CD4+ T cells was studied in vitro. RESULTS: AIT...... combined with short-term TOFA administration was significantly more effective in suppressing total cell and eosinophil infiltration into the lung, local cytokine production including IL-1β and CXCL1 and showed a trend for the reduction of IL-4, IL-13, TNF-α and IL-6 compared to AIT alone. Furthermore, TOFA...... co-administration significantly reduced systemic IL-6, IL-1β and OVA-specific IgE levels and induced IgG1 to the same extent as AIT alone. Additionally, TOFA enhanced the induction of human FOXP3+CD4+ T cells. CONCLUSIONS: This proof of concept study shows that JAK inhibition did not inhibit...

Inhibitory effects of a selective Jak2 inhibitor on adrenocorticotropic hormone production and proliferation of corticotroph tumor AtT20 cells

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Asari Y

2017-09-01

Full Text Available Yuko Asari, Kazunori Kageyama, Yuki Nakada, Mizuki Tasso, Shinobu Takayasu, Kanako Niioka, Noriko Ishigame, Makoto Daimon Department of Endocrinology and Metabolism, Graduate School of Medicine, Hirosaki University, Hirosaki, Japan Purpose: The primary cause of Cushing’s disease is adrenocorticotropic hormone (ACTH-producing pituitary adenomas. EGFR signaling induces POMC mRNA-transcript levels and ACTH secretion from corticotroph tumors. The Jak–STAT pathway is located downstream of EGFR signaling; therefore, a Jak2 inhibitor could be an effective therapy for EGFR-related tumors. In this study, we determined the effect of a potent and selective Jak2 inhibitor, SD1029, on ACTH production and proliferation in mouse AtT20 corticotroph tumor cells.Materials and methods: AtT20 pituitary corticotroph tumor cells were cultured after transfection with PTTG1- or GADD45β-specific siRNA. Expression levels of mouse POMC, PTTG1, and GADD45β mRNAs were evaluated using quantitative real-time polymerase chain reaction. ACTH levels were measured using ACTH ELISA. Western blot analysis was performed to examine protein expression of phosphorylated STAT3/STAT3. Viable cells and DNA fragmentation were measured using a cell-proliferation assay and cell-death detection ELISA, respectively. Cellular DNA content was analyzed using fluorescence-activated cell sorting.Results: SD1029 decreased POMC and PTTG1 mRNA and ACTH levels, while increasing GADD45β levels. The drug also decreased AtT20-cell proliferation and induced apoptosis, but did not alter cell-cycle progression. SD1029 also inhibited STAT3 phosphorylation. PTTG1 knockdown inhibited POMC mRNA levels and cell proliferation. However, combined treatment with PTTG1 knockdown and SD1029 had no additive effect on POMC mRNA levels or cell proliferation. GADD45β knockdown inhibited the SD1029-induced decrease in POMC mRNA levels and also partially inhibited the decrease in cell proliferation.Conclusion: Both

Arctigenin inhibits lipopolysaccharide-induced iNOS expression in RAW264.7 cells through suppressing JAK-STAT signal pathway.

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Kou, Xianjuan; Qi, Shimei; Dai, Wuxing; Luo, Lan; Yin, Zhimin

2011-08-01

Arctigenin has been demonstrated to have an anti-inflammatory function, but the precise mechanisms of its action remain to be fully defined. In the present study, we determined the effects of arctigenin on lipopolysaccharide (LPS)-induced production of proinflammatory mediators and the underlying mechanisms involved in RAW264.7 cells. Our results indicated that arctigenin exerted its anti-inflammatory effect by inhibiting ROS-dependent STAT signaling through its antioxidant activity. Arctigenin also significantly reduced the phosphorylation of STAT1 and STAT 3 as well as JAK2 in LPS-stimulated RAW264.7 cells. The inhibitions of STAT1 and STAT 3 by arctigenin prevented their translocation to the nucleus and consequently inhibited expression of iNOS, thereby suppressing the expression of inflammation-associated genes, such as IL-1β, IL-6 and MCP-1, whose promoters contain STAT-binding elements. However, COX-2 expression was slightly inhibited at higher drug concentrations (50 μM). Our data demonstrate that arctigenin inhibits iNOS expression via suppressing JAK-STAT signaling pathway in macrophages. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

Inhibition of the JAK2/STAT3/SOSC1 Signaling Pathway Improves Secretion Function of Vascular Endothelial Cells in a Rat Model of Pregnancy-Induced Hypertension

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Jian-Ying Luo

2016-11-01

Full Text Available Background/Aims: The present study aimed to investigate the effects of the JAK2/STAT3/SOSC1 signaling pathway on the secretion function of vascular endothelial cells (VECs in a rat model of pregnancy-induced hypertension (PIH. Methods: A PIH rat model was established. Forty-eight pregnant Sprague-Dawley female rats were selected and assigned into four groups: the normal group (normal non-pregnant rats, the non-PIH group (pregnant rats without PIH, the PIH group (pregnant rats with PIH and the AG490 group (pregnant rats with PIH treated with AG490. Systolic blood pressure (SBP and urinary protein (UP were measured. The expressions of JAK2/STAT3/SOSC1 signaling pathway-related proteins in placenta tissues were detect by Western blotting. Radioimmunoassay was applied to detect serum levels of nitric oxide (NO, super oxide dismutase (SOD, placental growth factor (PGF, thromboxane B2 (TXB2 and endothelin (ET. Enzyme-linked immunosorbent assay (ELISA was used to determine serum levels of interleukin-6 (IL-6, interleukin-10 (IL-10 and tumor necrosis factor-α (TNF-α. Results: Compared with the normal and non-PIH groups, the PIH and AG490 groups had higher SBP and UP levels at 17th and 25th day of pregnancy. The expressions of p/t-JAK2, p/t-STAT3 and SOSC1 in the PIH and AG490 groups were higher than those in the non-PIH group, while the expressions of p/t-JAK2, p/t-STAT3 and SOSC1 in the AG490 group were lower than those in the PIH group. Compared with the non-PIH group, serum levels of ET, TXB2, IL-6 and TNF-α were increased in the PIH and AG490 groups, while serum levels of NO, SOD, 6-keto-PGF1a and IL-10 levels were reduced. Furthermore, the AG490 had lower serum levels of ET, TXB2, IL-6 and TNF-α and higher serum levels of NO, SOD, 6-keto-PGF1a and IL-10 than those in the PIH group. Conclusion: Our study provides evidence that inhibition of the JAK2/STAT3/SOSC1 signaling pathway could improve the secretion function of VECs in PIH rats.

The IL-6/JAK/Stat3 feed-forward loop drives tumorigenesis and metastasis.

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Chang, Qing; Bournazou, Eirini; Sansone, Pasquale; Berishaj, Marjan; Gao, Sizhi Paul; Daly, Laura; Wels, Jared; Theilen, Till; Granitto, Selena; Zhang, Xinmin; Cotari, Jesse; Alpaugh, Mary L; de Stanchina, Elisa; Manova, Katia; Li, Ming; Bonafe, Massimiliano; Ceccarelli, Claudio; Taffurelli, Mario; Santini, Donatella; Altan-Bonnet, Gregoire; Kaplan, Rosandra; Norton, Larry; Nishimoto, Norihiro; Huszar, Dennis; Lyden, David; Bromberg, Jacqueline

2013-07-01

We have investigated the importance of interleukin-6 (IL-6) in promoting tumor growth and metastasis. In human primary breast cancers, increased levels of IL-6 were found at the tumor leading edge and positively correlated with advanced stage, suggesting a mechanistic link between tumor cell production of IL-6 and invasion. In support of this hypothesis, we showed that the IL-6/Janus kinase (JAK)/signal transducer and activator of transcription 3 (Stat3) pathway drives tumor progression through the stroma and metastatic niche. Overexpression of IL-6 in tumor cell lines promoted myeloid cell recruitment, angiogenesis, and induced metastases. We demonstrated the therapeutic potential of interrupting this pathway with IL-6 receptor blockade or by inhibiting its downstream effectors JAK1/2 or Stat3. These clinically relevant interventions did not inhibit tumor cell proliferation in vitro but had profound effects in vivo on tumor progression, interfering broadly with tumor-supportive stromal functions, including angiogenesis, fibroblast infiltration, and myeloid suppressor cell recruitment in both the tumor and pre-metastatic niche. This study provides the first evidence for IL-6 expression at the leading edge of invasive human breast tumors and demonstrates mechanistically that IL-6/JAK/Stat3 signaling plays a critical and pharmacologically targetable role in orchestrating the composition of the tumor microenvironment that promotes growth, invasion, and metastasis.

The IL-6/JAK/Stat3 Feed-Forward Loop Drives Tumorigenesis and Metastasis

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Qing Chang

2013-07-01

Full Text Available We have investigated the importance of interleukin-6 (IL-6 in promoting tumor growth and metastasis. In human primary breast cancers, increased levels of IL-6 were found at the tumor leading edge and positively correlated with advanced stage, suggesting a mechanistic link between tumor cell production of IL-6 and invasion. In support of this hypothesis, we showed that the IL-6/Janus kinase (JAK/signal transducer and activator of transcription 3 (Stat3 pathway drives tumor progression through the stroma and metastatic niche. Overexpression of IL-6 in tumor cell lines promoted myeloid cell recruitment, angiogenesis, and induced metastases. We demonstrated the therapeutic potential of interrupting this pathway with IL-6 receptor blockade or by inhibiting its downstream effectors JAK1/2 or Stat3. These clinically relevant interventions did not inhibit tumor cell proliferation in vitro but had profound effects in vivo on tumor progression, interfering broadly with tumor-supportive stromal functions, including angiogenesis, fibroblast infiltration, and myeloid suppressor cell recruitment in both the tumor and pre-metastatic niche. This study provides the first evidence for IL-6 expression at the leading edge of invasive human breast tumors and demonstrates mechanistically that IL-6/JAK/Stat3 signaling plays a critical and pharmacologically targetable role in orchestrating the composition of the tumor microenvironment that promotes growth, invasion, and metastasis.

Hierarchy of protein tyrosine kinases in interleukin-2 (IL-2) signaling: activation of syk depends on Jak3; however, neither Syk nor Lck is required for IL-2-mediated STAT activation.

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Zhou, Y J; Magnuson, K S; Cheng, T P; Gadina, M; Frucht, D M; Galon, J; Candotti, F; Geahlen, R L; Changelian, P S; O'Shea, J J

2000-06-01

Interleukin-2 (IL-2) activates several different families of tyrosine kinases, but precisely how these kinases interact is not completely understood. We therefore investigated the functional relationships among Jak3, Lck, and Syk in IL-2 signaling. We first observed that in the absence of Jak3, both Lck and Syk had the capacity to phosphorylate Stat3 and Stat5a. However, neither supported IL-2-induced STAT activation, nor did dominant negative alleles of these kinases inhibit. Moreover, pharmacological abrogation of Lck activity did not inhibit IL-2-mediated phosphorylation of Jak3 and Stat5a. Importantly, ligand-dependent Syk activation was dependent on the presence of catalytically active Jak3, whereas Lck activation was not. Interestingly, Syk functioned as a direct substrate of Jak1 but not Jak3. Additionally, Jak3 phosphorylated Jak1, whereas the reverse was not the case. Taken together, our data support a model in which Lck functions in parallel with Jak3, while Syk functions as a downstream element of Jaks in IL-2 signaling. Jak3 may regulate Syk catalytic activity indirectly via Jak1. However, IL-2-mediated Jak3/Stat activation is not dependent on Lck or Syk. While the essential roles of Jak1 and Jak3 in signaling by gammac-utilizing cytokines are clear, it will be important to dissect the exact contributions of Lck and Syk in mediating the effects of IL-2 and related cytokines.

The IL-6/JAK/Stat3 Feed-Forward Loop Drives Tumorigenesis and Metastasis12

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Chang, Qing; Bournazou, Eirini; Sansone, Pasquale; Berishaj, Marjan; Gao, Sizhi Paul; Daly, Laura; Wels, Jared; Theilen, Till; Granitto, Selena; Zhang, Xinmin; Cotari, Jesse; Alpaugh, Mary L; de Stanchina, Elisa; Manova, Katia; Li, Ming; Bonafe, Massimiliano; Ceccarelli, Claudio; Taffurelli, Mario; Santini, Donatella; Altan-Bonnet, Gregoire; Kaplan, Rosandra; Norton, Larry; Nishimoto, Norihiro; Huszar, Dennis; Lyden, David; Bromberg, Jacqueline

2013-01-01

We have investigated the importance of interleukin-6 (IL-6) in promoting tumor growth and metastasis. In human primary breast cancers, increased levels of IL-6 were found at the tumor leading edge and positively correlated with advanced stage, suggesting a mechanistic link between tumor cell production of IL-6 and invasion. In support of this hypothesis, we showed that the IL-6/Janus kinase (JAK)/signal transducer and activator of transcription 3 (Stat3) pathway drives tumor progression through the stroma and metastatic niche. Overexpression of IL-6 in tumor cell lines promoted myeloid cell recruitment, angiogenesis, and induced metastases. We demonstrated the therapeutic potential of interrupting this pathway with IL-6 receptor blockade or by inhibiting its downstream effectors JAK1/2 or Stat3. These clinically relevant interventions did not inhibit tumor cell proliferation in vitro but had profound effects in vivo on tumor progression, interfering broadly with tumor-supportive stromal functions, including angiogenesis, fibroblast infiltration, and myeloid suppressor cell recruitment in both the tumor and pre-metastatic niche. This study provides the first evidence for IL-6 expression at the leading edge of invasive human breast tumors and demonstrates mechanistically that IL-6/JAK/Stat3 signaling plays a critical and pharmacologically targetable role in orchestrating the composition of the tumor microenvironment that promotes growth, invasion, and metastasis. PMID:23814496

JAK inhibitors suppress t(8;21) fusion protein-induced leukemia

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Lo, Miao-Chia; Peterson, Luke F.; Yan, Ming; Cong, Xiuli; Hickman, Justin H.; DeKelver, Russel C.; Niewerth, Denise; Zhang, Dong-Er

2014-01-01

Oncogenic mutations in components of the JAK/STAT pathway, including those in cytokine receptors and JAKs, lead to increased activity of downstream signaling and are frequently found in leukemia and other hematological disorders. Thus, small-molecule inhibitors of this pathway have been the focus of targeted therapy in these hematological diseases. We previously showed that t(8;21) fusion protein AML1-ETO and its alternatively spliced variant AML1-ETO9a (AE9a) enhance the JAK/STAT pathway via down-regulation of CD45, a negative regulator of this pathway. To investigate the therapeutic potential of targeting JAK/STAT in t(8;21) leukemia, we examined the effects of a JAK2-selective inhibitor TG101209 and a JAK1/2-selective inhibitor INCB18424 on t(8;21) leukemia cells. TG101209 and INCB18424 inhibited proliferation and promoted apoptosis of these cells. Furthermore, TG101209 treatment in AE9a leukemia mice reduced tumor burden and significantly prolonged survival. TG101209 also significantly impaired the leukemia-initiating potential of AE9a leukemia cells in secondary recipient mice. These results demonstrate the potential therapeutic efficacy of JAK inhibitors in treating t(8;21) AML. PMID:23812420

Inhibition of the JAK2/STAT3 pathway in ovarian cancer results in the loss of cancer stem cell-like characteristics and a reduced tumor burden

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Abubaker, Khalid; Luwor, Rodney B; Zhu, Hongjian; McNally, Orla; Quinn, Michael A; Burns, Christopher J; Thompson, Erik W; Findlay, Jock K; Ahmed, Nuzhat

2014-01-01

Current treatment of ovarian cancer patients with chemotherapy leaves behind a residual tumor which results in recurrent ovarian cancer within a short time frame. We have previously demonstrated that a single short-term treatment of ovarian cancer cells with chemotherapy in vitro resulted in a cancer stem cell (CSC)-like enriched residual population which generated significantly greater tumor burden compared to the tumor burden generated by control untreated cells. In this report we looked at the mechanisms of the enrichment of CSC-like residual cells in response to paclitaxel treatment. The mechanism of survival of paclitaxel-treated residual cells at a growth inhibitory concentration of 50% (GI50) was determined on isolated tumor cells from the ascites of recurrent ovarian cancer patients and HEY ovarian cancer cell line by in vitro assays and in a mouse xenograft model. Treatment of isolated tumor cells from the ascites of ovarian cancer patients and HEY ovarian cancer cell line with paclitaxel resulted in a CSC-like residual population which coincided with the activation of Janus activated kinase 2 (JAK2) and signal transducer and activation of transcription 3 (STAT3) pathway in paclitaxel surviving cells. Both paclitaxel-induced JAK2/STAT3 activation and CSC-like characteristics were inhibited by a low dose JAK2-specific small molecule inhibitor CYT387 (1 μM) in vitro. Subsequent, in vivo transplantation of paclitaxel and CYT387-treated HEY cells in mice resulted in a significantly reduced tumor burden compared to that seen with paclitaxel only-treated transplanted cells. In vitro analysis of tumor xenografts at protein and mRNA levels demonstrated a loss of CSC-like markers and CA125 expression in paclitaxel and CYT387-treated cell-derived xenografts, compared to paclitaxel only-treated cell-derived xenografts. These results were consistent with significantly reduced activation of JAK2 and STAT3 in paclitaxel and CYT387-treated cell-derived xenografts

Trichostatin A, a histone deacetylase inhibitor, suppresses JAK2/STAT3 signaling via inducing the promoter-associated histone acetylation of SOCS1 and SOCS3 in human colorectal cancer cells.

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Xiong, Hua; Du, Wan; Zhang, Yan-Jie; Hong, Jie; Su, Wen-Yu; Tang, Jie-Ting; Wang, Ying-Chao; Lu, Rong; Fang, Jing-Yuan

2012-02-01

Aberrant janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling is involved in the oncogenesis of several cancers. Suppressors of cytokine signaling (SOCS) genes and SH2-containing protein tyrosine phosphatase 1 (SHP1) proteins, which are negative regulators of JAK/STAT signaling, have been reported to have tumor suppressor functions. However, in colorectal cancer (CRC) cells, the mechanisms that regulate SOCS and SHP1 genes, and the cause of abnormalities in the JAK/STAT signaling pathway, remain largely unknown. The present study shows that trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, leads to the hyperacetylation of histones associated with the SOCS1 and SOCS3 promoters, but not the SHP1 promoter in CRC cells. This indicates that histone modifications are involved in the regulation of SOCS1 and SOCS3. Moreover, upregulation of SOCS1 and SOCS3 expression was achieved using TSA, which also significantly downregulated JAK2/STAT3 signaling in CRC cells. We also demonstrate that TSA suppresses the growth of CRC cells, and induces G1 cell cycle arrest and apoptosis through the regulation of downstream targets of JAK2/STAT3 signaling, including Bcl-2, survivin and p16(ink4a) . Therefore, our data demonstrate that TSA may induce SOCS1 and SOCS3 expression by inducing histone modifications and consequently inhibits JAK2/STAT3 signaling in CRC cells. These results also establish a mechanistic link between the inhibition of JAK2/STAT3 signaling and the anticancer action of TSA in CRC cells. Copyright © 2011 Wiley Periodicals, Inc.

The Jak2 Inhibitor, G6, Alleviates Jak2-V617F–Mediated Myeloproliferative Neoplasia by Providing Significant Therapeutic Efficacy to the Bone Marrow

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Annet Kirabo

2011-11-01

Full Text Available We recently developed a Janus kinase 2 (Jak2 small-molecule inhibitor called G6 and found that it inhibits Jak2-V617F– mediated pathologic cell growth in vitro, ex vivo, and in vivo. However, its ability to inhibit Jak2-V617F–mediated myeloproliferative neoplasia, with particular emphasis in the bone marrow, has not previously been examined. Here, we investigated the efficacy of G6 in a transgenic mouse model of Jak2-V617F–mediated myeloproliferative neoplasia. We found that G6 provided therapeutic benefit to the peripheral blood as determined by elimination of leukocytosis, thrombocytosis, and erythrocytosis. G6 normalized the pathologically high plasma concentrations of interleukin 6 (IL-6. In the liver, G6 eliminated Jak2-V617F–driven extramedullary hematopoiesis. With respect to the spleen, G6 significantly reduced both the spleno-megaly and megakaryocytic hyperplasia. In the critically important bone marrow, G6 normalized the pathologically high levels of phospho-Jak2 and phospho–signal transducer and activator of transcription 5 (STAT5. It significantly reduced the megakaryocytic hyperplasia in the marrow and completely normalized the M/E ratio. Most importantly, G6 selectively reduced the mutant Jak2 burden by 67% on average, with virtual elimination of mutant Jak2 cells in one third of all treated mice. Lastly, clonogenic assays using marrow stem cells from the myeloproliferative neoplasm mice revealed a time-dependent elimination of the clonogenic growth potential of these cells by G6. Collectively, these data indicate that G6 exhibits exceptional efficacy in the peripheral blood, liver, spleen, and, most importantly, in the bone marrow, thereby raising the possibility that this compound may alter the natural history of Jak2-V617F–mediated myeloproliferative neoplasia.

Elevated serum IL-10 levels in diffuse large B-cell lymphoma: a mechanism of aberrant JAK2 activation.

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Gupta, Mamta; Han, Jing Jing; Stenson, Mary; Maurer, Matthew; Wellik, Linda; Hu, Guangzhen; Ziesmer, Steve; Dogan, Ahmet; Witzig, Thomas E

2012-03-22

Cytokines are deregulated in cancers and can contribute to tumor growth. In patients with diffuse large-cell lymphoma (DLBCL), we observed higher levels of JAK/STAT pathway-related serum cytokines (ie, IL-6, IL-10, epidermal growth factor, and IL-2) compared with controls. Of these, only IL-10 activated the JAK2 pathway in lymphoma cells in vitro. Patients with high serum IL-10 had shorter event-free survival (EFS) than patients with low levels (P > .01) and high IL-10 was correlated with high lactase dehydrogenase (P = .0085) and higher International Prognostic Index scores (P = .01). To explore the mechanism by which IL-10 may contribute to an inferior EFS, we investigated the effect of IL-10 on the JAK2 pathway and found that the IL-10/IL-10 receptor complex up-regulated JAK2 signaling. Neutralizing Ab to IL-10 inhibited constitutive and IL-10-induced JAK2/STAT3 phosphorylation. JAK2 inhibition dephosphorylated JAK2 and STAT3 and caused an inhibitory effect on phospho-JAK2-positive DLBCL cells; there was a minimal effect on phospho-JAK2-negative cells. Apoptosis induced by JAK2 inhibition was dependent on inhibition of autocrine IL-10 and c-myc expression and independent of Bcl-2 family expression. These results provide the rationale for testing JAK2 inhibitors in DLBCL patients, and indicate that serum IL-10 may be a biomarker to identify patients more likely to respond to JAK2-targeted therapy.

Ginkgolide B Suppresses TLR4-Mediated Inflammatory Response by Inhibiting the Phosphorylation of JAK2/STAT3 and p38 MAPK in High Glucose-Treated HUVECs

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Kun Chen

2017-01-01

Full Text Available Aim. Ginkgolide B is a Ginkgo biloba leaf extract that has been identified as a natural platelet-activating factor receptor (PAFR antagonist. We investigated the effect of ginkgolide B on high glucose-induced TLR4 activation in human umbilical vein endothelial cells (HUVECs. Methods. Protein expression was analyzed by immunoblotting. Small-interfering RNA (siRNA was used to knock down PAFR and TLR4 expression. Results. Ginkgolide B suppressed the expression of TLR4 and MyD88 that was induced by high glucose. Ginkgolide B also reduced the levels of platelet endothelial cell adhesion molecule-1, interleukin-6, and monocyte chemotactic protein 1. Further, we examined the association between PAFR and TLR4 by coimmunoprecipitation. The result showed that high glucose treatment caused the binding of PAFR and TLR4, whereas ginkgolide B abolished this binding. The functional analysis indicated that PAFR siRNA treatment reduced TLR4 expression, and TLR4 siRNA treatment decreased PAFR expression in high glucose-treated HUVECs, further supporting the coimmunoprecipitation data. Ginkgolide B inhibited the phosphorylation of Janus kinase 2 (JAK2/signal transducer and activator of transcription 3 (STAT3 and p38 mitogen-activated protein kinase (MAPK. Conclusion. Ginkgolide B exerted protective effects by inhibiting the TLR4-mediated inflammatory response in high glucose-treated endothelial cells. The mechanism of action of ginkgolide B might be associated with inhibition of the JAK2/STAT3 and p38 MAPK phosphorylation.

Cell type-specific roles of Jak3 in IL-2-induced proliferative signal transduction

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Fujii, Hodaka

2007-01-01

Binding of interleukin-2 (IL-2) to its specific receptor induces activation of two members of Jak family protein tyrosine kinases, Jak1 and Jak3. An IL-2 receptor (IL-2R)-reconstituted NIH 3T3 fibroblast cell line proliferates in response to IL-2 only when hematopoietic lineage-specific Jak3 is ectopically expressed. However, the mechanism of Jak3-dependent proliferation in the fibroblast cell line is not known. Here, I showed that Jak3 expression is dispensable for IL-2-induced activation of Jak1 and Stat proteins and expression of nuclear proto-oncogenes in the IL-2R-reconstituted fibroblast cell line. Jak3 expression markedly enhanced these IL-2-induced signaling events. In contrast, Jak3 expression was essential for induction of cyclin genes involved in the G1-S transition. These data suggest a critical role of Jak3 in IL-2 signaling in the fibroblast cell line and may provide further insight into the cell type-specific mechanism of cytokine signaling

Cell type-specific roles of Jak3 in IL-2-induced proliferative signal transduction

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Fujii, Hodaka

2007-01-01

Binding of IL-2 to its specific receptor induces activation of two members of Jak family protein tyrosine kinases, Jak1 and Jak3. An IL-2R-reconstituted NIH 3T3 fibroblast cell line proliferates in response to IL-2 only when hematopoietic lineage-specific Jak3 is ectopically expressed. However, the mechanism of Jak3-dependent proliferation in the fibroblast cell line is not known. Here, I showed that Jak3 expression is dispensable for IL-2-induced activation of Jak1 and Stat proteins and expression of nuclear proto-oncogenes in the IL-2R-reconstituted fibroblast cell line. However, Jak3 expression markedly enhanced these IL-2-induced signaling events. In contrast, Jak3 expression was essential for induction of cyclin genes involved in the G1-S transition. These data suggest a critical role of Jak3 in IL-2 signaling in the fibroblast cell line and may provide further insight into the cell type-specific mechanism of cytokine signaling. PMID:17266928

Activation of JAK3, but not JAK1, is critical to interleukin-4 (IL4) stimulated proliferation and requires a membrane-proximal region of IL4 receptor alpha.

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Malabarba, M G; Kirken, R A; Rui, H; Koettnitz, K; Kawamura, M; O'Shea, J J; Kalthoff, F S; Farrar, W L

1995-04-21

The tyrosine kinases JAK1 and JAK3 have been shown to undergo tyrosine phosphorylation in response to interleukin-2 (IL), IL4, IL7, and IL9, cytokines which share the common IL2 receptor gamma-chain (IL2R gamma), and evidence has been found for a preferential coupling of JAK3 to IL2R gamma and JAK1 to IL2R beta. Here we show, using human premyeloid TF-1 cells, that IL4 stimulates JAK3 to a larger extent than JAK1, based upon three different evaluation criteria. These include a more vigorous tyrosine phosphorylation of JAK3 as measured by anti-phosphotyrosine immunoblotting, a more marked activation of JAK3 as determined by in vitro tyrosine kinase assays and a more manifest presence of JAK3 in activated IL4-receptor complexes. These observations suggest that IL4 receptor signal transduction does not depend on equimolar heterodimerization of JAK1 and JAK3 following IL4-induced heterodimerization of IL4R alpha and IL2R gamma. Indeed, when human IL4R alpha was stably expressed in mouse BA/F3 cells, robust IL4-induced proliferation and JAK3 activation occurred without detectable involvement of JAK1, JAK2, or TYK2. The present study suggests that JAK1 plays a subordinate role in IL4 receptor signaling, and that in certain cells exclusive JAK3 activation may mediate IL4-induced cell growth. Moreover, mutational analysis of human IL4R alpha showed that a membrane-proximal cytoplasmic region was critical for JAK3 activation, while the I4R motif was not, which is compatible with a role of JAK3 upstream of the recruitment of the insulin receptor substrate-1/4PS signaling proteins by IL4 receptors.

Improved efficacy of allergen-specific immunotherapy by JAK inhibition in a murine model of allergic asthma.

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Aguilar-Pimentel, Antonio; Graessel, Anke; Alessandrini, Francesca; Fuchs, Helmut; Gailus-Durner, Valerie; Hrabě de Angelis, Martin; Russkamp, Dennis; Chaker, Adam; Ollert, Markus; Blank, Simon; Gutermuth, Jan; Schmidt-Weber, Carsten B

2017-01-01

Allergen-specific immunotherapy (AIT) is the only curative treatment for type-1 allergies, but sometimes shows limited therapeutic response as well as local and systemic side effects. Limited control of local inflammation and patient symptoms hampers its widespread use in severe allergic asthma. Our aim was to evaluate whether AIT is more effective in suppression of local inflammation if performed under the umbrella of short-term non-specific immunomodulation using a small molecule inhibitor of JAK pathways. In C57BL/6J mice, a model of ovalbumin (OVA)-induced allergic airway inflammation and allergen-specific immunotherapy was combined with the administration of Tofacitinib (TOFA, a FDA-approved JAK inhibitor) from 48 hours prior to 48 hours after therapeutic OVA-injection. The effect of TOFA on human FOXP3+CD4+ T cells was studied in vitro. AIT combined with short-term TOFA administration was significantly more effective in suppressing total cell and eosinophil infiltration into the lung, local cytokine production including IL-1β and CXCL1 and showed a trend for the reduction of IL-4, IL-13, TNF-α and IL-6 compared to AIT alone. Furthermore, TOFA co-administration significantly reduced systemic IL-6, IL-1β and OVA-specific IgE levels and induced IgG1 to the same extent as AIT alone. Additionally, TOFA enhanced the induction of human FOXP3+CD4+ T cells. This proof of concept study shows that JAK inhibition did not inhibit tolerance induction, but improved experimental AIT at the level of local inflammation. The improved control of local inflammation might extend the use of AIT in more severe conditions such as polyallergy, asthma and high-risk patients suffering from mastocytosis or anaphylaxis.

Improved efficacy of allergen-specific immunotherapy by JAK inhibition in a murine model of allergic asthma.

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Antonio Aguilar-Pimentel

Full Text Available Allergen-specific immunotherapy (AIT is the only curative treatment for type-1 allergies, but sometimes shows limited therapeutic response as well as local and systemic side effects. Limited control of local inflammation and patient symptoms hampers its widespread use in severe allergic asthma.Our aim was to evaluate whether AIT is more effective in suppression of local inflammation if performed under the umbrella of short-term non-specific immunomodulation using a small molecule inhibitor of JAK pathways.In C57BL/6J mice, a model of ovalbumin (OVA-induced allergic airway inflammation and allergen-specific immunotherapy was combined with the administration of Tofacitinib (TOFA, a FDA-approved JAK inhibitor from 48 hours prior to 48 hours after therapeutic OVA-injection. The effect of TOFA on human FOXP3+CD4+ T cells was studied in vitro.AIT combined with short-term TOFA administration was significantly more effective in suppressing total cell and eosinophil infiltration into the lung, local cytokine production including IL-1β and CXCL1 and showed a trend for the reduction of IL-4, IL-13, TNF-α and IL-6 compared to AIT alone. Furthermore, TOFA co-administration significantly reduced systemic IL-6, IL-1β and OVA-specific IgE levels and induced IgG1 to the same extent as AIT alone. Additionally, TOFA enhanced the induction of human FOXP3+CD4+ T cells.This proof of concept study shows that JAK inhibition did not inhibit tolerance induction, but improved experimental AIT at the level of local inflammation. The improved control of local inflammation might extend the use of AIT in more severe conditions such as polyallergy, asthma and high-risk patients suffering from mastocytosis or anaphylaxis.

Overexpression of B7-H3 augments anti-apoptosis of colorectal cancer cells by Jak2-STAT3.

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Zhang, Ting; Jiang, Bo; Zou, Shi-Tao; Liu, Fen; Hua, Dong

2015-02-14

To investigate the role of the overexpression of B7-H3 in apoptosis in colorectal cancer cell lines and the underlying molecular mechanisms. SW620 cells that highly overexpressed B7-H3 (SW620-B7-H3-EGFP) and HCT8 cells stably transfected with B7-H3 shRNA (HCT8-shB7-H3) were previously constructed in our laboratory. Cells transfected with pIRES2-EGFP were used as negative controls (SW620-NC and HCT8-NC). Real-time PCR and western blotting analysis were used to detect the mRNA and protein expressions of the apoptosis regulator proteins Bcl-2, Bcl-xl and Bax. A cell proliferation assay was used to evaluate the survival rate and drug sensitivity of the cells. The effect of drug resistance was detected by a cell cycle assay. Active caspase-3 western blotting was used to reflect the anti-apoptotic ability of cells. Western blotting was also performed to determine the expression of proteins associated with the Jak2-STAT3 signaling pathway and the apoptosis regulator proteins after the treatment with AG490, a Jak2 specific inhibitor, in B7-H3 overexpressing cells. The data were analyzed by GraphPad Prism 6 using a non-paired t-test. Whether by overexpression in SW620 cells or downregulation in HCT8, B7-H3 significantly affected the expression of anti- and pro-apoptotic proteins, at both the transcriptional and translational levels, compared with the negative control (P overexpression increased the drug resistance of cells and resulted in a higher survival rate (P overexpression inhibited apoptosis in colorectal cancer cell lines (P overexpression improved Jak2 and STAT3 phosphorylation and, in turn, increased the expression of the downstream anti-apoptotic proteins B-cell CLL/lymphoma 2 (Bcl-2) and Bcl-xl, based on western blotting (P overexpressing cells with the Jak2-specific inhibitor AG490, the phosphorylation of Jak2 and STAT3, and the expression of Bcl-2 and Bcl-xl, decreased accordingly (P overexpression of B7-H3 induces resistance to apoptosis in colorectal cancer

JAK inhibitors in autoinflammation.

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Hoffman, Hal M; Broderick, Lori

2018-06-11

Interferonopathies are a subset of autoinflammatory disorders with a prominent type I IFN gene signature. Treatment of these patients has been challenging, given the lack of response to common autoinflammatory therapeutics including IL-1 and TNF blockade. JAK inhibitors (Jakinibs) are a family of small-molecule inhibitors that target the JAK/STAT signaling pathway and have shown clinical efficacy, with FDA and European Medicines Agency (EMA) approval for arthritic and myeloproliferative syndromes. Sanchez and colleagues repurposed baricitinib to establish a significant role for JAK inhibition as a novel therapy for patients with interferonopathies, demonstrating the power of translational rare disease research with lifesaving effects.

HSP90 is essential for Jak-STAT signaling in classical Hodgkin lymphoma cells

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Kube Dieter

2009-07-01

Full Text Available Abstract In classical Hodgkin lymphoma (cHL chemotherapeutic regimens are associated with stagnant rates of secondary malignancies requiring the development of new therapeutic strategies. We and others have shown that permanently activated Signal Transducer and Activator of Transcription (STAT molecules are essential for cHL cells. Recently an overexpression of heat-shock protein 90 (HSP90 in cHL cells has been shown and inhibition of HSP90 seems to affect cHL cell survival. Here we analysed the effects of HSP90 inhibition by geldanamycin derivative 17-AAG or RNA interference (RNAi on aberrant Jak-STAT signaling in cHL cells. Treatment of cHL cell lines with 17-AAG led to reduced cell proliferation and a complete inhibition of STAT1, -3, -5 and -6 tyrosine phosphorylation probably as a result of reduced protein expression of Janus kinases (Jaks. RNAi-mediated inhibition of HSP90 showed similar effects on Jak-STAT signaling in L428 cHL cells. These results suggest a central role of HSP90 in permanently activated Jak-STAT signaling in cHL cells. Therapeutics targeting HSP90 may be a promising strategy in cHL and other cancer entities associated with deregulated Jak-STAT pathway activation.

JAK Inhibitors: Treatment Efficacy and Safety Profile in Patients with Psoriasis

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Leeyen Hsu

2014-01-01

Full Text Available Janus kinase (JAK pathways are key mediators in the immunopathogenesis of psoriasis. Psoriasis treatment has evolved with the advent of targeted therapies, which inhibit specific components of the psoriasis proinflammatory cascade. JAK inhibitors have been studied in early phase trials for psoriasis patients, and the data are promising for these agents as potential treatment options. Tofacitinib, an oral or topically administered JAK1 and JAK3 inhibitor, and ruxolitinib, a topical JAK1 and JAK2 inhibitor, have been most extensively studied in psoriasis, and both improved clinical symptoms of psoriasis. Additional JAK1 or JAK3 inhibitors are being studied in clinical trials. In phase III trials for rheumatoid arthritis, tofacitinib was efficacious in patients with inadequate responses to tumor necrosis factor inhibitors, methotrexate monotherapy, or disease-modifying antirheumatic drugs. The results of phase III trials are pending for these therapies in psoriasis, and these agents may represent important alternatives for patients with inadequate responses to currently available agents. Further investigations with long-term clinical trials are necessary to verify their utility in psoriasis treatment and assess their safety in this patient population.

Secoisolariciresinol diglucoside prevents the oxidative stress-induced apoptosis of myocardial cells through activation of the JAK2/STAT3 signaling pathway.

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Huang, Guiqiong; Huang, Xiaofang; Liu, Min; Hua, Yue; Deng, Bo; Jin, Wen; Yan, Wen; Tan, Zhangbin; Wu, Yifen; Liu, Bin; Zhou, Yingchun

2018-06-01

Myocardial cell apoptosis mediated by oxidative stress has previously been identified as a key process in ischemic heart disease. Secoisolariciresinol diglucoside (SDG), a polyphenolic plant lignan primarily found in flaxseed, has been demonstrated to effectively protect myocardial cells from apoptosis. In the present study, the role of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) was investigated in mediating the protective effect of SDG. Findings of the present study revealed that treatment with H2O2 reduced cell viability and induced apoptosis in H9C2 rat cardiomyocytes. However, SDG was able to reduce the effect of H2O2 in a dose‑dependent manner. H2O2 reduced the expression level of phosphorylated STAT3 and inhibited the levels of B‑cell lymphoma‑extra‑large and induced myeloid leukemia cell differentiation protein, which are the STAT3 target genes. Conversely, SDG rescued phosphorylation of STAT3 and increased the levels of STAT3 target genes. Treatment with SDG alone led to a dose‑dependent increased phosphorylation of JAK2 and STAT3, without activating Src. Furthermore, the anti‑apoptotic effects of SDG were partially abolished by a JAK2/STAT3 inhibitor. In addition, molecular docking revealed that SDG may bind to the protein kinase domain of JAK2, at a binding energy of ‑8.258 kcal/mol. Molecular dynamics simulations revealed that JAK2‑SDG binding was stable. In conclusion, activation of the JAK2/STAT3 signaling pathway contributed to the anti‑apoptotic activity of SDG, which may be a potential JAK2 activator.

The role of JAK/STAT3 signaling pathway on apoptosis of lung adenocarcinoma cell line PC-9 induced by icotinib.

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Zhang, Yuping; Meng, Xia; Shi, Hongyang; Li, Wei; Ming, Zongjuan; Zhong, Yujie; Deng, Wenjing; Zhang, Qiuhong; Fan, Na; Niu, Zequn; Chen, Guo'an; Yang, Shuanying

2016-01-01

The aim of this study is to estimate the role of JAK/STAT3 signaling pathway on apoptosis of lung adenocarcinoma induced by icotinib. EGFR mutation was detected in lung adenocarcinoma cell line PC-9 by ARMS assay; The inhibitory rates of cell proliferation of PC-9 cells which were exposed to different concentrations of icotinib (0~100 μMol/L) for different time (24~72 h) respectively were evaluated by MTT assay; Apoptosis of PC-9 cells exposed to different concentrations of icotinib (0, 0.1, 1 and 10 μMol/L) for 48 h were evaluated by TUNEL assay; JAK2, STAT3, Bcl-2, Bax mRNA expressions were evaluated by Real-time PCR assay; The protein levels of P-STAT3 and IL-6 were evaluated by Western-blot assay. Human lung adenocarcinoma cell line PC-9 had an exon 19 deletion mutation in EGFR gene; Followed by treatment of icotinib, the proliferation of PC-9 cells were all inhibited significantly, especially in 48 and 72 h (Picotinib. The most likely mechanism is icotinib inhibited the gene expression levels of JAK2, STAT3 and Bcl-2, so with the P-STAT3 and IL-6 protein levels, and mediated gene Bax overexpression.

Triptolide downregulates Rac1 and the JAK/STAT3 pathway and inhibits colitis-related colon cancer progression

DEFF Research Database (Denmark)

Wang, Zhipeng; Jin, Haifeng; Xu, Ruodan

2009-01-01

ability to block progress of colitis to colon cancer, and its molecular mechanism of action are investigated. A mouse model for colitis-induced colorectal cancer was used to test the effect of triptolide on cancer progression. Treatment of mice with triptolide decreased the incidence of colon cancer...... formation, and increased survival rate. Moreover, triptolide decreased the incidence of tumors in nude mice inoculated with cultured colon cancer cells dose-dependently. In vitro, triptolide inhibited the proliferation, migration and colony formation of colon cancer cells. Secretion of IL6 and levels of JAK....... This suggests that triptolide might be a candidate for prevention of colitis induced colon cancer because it reduces inflammation and prevents tumor formation and development....

Sesquiterpene dimmer (DSF-27) inhibits the release of neuroinflammatory mediators from microglia by targeting spleen tyrosine kinase (Syk) and Janus kinase 2 (Jak2): Two major non-receptor tyrosine signaling proteins involved in inflammatory events

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Zeng, Ke-Wu [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China); Wang, Shu [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China); Department of Medicinal Chemistry and Pharmaceutical Analysis, Logistics College of Chinese People' s Armed Police Forces, Tianjin 300162 (China); Dong, Xin; Jiang, Yong; Jin, Hong-Wei [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China); Tu, Peng-Fei, E-mail: pengfeitu@vip.163.com [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China)

2014-03-15

Non-receptor protein tyrosine kinases (NRPTKs)-dependent inflammatory signal transduction cascades play key roles in immunoregulation. However, drug intervention through NRPTKs-involved immunoregulation mechanism in microglia (the major immune cells of the central nervous system) has not been widely investigated. A main aim of the present study is to elucidate the contribution of two major NRPTKs (Syk and Jak2) in neuroinflammation suppression by a bioactive sesquiterpene dimmer (DSF-27). We found that LPS-stimulated BV-2 cells activated Syk and further initiated Akt/NF-κB inflammatory pathway. This Syk-dependent Akt/NF-κB inflammatory pathway can be effectively ameliorated by DSF-27. Moreover, Jak2 was activated by LPS, which was followed by transcriptional factor Stat3 activation. The Jak2/Stat3 signal was suppressed by DSF-27 through inhibition of Jak2 and Stat3 phosphorylation, promotion of Jak/Stat3 inhibitory factors PIAS3 expression, and down-regulation of ERK and p38 MAPK phosphorylation. Furthermore, DSF-27 protected cortical and mesencephalic dopaminergic neurons against neuroinflammatory injury. Taken together, our findings indicate NRPTK signaling pathways including Syk/NF-κB and Jak2/Stat3 cascades are potential anti-neuroinflammatory targets in microglia, and may also set the basis for the use of sesquiterpene dimmer as a therapeutic approach for neuroinflammation via interruption of these pathways. - Highlights: • Sesquiterpene dimmer DSF-27 inhibits inflammatory mediators' production in microglia. • Syk-dependent Akt/NF-κB pathway is important for DSF-27's anti-inflammation activity. • Jak2/Stat3 pathway is important for DSF-27's anti-inflammation activity. • Jak2/Stat3 signaling pathway is partly regulated by ERK and p38 MAPKs and PIAS3. • DSF-27 protects neurons against microglia-mediated neuroinflammatory injury.

LOSS OF JAK2 REGULATION VIA VHL-SOCS1 E3 UBIQUITIN HETEROCOMPLEX UNDERLIES CHUVASH POLYCYTHEMIA

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Russell, Ryan C.; Sufan, Roxana I.; Zhou, Bing; Heir, Pardeep; Bunda, Severa; Sybingco, Stephanie S.; Greer, Samantha N.; Roche, Olga; Heathcote, Samuel A.; Chow, Vinca W.K.; Boba, Lukasz M.; Richmond, Terri D.; Hickey, Michele M.; Barber, Dwayne L.; Cheresh, David A.; Simon, M. Celeste; Irwin, Meredith S.; Kim, William Y.; Ohh, Michael

2011-01-01

SUMMARY Chuvash polycythemia (CP) is a rare congenital form of polycythemia caused by homozygous R200W and H191D mutations in the von Hippel-Lindau (VHL) gene whose gene product is the principal negative regulator of hypoxia-inducible factor. However, the molecular mechanisms underlying some of the hallmark features of CP such as hypersensitivity to erythropoietin are unclear. Here, we show that VHL directly binds suppressor of cytokine signalling 1 (SOCS1) to form a heterodimeric E3 ligase that targets phosphorylated (p)JAK2 for ubiquitin-mediated destruction. In contrast, CP-associated VHL mutants have altered affinity for SOCS1 and fail to engage and degrade pJAK2. Systemic administration of a highly selective JAK2 inhibitor, TG101209, reverses the disease phenotype in vhlR200W/R200W knock-in mice, a model that faithfully recapitulates human CP. These results reveal VHL as a SOCS1-cooperative negative regulator of JAK2 and provide compelling biochemical and preclinical evidence for JAK2- targeted therapy in CP patients. PMID:21685897

Structurally modified curcumin analogs inhibit STAT3 phosphorylation and promote apoptosis of human renal cell carcinoma and melanoma cell lines.

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Matthew A Bill

Full Text Available The Janus kinase-2 (Jak2-signal transducer and activator of transcription-3 (STAT3 pathway is critical for promoting an oncogenic and metastatic phenotype in several types of cancer including renal cell carcinoma (RCC and melanoma. This study describes two small molecule inhibitors of the Jak2-STAT3 pathway, FLLL32 and its more soluble analog, FLLL62. These compounds are structurally distinct curcumin analogs that bind selectively to the SH2 domain of STAT3 to inhibit its phosphorylation and dimerization. We hypothesized that FLLL32 and FLLL62 would induce apoptosis in RCC and melanoma cells and display specificity for the Jak2-STAT3 pathway. FLLL32 and FLLL62 could inhibit STAT3 dimerization in vitro. These compounds reduced basal STAT3 phosphorylation (pSTAT3, and induced apoptosis in four separate human RCC cell lines and in human melanoma cell lines as determined by Annexin V/PI staining. Apoptosis was also confirmed by immunoblot analysis of caspase-3 processing and PARP cleavage. Pre-treatment of RCC and melanoma cell lines with FLLL32/62 did not inhibit IFN-γ-induced pSTAT1. In contrast to FLLL32, curcumin and FLLL62 reduced downstream STAT1-mediated gene expression of IRF1 as determined by Real Time PCR. FLLL32 and FLLL62 significantly reduced secretion of VEGF from RCC cell lines in a dose-dependent manner as determined by ELISA. Finally, each of these compounds inhibited in vitro generation of myeloid-derived suppressor cells. These data support further investigation of FLLL32 and FLLL62 as lead compounds for STAT3 inhibition in RCC and melanoma.

Blueberry and malvidin inhibit cell cycle progression and induce mitochondrial-mediated apoptosis by abrogating the JAK/STAT-3 signalling pathway.

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Baba, Abdul Basit; Nivetha, Ramesh; Chattopadhyay, Indranil; Nagini, Siddavaram

2017-11-01

Blueberries, a rich source of anthocyanins have attracted considerable attention as functional foods that confer immense health benefits including anticancer properties. Herein, we assessed the potential of blueberry and its major constituent malvidin to target STAT-3, a potentially druggable oncogenic transcription factor with high therapeutic index. We demonstrate that blueberry abrogates the JAK/STAT-3 pathway and modulates downstream targets that influence cell proliferation and apoptosis in a hamster model of oral oncogenesis. Further, we provide mechanistic evidence that blueberry and malvidin function as STAT-3 inhibitors in the oral cancer cell line SCC131. Blueberry and malvidin suppressed STAT-3 phosphorylation and nuclear translocation thereby inducing cell cycle arrest and mitochondrial-mediated apoptosis. However, the combination of blueberry and malvidin with the STAT-3 inhibitor S3I-201 was more efficacious in STAT-3 inhibition relative to single agents. The present study has provided leads for the development of novel combinations of compounds that can serve as inhibitors of STAT-mediated oncogenic signalling. Copyright © 2017 Elsevier Ltd. All rights reserved.

Tyrosine Phosphorylation of Jak2 in the JH2 Domain Inhibits Cytokine Signaling

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Feener, Edward P.; Rosario, Felicia; Dunn, Sarah L.; Stancheva, Zlatina; Myers, Martin G.

2004-01-01

Jak family tyrosine kinases mediate signaling by cytokine receptors to regulate diverse biological processes. Although Jak2 and other Jak kinase family members are phosphorylated on numerous sites during cytokine signaling, the identity and function of most of these sites remains unknown. Using tandem mass spectroscopic analysis of activated Jak2 protein from intact cells, we identified Tyr221 and Tyr570 as novel sites of Jak2 phosphorylation. Phosphorylation of both sites was stimulated by c...

Efficacy of the JAK2 inhibitor INCB16562 in a murine model of MPLW515L-induced thrombocytosis and myelofibrosis.

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Koppikar, Priya; Abdel-Wahab, Omar; Hedvat, Cyrus; Marubayashi, Sachie; Patel, Jay; Goel, Aviva; Kucine, Nicole; Gardner, Jeffrey R; Combs, Andrew P; Vaddi, Kris; Haley, Patrick J; Burn, Timothy C; Rupar, Mark; Bromberg, Jacqueline F; Heaney, Mark L; de Stanchina, Elisa; Fridman, Jordan S; Levine, Ross L

2010-04-08

The discovery of JAK2 and MPL mutations in patients with myeloproliferative neoplasms (MPNs) provided important insight into the genetic basis of these disorders and led to the development of JAK2 kinase inhibitors for MPN therapy. Although recent studies have shown that JAK2 kinase inhibitors demonstrate efficacy in a JAK2V617F murine bone marrow transplantation model, the effects of JAK2 inhibitors on MPLW515L-mediated myeloproliferation have not been investigated. In this report, we describe the in vitro and in vivo effects of INCB16562, a small-molecule JAK2 inhibitor. INCB16562 inhibited proliferation and signaling in cell lines transformed by JAK2 and MPL mutations. Compared with vehicle treatment, INCB16562 treatment improved survival, normalized white blood cell counts and platelet counts, and markedly reduced extramedullary hematopoeisis and bone marrow fibrosis. We observed inhibition of STAT3 and STAT5 phosphorylation in vivo consistent with potent inhibition of JAK-STAT signaling. These data suggest JAK2 inhibitor therapy may be of value in the treatment of JAK2V617F-negative MPNs. However, we did not observe a decrease in the size of the malignant clone in the bone marrow of treated mice at the end of therapy, which suggests that JAK2 inhibitor therapy, by itself, was not curative in this MPN model.

JAK and MPL mutations in myeloid malignancies.

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Tefferi, Ayalew

2008-03-01

The Janus family of non-receptor tyrosine kinases (JAK1, JAK2, JAK3 and tyrosine kinase 2) transduces signals downstream of type I and II cytokine receptors via signal transducers and activators of transcription (STATs). JAK3 is important in lymphoid and JAK2 in myeloid cell proliferation and differentiation. The thrombopoietin receptor MPL is one of several JAK2 cognate receptors and is essential for myelopoiesis in general and megakaryopoiesis in particular. Germline loss-of-function (LOF) JAK3 and MPL mutations cause severe combined immunodeficiency and congenital amegakaryocytic thrombocytopenia, respectively. Germline gain-of-function (GOF) MPL mutation (MPLS505N) causes familial thrombocytosis. Somatic JAK3 (e.g. JAK3A572V, JAK3V722I, JAK3P132T) and fusion JAK2 (e.g. ETV6-JAK2, PCM1-JAK2, BCR-JAK2) mutations have respectively been described in acute megakaryocytic leukemia and acute leukemia/chronic myeloid malignancies. However, current attention is focused on JAK2 (e.g. JAK2V617F, JAK2 exon 12 mutations) and MPL (e.g. MPLW515L/K/S, MPLS505N) mutations associated with myeloproliferative neoplasms (MPNs). A JAK2 mutation, primarily JAK2V617F, is invariably associated with polycythemia vera (PV). The latter mutation also occurs in the majority of patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF). MPL mutational frequency in MPNs is substantially less (<10%). In general, despite a certain degree of genotype - phenotype correlations, the prognostic relevance of harbouring one of these mutations, or their allele burden when present, remains dubious. Regardless, based on the logical assumption that amplified JAK-STAT signalling is central to the pathogenesis of PV, ET and PMF, several anti-JAK2 tyrosine kinase inhibitors have been developed and are currently being tested in humans with these disorders.

The Chemopreventive Phytochemical Moringin Isolated from Moringa oleifera Seeds Inhibits JAK/STAT Signaling.

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Carina Michl

Full Text Available Sulforaphane (SFN and moringin (GMG-ITC are edible isothiocyanates present as glucosinolate precursors in cruciferous vegetables and in the plant Moringa oleifera respectively, and recognized for their chemopreventive and medicinal properties. In contrast to the well-studied SFN, little is known about the molecular pathways targeted by GMG-ITC. We investigated the ability of GMG-ITC to inhibit essential signaling pathways that are frequently upregulated in cancer and immune disorders, such as JAK/STAT and NF-κB. We report for the first time that, similarly to SFN, GMG-ITC in the nanomolar range suppresses IL-3-induced expression of STAT5 target genes. GMG-ITC, like SFN, does not inhibit STAT5 phosphorylation, suggesting a downstream inhibitory event. Interestingly, treatment with GMG-ITC or SFN had a limited inhibitory effect on IFNα-induced STAT1 and STAT2 activity, indicating that both isothiocyanates differentially target JAK/STAT signaling pathways. Furthermore, we showed that GMG-ITC in the micromolar range is a more potent inhibitor of TNF-induced NF-κB activity than SFN. Finally, using a cellular system mimicking constitutive active STAT5-induced cell transformation, we demonstrated that SFN can reverse the survival and growth advantage mediated by oncogenic STAT5 and triggers cell death, therefore providing experimental evidence of a cancer chemopreventive activity of SFN. This work thus identified STAT5, and to a lesser extent STAT1/STAT2, as novel targets of moringin. It also contributes to a better understanding of the biological activities of the dietary isothiocyanates GMG-ITC and SFN and further supports their apparent beneficial role in the prevention of chronic illnesses such as cancer, inflammatory diseases and immune disorders.

Pleiotropy of the Drosophila JAK pathway cytokine Unpaired 3 in development and aging.

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Wang, Liqun; Sexton, Travis R; Venard, Claire; Giedt, Michelle; Guo, Qian; Chen, Qian; Harrison, Douglas A

2014-11-15

The Janus kinase (JAK) pathway is an essential, highly re-utilized developmental signaling cascade found in most metazoans. In vertebrates, the JAK intracellular cascade mediates signaling by dozens of cytokines and growth factors. In Drosophila, the Unpaired (Upd) family, encoded by three tandemly duplicated genes, is the only class of ligands associated with JAK stimulation. Unpaired has a central role in activation of JAK for most pathway functions, while Unpaired 2 regulates body size through insulin signaling. We show here that the third member of the family, unpaired 3 (upd3), overlaps upd in expression in some tissues and is essential for a subset of JAK-mediated developmental functions. First, consistent with the known requirements of JAK signaling in gametogenesis, we find that mutants of upd3 show an age-dependent impairment of fertility in both sexes. In oogenesis, graded JAK activity stimulated by Upd specifies the fates of the somatic follicle cells. As upd3 mutant females age, defects arise that can be attributed to perturbations of the terminal follicle cells, which require the highest levels of JAK activation. Therefore, in oogenesis, the activities of Upd and Upd3 both appear to quantitatively contribute to specification of those follicle cell fates. Furthermore, the sensitization of upd3 mutants to age-related decline in fertility can be used to investigate reproductive senescence. Second, loss of Upd3 during imaginal development results in defects of adult structures, including reduced eye size and abnormal wing and haltere posture. The outstretched wing and small eye phenotypes resemble classical alleles referred to as outstretched (os) mutations that have been previously ascribed to upd. However, we show that os alleles affect expression of both upd and upd3 and map to untranscribed regions, suggesting that they disrupt regulatory elements shared by both genes. Thus the upd region serves as a genetically tractable model for coordinate

The IGF-I/JAK2-STAT3/miR-21 signaling pathway may be associated with human renal cell carcinoma cell growth.

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Su, Ying; Zhao, An; Cheng, Guoping; Xu, Jingjing; Ji, Enming; Sun, Wenyong

2017-07-04

Renal cell carcinoma (RCC) is the highest mortality rate of the genitourinary cancers, and the treatment options are very limited. Thus, identification of molecular mechanisms underlying RCC tumorigenesis, is critical for identifying biomarkers for RCC diagnosis and prognosis. To validate whether the IGF-I/JAK2-STAT3/miR-21 signaling pathway is associated with human RCC cell growth. qRT-PCR and Western blotting were used to detect the mRNA and protein expression levels, respectively. The MTT assay was performed to determine cell survival rate. The Annexin V-FITC/PI apoptosis detection kit was used to detect cell apoptosis. We employed RCC tissues and cell lines (A498; ACHN; Caki-1; Caki-2 and 786-O) in the study. IGF-I, and its inhibitor (NT-157) were administrated to detect the effects of IGF-I on the expression of miR-21 and p-JAK2. JAK2 inhibitor (AG490), and si-STAT3 were used to detect the effects of JAK2/STAT3 signaling pathway on the expression of miR-21. In our study, we firstly showed that the expression levels of IGF-I and miR-21 were up-regulated in RCC tissues and cell lines. After exogenous IGF-I treatment, the expression levels of miR-21, p-IGF-IR and p-JAK2 were significantly increased, whereas NT-157 treatment showed the reversed results. Further study indicated that JAK2 inhibitor or si-STAT3 significantly reversed the IGF-I-induced miR-21 expression level. Finally, we found that IGF-I treatment significantly prompted human RCC cell survival and inhibited cell apoptosis, and NT-157 treatment showed the reversed results. The IGF-I/JAK2-STAT3/miR-21 signaling pathway may be associated with human RCC cell growth.

Inhibition of JAK3 and PKC via Immunosuppressive Drugs Tofacitinib and Sotrastaurin Inhibits Proliferation of Human B Lymphocytes In Vitro.

Science.gov (United States)

Martina, M N; Ramirez Bajo, M J; Bañon-Maneus, E; Moya Rull, D; Hierro-Garcia, N; Revuelta, I; Campistol, J M; Rovira, J; Diekmann, F

2016-11-01

Antibody-mediated response in solid organ transplantation is critical for graft dysfunction and loss. The use of immunosuppressive agents partially inhibits the B-lymphocyte response leading to a risk of acute and chronic antibody-mediated rejection. This study evaluated the impact of JAK3 and PKC inhibitors tofacitinib (Tofa) and sotrastaurin (STN), respectively, on B-cell proliferation, apoptosis, and activation in vitro. Human B cells isolated from peripheral blood of healthy volunteers were cocultured with CD40 ligand-transfected fibroblasts as feeder cells in the presence of interleukin (IL) 2, IL-10, and IL-21. The cocultures were treated with immunosuppressants Tofa, STN, and rapamycin (as a control), to analyze the proliferation and apoptosis of B cells by means of Cyquant and flow cytometry, respectively. CD27 and IgG staining were applied to evaluate whether treatments modified the activation of B cells. Tofa and STN were able to inhibit B-cell proliferation to the same extent as rapamycin, without inducing cell apoptosis. After 6 days in coculture with feeder cells, all B cells showed CD27 memory B-cell phenotype. None of the immunosuppressive treatments modified the proportion between class-switched and non-class-switched memory B cells observed in nontreated cultures. The high predominance of CD27 + CD24 + phenotype was not modified by any immunosuppressive treatment. Our results show that Tofa and STN can suppress B-cell antibody responses to an extent similar to rapamycin, in vitro; therefore these compounds may be a useful therapy against antibody-mediated rejection in transplantation. Copyright © 2016. Published by Elsevier Inc.

Inactivation of JAK2/STAT3 Signaling Axis and Downregulation of M1 mAChR Cause Cognitive Impairment in klotho Mutant Mice, a Genetic Model of Aging

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Park, Seok-Joo; Shin, Eun-Joo; Min, Sun Seek; An, Jihua; Li, Zhengyi; Hee Chung, Yoon; Hoon Jeong, Ji; Bach, Jae-Hyung; Nah, Seung-Yeol; Kim, Won-Ki; Jang, Choon-Gon; Kim, Yong-Sun; Nabeshima, Yo-ichi; Nabeshima, Toshitaka; Kim, Hyoung-Chun

2013-01-01

We previously reported cognitive dysfunction in klotho mutant mice. In the present study, we further examined novel mechanisms involved in cognitive impairment in these mice. Significantly decreased janus kinase 2 (JAK2) and signal transducer and activator of transcription3 (STAT3) phosphorylation were observed in the hippocampus of klotho mutant mice. A selective decrease in protein expression and binding density of the M1 muscarinic cholinergic receptor (M1 mAChR) was observed in these mice. Cholinergic parameters (ie, acetylcholine (ACh), choline acetyltransferase (ChAT), and acetylcholinesterase (AChE)) and NMDAR-dependent long-term potentiation (LTP) were significantly impaired in klotho mutant mice. McN-A-343 (McN), an M1 mAChR agonist, significantly attenuated these impairments. AG490 (AG), a JAK2 inhibitor, counteracted the attenuating effects of McN, although AG did not significantly alter the McN-induced effect on AChE. Furthermore, AG significantly inhibited the attenuating effects of McN on decreased NMDAR-dependent LTP, protein kinase C βII, p-ERK, p-CREB, BDNF, and p-JAK2/p-STAT3-expression in klotho mutant mice. In addition, k252a, a BDNF receptor tyrosine kinase B (TrkB) inhibitor, significantly counteracted McN effects on decreased ChAT, ACh, and M1 mAChR and p-JAK2/p-STAT3 expression. McN-induced effects on cognitive impairment in klotho mutant mice were consistently counteracted by either AG or k252a. Our results suggest that inactivation of the JAK2/STAT3 signaling axis and M1 mAChR downregulation play a critical role in cognitive impairment observed in klotho mutant mice. PMID:23389690

[HSP90 inhibitor 17-AAG plays an important role in JAK3/STAT5 signaling pathways in HTLV-1 infection cell line HUT-102].

Science.gov (United States)

Yang, Q Q; Tan, H; Fu, Z P; Ma, Q; Song, J L

2017-08-14

Objective: To analyze whether heat-shock protein 90 (HSP90) be involved in a permanently abnormal activated JAK/STAT signaling in ATL cells in vitro. Methods: The effect of 17-AAG on proliferation of ATL cell lines HUT-102 was assessed using CCK8 at different time points. Cell apoptosis was measured by flow cytometry. The specific proteins HSP90, STAT5, p-STAT5 and JAK3 were detected by Western blotting. Results: Overexpression of HSP90 in HUT-102 cell lines was disclosed ( P AAG led to reduced cell proliferation, but there was no significant change in terms of cell proliferation when the concentration of 17-AAG between 2 000-8 000 nmol/L ( P >0.05) . 17-AAG induced cell apoptosis in different time-points and concentrations. 17-AAG don't affect the expression of JAK3 gene. Conclusion: This study indicated that JAK3 as HSP90 client protein was aberrantly activated in HTLV-1-infected T-cell lines, leading to constitutive activation of p-STAT5 in JAK/STAT signal pathway, which demonstrated that HSP90-inhibitors 17-AAG inhibited the growth of HTLV-1-infected T-cell lines by reducing cell proliferation and inducing cell apoptosis.

Identification of JAK2 as a mediator of FIP1L1-PDGFRA-induced eosinophil growth and function in CEL.

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Bin Li

Full Text Available The Fip1-like1 (FIP1L1-platelet-derived growth factor receptor alpha fusion gene (F/P arising in the pluripotent hematopoietic stem cell (HSC,causes 14% to 60% of patients with hypereosinophilia syndrome (HES. These patients, classified as having F/P (+ chronic eosinophilic leukemia (CEL, present with clonal eosinophilia and display a more aggressive disease phenotype than patients with F/P (- HES patients. The mechanisms underlying predominant eosinophil lineage targeting and the cytotoxicity of eosinophils in this leukemia remain unclear. Given that the Janus tyrosine kinase (JAK/signal transducers and activators of transcription (Stat signaling pathway is key to cytokine receptor-mediated eosinophil development and activated Stat3 and Stat5 regulate the expression of genes involved in F/P malignant transformation, we investigated whether and how JAK proteins were involved in the pathogenesis of F/P-induced CEL. F/P activation of JAK2, Stat3 and Stat5, were confirmed in all the 11 F/P (+ CEL patients examined. In vitro inhibition of JAK2 in EOL-1, primary F/P(+ CEL cells (PC and T674I F/P Imatinib resistant cells(IR by either JAK2-specific short interfering RNA (siRNA or the tryphostin derivative AG490(AG490, significantly reduced cellular proliferation and induced cellular apoptosis. The F/P can enhance the IL-5-induced JAK2 activation, and further results indicated that JAK2 inhibition blocked IL-5-induced cellular migration and activation of the EOL-1 and PC cells in vitro. F/P-stimulation of the JAK2 suppressed cells led to a significantly reduction in Stat3 activation, but relatively normal induction of Stat5 activation. Interestingly, JAK2 inhibition also reduced PI3K, Akt and NF-κB activity in a dose-dependent manner, and suppressed expression levels of c-Myc and Survivin. These results strongly suggest that JAK2 is activated by F/P and is required for F/P stimulation of cellular proliferation and infiltration, possibly through

RANKL downregulates cell surface CXCR6 expression through JAK2/STAT3 signaling pathway during osteoclastogenesis

Energy Technology Data Exchange (ETDEWEB)

Li, Changhong; Zhao, Jinxia; Sun, Lin; Yao, Zhongqiang; Liu, Rui [Department of Rheumatology and Immunology, Peking University Third Hospital, Beijing 100191 (China); Huang, Jiansheng [Department of Pediatrics, Washington University School of Medicine, St. Louis, MO (United States); Liu, Xiangyuan, E-mail: liu-xiangyuan@263.net [Department of Rheumatology and Immunology, Peking University Third Hospital, Beijing 100191 (China)

2012-12-14

Highlights: Black-Right-Pointing-Pointer CXCR6 is down-regulated during RANKL-induced osteoclastogenesis in RAW264.7 cells. Black-Right-Pointing-Pointer CXCR6 reduction was nearly reversed by inhibition of JAK2/STAT3 signaling pathway. Black-Right-Pointing-Pointer CXCL16 alone does not positively regulate osteoclastogenesis. -- Abstract: The receptor activator of nuclear factor-{kappa}B ligand (RANKL), as a member of the tumor necrosis factor (TNF) family, plays an essential role in osteoclast differentiation and function. Chemokines and their receptors have recently been shown to play critical roles in osteoclastogenesis, however, whether CXCL16-CXCR6 plays role in RANKL-mediated osteoclastogenesis is unknown. In this study, we first reported that RANKL decreased CXCR6 in a dose-dependent manner, which may be through deactivation of Akt and STAT3 signaling induced by CXCL16. Interestingly, RANKL-mediated CXCR6 reduction may be associated to the activation of STAT3 by phosphorylation. When STAT3 activation was blocked by JAK2/STAT3 inhibitor AG490, RANKL failed to shut down CXCR6 expression during osteoclastogenesis. However, CXCL16 alone did not augment RANKL-mediated osteoclast differentiation and did not alter RANKL-receptor RANK mRNA expression. These results demonstrate that reduction of CXCL16-CXCR6 is critical in RANKL-mediated osteoclastogenesis, which is mainly through the activation of JAK2/STAT3 signaling. CXCL16-CXCR6 axis may become a novel target for the therapeutic intervention of bone resorbing diseases such as rheumatoid arthritis and osteoporosis.

Anti-cancer effect of bee venom toxin and melittin in ovarian cancer cells through induction of death receptors and inhibition of JAK2/STAT3 pathway.

Science.gov (United States)

Jo, Miran; Park, Mi Hee; Kollipara, Pushpa Saranya; An, Byeong Jun; Song, Ho Sueb; Han, Sang Bae; Kim, Jang Heub; Song, Min Jong; Hong, Jin Tae

2012-01-01

We investigated whether bee venom and melittin, a major component of bee venom, inhibit cell growth through enhancement of death receptor expressions in the human ovarian cancer cells, SKOV3 and PA-1. Bee venom (1-5 μg/ml) and melittin (0.5-2 μg/ml) inhibited the growth of SKOV3 and PA-1 ovarian cancer cells by the induction of apoptotic cell death in a dose dependent manner. Consistent with apoptotic cell death, expression of death receptor (DR) 3 and DR6 was increased in both cancer cells, but expression of DR4 was increased only in PA-1 cells. Expression of DR downstream pro-apoptotic proteins including caspase-3, 8, and Bax was concomitantly increased, but the phosphorylation of JAK2 and STAT3 and the expression of Bcl-2 were inhibited by treatment with bee venom and melittin in SKOV3 and PA-1 cells. Expression of cleaved caspase-3 was increased in SKOV3, but cleaved caspase-8 was increased in PA-1 cells. Moreover, deletion of DR3, DR4, and DR6 by small interfering RNA significantly reversed bee venom and melittin-induced cell growth inhibitory effect as well as down regulation of STAT3 by bee venom and melittin in SKOV3 and PA-1 ovarian cancer cell. These results suggest that bee venom and melittin induce apoptotic cell death in ovarian cancer cells through enhancement of DR3, DR4, and DR6 expression and inhibition of STAT3 pathway. Copyright © 2011 Elsevier Inc. All rights reserved.

Excess thyroid hormone inhibits embryonic neural stem/progenitor cells proliferation and maintenance through STAT3 signalling pathway.

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Chen, Chunhai; Zhou, Zhou; Zhong, Min; Li, Maoquan; Yang, Xuesen; Zhang, Yanwen; Wang, Yuan; Wei, Aimin; Qu, Mingyue; Zhang, Lei; Xu, Shangcheng; Chen, Shude; Yu, Zhengping

2011-07-01

Hyperthyroidism is prevalent during pregnancy, but little is known about the effects of excess thyroid hormone on the development of embryonic neural stem/progenitor cells (NSCs), and the mechanisms underlying these effects. Previous studies indicate that STAT3 plays a crucial role in determining NSC fate during neurodevelopment. In this study, we investigated the effects of a supraphysiological dose of 3,5,3'-L-triiodothyronine (T3) on the proliferation and maintenance of NSCs derived from embryonic day 13.5 mouse neocortex, and the involvement of STAT3 in this process. Our results suggest that excess T3 treatment inhibits NSC proliferation and maintenance. T3 decreased tyrosine phosphorylation of JAK1, JAK2 and STAT3, and subsequently inhibited STAT3-DNA binding activity. Furthermore, proliferation and maintenance of NSCs were decreased by inhibitors of JAKs and STAT3, indicating that the STAT3 signalling pathway is involved in the process of NSC proliferation and maintenance. Taken together, these results suggest that the STAT3 signalling pathway is involved in the process of T3-induced inhibition of embryonic NSC proliferation and maintenance. These findings provide data for understanding the effects of hyperthyroidism during pregnancy on fetal brain development, and the mechanisms underlying these effects.

Constitutive activation of a slowly migrating isoform of Stat3 in mycosis fungoides: tyrphostin AG490 inhibits Stat3 activation and growth of mycosis fungoides tumor cell lines

DEFF Research Database (Denmark)

Nielsen, M; Kaltoft, K; Nordahl, M

1997-01-01

. Jaks link cytokine receptors to Stats, and abnormal Jak/Stat signaling has been observed in some hemopoietic cancers. In MF tumor cells, a slowly migrating isoform of Stat3, Stat3(sm), was found to be constitutively activated, i.e., (i) Stat3(sm) was constitutively phosphorylated on tyrosine residues...... specific. Thus, neither the fast migrating isoform of Stat3 (Stat3(fm)) nor other Stats (Stat1, Stat2, and Stat4 through Stat6) were constitutively activated. The Jak kinase inhibitor, tyrphostin AG490, blocked the constitutive activation of Stat3(sm) and inhibited spontaneous as well as interleukin 2...

The effect of quercetin nanoparticle on cervical cancer progression by inducing apoptosis, autophagy and anti-proliferation via JAK2 suppression.

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Luo, Cheng-Lin; Liu, Yu-Qiong; Wang, Peng; Song, Chun-Hua; Wang, Kai-Juan; Dai, Li-Ping; Zhang, Jian-Ying; Ye, Hua

2016-08-01

Cervical cancer is a cause of cancer death, making it as the one of the most common cause for death among women globally. Though many studies before have explored a lot for cervical cancer prevention and treatment, there are still a lot far from to know based on the molecular mechanisms. Janus kinase 2 (JAK2) has been reported to play an essential role in the progression of apoptosis, autophagy and proliferation for cells. We loaded gold-quercetin into poly (dl-lactide-co-glycolide) nanoparticles to cervical cancer cells due to the propertities of quercetin in ameliorating cellular processes and the easier absorbance of nanoparticles. Here, in our study, quercetin nanoparticles (NQ) were administrated to cells to investigate the underlying mechanism by which the cervical cancer was regulated. First, JAK2-inhibited carvical cancer cell lines were involved for our experiments in vitro and in vivo. Western blotting, quantitative RT-PCR (qRT-PCR), ELISA, Immunohistochemistry, and flow-cytometric analysis were used to determine the key signaling pathway regulated by JAK2 for cervical cancer progression. And the role of quercetin nanoparticles was determined during the process. Data here indicated that JAK2, indeed, expressed highly in cancer cell lines compared to the normal cervical cells. And apoptosis and autophagy were found in JAK2-inhibited cancer cells through activating Caspase-3, and suppressing Cyclin-D1 and mTOR regulated by Signal Transducer and Activator of Transcription (STAT) 3/5 and phosphatidylinositide 3-kinase/protein kinases (PI3K/AKT) signaling pathway. The cervical cancer cells proliferation was inhibited. Further, tumor size and weight were reduced by inhibition of JAK2 in vivo experiments. Notably, administration with quercetin nanoparticles displayed similar role with JAK2 suppression, which could inhibit cervical cancer cells proliferation, invasion and migration. In addition, autophogy and apoptosis were induced, promoting cervical cancer cell

JAK2 aberrations in childhood B-cell precursor acute lymphoblastic leukemia

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de Goffau-Nobel, Willemieke; Hoogkamer, Alex Q.; Boer, Judith M.; Boeree, Aurélie; van de Ven, Cesca; Koudijs, Marco J.; Besselink, Nicolle J.M.; de Groot-Kruseman, Hester A.; Zwaan, Christian Michel; Horstmann, Martin A.; Pieters, Rob; den Boer, Monique L.

2017-01-01

JAK2 abnormalities may serve as target for precision medicines in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). In the current study we performed a screening for JAK2 mutations and translocations, analyzed the clinical outcome and studied the efficacy of two JAK inhibitors in primary BCP-ALL cells. Importantly, we identify a number of limitations of JAK inhibitor therapy. JAK2 mutations mainly occurred in the poor prognostic subtypes BCR-ABL1-like and non- BCR-ABL1-like B-other (negative for sentinel cytogenetic lesions). JAK2 translocations were restricted to BCR-ABL1-like cases. Momelotinib and ruxolitinib were cytotoxic in both JAK2 translocated and JAK2 mutated cells, although efficacy in JAK2 mutated cells highly depended on cytokine receptor activation by TSLP. However, our data also suggest that the effect of JAK inhibition may be compromised by mutations in alternative survival pathways and microenvironment-induced resistance. Furthermore, inhibitors induced accumulation of phosphorylated JAK2Y1007, which resulted in a profound re-activation of JAK2 signaling upon release of the inhibitors. This preclinical evidence implies that further optimization and evaluation of JAK inhibitor treatment is necessary prior to its clinical integration in pediatric BCP-ALL. PMID:29163799

Anti-cancer effect of bee venom toxin and melittin in ovarian cancer cells through induction of death receptors and inhibition of JAK2/STAT3 pathway

International Nuclear Information System (INIS)

Jo, Miran; Park, Mi Hee; Kollipara, Pushpa Saranya; An, Byeong Jun; Song, Ho Sueb; Han, Sang Bae; Kim, Jang Heub; Song, Min Jong; Hong, Jin Tae

2012-01-01

We investigated whether bee venom and melittin, a major component of bee venom, inhibit cell growth through enhancement of death receptor expressions in the human ovarian cancer cells, SKOV3 and PA-1. Bee venom (1–5 μg/ml) and melittin (0.5–2 μg/ml) inhibited the growth of SKOV3 and PA-1 ovarian cancer cells by the induction of apoptotic cell death in a dose dependent manner. Consistent with apoptotic cell death, expression of death receptor (DR) 3 and DR6 was increased in both cancer cells, but expression of DR4 was increased only in PA-1 cells. Expression of DR downstream pro-apoptotic proteins including caspase-3, 8, and Bax was concomitantly increased, but the phosphorylation of JAK2 and STAT3 and the expression of Bcl-2 were inhibited by treatment with bee venom and melittin in SKOV3 and PA-1 cells. Expression of cleaved caspase-3 was increased in SKOV3, but cleaved caspase-8 was increased in PA-1 cells. Moreover, deletion of DR3, DR4, and DR6 by small interfering RNA significantly reversed bee venom and melittin-induced cell growth inhibitory effect as well as down regulation of STAT3 by bee venom and melittin in SKOV3 and PA-1 ovarian cancer cell. These results suggest that bee venom and melittin induce apoptotic cell death in ovarian cancer cells through enhancement of DR3, DR4, and DR6 expression and inhibition of STAT3 pathway. -- Highlights: ► Some studies have showed that bee venom and/or melittin have anti-cancer effects. ► We found that bee venom and melittin inhibited cell growth in ovarian cancer cells. ► Bee venom and melittin induce apoptosis in SKOV3 and PA-1.

Bim and Mcl-1 exert key roles in regulating JAK2V617F cell survival

International Nuclear Information System (INIS)

Rubert, Joëlle; Qian, Zhiyan; Andraos, Rita; Guthy, Daniel A; Radimerski, Thomas

2011-01-01

The JAK2 V617F mutation plays a major role in the pathogenesis of myeloproliferative neoplasms and is found in the vast majority of patients suffering from polycythemia vera and in roughly every second patient suffering from essential thrombocythemia or from primary myelofibrosis. The V617F mutation is thought to provide hematopoietic stem cells and myeloid progenitors with a survival and proliferation advantage. It has previously been shown that activated JAK2 promotes cell survival by upregulating the anti-apoptotic STAT5 target gene Bcl-xL. In this study, we have investigated the role of additional apoptotic players, the pro-apoptotic protein Bim as well as the anti-apoptotic protein Mcl-1. Pharmacological inhibition of JAK2/STAT5 signaling in JAK2 V617F mutant SET-2 and MB-02 cells was used to study effects on signaling, cell proliferation and apoptosis by Western blot analysis, WST-1 proliferation assays and flow cytometry. Cells were transfected with siRNA oligos to deplete candidate pro- and anti-apoptotic proteins. Co-immunoprecipitation assays were performed to assess the impact of JAK2 inhibition on complexes of pro- and anti-apoptotic proteins. Treatment of JAK2 V617F mutant cell lines with a JAK2 inhibitor was found to trigger Bim activation. Furthermore, Bim depletion by RNAi suppressed JAK2 inhibitor-induced cell death. Bim activation following JAK2 inhibition led to enhanced sequestration of Mcl-1, besides Bcl-xL. Importantly, Mcl-1 depletion by RNAi was sufficient to compromise JAK2 V617F mutant cell viability and sensitized the cells to JAK2 inhibition. We conclude that Bim and Mcl-1 have key opposing roles in regulating JAK2 V617F cell survival and propose that inactivation of aberrant JAK2 signaling leads to changes in Bim complexes that trigger cell death. Thus, further preclinical evaluation of combinations of JAK2 inhibitors with Bcl-2 family antagonists that also tackle Mcl-1, besides Bcl-xL, is warranted to assess the therapeutic potential

JAK inhibitors for the treatment of myeloproliferative neoplasms and other disorders [version 1; referees: 2 approved

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William Vainchenker

2018-01-01

Full Text Available JAK inhibitors have been developed following the discovery of the JAK2V617F in 2005 as the driver mutation of the majority of non-BCR-ABL1 myeloproliferative neoplasms (MPNs. Subsequently, the search for JAK2 inhibitors continued with the discovery that the other driver mutations (CALR and MPL also exhibited persistent JAK2 activation. Several type I ATP-competitive JAK inhibitors with different specificities were assessed in clinical trials and exhibited minimal hematologic toxicity. Interestingly, these JAK inhibitors display potent anti-inflammatory activity. Thus, JAK inhibitors targeting preferentially JAK1 and JAK3 have been developed to treat inflammation, autoimmune diseases, and graft-versus-host disease. Ten years after the beginning of clinical trials, only two drugs have been approved by the US Food and Drug Administration: one JAK2/JAK1 inhibitor (ruxolitinib in intermediate-2 and high-risk myelofibrosis and hydroxyurea-resistant or -intolerant polycythemia vera and one JAK1/JAK3 inhibitor (tofacitinib in methotrexate-resistant rheumatoid arthritis. The non-approved compounds exhibited many off-target effects leading to neurological and gastrointestinal toxicities, as seen in clinical trials for MPNs. Ruxolitinib is a well-tolerated drug with mostly anti-inflammatory properties. Despite a weak effect on the cause of the disease itself in MPNs, it improves the clinical state of patients and increases survival in myelofibrosis. This limited effect is related to the fact that ruxolitinib, like the other type I JAK2 inhibitors, inhibits equally mutated and wild-type JAK2 (JAK2WT and also the JAK2 oncogenic activation. Thus, other approaches need to be developed and could be based on either (1 the development of new inhibitors specifically targeting JAK2V617F or (2 the combination of the actual JAK2 inhibitors with other therapies, in particular with molecules targeting pathways downstream of JAK2 activation or the stability of JAK2

Loss of function mutations in PTPN6 promote STAT3 deregulation via JAK3 kinase in diffuse large B-cell lymphoma

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Demosthenous, Christos; Han, Jing Jing; Hu, Guangzhen; Stenson, Mary; Gupta, Mamta

2015-01-01

PTPN6 (SHP1) is a tyrosine phosphatase that negatively controls the activity of multiple signaling pathways including STAT signaling, however role of mutated PTPN6 is not much known. Here we investigated whether PTPN6 might also be a potential target for diffuse large B cell lymphoma (DLBCL) and performed Sanger sequencing of the PTPN6 gene. We have identified missense mutations within PTPN6 (N225K and A550V) in 5% (2/38) of DLBCL tumors. Site directed mutagenesis was performed to mutate wild type (WT) PTPN6 and stable cell lines were generated by lentiviral transduction of PTPN6WT, PTPN6N225K and PTPN6A550V constructs, and effects of WT or mutated PTPN6 on STAT3 signaling were analyzed. WT PTPN6 dephosphorylated STAT3, but had no effect on STAT1, STAT5 or STAT6 phosphorylation. Both PTPN6 mutants were unable to inhibit constitutive, as well as cytokines induced STAT3 activation. Both PTPN6 mutants also demonstrated reduced tyrosine phosphatase activity and exhibited enhanced STAT3 transactivation activity. Intriguingly, a lack of direct binding between STAT3 and WT or mutated PTPN6 was observed. However, compared to WT PTPN6, cells expressing PTPN6 mutants exhibited increased binding between JAK3 and PTPN6 suggesting a more dynamic interaction of PTPN6 with upstream regulators of STAT3. Consistent with this notion, both the mutants demonstrated increased resistance to JAK3 inhibitor, WHIP-154 relative to WT PTPN6. Overall, this is the first study, which demonstrates that N225K and A550V PTPN6 mutations cause loss-of-function leading to JAK3 mediated deregulation of STAT3 pathway and uncovers a mechanism that tumor cells can use to control PTPN6 substrate specificity. PMID:26565811

Parathyroid hormone inhibition of Na{sup +}/H{sup +} exchanger 3 transcription: Intracellular signaling pathways and transcription factor expression

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Neri, Elida Adalgisa; Bezerra, Camila Nogueira Alves, E-mail: camilab@icb.usp.br; Queiroz-Leite, Gabriella Duarte; Polidoro, Juliano Zequini; Rebouças, Nancy Amaral

2015-06-12

The main transport mechanism of reabsorption of sodium bicarbonate and fluid in the renal proximal tubules involves Na{sup +}/H{sup +} exchanger 3 (NHE3), which is acutely and chronically downregulated by parathyroid hormone (PTH). Although PTH is known to exert an inhibitory effect on NHE3 expression and transcription, the molecular mechanisms involved remain unclear. Here, we demonstrated that, in opossum kidney proximal tubule (OKP) cells, PTH-induced inhibition of Nhe3 gene promoter occurs even in the core promoter that controls expression of the reporter gene. We found that inhibition of the protein kinase A (PKA) and Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways transformed PTH from an inhibitor of promoter activity into an activator of that same activity, as did point mutations in the EGR1, Sp1, and Sp3 binding consensus elements in the promoter. In nuclear extracts of PTH-treated OKP cells, we also observed increased expression of EGR1 mRNA and of some Sp3 isoforms. Electrophoretic mobility shift assay showed a supershift of the −61 to −42-bp probe with an anti-EGR1 antibody in PTH-treated cells, suggesting that EGR1 binding is relevant for the inhibitory activity of PTH. We conclude that PTH-induced inhibition of NHE3 transcription is related to higher EGR1 expression; to EGR1 binding to the proximal and core promoters; and to PKA and JAK/STAT pathway activation. This mechanism might be responsible, at least in part, for lower NHE3 expression and sodium reabsorption in renal proximal tubules in the presence of high PTH levels. - Highlights: • PTH regulation of Nhe3 promoter depends on EGR1 binding. • EGR1, PKA and JAK/STAT are involved in PTH inhibition of the Nhe3 promoter. • PTH alters expression of EGR1 and Sp3. • PTH inhibits the Nhe3 promoter by regulating PKA and JAK/STAT signaling.

LIF-activated Jak signaling determines Esrrb expression during late-stage reprogramming

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Delun Huang

2018-01-01

Full Text Available The regulatory process of naïve-state induced pluripotent stem cell (iPSC generation is not well understood. Leukemia inhibitory factor (LIF-activated Janus kinase/signal transducer and activator of transcription 3 (Jak/Stat3 is the master regulator for naïve-state pluripotency achievement and maintenance. The estrogen-related receptor beta (Esrrb serves as a naïve-state marker gene regulating self-renewal of embryonic stem cells (ESCs. However, the interconnection between Esrrb and LIF signaling for pluripotency establishment in reprogramming is unclear. We screened the marker genes critical for complete reprogramming during mouse iPSC generation, and identified genes including Esrrb that are responsive to LIF/Jak pathway signaling. Overexpression of Esrrb resumes the reprogramming halted by inhibition of Jak activity in partially reprogrammed cells (pre-iPSCs, and leads to the generation of pluripotent iPSCs. We further show that neither overexpression of Nanog nor stimulation of Wnt signaling, two upstream regulators of Esrrb in ESCs, stimulates the expression of Esrrb in reprogramming when LIF or Jak activity is blocked. Our study demonstrates that Esrrb is a specific reprogramming factor regulated downstream of the LIF/Jak signaling pathway. These results shed new light on the regulatory role of LIF pathway on complete pluripotency establishment during iPSC generation.

The effect of curcumol on protein expression of JAK2/STAT3 signaling pathway in human ovarian cancer line SKOV3

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Feng-juan HAN

2013-12-01

Full Text Available Objective: To study the effects of curcumol on the protein expression of JAK2 and STAT3 in SKOV3 and to investigate its treatment on molecular mechanism of ovarian cancer. Methods: Choose curcumol of different concentrations to act on human ovarian cancer cell line SKOV3, and extract the corresponding cell protein, and detect the protein expression of JAK2 and STAT3 by western blotting. Results: The protein expression of JAK2 and STAT3 in SKOV3 are significantly inhabited by curcumol, and its strength will enhance with the increase in drug concentration, and it shows in a dose-dependent manner. Conclusion: Curcumol can significantly inhabit the proliferation of SKOV3 cells, and induce apoptosis, and achieve its mechanism by regulating the protein expression of JAK2 and STAT3.

mTOR inhibitors alone and in combination with JAK2 inhibitors effectively inhibit cells of myeloproliferative neoplasms.

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Costanza Bogani

Full Text Available BACKGROUND: Dysregulated signaling of the JAK/STAT pathway is a common feature of chronic myeloproliferative neoplasms (MPN, usually associated with JAK2V617F mutation. Recent clinical trials with JAK2 inhibitors showed significant improvements in splenomegaly and constitutional symptoms in patients with myelofibrosis but meaningful molecular responses were not documented. Accordingly, there remains a need for exploring new treatment strategies of MPN. A potential additional target for treatment is represented by the PI3K/AKT/mammalian target of rapamycin (mTOR pathway that has been found constitutively activated in MPN cells; proof-of-evidence of efficacy of the mTOR inhibitor RAD001 has been obtained recently in a Phase I/II trial in patients with myelofibrosis. The aim of the study was to characterize the effects in vitro of mTOR inhibitors, used alone and in combination with JAK2 inhibitors, against MPN cells. FINDINGS: Mouse and human JAK2V617F mutated cell lines and primary hematopoietic progenitors from MPN patients were challenged with an allosteric (RAD001 and an ATP-competitive (PP242 mTOR inhibitor and two JAK2 inhibitors (AZD1480 and ruxolitinib. mTOR inhibitors effectively reduced proliferation and colony formation of cell lines through a slowed cell division mediated by changes in cell cycle transition to the S-phase. mTOR inhibitors also impaired the proliferation and prevented colony formation from MPN hematopoietic progenitors at doses significantly lower than healthy controls. JAK2 inhibitors produced similar antiproliferative effects in MPN cell lines and primary cells but were more potent inducers of apoptosis, as also supported by differential effects on cyclinD1, PIM1 and BcLxL expression levels. Co-treatment of mTOR inhibitor with JAK2 inhibitor resulted in synergistic activity against the proliferation of JAK2V617F mutated cell lines and significantly reduced erythropoietin-independent colony growth in patients with

IGF-1-induced MMP-11 expression promotes the proliferation and invasion of gastric cancer cells through the JAK1/STAT3 signaling pathway.

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Su, Chao; Wang, Wenchang; Wang, Cunchuan

2018-05-01

The present study aimed to investigate the association between insulin-like growth factor-1 (IGF-1) and matrix metalloproteinase-11 (MMP-11) expression in gastric cancer (GC) and the underlying mechanisms in SGC-7901 cells. Reverse transcription-quantitative polymerase chain reaction analysis revealed that the expression of IGF-1 and MMP-11 was significantly upregulated in GC tissues compared with normal gastric tissue. Furthermore, IGF-1 significantly and dose-dependently promoted MMP-11. Western blotting revealed that the addition of IGF-1 to SGC-7901 cells led to an evident enhancement in signal transducer and activator of transcription 3 (STAT3), IGF-1R and Janus kinase 1 (JAK1) phosphorylation at 20 and 40 min. A decrease in the extent of the elevated expression of MMP-11 and the enhanced phosphorylation of STAT3, JAK1 and IGF-1 receptor (IGF-1R) induced by IGF-1 in SGC-7901 cells were observed following treatment with NT157 (an IGF-1R inhibitor). Furthermore, piceatannol (a JAK1 inhibitor) or small interfering RNA against STAT3 reduced the extent of the increased expression of MMP-11 induced by IGF-1 in SGC-7901 cells. Piceatannol treatment induced the dose-dependent decline in the enhancement of STAT3 phosphorylation induced by IGF-1, indicating that the JAK1/STAT3 pathway may be implicated in the elevated expression of MMP-11 induced by IGF-1 in SGC-7901 cells. Finally, IGF-1 treatment significantly promoted the proliferation and invasion of SGC-7901 cells, which was inhibited following NT157, piceatannol or si-STAT3 treatment. The present study therefore demonstrated that IGF-1-induced MMP-11 may have facilitated the proliferation and invasion of SGC-7901 cells via the JAK1/STAT3 pathway.

Ekspresja kinazy Jak3 i aktywacja białka Stat3 u chorych na reumatoidalne zapalenie stawów i spondyloartropatie zapalne

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Andrzej Steciwko

2010-08-01

Full Text Available Wstęp: Układ przekaźnikowy Jak/Stat (kinaza tyrozynowaJanus/sygnał transdukcji i aktywacji transkrypcji jest wykorzystywanyprzez wiele cytokin, czynników wzrostu i hormonów regulującychmechanizmy transkrypcji genów oraz aktywacji, proliferacji,różnicowania i apoptozy komórek. Wyniki dotychczasowych badańwskazują, że kinaza Jak3 odgrywa istotną rolę w patogeneziereumatoidalnego zapalenia stawów (RZS. Cel pracy: Ocena ekspresji Jak3 oraz aktywacji Stat3 w leukocytachkrwi obwodowej (LKO i komórkach płynu stawowego (KPSu chorych na RZS i spondyloartropatie zapalne (SpaZ oraz analizazwiązku badanych parametrów ze wskaźnikami aktywności chorobyużywanymi w praktyce klinicznej. Ponadto analizie poddanozależności między ekspresją Jak3 a aktywacją Stat3. Materiał i metody: Do badania zakwalifikowano 19 chorych na RZSoraz 22 chorych na SpaZ (zesztywniające zapalenie stawów, łuszczycowezapalenie stawów, spondyloartropatia niezróżnico wana.Grupę kontrolną stanowiły 23 zdrowe osoby. W badanych grupachw LKO metodą immunocytochemiczną oznaczono ekspresję kinazyJak3 i aktywację białka Stat3. Tą samą metodą oznaczono ekspresjęJak3 i aktywację Stat3 u 11 chorych na RZS i u 12 chorych na SpaZw KPS. U chorych zostały oznaczone warto ści parametrów stanuzapalnego oraz wskaźników aktywności choroby DAS28 i BASDAI.Wykonano rentgenogramy stawów zajętych procesem chorobowym. Wyniki: Ekspresja Jak3 oraz aktywacja Stat3 były znacząco wyższeu chorych na RZS i SpaZ w porównaniu z grupą kontrolną. War to -ści te były wyższe w KPS niż w LKO. U chorych na RZS zaobserwowanododatnią korelację między aktywnością Stat3 w KPSa wartością CRP. Nie wykazano korelacji między ekspresją Jak3a aktywacją Stat3. Wnioski: Funkcja Jak3 i Stat3 jest związana z procesem immunolo -gicznym w przebiegu RZS i SpaZ. Wydaje się, że zablokowanie ichfunkcji może stanowić cel terapeutyczny w obydwu grupach chorych.

Novel compounds TAD-1822-7-F2 and F5 inhibited HeLa cells growth through the JAK/Stat signaling pathway.

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Yang, Tianfeng; Shi, Xianpeng; Kang, Yuan; Zhu, Man; Fan, Mengying; Zhang, Dongdong; Zhang, Yanmin

2018-07-01

Cervical carcinoma remains the second most common malignancy with a high mortality rate among women worldwide. TAD-1822-7-F2 (F2) and TAD-1822-7-F5 (F5) are novel compounds synthesized on the chemical structure of taspine derivatives, and show an effective suppression for HeLa cells. Our study aims to confirm the potential targets of F2 and F5, and investigate the underlying mechanism of the inhibitory effect on HeLa cells. In this study, Real Time Cell Analysis and crystal violet staining assay were conducted to investigate the effect of F2 and F5 on HeLa cells proliferation. And the analytical methods of surface plasmon resonance and quartz crystal microbalance were established and employed to study the interaction between F2 and F5 and potential target protein JAK2, suggesting that both compounds have strong interaction with the JAK2 protein. Western blot analysis, immunofluorescence staining study and PCR was conducted to investigate the molecules of JAK/Stat signaling pathway. Interestingly, F2 and F5 showed diverse regulation for signaling molecules because of their different chemical structure. F2 increased the expression of JAK2 and downregulated the level of P-JAK1 and P-JAK2, and decreased P-Stat3 (Ser727). While F5 could increase the expression of JAK2 and naturally decrease the phosphorylation of JAK1 and Tyk2, and decreased the expression of P-Stat6. Moreover, F2 and F5 showed the same downregulation on the P-Stat3 (Tyr705). Therefore, F2 and F5 could target the JAK2 protein and prevent the phosphorylation of JAKs to suppress the phosphorylation of the downstream effector Stats, which suggested that F2 and F5 have great potential to be the inhibitors of the JAK/Stat signaling pathway. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Genetic variants in IL-6/JAK/STAT3 pathway and the risk of CRC.

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Wang, Shuwei; Zhang, Weidong

2016-05-01

Interleukin (IL)-6 and the downstream Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway have previously been reported to be important in the development of colorectal cancer (CRC), and several studies have shown the relationship between the polymorphisms of related genes in this pathway with the risk of CRC. However, the findings of these related studies are inconsistent. Moreover, there has no systematic review and meta-analysis to evaluate the relationship between genetic variants in IL-6/JAK/STAT3 pathway and CRC susceptibility. Hence, we conducted a meta-analysis to explore the relationship between polymorphisms in IL-6/JAK/STAT3 pathway genes and CRC risk. Eighteen eligible studies with a total of 13,795 CRC cases and 18,043 controls were identified by searching PubMed, Web of Science, Embase, and the Cochrane Library databases for the period up to September 15, 2015. Odds ratios (ORs) and their 95 % confidence intervals (CIs) were used to calculate the strength of the association. Our results indicated that IL-6 genetic variants in allele additive model (OR = 1.05, 95 % CI = 1.00, 1.09) and JAK2 genetic variants (OR = 1.40, 95 % CI = 1.15, 1.65) in genotype recessive model were significantly associated with CRC risk. Moreover, the pooled data revealed that IL-6 rs1800795 polymorphism significantly increased the risk of CRC in allele additive model in Europe (OR = 1.07, 95 % CI = 1.01, 1.14). In conclusion, the present findings indicate that IL-6 and JAK2 genetic variants are associated with the increased risk of CRC while STAT3 genetic variants not. We need more well-designed clinical studies covering more countries and population to definitively establish the association between genetic variants in IL-6/JAK/STAT3 pathway and CRC susceptibility.

Hepatic Stellate Cell Coculture Enables Sorafenib Resistance in Huh7 Cells through HGF/c-Met/Akt and Jak2/Stat3 Pathways

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Weibo Chen

2014-01-01

Full Text Available Purpose. Tumor microenvironment confers drug resistance to kinase inhibitors by increasing RKT ligand levels that result in the activation of cell-survival signaling including PI3K and MAPK signals. We assessed whether HSC-LX2 coculture conferred sorafenib resistance in Huh7 and revealed the mechanism underlying the drug resistance. Experimental Design. The effect of LX2 on sorafenib resistance was determined by coculture system with Huh7 cells. The rescue function of LX2 supernatants was assessed by MTT assay and fluorescence microscopy. The underlying mechanism was tested by administration of pathway inhibitors and manifested by Western blotting. Results. LX2 coculture significantly induced sorafenib resistance in Huh7 by activating p-Akt that led to reactivation of p-ERK. LX2 secreted HGF into the culture medium that triggered drug resistance, and exogenous HGF could also induce sorafenib resistance. The inhibition of p-Akt blocked sorafenib resistance caused by LX2 coculture. Increased phosphorylation of Jak2 and Stat3 was also detected in LX2 cocultured Huh7 cells. The Jak inhibitor tofacitinib reversed sorafenib resistance by blocking Jak2 and Stat3 activation. The combined administration of sorafenib and p-Stat3 inhibitor S3I-201 augmented induced apoptosis even in the presence of sorafenib resistance. Conclusions. HSC-LX2 coculture induced sorafenib resistance in Huh7 through multiple pathways: HGF/c-Met/Akt pathway and Jak2/Stat3 pathway. A combined administration of sorafenib and S3I-201 was able to augment sorafenib-induced apoptosis even in the presence of LX2 coculture.

Autophagy Facilitates IFN-γ-induced Jak2-STAT1 Activation and Cellular Inflammation*

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Chang, Yu-Ping; Tsai, Cheng-Chieh; Huang, Wei-Ching; Wang, Chi-Yun; Chen, Chia-Ling; Lin, Yee-Shin; Kai, Jui-In; Hsieh, Chia-Yuan; Cheng, Yi-Lin; Choi, Pui-Ching; Chen, Shun-Hua; Chang, Shih-Ping; Liu, Hsiao-Sheng; Lin, Chiou-Feng

2010-01-01

Autophagy is regulated for IFN-γ-mediated antimicrobial efficacy; however, its molecular effects for IFN-γ signaling are largely unknown. Here, we show that autophagy facilitates IFN-γ-activated Jak2-STAT1. IFN-γ induces autophagy in wild-type but not in autophagy protein 5 (Atg5−/−)-deficient mouse embryonic fibroblasts (MEFs), and, autophagy-dependently, IFN-γ induces IFN regulatory factor 1 and cellular inflammatory responses. Pharmacologically inhibiting autophagy using 3-methyladenine, a known inhibitor of class III phosphatidylinositol 3-kinase, confirms these effects. Either Atg5−/− or Atg7−/− MEFs are, independent of changes in IFN-γ receptor expression, resistant to IFN-γ-activated Jak2-STAT1, which suggests that autophagy is important for IFN-γ signal transduction. Lentivirus-based short hairpin RNA for Atg5 knockdown confirmed the importance of autophagy for IFN-γ-activated STAT1. Without autophagy, reactive oxygen species increase and cause SHP2 (Src homology-2 domain-containing phosphatase 2)-regulated STAT1 inactivation. Inhibiting SHP2 reversed both cellular inflammation and the IFN-γ-induced activation of STAT1 in Atg5−/− MEFs. Our study provides evidence that there is a link between autophagy and both IFN-γ signaling and cellular inflammation and that autophagy, because it inhibits the expression of reactive oxygen species and SHP2, is pivotal for Jak2-STAT1 activation. PMID:20592027

Regulation of apoptosis by resveratrol through JAK/STAT and mitochondria mediated pathway in human epidermoid carcinoma A431 cells

International Nuclear Information System (INIS)

Madan, Esha; Prasad, Sahdeo; Roy, Preeti; George, Jasmine; Shukla, Yogeshwer

2008-01-01

Resveratrol (trans-3,4',5-trihydroxystilbene), a polyphenolic phytoalexin present mainly in grapes, red wine and berries, is known to possess strong chemopreventive and anticancer properties. Here, we demonstrated the anti-proliferative and apoptosis-inducing activities of resveratrol in human epidermoid carcinoma A431 cells. Resveratrol has cytotoxic effects through inhibiting cellular proliferation of A431 cells, which leads to the induction of apoptosis, as evident by an increase in the fraction of cells in the sub-G 1 phase of the cell cycle and Annexin-V binding of externalized phosphatidylserine. Results revealed that inhibition of proliferation is associated with regulation of the JAK/STAT pathway, where resveratrol prevents phosphorylation of JAK, thereby inhibiting STAT1 phosphorylation. Furthermore, resveratrol treatment actively stimulated reactive oxygen species (ROS) and mitochondrial membrane depolarization. Consequently, an imbalance in the Bax/Bcl-2 ratio triggered the caspase cascade and subsequent cleavage of PARP, thereby shifting the balance in favor of apoptosis. These observations indicate that resveratrol treatment inhibits JAK/STAT-mediated gene transcription and induce the mitochondrial cell death pathway.

Inhibitors of JAK-family kinases: an update on the patent literature 2013-2015, part 2.

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Kettle, Jason G; Åstrand, Annika; Catley, Matthew; Grimster, Neil P; Nilsson, Magnus; Su, Qibin; Woessner, Richard

2017-02-01

Janus kinases (JAKs) are a family of four enzymes; JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2) that are critical in cytokine signalling and are strongly linked to both cancer and inflammatory diseases. There are currently two launched JAK inhibitors for the treatment of human conditions: tofacitinib for Rheumatoid arthritis (RA) and ruxolitinib for myeloproliferative neoplasms including intermediate or high risk myelofibrosis and polycythemia vera. Areas covered: This review covers patents claiming activity against one or more JAK family members in the period 2013-2015 inclusive, and covers 95 patents from 42 applicants, split over two parts. The authors have ordered recent patents according to the primary applicant's name, with part 2 covering J through Z. Expert opinion: Inhibition of JAK-family kinases is an area of growing interest, catalysed by the maturity of data on marketed inhibitors ruxolitinib and tofacitinib in late stage clinical trials. Many applicants are pursuing traditional fast-follower strategies around these inhibitors, with a range of chemical strategies adopted. The challenge will be to show sufficient differentiation to the originator compounds, since dose limiting toxicities with such agents appear to be on target and mechanism-related and also considering that such agents may be available as generic compounds by the time follower agents reach market.

Inhibitors of JAK-family kinases: an update on the patent literature 2013-2015, part 1.

Science.gov (United States)

Kettle, Jason G; Åstrand, Annika; Catley, Matthew; Grimster, Neil P; Nilsson, Magnus; Su, Qibin; Woessner, Richard

2017-02-01

Janus kinases (JAKs) are a family of four enzymes; JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2) that are critical in cytokine signalling and are strongly linked to both cancer and inflammatory diseases. There are currently two launched JAK inhibitors for the treatment of human conditions: tofacitinib for Rheumatoid arthritis (RA) and ruxolitinib for myeloproliferative neoplasms including intermediate or high risk myelofibrosis and polycythemia vera. Areas covered: This review covers patents claiming activity against one or more JAK family members in the period 2013-2015 inclusive, and covers 95 patents from 42 applicants, split over two parts. The authors have ordered recent patents according to the primary applicant's name, with part 1 covering A through to I. Expert opinion: Inhibition of JAK-family kinases is an area of growing interest, catalysed by the maturity of data on marketed inhibitors ruxolitinib and tofacitinib in late stage clinical trials. Many applicants are pursuing traditional fast-follower strategies around these inhibitors, with a range of chemical strategies adopted. The challenge will be to show sufficient differentiation to the originator compounds, since dose limiting toxicities with such agents appear to be on target and mechanism-related and also considering that such agents may be available as generic compounds by the time follower agents reach market.

Role of JAK-STAT pathway in reducing cardiomyocytes hypoxia/reoxygenation injury induced by S1P postconditioning.

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Wang, Yuqing; Wang, Dongfei; Zhang, Lizhi; Ye, Fangyu; Li, Mengmeng; Wen, Ke

2016-08-05

This experiment was designed to explore the protection of sphingosine1-phosphate (S1P) postconditioning on rat myocardial cells injured by hypoxia/reoxygenation acting via the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signal pathway. The data showed that S1P could significantly increase cell viability, lower the rate of apoptosis, decrease the content of lactate dehydrogenase (LDH) and caspase3 activity in the culture medium, increase the activity of total superoxide dismutase (T-SOD) and manganese superoxide dismutase (Mn-SOD), reduce the loss of mitochondrial membrane potential and the fluorescence intensity of intracellular calcium, as well as increase the phosphorylation of JAK2 and STAT3 in comparison with the H/R group. When the JAK inhibitor AG490 or the STAT inhibitor stattic were added, the effects of S1P were inhibited. Our date shows that S1P protects H9c2 cells from hypoxia/reoxygenation injury and that the protection by S1P was inhibited by AG490 and stattic. Therefore S1P protects H9c2 cells against hypoxia/reoxygenation injury via the JAK-STAT pathway. Copyright © 2016 Elsevier B.V. All rights reserved.

Honokiol inhibits sphere formation and xenograft growth of oral cancer side population cells accompanied with JAK/STAT signaling pathway suppression and apoptosis induction

International Nuclear Information System (INIS)

Huang, Jhy-Shrian; Yao, Chih-Jung; Chuang, Shuang-En; Yeh, Chi-Tai; Lee, Liang-Ming; Chen, Ruei-Ming; Chao, Wan-Ju; Whang-Peng, Jacqueline; Lai, Gi-Ming

2016-01-01

Eliminating cancer stem cells (CSCs) has been suggested for prevention of tumor recurrence and metastasis. Honokiol, an active compound of Magnolia officinalis, had been proposed to be a potential candidate drug for cancer treatment. We explored its effects on the elimination of oral CSCs both in vitro and in vivo. By using the Hoechst side population (SP) technique, CSCs-like SP cells were isolated from human oral squamous cell carcinoma (OSCC) cell lines, SAS and OECM-1. Effects of honokiol on the apoptosis and signaling pathways of SP-derived spheres were examined by Annexin V/Propidium iodide staining and Western blotting, respectively. The in vivo effectiveness was examined by xenograft mouse model and immunohistochemical tissue staining. The SP cells possessed higher stemness marker expression (ABCG2, Ep-CAM, Oct-4 and Nestin), clonogenicity, sphere formation capacity as well as tumorigenicity when compared to the parental cells. Treatment of these SP-derived spheres with honokiol resulted in apoptosis induction via Bax/Bcl-2 and caspase-3-dependent pathway. This apoptosis induction was associated with marked suppression of JAK2/STAT3, Akt and Erk signaling pathways in honokiol-treated SAS spheres. Consistent with its effect on JAK2/STAT3 suppression, honokiol also markedly inhibited IL-6-mediated migration of SAS cells. Accordingly, honokiol dose-dependently inhibited the growth of SAS SP xenograft and markedly reduced the immunohistochemical staining of PCNA and endothelial marker CD31 in the xenograft tumor. Honokiol suppressed the sphere formation and xenograft growth of oral CSC-like cells in association with apoptosis induction and inhibition of survival/proliferation signaling pathways as well as angiogenesis. These results suggest its potential as an integrative medicine for combating oral cancer through targeting on CSCs. The online version of this article (doi:10.1186/s12885-016-2265-6) contains supplementary material, which is available to

TARGETED ANALYSIS OF JAK-STAT-SOCS GENES IN DAIRY CATTLE

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Arun Sondur Jayappa

2015-12-01

Full Text Available The Janus kinase and signal transducer and activator of transcription (JAK-STAT pathway genes along with suppressors of cytokine signalling (SOCS family genes play a crucial role in controlling cytokine signals in the mammary gland and thus mammary gland development. Mammary gene expression studies showed differential expression patterns for all the JAK-STAT pathway genes. Gene expression studies using qRT-PCR revealed differential expression of SOCS2, SOCS4 and SOCS5 genes across the lactation cycle in dairy cows. Using genotypes from 1,546 Australian Holstein- Friesian bulls, a statistical model based on SNPs within 500kb of JAK-STAT pathway genes, and SOCS genes alone was carried out. The analysis suggested that these genes and pathways make a significant contribution to the Australian milk production traits. Selection of 24 SNPs close to SOCS1, SOCS3, SOCS5, SOCS7 and CISH genes were significantly associated with, Australian Profit Ranking (APR, Australian Selection Index (ASI and protein yield (PY. This study supports the view that there may be some merit in choosing SNPs around functionally relevant genes for the selection and genetic improvement schemes for dairy production traits.

The cytomegalovirus homolog of interleukin-10 requires phosphatidylinositol 3-kinase activity for inhibition of cytokine synthesis in monocytes.

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Spencer, Juliet V

2007-02-01

Human cytomegalovirus (CMV) has evolved numerous strategies for evading host immune defenses, including piracy of cellular cytokines. A viral homolog of interleukin-10, designated cmvIL-10, binds to the cellular IL-10 receptor and effects potent immune suppression. The signaling pathways employed by cmvIL-10 were investigated, and the classic IL-10R/JAK1/Stat3 pathway was found to be activated in monocytes. However, inhibition of JAK1 had little effect on cmvIL-10-mediated suppression of tumor necrosis factor alpha (TNF-alpha) production. Inhibition of the phosphatidylinositol 3-kinase/Akt pathway had a more significant impact on TNF-alpha levels but did not completely relieve the immune suppression, demonstrating that cmvIL-10 stimulates multiple signaling pathways to modulate cell function.

Proteasome Inhibitor YSY01A Abrogates Constitutive STAT3 Signaling via Down-regulation of Gp130 and JAK2 in Human A549 Lung Cancer Cells

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Wei Huang

2017-08-01

Full Text Available Proteasome inhibition interfering with many cell signaling pathways has been extensively explored as a therapeutic strategy for cancers. Proteasome inhibitor YSY01A is a novel agent that has shown remarkable anti-tumor effects; however, its mechanisms of action are not fully understood. Here we report that YSY01A is capable of suppressing cancer cell survival by induction of apoptosis. Paradoxically, we find that YSY01A abrogates constitutive activation of STAT3 via proteasome-independent degradation of gp130 and JAK2, but not transcriptional regulation, in human A549 non-small cell lung cancer cells. The reduction in gp130 and JAK2 can be restored by co-treatment with 3-methyladenine, an early-stage autophagy lysosome and type I/III PI3K inhibitor. YSY01A also effectively inhibits cancer cell migration and lung xenograft tumor growth with little adverse effect on animals. Thus, our findings suggest that YSY01A represents a promising candidate for further development of novel anticancer therapeutics targeting the proteasome.

Karel IV. a dvojí Te Deum aneb Jak to bylo tehdy a jak dnes

Czech Academy of Sciences Publication Activity Database

Maňour, Ondřej

2016-01-01

Roč. 3, č. 2 (2016) ISSN 2533-6940 Institutional support: RVO:68378076 Keywords : Czech medieval music * Charles IV * society Subject RIV: AL - Art, Architecture, Cultural Heritage http://blog.ceskafilharmonie.cz/karel-iv-a-dvoji-te-deum-aneb-jak-to-bylo-tehdy-a-jak-dnes/

Novel 1,3,4-thiadiazoles inhibit colorectal cancer via blockade of IL-6/COX-2 mediated JAK2/STAT3 signals as evidenced through data-based mathematical modeling.

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Raj, Vinit; Bhadauria, Archana S; Singh, Ashok K; Kumar, Umesh; Rai, Amit; Keshari, Amit K; Kumar, Pranesh; Kumar, Dinesh; Maity, Biswanath; Nath, Sneha; Prakash, Anand; Ansari, Kausar M; Jat, Jawahar L; Saha, Sudipta

2018-03-23

We attempted a preclinical study using DMH-induced CRC rat model to evaluate the antitumor potential of our recently synthesized 1,3,4-thiadiazoles. The molecular insights were confirmed through ELISA, qRT-PCR and western blot analyses. The CRC condition was produced in response to COX-2 and IL-6 induced activation of JAK2/STAT3 which, in turn, was due to the enhanced phosphorylation of JAK2 and STAT3. The treatment with 1,3,4-thiadiazole derivatives (VR24 and VR27) caused the significant blockade of this signaling pathway. The behavior of STAT3 populations in response to IL-6 and COX-2 stimulations was further confirmed through data-based mathematical modeling using the quantitative western blot data. Finally, VR24 and VR27 restored the perturbed metabolites associated to DMH-induced CRC as evidenced through 1 H NMR based serum metabolomics. The tumor protecting ability of VR24 and VR27 was found comparable or to some degree better than the marketed chemotherapeutics, 5-flurouracil. Copyright © 2018 Elsevier Ltd. All rights reserved.

JAK2 mediates lung fibrosis, pulmonary vascular remodelling and hypertension in idiopathic pulmonary fibrosis: an experimental study.

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Milara, Javier; Ballester, Beatriz; Morell, Anselm; Ortiz, José L; Escrivá, Juan; Fernández, Estrella; Perez-Vizcaino, Francisco; Cogolludo, Angel; Pastor, Enrique; Artigues, Enrique; Morcillo, Esteban; Cortijo, Julio

2018-06-01

Pulmonary hypertension (PH) is a common disorder in patients with idiopathic pulmonary fibrosis (IPF) and portends a poor prognosis. Recent studies using vasodilators approved for PH have failed in improving IPF mainly due to ventilation ( V )/perfusion ( Q ) mismatching and oxygen desaturation. Janus kinase type 2 (JAK2) is a non-receptor tyrosine kinase activated by a broad spectrum of profibrotic and vasoactive mediators, but its role in PH associated to PH is unknown. The study of JAK2 as potential target to treat PH in IPF. JAK2 expression was increased in pulmonary arteries (PAs) from IPF (n=10; 1.93-fold; P=0.0011) and IPF+PH (n=9; 2.65-fold; Ppulmonary artery endothelial cells (HPAECs) and human pulmonary artery smooth muscle cells (HPASMCs) from patients with IPF in vitro treated with the JAK2 inhibitor JSI-124 or siRNA-JAK2 and stimulated with transforming growth factor beta. Both JSI-124 and siRNA-JAK2 inhibited the HPAEC to mesenchymal transition and the HPASMCs to myofibroblast transition and proliferation. JAK2 inhibition induced small PA relaxation in precision-cut lung slice experiments. PA relaxation was dependent of the large conductance calcium-activated potassium channel (BK Ca ). JAK2 inhibition activated BK Ca channels and reduced intracellular Ca 2+ . JSI-124 1 mg/kg/day, reduced bleomycin-induced lung fibrosis, PA remodelling, right ventricular hypertrophy, PA hypertension and V / Q mismatching in rats. The animal studies followed the ARRIVE guidelines. JAK2 participates in PA remodelling and tension and may be an attractive target to treat IPF associated to PH. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Intracellular expression of IRF9 Stat fusion protein overcomes the defective Jak-Stat signaling and inhibits HCV RNA replication

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Balart Luis A

2010-10-01

Full Text Available Abstract Interferon alpha (IFN-α binds to a cell surface receptor that activates the Jak-Stat signaling pathway. A critical component of this pathway is the translocation of interferon stimulated gene factor 3 (a complex of three proteins Stat1, Stat2 and IRF9 to the nucleus to activate antiviral genes. A stable sub-genomic replicon cell line resistant to IFN-α was developed in which the nuclear translocation of Stat1 and Stat2 proteins was prevented due to the lack of phosphorylation; whereas the nuclear translocation of IRF9 protein was not affected. In this study, we sought to overcome defective Jak-Stat signaling and to induce an antiviral state in the IFN-α resistant replicon cell line by developing a chimera IRF9 protein fused with the trans activating domain (TAD of either a Stat1 (IRF9-S1C or Stat2 (IRF9-S2C protein. We show here that intracellular expression of fusion proteins using the plasmid constructs of either IRF9-S1C or IRF9-S2C, in the IFN-α resistant cells, resulted in an increase in Interferon Stimulated Response Element (ISRE luciferase promoter activity and significantly induced HLA-1 surface expression. Moreover, we show that transient transfection of IRF9-S1C or IRF9-S2C plasmid constructs into IFN-α resistant replicon cells containing sub-genomic HCV1b and HCV2a viruses resulted in an inhibition of viral replication and viral protein expression independent of IFN-α treatment. The results of this study indicate that the recombinant fusion proteins of IRF9-S1C, IRF9-S2C alone, or in combination, have potent antiviral properties against the HCV in an IFN-α resistant cell line with a defective Jak-Stat signaling.

Transforming and tumorigenic activity of JAK2 by fusion to BCR: molecular mechanisms of action of a novel BCR-JAK2 tyrosine-kinase.

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Álvaro Cuesta-Domínguez

Full Text Available Chromosomal translocations in tumors frequently produce fusion genes coding for chimeric proteins with a key role in oncogenesis. Recent reports described a BCR-JAK2 fusion gene in fatal chronic and acute myeloid leukemia, but the functional behavior of the chimeric protein remains uncharacterized. We used fluorescence in situ hybridization and reverse transcription polymerase chain reaction (RT-PCR assays to describe a BCR-JAK2 fusion gene from a patient with acute lymphoblastic leukemia. The patient has been in complete remission for six years following treatment and autologous transplantation, and minimal residual disease was monitored by real-time RT-PCR. BCR-JAK2 codes for a protein containing the BCR oligomerization domain fused to the JAK2 tyrosine-kinase domain. In vitro analysis of transfected cells showed that BCR-JAK2 is located in the cytoplasm. Transduction of hematopoietic Ba/F3 cells with retroviral vectors carrying BCR-JAK2 induced IL-3-independent cell growth, constitutive activation of the chimeric protein as well as STAT5 phosphorylation and translocation to the nuclei, where Bcl-xL gene expression was elicited. Primary mouse progenitor cells transduced with BCR-JAK2 also showed increased proliferation and survival. Treatment with the JAK2 inhibitor TG101209 abrogated BCR-JAK2 and STAT5 phosphorylation, decreased Bcl-xL expression and triggered apoptosis of transformed Ba/F3 cells. Therefore, BCR-JAK2 is a novel tyrosine-kinase with transforming activity. It deregulates growth factor-dependent proliferation and cell survival, which can be abrogated by the TG101209 inhibitor. Moreover, transformed Ba/F3 cells developed tumors when injected subcutaneously into nude mice, thus proving the tumorigenic capacity of BCR-JAK2 in vivo. Together these findings suggest that adult and pediatric patients with BCR-ABL-negative leukemia and JAK2 overexpression may benefit from targeted therapies.

JAK2 and MPL protein levels determine TPO-induced megakaryocyte proliferation vs differentiation.

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Besancenot, Rodolphe; Roos-Weil, Damien; Tonetti, Carole; Abdelouahab, Hadjer; Lacout, Catherine; Pasquier, Florence; Willekens, Christophe; Rameau, Philippe; Lecluse, Yann; Micol, Jean-Baptiste; Constantinescu, Stefan N; Vainchenker, William; Solary, Eric; Giraudier, Stéphane

2014-09-25

Megakaryopoiesis is a 2-step differentiation process, regulated by thrombopoietin (TPO), on binding to its cognate receptor myeloproliferative leukemia (MPL). This receptor associates with intracytoplasmic tyrosine kinases, essentially janus kinase 2 (JAK2), which regulates MPL stability and cell-surface expression, and mediates TPO-induced signal transduction. We demonstrate that JAK2 and MPL mediate TPO-induced proliferation arrest and megakaryocytic differentiation of the human megakaryoblastic leukemia cell line UT7-MPL. A decrease in JAK2 or MPL protein expression, and JAK2 chemical inhibition, suppress this antiproliferative action of TPO. The expression of JAK2 and MPL, which progressively increases along normal human megakaryopoiesis, is decreased in platelets of patients diagnosed with JAK2- or MPL-mutated essential thrombocytemia and primary myelofibrosis, 2 myeloproliferative neoplasms in which megakaryocytes (MKs) proliferate excessively. Finally, low doses of JAK2 chemical inhibitors are shown to induce a paradoxical increase in MK production, both in vitro and in vivo. We propose that JAK2 and MPL expression levels regulate megakaryocytic proliferation vs differentiation in both normal and pathological conditions, and that JAK2 chemical inhibitors could promote a paradoxical thrombocytosis when used at suboptimal doses. © 2014 by The American Society of Hematology.

Resolution of bone marrow fibrosis in a patient receiving JAK1/JAK2 inhibitor treatment with ruxolitinib.

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Wilkins, Bridget S; Radia, Deepti; Woodley, Claire; Farhi, Sarah El; Keohane, Clodagh; Harrison, Claire N

2013-12-01

Ruxolitinib, a JAK1/JAK2 inhibitor, is currently the only pharmacological agent approved for the treatment of myelofibrosis. Approval was based on findings from two phase 3 trials comparing ruxolitinib with placebo (COMFORT-I) and with best available therapy (COMFORT-II) for the treatment of primary or secondary myelofibrosis. In those pivotal trials, ruxolitinib rapidly improved splenomegaly, disease-related symptoms, and quality of life and prolonged survival compared with both placebo and conventional treatments. However, for reasons that are currently unclear, there were only modest histomorphological changes in the bone marrow, and only a subset of patients had significant reductions in JAK2 V617F clonal burden. Here we describe a patient with post-polycythemia vera myelofibrosis who received ruxolitinib at our institution (Guy's and St. Thomas' NHS Foundation Trust, London, United Kingdom) as part of the COMFORT-II study. While on treatment, the patient had dramatic improvements in splenomegaly and symptoms shortly after starting ruxolitinib. With longer treatment, the patient had marked reductions in JAK2 V617F allele burden, and fibrosis of the bone marrow resolved after approximately 3 years of ruxolitinib treatment. To our knowledge, this is the first detailed case report of resolution of fibrosis with a JAK1/JAK2 inhibitor. ClinicalTrials.gov Identifier: NCT00934544.

Inhibitory Effect of Curcumol on Jak2-STAT Signal Pathway Molecules of Fibroblast-Like Synoviocytes in Patients with Rheumatoid Arthritis

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Heng Wang

2012-01-01

Full Text Available Hyperplasia of synovial membrane in rheumatoid arthritis (RA is a critical pathological foundation for inducing articular injury. The janus kinase and signal transducer and activator of transcription (Jak-STAT pathway plays a critical role in synovial membrane proliferation induced by platelet-derived growth factor (PDGF. To explore the anti-cell proliferation mechanism of curcumol, a pure monomer extracted from Chinese medical plant zedoary rhizome, the changes of Jak2-STAT1/3 signal pathway-related molecules in synoviocytes were observed in vitro. In this study, the fibroblast-like synoviocytes (FLS in patients with RA were collected and cultured. The following parameters were measured: cell proliferation (WST-1 assay, cell cycles (fluorescence-activated cell sorting, FACS, STAT1 and STAT3 activities (electrophoretic mobility shift assay, EMSA, and the protein expressions of phosphorylated Jak2, STAT1, and STAT3 (Western blot. It was shown that curcumol could inhibit the RA-FLS proliferation and DNA synthesis induced by PDGF-BB in a dose-dependent manner in vitro. The transcription factors activities of STAT1 and STAT3 were obviously elevated after PDGF-BB stimulation (P<0.05. Super-shift experiments identified the STAT1 or STAT3 proteins in the complex. Furthermore, the different concentration curcumol could downregulate the DNA binding activities of STAT1 and STAT3 (P<0.05 and inhibit the phosphorylation of Jak2 while it had no effect on the protein expressions of STAT1 and STAT3. Positive correlations were found between changes of cell proliferation and DNA-binding activities of STAT1 and STAT3, respectively (P<0.01. In conclusion, curcumol might suppress the FLS proliferation and DNA synthesis induced by PDGF-BB through attenuating Jak2 phosphorylation, downregulating STAT1 and STAT3 DNA-binding activities, which could provide theoretical foundation for clinical treatment of RA.

Epigallocatechin-3-gallate attenuates apoptosis and autophagy in concanavalin A-induced hepatitis by inhibiting BNIP3

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Li S

2016-02-01

Full Text Available Sainan Li, Yujing Xia, Kan Chen, Jingjing Li, Tong Liu, Fan Wang, Jie Lu, Yingqun Zhou, Chuanyong Guo Department of Gastroenterology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai, People’s Republic of China Background: Epigallocatechin-3-gallate (EGCG is the most effective compound in green tea, and possesses a wide range of beneficial effects, including anti-inflammatory, antioxidant, antiobesity, and anticancer effects. In this study, we investigated the protective effects of EGCG in concanavalin A (ConA-induced hepatitis in mice and explored the possible mechanisms involved in these effects.Methods: Balb/C mice were injected with ConA (25 mg/kg to induce acute autoimmune hepatitis, and EGCG (10 or 30 mg/kg was administered orally twice daily for 10 days before ConA injection. Serum liver enzymes, proinflammatory cytokines, and other marker proteins were determined 2, 8, and 24 hours after the ConA administration.Results: BNIP3 mediated cell apoptosis and autophagy in ConA-induced hepatitis. EGCG decreased the immunoreaction and pathological damage by reducing inflammatory factors, such as TNF-α, IL-6, IFN-γ, and IL-1β. EGCG also exhibited an antiapoptotic and antiautophagic effect by inhibiting BNIP3 via the IL-6/JAKs/STAT3 pathway.Conclusion: EGCG attenuated liver injury in ConA-induced hepatitis by downregulating IL-6/JAKs/STAT3/BNIP3-mediated apoptosis and autophagy. Keywords: concanavalin A, hepatitis, EGCG, autophagy, apoptosis, BNIP3, STAT3, JAKs, IL-6

Suppressor of Cytokine Signaling (SOCS 5 utilises distinct domains for regulation of JAK1 and interaction with the adaptor protein Shc-1.

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Edmond M Linossi

Full Text Available Suppressor of Cytokine Signaling (SOCS5 is thought to act as a tumour suppressor through negative regulation of JAK/STAT and epidermal growth factor (EGF signaling. However, the mechanism/s by which SOCS5 acts on these two distinct pathways is unclear. We show for the first time that SOCS5 can interact directly with JAK via a unique, conserved region in its N-terminus, which we have termed the JAK interaction region (JIR. Co-expression of SOCS5 was able to specifically reduce JAK1 and JAK2 (but not JAK3 or TYK2 autophosphorylation and this function required both the conserved JIR and additional sequences within the long SOCS5 N-terminal region. We further demonstrate that SOCS5 can directly inhibit JAK1 kinase activity, although its mechanism of action appears distinct from that of SOCS1 and SOCS3. In addition, we identify phosphoTyr317 in Shc-1 as a high-affinity substrate for the SOCS5-SH2 domain and suggest that SOCS5 may negatively regulate EGF and growth factor-driven Shc-1 signaling by binding to this site. These findings suggest that different domains in SOCS5 contribute to two distinct mechanisms for regulation of cytokine and growth factor signaling.

CRH promotes human colon cancer cell proliferation via IL-6/JAK2/STAT3 signaling pathway and VEGF-induced tumor angiogenesis.

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Fang, Xianjun; Hong, Yali; Dai, Li; Qian, Yuanyuan; Zhu, Chao; Wu, Biao; Li, Shengnan

2017-11-01

Corticotrophin-releasing hormone (CRH) has been demonstrated to participate in various diseases. Our previous study showed that its receptor CRHR1 mediated the development of colitis-associated cancer in mouse model. However, the detailed mechanisms remain unclear. In this study, we explored the oncogenetic role of CRH/CRHR1 signaling in colon cancer cells. Cell proliferation and colony formation assays revealed that CRH contributed to cell proliferation. Moreover, tube formation assay showed that CRH-treated colon cancer cell supernatant significantly promoted tube formation of human umbilical vein endothelial cells (HUVECs). And these effects could be reversed by the CRHR1 specific antagonist Antalarmin. Further investigation showed that CRH significantly upregulated the expressions of interlukin-6 (IL-6) and vascular endothelial growth factor (VEGF) through activating nuclear factor-kappa B (NF-κB). The CRH-induced IL-6 promoted phosphorylation of janus kinase 2 (JAK2) and signal transducers and activators of transcription 3 (STAT3). STAT3 inhibition by Stattic significantly inhibited the CRH-induced cell proliferation. In addition, silence of VEGF resulted in declined tube formation induced by CRH. Taken together, CRH/CRHR1 signaling promoted human colon cancer cell proliferation via NF-κB/IL-6/JAK2/STAT3 signaling pathway and tumor angiogenesis via NF-κB/VEGF signaling pathway. Our results provide evidence to support a critical role for the CRH/CRHR1 signaling in colon cancer progression and suggest its potential utility as a new therapeutic target for colon cancer. © 2017 Wiley Periodicals, Inc.

Curcumin induces growth-arrest and apoptosis in association with the inhibition of constitutively active JAK-STAT pathway in T cell leukemia

International Nuclear Information System (INIS)

Rajasingh, Johnson; Raikwar, Himanshu P.; Muthian, Gladson; Johnson, Caroline; Bright, John J.

2006-01-01

Adult T cell leukemia is an aggressive and frequently fatal malignancy that expressess constitutively activated growth-signaling pathways in association with deregulated growth and resistance to apoptosis. Curcumin (diferuloylmethane) is a naturally occurring yellow pigment, isolated from the rhizomes of the plant Curcuma longa that has traditionally been used in the treatment of injury and inflammation. But the effect and mechanism of action of curcumin on T cell leukemia is not known. To investigate the antitumor activity of curcumin in T cell leukemia, we examined its effect on constitutive phosphorylation of JAK and STAT proteins, proliferation, and apoptosis in HTLV-I-transformed T cell lines. HTLV-I-transformed T cell leukemia lines, MT-2, HuT-102, and SLB-1, express constitutively phosphorylated JAK3, TYK2, STAT3, and STAT5 signaling proteins. In vitro treatment with curcumin induced a dose-dependent decrease in JAK and STAT phosphorylation resulting in the induction of growth-arrest and apoptosis in T cell leukemia. The induction of growth-arrest and apoptosis in association with the blockade of constitutively active JAK-STAT pathway suggests this be a mechanism by which curcumin induces antitumor activity in T cell leukemia

Arctigenin enhances chemosensitivity of cancer cells to cisplatin through inhibition of the STAT3 signaling pathway.

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Yao, Xiangyang; Zhu, Fenfen; Zhao, Zhihui; Liu, Chang; Luo, Lan; Yin, Zhimin

2011-10-01

Arctigenin is a dibenzylbutyrolactone lignan isolated from Bardanae fructus, Arctium lappa L, Saussureamedusa, Torreya nucifera, and Ipomea cairica. It has been reported to exhibit anti-inflammatory activities, which is mainly mediated through its inhibitory effect on nuclear transcription factor-kappaB (NF-κB). But the role of arctigenin in JAK-STAT3 signaling pathways is still unclear. In present study, we investigated the effect of arctigenin on signal transducer and activator of transcription 3 (STAT3) pathway and evaluated whether suppression of STAT3 activity by arctigenin could sensitize cancer cells to a chemotherapeutic drug cisplatin. Our results show that arctigenin significantly suppressed both constitutively activated and IL-6-induced STAT3 phosphorylation and subsequent nuclear translocation in cancer cells. Inhibition of STAT3 tyrosine phosphorylation was found to be achieved through suppression of Src, JAK1, and JAK2, while suppression of STAT3 serine phosphorylation was mediated by inhibition of ERK activation. Pervanadate reversed the arctigenin-induced downregulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase. Indeed, arctigenin can obviously induce the expression of the PTP SHP-2. Furthermore, the constitutive activation level of STAT3 was found to be correlated to the resistance of cancer cells to cisplatin-induced apoptosis. Arctigenin dramatically promoted cisplatin-induced cell death in cancer cells, indicating that arctigenin enhanced the sensitivity of cancer cells to cisplatin mainly via STAT3 suppression. These observations suggest a novel anticancer function of arctigenin and a potential therapeutic strategy of using arctigenin in combination with chemotherapeutic agents for cancer treatment. Copyright © 2011 Wiley-Liss, Inc.

Mutation Analysis of JAK2V617F, FLT3-ITD, NPM1, and DNMT3A in Chinese Patients with Myeloproliferative Neoplasms

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Min Wang

2014-01-01

Full Text Available Since the discovery of JAK2V617F tyrosine kinase-activating mutation, several genes have been found mutated in myeloproliferative neoplasms (MPNs. FLT3-ITD, NPM1, and DNMT3A mutations frequently occurred in AML patients and have been found conferred with myeloproliferative neoplasms in mouse model. Therefore, we sought to search for mutations in JAK2V617F, FLT3-ITD, NPM1, and DNMT3A in 129 cases including 120 classic MPN cases and 9 MDS/MPN cases. JAK2V617F mutation was found in 60% of the 120 classic MPNs. However, none of the patients displayed FLT3-ITD and NPM1 mutations; only 2 patients harbored DNMT3A R882 mutation. Further studies including whole-genome sequence will be conducted to investigate the possible involvement of these genes in MPN.

JAK2 mutations and clinical practice in myeloproliferative neoplasms.

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Tefferi, Ayalew

2007-01-01

With the discovery in the last 3 years of novel Janus kinase 2 (JAK2) and thrombopoietin receptor (MPL) mutations, the pathogenetic understanding of and clinical practice for myeloproliferative neoplasms (MPNs) have entered a new era. Each one of these newly discovered mutations, including JAK2V617F, MPLW515L, and a JAK2 exon 12 mutation, has been shown to result in constitutive activation of JAK-STAT signaling and also induce a MPN phenotype in mice. Thus, JAK2 is now considered to be a legitimate target for drug development in MPNs, and small molecule JAK2 inhibitors have already gone through successful preclinical testing, and early-phase human trials in primary myelofibrosis have already begun. Furthermore, JAK2 mutation screening has now become a front-line diagnostic test in the evaluation of both "erythrocytosis" and thrombocytosis and the 2001 World Health Organization diagnostic criteria for polycythemia vera, essential thrombocythemia, and primary myelofibrosis have now been revised to incorporate JAK2V617F mutation screening.

Controlling nuclear JAKs and STATs for specific gene activation by IFNγ

International Nuclear Information System (INIS)

Noon-Song, Ezra N.; Ahmed, Chulbul M.; Dabelic, Rea; Canton, Johnathan; Johnson, Howard M.

2011-01-01

Highlights: → Gamma interferon (IFNγ) and its receptor subunit, IFNGR1, interact with the promoter region of IFNγ-associated genes along with transcription factor STAT1α. → We show that activated Janus kinases pJAK2 and pJAK1 also associate with IFNGR1 in the nucleus. → The activated Janus kinases are responsible for phosphorylation of tyrosine 41 on histone H3, an important epigenetic event for specific gene activation. -- Abstract: We previously showed that gamma interferon (IFNγ) and its receptor subunit, IFNGR1, interacted with the promoter region of IFNγ-activated genes along with transcription factor STAT1α. Recent studies have suggested that activated Janus kinases pJAK2 and pJAK1 also played a role in gene activation by phosphorylation of histone H3 on tyrosine 41. This study addresses the question of the role of activated JAKs in specific gene activation by IFNγ. We carried out chromatin immunoprecipitation (ChIP) followed by PCR in IFNγ treated WISH cells and showed association of pJAK1, pJAK2, IFNGR1, and STAT1 on the same DNA sequence of the IRF-1 gene promoter. The β-actin gene, which is not activated by IFNγ, did not show this association. The movement of activated JAK to the nucleus and the IRF-1 promoter was confirmed by the combination of nuclear fractionation, confocal microscopy and DNA precipitation analysis using the biotinylated GAS promoter. Activated JAKs in the nucleus was associated with phosphorylated tyrosine 41 on histone H3 in the region of the GAS promoter. Unphosphorylated JAK2 was found to be constitutively present in the nucleus and was capable of undergoing activation in IFNγ treated cells, most likely via nuclear IFNGR1. Association of pJAK2 and IFNGR1 with histone H3 in IFNγ treated cells was demonstrated by histone H3 immunoprecipitation. Unphosphorylated STAT1 protein was associated with histone H3 of untreated cells. IFNγ treatment resulted in its disassociation and then re-association as pSTAT1. The

Crystal Structure of the FERM-SH2 Module of Human Jak2.

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McNally, Randall; Toms, Angela V; Eck, Michael J

2016-01-01

Jak-family tyrosine kinases mediate signaling from diverse cytokine receptors. Binding of Jaks to their cognate receptors is mediated by their N-terminal region, which contains FERM and SH2 domains. Here we describe the crystal structure of the FERM-SH2 region of Jak2 at 3.0Å resolution. The structure reveals that these domains and their flanking linker segments interact intimately to form an integrated structural module. The Jak2 FERM-SH2 structure closely resembles that recently described for Tyk2, another member of the Jak family. While the overall architecture and interdomain orientations are preserved between Jak2 and Tyk2, we identify residues in the putative receptor-binding groove that differ between the two and may contribute to the specificity of receptor recognition. Analysis of Jak mutations that are reported to disrupt receptor binding reveals that they lie in the hydrophobic core of the FERM domain, and are thus expected to compromise the structural integrity of the FERM-SH2 unit. Similarly, analysis of mutations in Jak3 that are associated with severe combined immunodeficiency suggests that they compromise Jak3 function by destabilizing the FERM-SH2 structure.

MPL mutation profile in JAK2 mutation-negative patients with myeloproliferative disorders.

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Ma, Wanlong; Zhang, Xi; Wang, Xiuqiang; Zhang, Zhong; Yeh, Chen-Hsiung; Uyeji, Jennifer; Albitar, Maher

2011-03-01

Mutations in the thrombopoietin receptor gene (myeloproliferative leukemia, MPL) have been reported in patients with JAK2 V617F-negative chronic myeloproliferative disorders (MPDs). We evaluated the prevalence of MPL mutations relative to JAK2 mutations in patients with suspected MPDs. A total of 2790 patient samples submitted for JAK2 mutation analysis were tested using real-time polymerase chain reaction and bidirectional sequencing of plasma RNA. JAK2 V617F-negative samples were tested for JAK2 exons 12 to 14 mutations, and those with negative results were then tested for mutations in MPL exons 10 and 11. Of the 2790 patients, 529 (18.96%) had V617F, 12 (0.43%) had small insertions or deletions in exon 12, and 7 (0.25%) had other JAK2 mutations in exons 12 to 14. Of the 2242 JAK2 mutation-negative patients, 68 (3.03%) had MPL mutations. W515L was the predominant MPL mutation (n=46; 68%), and 10 (15%) patients had other W515 variants. The remaining MPL mutations (n=12, 17%) were detected at other locations in exons 10 and 11 and included 3 insertion/deletion mutations. The S505N mutation, associated with familial MPD, was detected in 3 patients. Overall, for every 100 V617F mutations in patients with suspected MPDs, there were 12.9 MPL mutations, 2.3 JAK2 exon 12 mutations, and 1.3 JAK2 exons 13 to 14 mutations. These findings suggest that MPL mutation screening should be performed before JAK2 exons 12 to 14 testing in JAK2 V617F-negative patients with suspected MPDs.

The pseudokinase domain of JAK2 is a dual-specificity protein kinase that negatively regulates cytokine signaling

DEFF Research Database (Denmark)

Ungureanu, Daniela; Wu, Jinhua; Pekkala, Tuija

2011-01-01

Human JAK2 tyrosine kinase mediates signaling through numerous cytokine receptors. The JAK2 JH2 domain functions as a negative regulator and is presumed to be a catalytically inactive pseudokinase, but the mechanism(s) for its inhibition of JAK2 remains unknown. Mutations in JH2 lead to increased...... JAK2 activity, contributing to myeloproliferative neoplasms (MPNs). Here we show that JH2 is a dual-specificity protein kinase that phosphorylates two negative regulatory sites in JAK2: Ser523 and Tyr570. Inactivation of JH2 catalytic activity increased JAK2 basal activity and downstream signaling....... Notably, different MPN mutations abrogated JH2 activity in cells, and in MPN (V617F) patient cells phosphorylation of Tyr570 was reduced, suggesting that loss of JH2 activity contributes to the pathogenesis of MPNs. These results identify the catalytic activity of JH2 as a previously unrecognized...

Role of JAK/STAT signaling in neuroepithelial stem cell maintenance and proliferation in the Drosophila optic lobe

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Wang, Wei; Li, Yonggang; Zhou, Liya; Yue, Haitao [School of Life Sciences, Tsinghua University, Beijing 100084 (China); Luo, Hong, E-mail: luohong@mail.tsinghua.edu.cn [School of Life Sciences, Tsinghua University, Beijing 100084 (China)

2011-07-15

Highlights: {yields} JAK/STAT activity is graded in the Drosophila optic lobe neuroepithelium. {yields} Inactivation of JAK signaling causes disintegration of the optic lobe neuroepithelium and depletion of the neuroepithelial stem cells. {yields} JAK pathway overactivation promotes neuroepithelial overgrowth. {yields} Notch signaling acts downstream of JAK/STAT to promote neuroepithelial growth and expansion. -- Abstract: During Drosophila optic lobe development, proliferation and differentiation must be tightly modulated to reach its normal size for proper functioning. The JAK/STAT pathway plays pleiotropic roles in Drosophila development and in the larval brain, has been shown to inhibit medulla neuroblast formation. In this study, we find that JAK/STAT activity is required for the maintenance and proliferation of the neuroepithelial stem cells in the optic lobe. In loss-of-function JAK/STAT mutant brains, the neuroepithelial cells lose epithelial cell characters and differentiate prematurely while ectopic activation of this pathway is sufficient to induce neuroepithelial overgrowth in the optic lobe. We further show that Notch signaling acts downstream of JAK/STAT to control the maintenance and growth of the optic lobe neuroepithelium. Thus, in addition to its role in suppression of neuroblast formation, the JAK/STAT pathway is necessary and sufficient for optic lobe neuroepithelial growth.

Synergistic effect of pacritinib with erlotinib on JAK2-mediated resistance in epidermal gowth factor receptor mutation-positive non-small cell lung Cancer.

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Ochi, Nobuaki; Isozaki, Hideko; Takeyama, Masami; Singer, Jack W; Yamane, Hiromichi; Honda, Yoshihiro; Kiura, Katsuyuki; Takigawa, Nagio

2016-06-10

The combination effect of pacritinib, a novel JAK2/FLT3 inhibitor, with erlotinib, the epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), on non-small cell lung cancer cells with EGFR activating mutations was investigated. The combination showed synergistic effects on JAK2-mediated EGFR TKI-resistant PC-9/ER3 cells in some cases. The combination markedly suppressed pAKT and pERK although pSTAT3 expression was similar regardless of treatment with the pacritinib, pacritinib + erlotinib, or control in PC-9/ER3 cells. Receptor tyrosine kinase array profiling demonstrated that pacritinib suppressed MET in the PC-9/ER3 cells. The combined treatment of pacritinib and erlotinib in PC-9/ER3 xenografts showed more tumor shrinkage compared with each drug as monotherapy. Western blotting revealed that pMET in tumor samples was inhibited. These results suggest MET suppression by pacritinib may play a role in overcoming the EGFR-TKI resistance mediated by JAK2 in the PC-9/ER3 cells. In conclusion, pacritinib combined with EGFR-TKI might be a potent strategy against JAK2-mediated EGFR-TKI resistance. Copyright © 2016 Elsevier Inc. All rights reserved.

CKLF-Like MARVEL Transmembrane Domain-Containing Member 3 (CMTM3) Inhibits the Proliferation and Tumorigenisis in Hepatocellular Carcinoma Cells.

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Li, Wujun; Zhang, Shaobo

2017-01-26

The CKLF-like MARVEL transmembrane domain-containing 3 (CMTM3), a member of the CMTM family, was found in several human tumors and plays an important role in the development and progression of tumors. However, the role of CMTM3 in hepatocellular carcinoma (HCC) remains largely unknown. Thus, in the present study, we explored its expression pattern in human HCC cell lines, as well as its functions in HCC cells. Our results demonstrated that the expression of CMTM3 is lowly expressed in HCC cell lines. In vitro, we found that overexpression of CMTM3 obviously inhibited the proliferation, invasion, and EMT process in HCC cells. Furthermore, overexpression of CMTM3 significantly downregulated the expression levels of phosphorylation of JAK2 and STAT3 in HepG2 cells. In vivo, overexpression of CMTM3 attenuated the tumor growth in Balb/c nude mice. In conclusion, we demonstrated that CMTM3 could play an important role in HCC metastasis by EMT induction via, at least partially, suppressing the JAK2/STAT3 signaling pathway. Therefore, CMTM3 may serve as a potential molecular target in the prevention and/or treatment of HCC invasion and metastasis.

Scoparone exerts anti-tumor activity against DU145 prostate cancer cells via inhibition of STAT3 activity.

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Jeong-Kook Kim

Full Text Available Scoparone, a natural compound isolated from Artemisia capillaris, has been used in Chinese herbal medicine to treat neonatal jaundice. Signal transducer and activator of transcription 3 (STAT3 contributes to the growth and survival of many human tumors. This study was undertaken to investigate the anti-tumor activity of scoparone against DU145 prostate cancer cells and to determine whether its effects are mediated by inhibition of STAT3 activity. Scoparone inhibited proliferation of DU145 cells via cell cycle arrest in G1 phase. Transient transfection assays showed that scoparone repressed both constitutive and IL-6-induced transcriptional activity of STAT3. Western blot and quantitative real-time PCR analyses demonstrated that scoparone suppressed the transcription of STAT3 target genes such as cyclin D1, c-Myc, survivin, Bcl-2, and Socs3. Consistent with this, scoparone decreased phosphorylation and nuclear accumulation of STAT3, but did not reduce phosphorylation of janus kinase 2 (JAK2 or Src, the major upstream kinases responsible for STAT3 activation. Moreover, transcriptional activity of a constitutively active mutant of STAT3 (STAT3C was inhibited by scoparone, but not by AG490, a JAK2 inhibitor. Furthermore, scoparone treatment suppressed anchorage-independent growth in soft agar and tumor growth of DU145 xenografts in nude mice, concomitant with a reduction in STAT3 phosphorylation. Computational modeling suggested that scoparone might bind the SH2 domain of STAT3. Our findings suggest that scoparone elicits an anti-tumor effect against DU145 prostate cancer cells in part through inhibition of STAT3 activity.

The Janus Kinase (JAK) FERM and SH2 Domains: Bringing Specificity to JAK-Receptor Interactions.

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Ferrao, Ryan; Lupardus, Patrick J

2017-01-01

The Janus kinases (JAKs) are non-receptor tyrosine kinases essential for signaling in response to cytokines and interferons and thereby control many essential functions in growth, development, and immune regulation. JAKs are unique among tyrosine kinases for their constitutive yet non-covalent association with class I and II cytokine receptors, which upon cytokine binding bring together two JAKs to create an active signaling complex. JAK association with cytokine receptors is facilitated by N-terminal FERM and SH2 domains, both of which are classical mediators of peptide interactions. Together, the JAK FERM and SH2 domains mediate a bipartite interaction with two distinct receptor peptide motifs, the proline-rich "Box1" and hydrophobic "Box2," which are present in the intracellular domain of cytokine receptors. While the general sidechain chemistry of Box1 and Box2 peptides is conserved between receptors, they share very weak primary sequence homology, making it impossible to posit why certain JAKs preferentially interact with and signal through specific subsets of cytokine receptors. Here, we review the structure and function of the JAK FERM and SH2 domains in light of several recent studies that reveal their atomic structure and elucidate interaction mechanisms with both the Box1 and Box2 receptor motifs. These crystal structures demonstrate how evolution has repurposed the JAK FERM and SH2 domains into a receptor-binding module that facilitates interactions with multiple receptors possessing diverse primary sequences.

JAK/STAT signaling pathway-mediated immune response in silkworm (Bombyx mori) challenged by Beauveria bassiana.

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Geng, Tao; Lv, Ding-Ding; Huang, Yu-Xia; Hou, Cheng-Xiang; Qin, Guang-Xing; Guo, Xi-Jie

2016-12-20

Innate immunity was critical in insects defensive system and able to be induced by Janus kinase/signal transducer and activator of transcription cascade transduction (JAK/STAT) signaling pathway. Currently, it had been identified many JAK/STAT signaling pathway-related genes in silkworm, but little function was known on insect innate immunity. To explore the roles of JAK/STAT pathway in antifungal immune response in silkworm (Bombyx mori) against Beauveria bassiana infection, the expression patterns of B. mori C-type lectin 5 (BmCTL5) and genes encoding 6 components of JAK/STAT signaling pathway in silkworm challenged by B. bassiana were analyzed using quantitative real time PCR. Meanwhile the activation of JAK/STAT signaling pathway by various pathogenic micro-organisms and the affect of JAK/STAT signaling pathway inhibitors on antifungal activity in silkworm hemolymph was also detected. Moreover, RNAi assay of BmCTL5 and the affect on expression levels of signaling factors were also analyzed. We found that JAK/STAT pathway could be obviously activated in silkworm challenged with B. bassiana and had no response to bacteria and B. mori cytoplasmic polyhedrosis virus (BmCPV). However, the temporal expression patterns of JAK/STAT signaling pathway related genes were significantly different. B. mori downstream receptor kinase (BmDRK) might be a positive regulator of JAK/STAT signaling pathway in silkworm against B. bassiana infection. Moreover, antifungal activity assay showed that the suppression of JAK/STAT signaling pathway by inhibitors could significantly inhibit the antifungal activity in hemolymph and resulted in increased sensitivity of silkworm to B. bassiana infection, indicating that JAK/STAT signaling pathway might be involved in the synthesis and secretion of antifungal substances. The results of RNAi assays suggested that BmCTL5 might be one pattern recognition receptors for JAK/STAT signaling pathway in silkworm. These findings yield insights for better

ETS transcription factor ELF5 induces lumen formation in a 3D model of mammary morphogenesis and its expression is inhibited by Jak2 inhibitor TG101348.

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Chean, Jennifer; Chen, Charng-Jui; Shively, John E

2017-10-01

The loss of expression of a single gene can revert normal tissue to a malignant phenotype. For example, while normal breast has high lumenal expression of CEACAM1, the majority of breast cancers exhibit the early loss of this gene with the concurrent loss of their lumenal phenotype. MCF7 cells that lack CEACAM1 expression and fail to form lumena in 3D culture, regain the normal phenotype when transfected with CEACAM1. In order to probe the mechanism of this gain of function, we treated these cells with the clinically relevant Jak2 inhibitor TG101348 (TG), expecting that disruption of the prolactin receptor signaling pathway would interfere with the positive effects of transfection of MCF7 cells with CEACAM1. Indeed, lumen formation was inhibited, resulting in the down regulation of a set of genes, likely involved in the complex process of lumen formation. As expected, inhibition of the expression of many of these genes also inhibited lumen formation, confirming their involvement in a single pathway. Among the genes identified by the inhibition assay, ETS transcription factor ELF5 stood out, since it has been identified as a master regulator of mammary morphogenesis, and is associated with prolactin receptor signaling. When ELF5 was transfected into the parental MCF7 cells that lack CEACAM1, lumen formation was restored, indicating that ELF5 can replace CEACAM1 in this model system of lumenogenesis. We conclude that the event(s) that led to the loss of expression of CEACAM1 is epistatic in that multiple genes associated with a critical pathway were affected, but that restoration of the normal phenotype can be achieved with reactivation of certain genes at various nodal points in tissue morphogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Proinflammatory Cytokine IL-6 and JAK-STAT Signaling Pathway in Myeloproliferative Neoplasms

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Vladan P. Čokić

2015-01-01

Full Text Available The recent JAK1/2 inhibitor trial in myeloproliferative neoplasms (MPNs showed that reducing inflammation can be more beneficial than targeting gene mutants. We evaluated the proinflammatory IL-6 cytokine and JAK-STAT signaling pathway related genes in circulating CD34+ cells of MPNs. Regarding laboratory data, leukocytosis has been observed in polycythemia vera (PV and JAK2V617F mutation positive versus negative primary myelofibrosis (PMF patients. Moreover, thrombocytosis was reduced by JAK2V617F allele burden in essential thrombocythemia (ET and PMF. 261 significantly changed genes have been detected in PV, 82 in ET, and 94 genes in PMF. The following JAK-STAT signaling pathway related genes had augmented expression in CD34+ cells of MPNs: CCND3 and IL23A regardless of JAK2V617F allele burden; CSF3R, IL6ST, and STAT1/2 in ET and PV with JAK2V617F mutation; and AKT2, IFNGR2, PIM1, PTPN11, and STAT3 only in PV. STAT5A gene expression was generally reduced in MPNs. IL-6 cytokine levels were increased in plasma, as well as IL-6 protein levels in bone marrow stroma of MPNs, dependent on JAK2V617F mutation presence in ET and PMF patients. Therefore, the JAK2V617F mutant allele burden participated in inflammation biomarkers induction and related signaling pathways activation in MPNs.

Egr2 enhances insulin resistance via JAK2/STAT3/SOCS-1 pathway in HepG2 cells treated with palmitate.

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Lu, Lin; Ye, Xinhua; Yao, Qing; Lu, Aijiao; Zhao, Zhen; Ding, Yang; Meng, Chuchen; Yu, Wenlong; Du, Yunfeng; Cheng, JinLuo

2018-05-01

Insulin resistance is generally responsible for the pathogenesis of type 2 diabetes mellitus (T2DM). Early growth response proteins-2 (Egr2) has been reported to be able to increase the expression of the suppressors of cytokine signaling-1 (SOCS-1), and impair insulin signaling pathway through suppression of insulin receptor substrates (IRS), including IRS-1 and IRS-2. However, whether Egr2 is directly involved in the development of insulin resistance, and how its potential contributions to insulin resistance still remain unknown. Here, our present investigation found that the expression levels of Egr2 were up-regulated when insulin resistance occurs, and knockdown of Egr2 abolished the effect of insulin resistance in HepG2 cells induced with palmitate (PA). Importantly, inhibition of Egr2 decreased the expression of SOCS-1 as well as reduced phosphorylation of JAK2 and STAT3. And, our data indicated that silencing of Egr2 accelerated hepatic glucose uptake and reversed the impaired lipid metabolism upon insulin resistance. In summary, the present study confirms that Egr2 could deteriorate insulin resistance via the pathway of JAK2/STAT3/SOCS-1 and may shed light on resolving insulin resistance and further the pathogenesis of T2DM. Copyright © 2017 Elsevier Inc. All rights reserved.

Macrophage JAK2 deficiency protects against high-fat diet-induced inflammation.

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Desai, Harsh R; Sivasubramaniyam, Tharini; Revelo, Xavier S; Schroer, Stephanie A; Luk, Cynthia T; Rikkala, Prashanth R; Metherel, Adam H; Dodington, David W; Park, Yoo Jin; Kim, Min Jeong; Rapps, Joshua A; Besla, Rickvinder; Robbins, Clinton S; Wagner, Kay-Uwe; Bazinet, Richard P; Winer, Daniel A; Woo, Minna

2017-08-09

During obesity, macrophages can infiltrate metabolic tissues, and contribute to chronic low-grade inflammation, and mediate insulin resistance and diabetes. Recent studies have elucidated the metabolic role of JAK2, a key mediator downstream of various cytokines and growth factors. Our study addresses the essential role of macrophage JAK2 in the pathogenesis to obesity-associated inflammation and insulin resistance. During high-fat diet (HFD) feeding, macrophage-specific JAK2 knockout (M-JAK2 -/- ) mice gained less body weight compared to wildtype littermate control (M-JAK2 +/+ ) mice and were protected from HFD-induced systemic insulin resistance. Histological analysis revealed smaller adipocytes and qPCR analysis showed upregulated expression of some adipogenesis markers in visceral adipose tissue (VAT) of HFD-fed M-JAK2 -/- mice. There were decreased crown-like structures in VAT along with reduced mRNA expression of some macrophage markers and chemokines in liver and VAT of HFD-fed M-JAK2 -/- mice. Peritoneal macrophages from M-JAK2 -/- mice and Jak2 knockdown in macrophage cell line RAW 264.7 also showed lower levels of chemokine expression and reduced phosphorylated STAT3. However, leptin-dependent effects on augmenting chemokine expression in RAW 264.7 cells did not require JAK2. Collectively, our findings show that macrophage JAK2 deficiency improves systemic insulin sensitivity and reduces inflammation in VAT and liver in response to metabolic stress.

The effects of electromagnetic irradiation on activation of microglia and JAKs in rat hippocampus

International Nuclear Information System (INIS)

Chen Chunhai; Yang Xuesen; Hao Yutong; Zhang Guangbin; Yu Zhengping

2008-01-01

Objective: To determine the activation of microglia and the phosphorylation of Jaks, the upstream factors of JAK/STAT(janus activated kinase/signal transducers and activators of transcription) signaling pathway, after electromagnetic irradiation. Methods: Rats were irradiated by 90 mW/cm 2 EMF for 20 min. The phosphorylation of Jaks was determined by western blot at different time after electromagnetic irradiation. The activation of microglia was determined by immuno- chemistry. Results: GSA-IB4 was upregulated in microglia, which indicated microglia was activated after electromagnetic irradiation. The phosphorylation of Jak1, Jak2 and Jak3 in rat hippocampus was upregulated after electromagnetic irradiation. The phosphorylation of Jakl was upregulated after microwave exposure and peaked at 12 h. Jak2 peaked at 0 h after electro-magnetic irradiation and sustained in a high level. Jak3 was slightly affected by electromagnetic irradiation. All the three members of JAKs return to normal at 72 h after electromagnetic irradiation. Conclusion: Microglia cells was activated after electromagnetic irradiation. The phosphorylation of Jaks was upregulated by electromagnetic irradiation. It suggested that JAK/ STAT singnaling pathway was activated after electromagnetic irradiation, which indicated that JAK/STAT signaling pathway may participate in brain microglia activation induced by electromagnetic irradiation. (authors)

JAK/Stat signaling regulates heart precursor diversification in Drosophila

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Johnson, Aaron N.; Mokalled, Mayssa H.; Haden, Tom N.; Olson, Eric N.

2011-01-01

Intercellular signal transduction pathways regulate the NK-2 family of transcription factors in a conserved gene regulatory network that directs cardiogenesis in both flies and mammals. The Drosophila NK-2 protein Tinman (Tin) was recently shown to regulate Stat92E, the Janus kinase (JAK) and Signal transducer and activator of transcription (Stat) pathway effector, in the developing mesoderm. To understand whether the JAK/Stat pathway also regulates cardiogenesis, we performed a systematic characterization of JAK/Stat signaling during mesoderm development. Drosophila embryos with mutations in the JAK/Stat ligand upd or in Stat92E have non-functional hearts with luminal defects and inappropriate cell aggregations. Using strong Stat92E loss-of-function alleles, we show that the JAK/Stat pathway regulates tin expression prior to heart precursor cell diversification. tin expression can be subdivided into four phases and, in Stat92E mutant embryos, the broad phase 2 expression pattern in the dorsal mesoderm does not restrict to the constrained phase 3 pattern. These embryos also have an expanded pericardial cell domain. We show the E(spl)-C gene HLHm5 is expressed in a pattern complementary to tin during phase 3 and that this expression is JAK/Stat dependent. In addition, E(spl)-C mutant embryos phenocopy the cardiac defects of Stat92E embryos. Mechanistically, JAK/Stat signals activate E(spl)-C genes to restrict Tin expression and the subsequent expression of the T-box transcription factor H15 to direct heart precursor diversification. This study is the first to characterize a role for the JAK/Stat pathway during cardiogenesis and identifies an autoregulatory circuit in which tin limits its own expression domain. PMID:21965617

A phase II study of the oral JAK1/JAK2 inhibitor ruxolitinib in advanced relapsed/refractory Hodgkin lymphoma

NARCIS (Netherlands)

E. van den Neste (Eric); J.-L. André (Jean-Luc); Gastinne, T. (Thomas); A. Stamatoullas (Aspasia); C. Haioun (Corinne); Belhabri, A. (Amine); O. Reman (Oumédaly); O. Casasnovas (O.); H. Ghesquieres; G.E.G. Verhoef (Gregor); Claessen, M.-J. (Marie-José); H.A. Poirel (Hélène A); M.-C. Copin; Dubois, R. (Romain); P. Vandenberghe (Peter); Stoian, I.-A. (Ioanna-Andrea); Cottereau, A.S. (Anne S.); Bailly, S. (Sarah); L. Knoops (Laurent); F. Morschhauser (Frank)

2018-01-01

textabstractJAK2 constitutive activation/overexpression is common in classical Hodgkin lymphoma, and several cytokines stimulate Hodgkin lymphoma cells by recognizing JAK1-/JAK2-bound receptors. JAK blockade may thus be therapeutically beneficial in Hodgkin lymphoma. In this phase II study we

JAK2 Exon 12 Mutations in Polycythemia Vera and Idiopathic Erythrocytosis

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Scott, Linda M.; Tong, Wei; Levine, Ross L.; Scott, Mike A.; Beer, Philip A.; Stratton, Michael R.; Futreal, P. Andrew; Erber, Wendy N.; McMullin, Mary Frances; Harrison, Claire N.; Warren, Alan J.; Gilliland, D. Gary; Lodish, Harvey F.; Green, Anthony R.

2010-01-01

BACKGROUND The V617F mutation, which causes the substitution of phenylalanine for valine at position 617 of the Janus kinase (JAK) 2 gene (JAK2), is often present in patients with polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. However, the molecular basis of these myeloproliferative disorders in patients without the V617F mutation is unclear. METHODS We searched for new mutations in members of the JAK and signal transducer and activator of transcription (STAT) gene families in patients with V617F-negative polycythemia vera or idiopathic erythrocytosis. The mutations were characterized biochemically and in a murine model of bone marrow transplantation. RESULTS We identified four somatic gain-of-function mutations affecting JAK2 exon 12 in 10 V617F-negative patients. Those with a JAK2 exon 12 mutation presented with an isolated erythrocytosis and distinctive bone marrow morphology, and several also had reduced serum erythropoietin levels. Erythroid colonies could be grown from their blood samples in the absence of exogenous erythropoietin. All such erythroid colonies were heterozygous for the mutation, whereas colonies homozygous for the mutation occur in most patients with V617F-positive polycythemia vera. BaF3 cells expressing the murine erythropoietin receptor and also carrying exon 12 mutations could proliferate without added interleukin-3. They also exhibited increased phosphorylation of JAK2 and extracellular regulated kinase 1 and 2, as compared with cells transduced by wild-type JAK2 or V617F JAK2. Three of the exon 12 mutations included a substitution of leucine for lysine at position 539 of JAK2. This mutation resulted in a myeloproliferative phenotype, including erythrocytosis, in a murine model of retroviral bone marrow transplantation. CONCLUSIONS JAK2 exon 12 mutations define a distinctive myeloproliferative syndrome that affects patients who currently receive a diagnosis of polycythemia vera or idiopathic erythrocytosis

JAK2 V617F, MPL W515L and JAK2 Exon 12 Mutations in Chinese Patients with Primary Myelofibrosis.

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Xia, Jun; Lu, Mi-Ze; Jiang, Yuan-Qiang; Yang, Guo-Hua; Zhuang, Yun; Sun, Hong-Li; Shen, Yun-Feng

2012-03-01

JAK2 V617F, MPL W515L and JAK2 exon 12 mutations are novel acquired mutations that induce constitutive cytokine-independent activation of the JAK-STAT pathway in myeloproliferative disorders (MPD). The discovery of these mutations provides novel mechanism for activation of signal transduction in hematopoietic malignancies. This research was to investigate their prevalence in Chinese patients with primary myelofibrosis (PMF). We introduced allele-specific PCR (AS-PCR) combined with sequence analysis to simultaneously screen JAK2 V617F, MPL W515L and JAK2 exon 12 mutations in 30 patients with PMF. Fifteen PMF patients (50.0%) carried JAK2 V617F mutation, and only two JAK2 V617F-negative patients (6.7%) harbored MPL W515L mutation. None had JAK2 exon 12 mutations. Furthermore, these three mutations were not detected in 50 healthy controls. MPL W515L and JAK2 V617F mutations existed in PMF patients but JAK2 exon 12 mutations not. JAK2 V617F and MPL W515L and mutations might contribute to the primary molecular pathogenesis in patients with PMF.

JAK inhibitor has the amelioration effect in lupus-prone mice: the involvement of IFN signature gene downregulation.

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Ikeda, Keigo; Hayakawa, Kunihiro; Fujishiro, Maki; Kawasaki, Mikiko; Hirai, Takuya; Tsushima, Hiroshi; Miyashita, Tomoko; Suzuki, Satoshi; Morimoto, Shinji; Tamura, Naoto; Takamori, Kenji; Ogawa, Hideoki; Sekigawa, Iwao

2017-08-22

We previously reported that JAK-STAT-pathway mediated regulation of IFN-regulatory factor genes could play an important role in SLE pathogenesis. Here, we evaluated the efficacy of the JAK inhibitor tofacitinib (TOFA) for controlling IFN signalling via the JAK-STAT pathway and as a therapeutic for SLE. We treated NZB/NZW F1 mice with TOFA and assessed alterations in their disease, pathological, and immunological conditions. Gene-expression results obtained from CD4 + T cells (SLE mice) and CD3 + T cells (human SLE patients) were measured by DNA microarray and qRT-PCR. TOFA treatment resulted in reduced levels of anti-dsDNA antibodies, decreased proteinuria, and amelioration of nephritis as compared with those observed in control animals. Moreover, we observed the rebalance in the populations of naïve CD4 + T cells and effector/memory cells in TOFA-treated mice; however, treatment with a combination of TOFA and dexamethasone (DEXA) elicited a stronger inhibitory effect toward the effector/memory cells than did TOFA or DEXA monotherapy. We also detected decreased expression of several IFN-signature genes Ifit3 and Isg15 in CD4 + from SLE-prone mice following TOFA and DEXA treatment, and IFIT3 in CD3 + T cells from human patients following immunosuppressant therapy including steroid, respectively. Modulation of type I IFN signalling via JAK-STAT inhibition may exert a beneficial effect in SLE patients, and our results suggest that TOFA could be utilised for the development of new SLE-specific therapeutic strategies.

A novel small molecule inhibits STAT3 phosphorylation and DNA binding activity and exhibits potent growth suppressive activity in human cancer cells

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Lin Li

2010-08-01

Full Text Available Abstract Background Targeting Signal Transducer and Activator of Transcription 3 (STAT3 signaling is an attractive therapeutic approach for most types of human cancers with constitutively activated STAT3. A novel small molecular STAT3 inhibitor, FLLL32 was specifically designed from dietary agent, curcumin to inhibit constitutive STAT3 signaling in multiple myeloma, glioblastoma, liver cancer, and colorectal cancer cells. Results FLLL32 was found to be a potent inhibitor of STAT3 phosphorylation, STAT3 DNA binding activity, and the expression of STAT3 downstream target genes in vitro, leading to the inhibition of cell proliferation as well as the induction of Caspase-3 and PARP cleavages in human multiple myeloma, glioblastoma, liver cancer, and colorectal cancer cell lines. However, FLLL32 exhibited little inhibition on some tyrosine kinases containing SH2 or both SH2 and SH3 domains, and other protein and lipid kinases using a kinase profile assay. FLLL32 was also more potent than four previously reported JAK2 and STAT3 inhibitors as well as curcumin to inhibit cell viability in these cancer cells. Furthermore, FLLL32 selectively inhibited the induction of STAT3 phosphorylation by Interleukin-6 but not STAT1 phosphorylation by IFN-γ. Conclusion Our findings indicate that FLLL32 exhibits potent inhibitory activity to STAT3 and has potential for targeting multiple myeloma, glioblastoma, liver cancer, and colorectal cancer cells expressing constitutive STAT3 signaling.

Hyperactivation of JAK1 tyrosine kinase induces stepwise, progressive pruritic dermatitis.

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Yasuda, Takuwa; Fukada, Toshiyuki; Nishida, Keigo; Nakayama, Manabu; Matsuda, Masashi; Miura, Ikuo; Dainichi, Teruki; Fukuda, Shinji; Kabashima, Kenji; Nakaoka, Shinji; Bin, Bum-Ho; Kubo, Masato; Ohno, Hiroshi; Hasegawa, Takanori; Ohara, Osamu; Koseki, Haruhiko; Wakana, Shigeharu; Yoshida, Hisahiro

2016-06-01

Skin homeostasis is maintained by the continuous proliferation and differentiation of epidermal cells. The skin forms a strong but flexible barrier against microorganisms as well as physical and chemical insults; however, the physiological mechanisms that maintain this barrier are not fully understood. Here, we have described a mutant mouse that spontaneously develops pruritic dermatitis as the result of an initial defect in skin homeostasis that is followed by induction of a Th2-biased immune response. These mice harbor a mutation that results in a single aa substitution in the JAK1 tyrosine kinase that results in hyperactivation, thereby leading to skin serine protease overexpression and disruption of skin barrier function. Accordingly, treatment with an ointment to maintain normal skin barrier function protected mutant mice from dermatitis onset. Pharmacological inhibition of JAK1 also delayed disease onset. Together, these findings indicate that JAK1-mediated signaling cascades in skin regulate the expression of proteases associated with the maintenance of skin barrier function and demonstrate that perturbation of these pathways can lead to the development of spontaneous pruritic dermatitis.

Upregulation of Nicotinic Acetylcholine Receptor alph4+beta2 through a Ligand-Independent PI3Kbeta Mechanism That Is Enhanced by TNFalpha and the Jak2/p38Mapk Pathways.

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Rogers, Scott W; Gahring, Lorise C

2015-01-01

High affinity nicotine-binding sites in the mammalian brain are neuronal nicotinic acetylcholine receptors (nAChR) assembled from at least alpha4 and beta2 subunits into pentameric ion channels. When exposed to ligands such as nicotine, these receptors respond by undergoing upregulation, a correlate of nicotine addiction. Upregulation can be measured using HEK293 (293) cells that stably express alpha4 and beta2 subunits using quantification of [3H]epibatidine ([3H]Eb) binding to measure mature receptors. Treatment of these cells with choline also produces upregulation through a hemicholinium3 (HC3)-sensitive (choline kinase) and an HC3-insensitive pathway which are both independent of the mechanism used by nicotine for upregulation. In both cases, upregulation is significantly enhanced by the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) which signals through its receptor Tnfr1 to activate p38Mapk. Here we report that the inhibition of class1 phosphoinositide 3-kinases isoform PI3Kbeta using the selective antagonist PI828 is alone sufficient to produce upregulation and enhance both nicotine and choline HC3-sensitive mediated upregulation. Further, these processes are impacted upon by an AG-490 sensitive Jak2-associated pathway. Both PI3Kbeta (negative) and Jak2 (positive) modulation of upregulation converge through p38Mapk and both overlap with TNFalpha enhancement of this process. Upregulation through the PI3Kbeta pathway did not require Akt. Collectively these findings support upregulation of endogenous alpha4beta2 as a balance among cellular signaling networks that are highly responsive to multiple environmental, inflammatory and metabolic agents. The findings also suggest how illness and metabolic stress could alter the expression of this important nicotinic receptor and novel avenues to intercede in modifying its expression.

Relationship of JAK2V617F gene mutation with cell proliferation and coagulation function in myeloproliferative neoplasms

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Xiao-Nan Zhang

2017-05-01

Full Text Available Objective: To study the relationship of JAK2V617F gene mutation with cell proliferation and coagulation function in myeloproliferative neoplasms. Methods: Patients who were diagnosed with BCR-ABL-negative myeloproliferative neoplasms in Anyang District Hospital between June 2014 and August 2016 were selected, JAK2V617F gene mutation was detected, and according to the test results, the patients were divided into mutation-positive group and mutation-negative group. The expression of JAK2/STATs signaling pathway molecules and cell proliferation genes in bone marrow fluid as well as the coagulation function indexes in peripheral blood were detected. Results: p-JAK2, p-STAT3, p-STAT5, Survivin, C-myc, CyclinD1 and ASXL1 protein expression in myeloproliferative neoplasms of mutation-positive group were significantly higher than those of mutation-negative group, and peripheral blood PT and APTT levels were significantly lower than those of mutation-negative group while TT and FIB levels were not significantly different from those of mutation-negative group. Conclusion: JAK2V617F gene mutation in myeloproliferative neoplasms can promote the cell proliferation and cause the hypercoagulable state.

Ruxolitinib and Tofacitinib Are Potent and Selective Inhibitors of HIV-1 Replication and Virus Reactivation In Vitro

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Gavegnano, Christina; Detorio, Mervi; Montero, Catherine; Bosque, Alberto; Planelles, Vicente

2014-01-01

The JAK-STAT pathway is activated in both macrophages and lymphocytes upon human immunodeficiency virus type 1 (HIV-1) infection and thus represents an attractive cellular target to achieve HIV suppression and reduced inflammation, which may impact virus sanctuaries. Ruxolitinib and tofacitinib are JAK1/2 inhibitors that are FDA approved for rheumatoid arthritis and myelofibrosis, respectively, but their therapeutic application for treatment of HIV infection was unexplored. Both drugs demonstrated submicromolar inhibition of infection with HIV-1, HIV-2, and a simian-human immunodeficiency virus, RT-SHIV, across primary human or rhesus macaque lymphocytes and macrophages, with no apparent significant cytotoxicity at 2 to 3 logs above the median effective antiviral concentration. Combination of tofacitinib and ruxolitinib increased the efficacy by 53- to 161-fold versus that observed for monotherapy, respectively, and each drug applied alone to primary human lymphocytes displayed similar efficacy against HIV-1 containing various polymerase substitutions. Both drugs inhibited virus replication in lymphocytes stimulated with phytohemagglutinin (PHA) plus interleukin-2 (IL-2), but not PHA alone, and inhibited reactivation of latent HIV-1 at low-micromolar concentrations across the J-Lat T cell latency model and in primary human central memory lymphocytes. Thus, targeted inhibition of JAK provided a selective, potent, and novel mechanism to inhibit HIV-1 replication in lymphocytes and macrophages, replication of drug-resistant HIV-1, and reactivation of latent HIV-1 and has the potential to reset the immunologic milieu in HIV-infected individuals. PMID:24419350

JAK2 46/1 haplotype is associated with JAK2 V617F--positive myeloproliferative neoplasms in Brazilian patients.

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Macedo, L C; Santos, B C; Pagliarini-e-Silva, S; Pagnano, K B B; Rodrigues, C; Quintero, F C; Ferreira, M E; Baraldi, E C; Ambrosio-Albuquerque, E P; Sell, A M; Visentainer, J E L

2015-10-01

This study aimed to verify the association between the JAK2 46/1 haplotype (V617F positive) and some hematological parameters in BCR-ABL-negative chronic myeloproliferative neoplasms (cMPNs) in our population. The blood samples obtained from the patients with cMPN were genotyped for the JAK2 V617F mutation and JAK2 rs10974944 SNP screening using a PCR-RFLP assay. The JAK2 V617F mutation was detected in 80.15% of patients. The G variant of rs10974944 was more frequent in all MPNs, especially those that were JAK2 V617F positive, than in the control population. We also compared the 46/1 haplotype status in each MPN disease entity, polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF), and MPNu with controls. The G allele frequency relative to controls was significantly enriched in patients with PV and ET, but not in those with PMF and MPNu. PV and ET patients especially, all of whom had the JAK2 V617F mutation, showed significant excess of the G allele. The frequency of JAK2 V617F mutation was associated with elevated hematological parameters, but when we analyze the occurrence of the mutation and the presence of the G allele, just the high hemoglobin was significantly. In agreement with previous reports, JAK2 46/1 haplotype for JAK2 V617F was associated with cMPN positive in Brazilian patients. © 2015 John Wiley & Sons Ltd.

Nuclear protein IκB-ζ inhibits the activity of STAT3

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Wu, Zhihao; Zhang, Xiaoai; Yang, Juntao; Wu, Guangzhou; Zhang, Ying; Yuan, Yanzhi; Jin, Chaozhi; Chang, Zhijie; Wang, Jian; Yang, Xiaoming; He, Fuchu

2009-01-01

STAT3 (Signal transducer and activator of transcription 3) is a key transcription factor of the JAK-STAT (Janus kinase/signal transducer and activator of transcription) pathway that regulates cell proliferation and apoptosis. Activation of STAT3 is under tight regulation, and yet the different signaling pathways and the mechanisms that regulate its activity remain to be elucidated. Using a yeast two-hybrid screening, we have identified a nuclear protein IκB-ζ that interacts in a novel way with STAT3. This physical interaction was further confirmed by co-immunoprecipitation assays. The interaction regions were mapped to the coiled-coil domain of STAT3 and the C-terminal of IκB-ζ. Overexpression of IκB-ζ inhibited the transcriptional activity of STAT3. It also suppressed cell growth and induced cell apoptosis in SRC-simulated cells, which is partially mediated by down-regulation of expression of a known STAT3 target gene, MCL1. Our results suggest that IκB-ζ is a negative regulator of STAT3, and demonstrate a novel mechanism in which a component of the NF-κB signaling pathway inhibits the activation of STAT3.

Leptin-induced cardioprotection involves JAK/STAT signaling that may be linked to the mitochondrial permeability transition pore

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Smith, Christopher C. T.; Dixon, Richard A.; Wynne, Abigail M.; Theodorou, Louise; Ong, Sang-Ging; Subrayan, Sapna; Davidson, Sean M.; Hausenloy, Derek J.; Yellon, Derek M.

2010-01-01

Leptin-induced protection against myocardial ischemia-reperfusion (I/R) injury involves the activation of the reperfusion injury salvage kinase pathway, incorporating phosphatidylinositol 3-kinase-Akt/protein kinase B and p44/42 MAPK, and the inhibition of the mitochondrial permeability transition pore (MPTP). Recently published data indicate that the JAK/STAT signaling pathway, which mediates the metabolic actions of leptin, also plays a pivotal role in cardioprotection. Consequently, in the...

Clinical utility of the oral JAK inhibitor tofacitinib in the treatment of rheumatoid arthritis

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Cutolo M

2013-11-01

Full Text Available Maurizio Cutolo, Marianna Meroni Research Laboratories and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Genova, Italy Abstract: Immune/inflammatory cells act in rheumatoid arthritis (RA-affected patients by synthesizing several inflammatory mediators, including cytokines that initiate intracellular signaling. Recently, small molecule inhibitors of transduction and transcription signals that influence the intracellular pathways (such as the Janus kinase [JAK] family of tyrosine kinases have been tested for RA treatment. Four members of the JAK family are known: JAK1, JAK2, JAK3, and TyK2. JAK1/JAK3 constitutively binds to the cytoplasmic portion of the cytokine receptor – the common gamma chain – that represents a common subunit of several cytokines involved in T-cell and natural killer cell development, as well as in B-cell activation. Tofacitinib is an oral JAK inhibitor that is now available and effective in RA treatment, as shown in multiple Phase II and Phase III clinical trials. However, long-term safety data and comparisons with other disease-modifying antirheumatic drugs and small molecule inhibitors are necessary to better determine the role of tofacitinib in RA. Keywords: Janus kinase inhibitors, tofacitinib, rheumatoid arthritis, kinases, small molecules inhibitors, intracellular signaling

Jak2 46/1 Haplotype Is Associated With Jak2 V617f--positive Myeloproliferative Neoplasms In Brazilian Patients.

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Macedo, L C; Santos, B C; Pagliarini-e-Silva, S; Pagnano, K B B; Rodrigues, C; Quintero, F C; Ferreira, M E; Baraldi, E C; Ambrosio-Albuquerque, E P; Sell, A M; Visentainer, J E L

2016-01-01

This study aimed to verify the association between the JAK2 46/1 haplotype (V617F positive) and some hematological parameters in BCR-ABL-negative chronic myeloproliferative neoplasms (cMPNs) in our population. The blood samples obtained from the patients with cMPN were genotyped for the JAK2 V617F mutation and JAK2 rs10974944 SNP screening using a PCR-RFLP assay. The JAK2 V617F mutation was detected in 80.15% of patients. The G variant of rs10974944 was more frequent in all MPNs, especially t...

Docosahexaenoic acid inhibits IL-6 expression via PPARγ-mediated expression of catalase in cerulein-stimulated pancreatic acinar cells.

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Song, Eun Ah; Lim, Joo Weon; Kim, Hyeyoung

2017-07-01

Cerulein pancreatitis mirrors human acute pancreatitis. In pancreatic acinar cells exposed to cerulein, reactive oxygen species (ROS) mediate inflammatory signaling by Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 3, and cytokine induction. Docosahexaenoic acid (DHA) acts as an agonist of peroxisome proliferator activated receptor γ (PPARγ), which mediates the expression of some antioxidant enzymes. We hypothesized that DHA may induce PPARγ-target catalase expression and reduce ROS levels, leading to the inhibition of JAK2/STAT3 activation and IL-6 expression in cerulein-stimulated acinar cells. Pancreatic acinar AR42J cells were treated with DHA in the presence or absence of the PPARγ antagonist GW9662, or treated with the PPARγ agonist troglitazone, and then stimulated with cerulein. Expression of IL-6 and catalase, ROS levels, JAK2/STAT3 activation, and nuclear translocation of PPARγ were assessed. DHA suppressed the increase in ROS, JAK2/STAT3 activation, and IL-6 expression induced nuclear translocation of PPARγ and catalase expression in cerulein-stimulated AR42J cells. Troglitazone inhibited the cerulein-induced increase in ROS and IL-6 expression, but induced catalase expression similar to DHA in AR42J cells. GW9662 abolished the inhibitory effect of DHA on cerulein-induced increase in ROS and IL-6 expression in AR42J cells. DHA-induced expression of catalase was suppressed by GW9662 in cerulein-stimulated AR42J cells. Thus, DHA induces PPARγ activation and catalase expression, which inhibits ROS-mediated activation of JAK2/STAT3 and IL-6 expression in cerulein-stimulated pancreatic acinar cells. Copyright © 2017. Published by Elsevier Ltd.

Non-cell-autonomous stimulation of stem cell proliferation following ablation of Tcf3

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Yi, Fei; Merrill, Bradley J.

2010-01-01

A combination of cell intrinsic factors and extracellular signals determine whether mouse embryonic stem cells (ESC) divide, self-renew, and differentiate. Here, we report a new interaction between cell intrinsic aspects of the canonical Wnt/Tcf/β-catenin signaling pathway and extracellular Lif/Jak/Stat3 stimulation that combines to promote self-renewal and proliferation of ESC. Mutant ESC lacking the Tcf3 transcriptional repressor continue to self-renew in the absence of exogenous Lif and through pharmacological inhibition of Lif/Jak/Stat3 signaling; however, proliferation rates of TCF3-/- ESC were significantly decreased by inhibiting Jak/Stat3 activity. Cell mixing experiments showed that stimulation of Stat3 phosphorylation in TCF3-/- ESC was mediated through secretion of paracrine acting factors, but did not involve elevated Lif or LifR transcription. The new interaction between Wnt and Lif/Jak/Stat3 signaling pathways has potential for new insights into the growth of tumors caused by aberrant activity of Wnt/Tcf/β-catenin signaling.

Engineered Aedes aegypti JAK/STAT Pathway-Mediated Immunity to Dengue Virus.

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Natapong Jupatanakul

2017-01-01

Full Text Available We have developed genetically modified Ae. aegypti mosquitoes that activate the conserved antiviral JAK/STAT pathway in the fat body tissue, by overexpressing either the receptor Dome or the Janus kinase Hop by the blood feeding-induced vitellogenin (Vg promoter. Transgene expression inhibits infection with several dengue virus (DENV serotypes in the midgut as well as systemically and in the salivary glands. The impact of the transgenes Dome and Hop on mosquito longevity was minimal, but it resulted in a compromised fecundity when compared to wild-type mosquitoes. Overexpression of Dome and Hop resulted in profound transcriptome regulation in the fat body tissue as well as the midgut tissue, pinpointing several expression signatures that reflect mechanisms of DENV restriction. Our transcriptome studies and reverse genetic analyses suggested that enrichment of DENV restriction factor and depletion of DENV host factor transcripts likely accounts for the DENV inhibition, and they allowed us to identify novel factors that modulate infection. Interestingly, the fat body-specific activation of the JAK/STAT pathway did not result in any enhanced resistance to Zika virus (ZIKV or chikungunya virus (CHIKV infection, thereby indicating a possible specialization of the pathway's antiviral role.

ONC201 selectively induces apoptosis in cutaneous T-cell lymphoma cells via activating pro-apoptotic integrated stress response and inactivating JAK/STAT and NF-κB pathways.

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Ni, Xiao; Zhang, Xiang; Hu, Cheng-Hui; Langridge, Timothy; Tarapore, Rohinton S; Allen, Joshua E; Oster, Wolfgang; Duvic, Madeleine

2017-09-22

Cutaneous T-cell lymphomas (CTCLs) are extremely symptomatic and still incurable, and more effective and less toxic therapies are urgently needed. ONC201, an imipridone compound, has shown efficacy in pre-clinical studies in multiple advanced cancers. This study was to evaluate the anti-tumor activity of ONC201 on CTCL cells. The effect of ONC201 on the cell growth and apoptosis were evaluated in CTCL cell lines (n=8) and primary CD4 + malignant T cells isolated from CTCL patients (n=5). ONC201 showed a time-dependent cell growth inhibition in all treated cell lines with a concentration range of 1.25-10.0 μM. ONC201 also induced apoptosis in tested cells with a narrow concentration range of 2.5-10.0 μM, evidenced by increased Annexin V + cells, accompanied by accumulated sub-G1 portions. ONC201 only induced apoptosis in CD4 + malignant T cells, not in normal CD4 + T cells. The activating transcription factor 4 (ATF4), a hallmark of integrated stress response, was upregulated in response to ONC201 whereas Akt was downregulated. In addition, molecules in JAK/STAT and NF-κB pathways, as well as IL-32β, were downregulated following ONC201 treatment. Thus, ONC201 exerts a potent and selective anti-tumor effect on CTCL cells. Its efficacy may involve activating integrated stress response through ATF4 and inactivating JAK/STAT and NF-κB pathways.

Tofacitinib and analogs as inhibitors of the histone kinase PRK1 (PKN1).

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Ostrovskyi, Dmytro; Rumpf, Tobias; Eib, Julia; Lumbroso, Alexandre; Slynko, Inna; Klaeger, Susan; Heinzlmeir, Stephanie; Forster, Michael; Gehringer, Matthias; Pfaffenrot, Ellen; Bauer, Silke Mona; Schmidtkunz, Karin; Wenzler, Sandra; Metzger, Eric; Kuster, Bernhard; Laufer, Stefan; Schüle, Roland; Sippl, Wolfgang; Breit, Bernhard; Jung, Manfred

2016-09-01

The histone kinase PRK1 has been identified as a potential target to combat prostate cancer but selective PRK1 inhibitors are lacking. The US FDA -approved JAK1-3 inhibitor tofacitinib also potently inhibits PRK1 in vitro. We show that tofacitinib also inhibits PRK1 in a cellular setting. Using tofacitinib as a starting point for structure-activity relationship studies, we identified a more potent and another more selective PRK1 inhibitor compared with tofacitinib. Furthermore, we found two potential PRK1/JAK3-selectivity hotspots. The identified inhibitors and the selectivity hotspots lay the basis for the development of selective PRK1 inhibitors. The identification of PRK1, but also of other cellular tofacitinib targets, has implications on its clinical use and on future development of tofacitinib-like JAK inhibitors. [Formula: see text].

JAK1/2 inhibition with baricitinib in the treatment of autoinflammatory interferonopathies.

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Sanchez, Gina A Montealegre; Reinhardt, Adam; Ramsey, Suzanne; Wittkowski, Helmut; Hashkes, Philip J; Berkun, Yackov; Schalm, Susanne; Murias, Sara; Dare, Jason A; Brown, Diane; Stone, Deborah L; Gao, Ling; Klausmeier, Thomas; Foell, Dirk; Jesus, Adriana A de; Chapelle, Dawn C; Kim, Hanna; Dill, Samantha; Colbert, Robert; Failla, Laura; Kost, Bahar; O'Brien, Michelle; Reynolds, James C; Folio, Les R; Calvo, Katherine R; Paul, Scott M; Weir, Nargues; Brofferio, Alessandra; Soldatos, Ariane; Biancotto, Angélique; Cowen, Edward W; Digiovanna, John G; Gadina, Massimo; Lipton, Andrew J; Hadigan, Colleen; Holland, Steven M; Fontana, Joseph; Alawad, Ahmad S; Brown, Rebecca J; Rother, Kristina I; Heller, Theo; Brooks, Kristina M; Kumar, Parag; Brooks, Stephen R; Waldman, Meryl; Singh, Harsharan K; Nickeleit, Volker; Silk, Maria; Prakash, Apurva; Janes, Jonathan M; Ozen, Seza; Wakim, Paul G; Brogan, Paul A; Macias, William L; Goldbach-Mansky, Raphaela

2018-04-12

Monogenic Interferon (IFN)-mediated autoinflammatory diseases present in infancy with systemic inflammation, an IFN-response-gene-signature (IRS), inflammatory organ damage and high mortality. We used the janus kinase (JAK) inhibitor baricitinib with IFN-blocking activity in vitro, to ameliorate disease. Between October 2011 and February 2017, 10 patients with CANDLE (chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperatures), 4 with SAVI (Stimulator of IFN genes (STING)-associated vasculopathy with onset in infancy), and 4 patients with other interferonopathies were enrolled in an Expanded Access Program. Patients underwent dose-escalation, benefit was assessed by reductions in daily disease symptoms and corticosteroid requirement. Quality-of-life, organ inflammation, changes in IFN-induced biomarkers, and safety were longitudinally assessed. 18 patients were treated for a mean duration of 3.0 years (1.5-4.9 years). The median daily symptom score decreased from 1.3 (IQR 0.93-1.78) to 0.25 (IQR 0.1-0.63) (P Eli Lilly. Eli Lilly is the sponsor of the compassionate use program.

Increased frequency of co-existing JAK2 exon-12 or MPL exon-10 mutations in patients with low JAK2(V617F) allelic burden.

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Nussenzveig, Roberto H; Pham, Ha T; Perkins, Sherrie L; Prchal, Josef T; Agarwal, Archana M; Salama, Mohamed E

2016-01-01

The frequency of co-existing JAK2(V617F)/MPL and JAK2(V617F)/JAK2 exon-12 mutations has not been previously investigated in MPNs. Poor survival was reported in primary myelofibrosis with low JAK2(V617F) allelic burden. However, mutational status of JAK2 exon-12 or MPL were not reported in these patients. This study developed a cost-effective multiplex high resolution melt assay that screens for mutations in JAK2 gene exons-12 and -14 ((V617F)) and MPL gene exon-10. Co-existing mutations with JAK2(V617F) were detected in 2.9% (6/208; two JAK2 exon-12 and four MPL exon-10) patient specimens with known JAK2(V617F) (allelic-burden range: 0.1-96.8%). Co-existing mutations were detected in specimens with MPL exon-10 mutation should be pursued.

Myeloproliferative neoplasms: JAK2 signaling pathway as a central target for therapy.

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Pasquier, Florence; Cabagnols, Xenia; Secardin, Lise; Plo, Isabelle; Vainchenker, William

2014-09-01

The discovery of the JAK2V617F mutation followed by the discovery of other genetic abnormalities allowed important progress in the understanding of the pathogenesis and management of myeloproliferative neoplasms (MPN)s. Classical Breakpoint cluster region-Abelson (BCR-ABL)-negative neoplasms include 3 main disorders: essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). Genomic studies have shown that these disorders are more heterogeneous than previously thought with 3 main entities corresponding to different gene mutations: the JAK2 disorder, essentially due to JAK2V617F mutation, which includes nearly all PVs and a majority of ETs and PMFs with a continuum between these diseases and the myeloproliferative leukemia (MPL) and calreticulin (CALR) disorders, which include a fraction of ET and PMF. All of these mutations lead to a JAK2 constitutive activation. Murine models either with JAK2V617F or MPLW515L, but also with JAK2 or MPL germ line mutations found in hereditary thrombocytosis, have demonstrated that they are drivers of myeloproliferation. However, the myeloproliferative driver mutation is still unknown in approximately 15% of ET and PMF, but appears to also target the JAK/Signal Transducer and Activator of Transcription (STAT) pathway. However, other mutations in genes involved in epigenetics or splicing also can be present and can predate or follow mutations in signaling. They are involved either in clonal dominance or in phenotypic changes, more particularly in PMF. They can be associated with leukemic progression and might have an important prognostic value such as additional sex comb-like 1 mutations. Despite this heterogeneity, it is tempting to target JAK2 and its signaling for therapy. However in PMF, Adenosine Tri-Phosphate (ATP)-competitive JAK2 inhibitors have shown their interest, but also their important limitations. Thus, other approaches are required, which are discussed in this review. Copyright © 2014

Quercetin inhibits the poly(dA:dT)-induced secretion of IL-18 via down-regulation of the expressions of AIM2 and pro-caspase-1 by inhibiting the JAK2/STAT1 pathway in IFN-γ-primed human keratinocytes.

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Lee, Kyung-Mi; Kang, Jung Hoon; Yun, Mihee; Lee, Seong-Beom

2018-06-05

Quercetin, a polyphenol, belongs to a class of flavonoids that exerts anti-inflammatory effects. Interleukin (IL)-18 is a member of the IL-1 family cytokine that regulates immune responses and is implicated in various inflammatory skin diseases. Absent in melanoma 2 (AIM2) is a cytosolic double-stranded (ds) DNA sensor that recognizes the dsDNA of a microbial or host origin. Binding of dsDNA to AIM2 simulates caspase-1-dependent inflammasome activity, which leads to the production of IL-1β and IL-18. Increased levels of AIM2 have been observed in patients with inflammatory skin diseases. In the current study, we investigated the issue of whether or how Quercetin attenuates poly (dA:dT), a synthetic analog of microbial dsDNA, -induced IL-18 secretion in IFN-γ-primed human keratinocytes. Treatment with 5 and 10 μM of Quercetin inhibited the poly (dA:dT)-induced secretion of IL-18 after IFN-γ priming and before poly (dA:dT)-induced AIM2 activation. In addition, treatment with Quercetin at 10 μM, significantly inhibited the phosphorylation of JAK2 and STAT1, and the nuclear translocation of phosphorylated STAT1 in poly (dA:dT)-treated and IFN-γ-primed keratinocytes. These results suggest that treatment with Quercetin inhibits the poly (dA:dT)-induced secretion of IL-18 via down-regulation of the expressions of AIM2 and pro-caspase-1 by inhibiting the JAK2/STAT1 pathway in IFN-γ-primed keratinocytes. Copyright © 2018 Elsevier Inc. All rights reserved.

The Amelioration of Myelofibrosis with Thrombocytopenia by a JAK1/2 Inhibitor, Ruxolitinib, in a Post-polycythemia Vera Myelofibrosis Patient with a JAK2 Exon 12 Mutation.

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Ikeda, Kazuhiko; Ueda, Koki; Sano, Takahiro; Ogawa, Kazuei; Ikezoe, Takayuki; Hashimoto, Yuko; Morishita, Soji; Komatsu, Norio; Ohto, Hitoshi; Takeishi, Yasuchika

2017-01-01

Less than 5% of patients with polycythemia vera (PV) show JAK2 exon 12 mutations. Although PV patients with JAK2 exon 12 mutations are known to develop post-PV myelofibrosis (MF) as well as PV with JAK2V617F, the role of JAK inhibitors in post-PV MF patients with JAK2 exon 12 mutations remains unknown. We describe how treatment with a JAK1/2 inhibitor, ruxolitinib, led to the rapid amelioration of marrow fibrosis, erythrocytosis and thrombocytopenia in a 77-year-old man with post-PV MF who carried a JAK2 exon 12 mutation (JAK2H538QK539L). This case suggests that ruxolitinib is a treatment option for post-PV MF in patients with thrombocytopenia or JAK2 exon 12 mutations.

Driver mutations (JAK2V617F, MPLW515L/K or CALR), pentraxin-3 and C-reactive protein in essential thrombocythemia and polycythemia vera.

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Lussana, Federico; Carobbio, Alessandra; Salmoiraghi, Silvia; Guglielmelli, Paola; Vannucchi, Alessandro Maria; Bottazzi, Barbara; Leone, Roberto; Mantovani, Alberto; Barbui, Tiziano; Rambaldi, Alessandro

2017-02-22

The driver mutations JAK2V617F, MPLW515L/K and CALR influence disease phenotype of myeloproliferative neoplasms (MPNs) and might sustain a condition of chronic inflammation. Pentraxin 3 (PTX3) and high-sensitivity C-reactive protein (hs-CRP) are inflammatory biomarkers potentially useful for refining prognostic classification of MPNs. We evaluated 305 with essential thrombocythemia (ET) and 172 polycythemia vera (PV) patients diagnosed according to the 2016 WHO criteria and with full molecular characterization for driver mutations. PTX3 levels were significantly increased in carriers of homozygous JAK2V617F mutation compared to all the other genotypes and triple negative ET patients, while hs-CRP levels were independent of the mutational profile. The risk of haematological evolution and death from any cause was about 2- and 1.5-fold increased in individuals with high PTX-3 levels, while the thrombosis rate tended to be lower. High hs-CRP levels were associated with risk of haematological evolution, death and also major thrombosis. After sequential adjustment for potential confounders (age, gender, diagnosis and treatments) and the presence of JAK2V617F homozygous status, high hs-CRP levels remained significant for all outcomes, while JAK2V617F homozygous status as well as treatments were the factors independently accounting for adverse outcomes among patients with high PTX3 levels. These results provide evidence that JAK2V617F mutation influences MPN-associated inflammation with a strong correlation between allele burden and PTX3 levels. Plasma levels of hs-CRP and PTX3 might be of prognostic value for patients with ET and PV, but their validation in future prospective studies is needed.

Hydrogen sulfide postconditioning protects isolated rat hearts against ischemia and reperfusion injury mediated by the JAK2/STAT3 survival pathway

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Heng-Fei Luan

2012-10-01

Full Text Available The JAK2/STAT3 signal pathway is an important component of survivor activating factor enhancement (SAFE pathway. The objective of the present study was to determine whether the JAK2/STAT3 signaling pathway participates in hydrogen sulfide (H2S postconditioning, protecting isolated rat hearts from ischemic-reperfusion injury. Male Sprague-Dawley rats (230-270 g were divided into 6 groups (N = 14 per group: time-matched perfusion (Sham group, ischemia/reperfusion (I/R group, NaHS postconditioning group, NaHS with AG-490 group, AG-490 (5 µM group, and dimethyl sulfoxide (DMSO; <0.2% group. Langendorff-perfused rat hearts, with the exception of the Sham group, were subjected to 30 min of ischemia followed by 90 min of reperfusion after 20 min of equilibrium. Heart rate, left ventricular developed pressure (LVDP, left ventricular end-diastolic pressure (LVEDP, and the maximum rate of increase or decrease of left ventricular pressure (± dp/dt max were recorded. Infarct size was determined using triphenyltetrazolium chloride (TTC staining. Myocardial TUNEL staining was used as the in situ cell death detection method and the percentage of TUNEL-positive nuclei to all nuclei counted was used as the apoptotic index. The expression of STAT3, bcl-2 and bax was determined by Western blotting. After reperfusion, compared to the I/R group, H2S significantly improved functional recovery and decreased infarct size (23.3 ± 3.8 vs 41.2 ± 4.7%, P < 0.05 and apoptotic index (22.1 ± 3.6 vs 43.0 ± 4.8%, P < 0.05. However, H2S-mediated protection was abolished by AG-490, the JAK2 inhibitor. In conclusion, H2S postconditioning effectively protects isolated I/R rat hearts via activation of the JAK2/STAT3 signaling pathway.

FLT3 and JAK2 Mutations in Acute Myeloid Leukemia Promote Interchromosomal Homologous Recombination and the Potential for Copy Neutral Loss of Heterozygosity.

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Gaymes, Terry J; Mohamedali, Azim; Eiliazadeh, Anthony L; Darling, David; Mufti, Ghulam J

2017-04-01

Acquired copy neutral LOH (CN-LOH) is a frequent occurrence in myeloid malignancies and is often associated with resistance to standard therapeutic modalities and poor survival. Here, we show that constitutive signaling driven by mutated FLT3 and JAK2 confers interchromosomal homologous recombination (iHR), a precedent for CN-LOH. Using a targeted recombination assay, we determined significant iHR activity in internal tandem duplication FLT3 (FLT3-ITD) and JAK2V617F-mutated cells. Sister chromatid exchanges, a surrogate measure of iHR, was significantly elevated in primary FLT3-ITD normal karyotype acute myeloid leukemia (NK-AML) compared with wild-type FLT3 NK-AML. HR was harmonized to S phase of the cell cycle to repair broken chromatids and prevent iHR. Increased HR activity in G 0 arrested primary FLT3-ITD NK-AML in contrast to wild-type FLT3 NK-AML. Cells expressing mutated FLT3-ITD demonstrated a relative increase in mutation frequency as detected by thymidine kinase (TK) gene mutation assay. Moreover, resistance was associated with CN-LOH at the TK locus. Treatment of FLT3-ITD- and JAK2V617F-mutant cells with the antioxidant N -acetylcysteine diminished reactive oxygen species (ROS), restoring iHR and HR levels. Our findings show that mutated FLT3-ITD and JAK2 augment ROS production and HR, shifting the cellular milieu toward illegitimate recombination events such as iHR and CN-LOH. Therapeutic reduction of ROS may thus prevent leukemic progression and relapse in myeloid malignancies. Cancer Res; 77(7); 1697-708. ©2017 AACR . ©2017 American Association for Cancer Research.

Canonical and Non-Canonical Aspects of JAK-STAT Signaling: Lessons from Interferons for Cytokine Responses.

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Majoros, Andrea; Platanitis, Ekaterini; Kernbauer-Hölzl, Elisabeth; Rosebrock, Felix; Müller, Mathias; Decker, Thomas

2017-01-01

Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signal transduction mediates cytokine responses. Canonical signaling is based on STAT tyrosine phosphorylation by activated JAKs. Downstream of interferon (IFN) receptors, activated JAKs cause the formation of the transcription factors IFN-stimulated gene factor 3 (ISGF3), a heterotrimer of STAT1, STAT2 and interferon regulatory factor 9 (IRF9) subunits, and gamma interferon-activated factor (GAF), a STAT1 homodimer. In recent years, several deviations from this paradigm were reported. These include kinase-independent JAK functions as well as extra- and intranuclear activities of U-STATs without phosphotyrosines. Additionally, transcriptional control by STAT complexes resembling neither GAF nor ISGF3 contributes to transcriptome changes in IFN-treated cells. Our review summarizes the contribution of non-canonical JAK-STAT signaling to the innate antimicrobial immunity imparted by IFN. Moreover, we touch upon functions of IFN pathway proteins beyond the IFN response. These include metabolic functions of IRF9 as well as the regulation of natural killer cell activity by kinase-dead TYK2 and different phosphorylation isoforms of STAT1.

Driver mutations (JAK2V617F, MPLW515L/K or CALR, pentraxin-3 and C-reactive protein in essential thrombocythemia and polycythemia vera

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Federico Lussana

2017-02-01

Full Text Available Abstract Background The driver mutations JAK2V617F, MPLW515L/K and CALR influence disease phenotype of myeloproliferative neoplasms (MPNs and might sustain a condition of chronic inflammation. Pentraxin 3 (PTX3 and high-sensitivity C-reactive protein (hs-CRP are inflammatory biomarkers potentially useful for refining prognostic classification of MPNs. Methods We evaluated 305 with essential thrombocythemia (ET and 172 polycythemia vera (PV patients diagnosed according to the 2016 WHO criteria and with full molecular characterization for driver mutations. Results PTX3 levels were significantly increased in carriers of homozygous JAK2V617F mutation compared to all the other genotypes and triple negative ET patients, while hs-CRP levels were independent of the mutational profile. The risk of haematological evolution and death from any cause was about 2- and 1.5-fold increased in individuals with high PTX-3 levels, while the thrombosis rate tended to be lower. High hs-CRP levels were associated with risk of haematological evolution, death and also major thrombosis. After sequential adjustment for potential confounders (age, gender, diagnosis and treatments and the presence of JAK2V617F homozygous status, high hs-CRP levels remained significant for all outcomes, while JAK2V617F homozygous status as well as treatments were the factors independently accounting for adverse outcomes among patients with high PTX3 levels. Conclusions These results provide evidence that JAK2V617F mutation influences MPN-associated inflammation with a strong correlation between allele burden and PTX3 levels. Plasma levels of hs-CRP and PTX3 might be of prognostic value for patients with ET and PV, but their validation in future prospective studies is needed.

Treatment With JAK Inhibitors in Myelofibrosis Patients Nullifies the Prognostic Impact of Unfavorable Cytogenetics.

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Ma, Vincent T; Boonstra, Philip S; Menghrajani, Kamal; Perkins, Cecelia; Gowin, Krisstina L; Mesa, Ruben A; Gotlib, Jason R; Talpaz, Moshe

2018-05-01

In the era before Janus kinase (JAK) inhibitors, cytogenetic information was used to predict survival in myelofibrosis patients. However, the prognostic value of cytogenetics in the setting of JAK inhibitor therapy remains unknown. We performed a retrospective analysis of 180 patients with bone marrow biopsy-proven myelofibrosis from 3 US academic medical centers. We fit Cox proportional hazards models for overall survival and transformation-free survival on the bases of 3 factors: JAK inhibitor therapy as a time-dependent covariate, dichotomized cytogenetic status (favorable vs. unfavorable), and statistical interaction between the two. The median follow-up time was 37.1 months. Among patients treated with best available therapy, unfavorable cytogenetic status was associated with decreased survival (hazard ratio = 2.31; P = .025). At initiation of JAK inhibitor therapy, unfavorable cytogenetics was (nonsignificantly) associated with increased survival compared to favorable cytogenetics (hazard ratio = 0.292; P = .172). The ratio of hazard ratios was 0.126 (P = .034). These findings were similar after adjusting for standard clinical prognostic factors as well as when measured against transformation-free survival. The initiation of JAK inhibitor therapy appears to change the association between cytogenetics and overall survival. There was little difference in survival between treatment types in patients with favorable cytogenetics. However, the use of JAK inhibitor therapy among patients with unfavorable cytogenetics was not associated with worse survival compared to favorable cytogenetics. Our analyses suggest that initiation of JAK inhibitor therapy nullifies the negative prognostic implication of unfavorable cytogenetics established in the pre-JAK inhibitor therapy era. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Anti-Inflammatory Inhibitors Targeting Jak and Ikk Have An Anabolic Effect on Type II Collagen Turnover ex Vivo

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Kjelgaard-Petersen, Cecilie Freja; Bay-Jensen, Anne-Christine; Karsdal, M.A.

2016-01-01

be beneficial for the selection of novel anti-inflammatory treatments for RA and iOA. Objectives The aim of this study was to investigate the direct effect of the anti-inflammatory inhibitors R406 (the active metabolite of Fostamatinib), Tofacitinib, TPCA-1 and SB203580 on the cartilage ECM turnover. Methods...... Full depth bovine cartilage ex vivo cultures were cultured for 3 weeks with OSM [10 ng/mL] and TNFα [2 ng/mL] (O+T) or together with R406, Tofacitinib or TPCA-1 at 10 μM and a two-fold dilution to 0.16 μM. SB203580 was tested at 3 μM, 1 μM and 0.3 μM. As negative control, untreated explants were...... R406, the Jak inhibitor Tofacitinib, and the IKK inhibitor TPCA-1 inhibited the release of ARGS or AGNx1, while the p38 inhibitor, SB203580, had no effect. The turnover of type II collagen was measured by the formation of type II collagen (ProC2) and MMP-mediated degradation of type II collagen (C2M...

Histamine H3 receptor activation selectively inhibits dopamine D1 receptor-dependent [3H]GABA release from depolarization-stimulated slices of rat substantia nigra pars reticulata

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Aceves, J.; Young, J.M.; Arias-Montano, J.A.; Floran, B.; Garcia, M.

1997-01-01

The release of [ 3 H]GABA from slices of rat substantia nigra pars reticulata induced by increasing extracellular K + from 6 to 15 mM in the presence of 10 μM sulpiride was inhibited by 73±3% by 1 μM SCH 23390, consistent with a large component of release dependent upon D 1 receptor activation. The histamine H 3 receptor-selective agonist immepip (1 μM) and the non-selective agonist histamine (100 μM) inhibited [ 3 H]GABA release by 78±2 and 80±2%, respectively. The inhibition by both agonists was reversed by the H 3 receptor antagonist thioperamide (1 μM). However, in the presence of 1 μM SCH 23390 depolarization-induced release of [ 3 H]GABA was not significantly decreased by 1 μM immepip. In rats depleted of dopamine by pretreatment with reserpine, immepip no longer inhibited control release of [ 3 H]GABA, but in the presence of 1 μM SKF 38393, which produced a 7±1-fold stimulation of release, immepip reduced the release to a level not statistically different from that in the presence of immepip alone. Immepip (1 μM) also inhibited the depolarization-induced release of [ 3 H]dopamine from substantia nigra pars reticulata slices, by 38±3%.The evidence is consistent with the proposition that activation of histamine H 3 receptors leads to the selective inhibition of the component of depolarization-induced [ 3 H]GABA release in substantia nigra pars reticulata slices which is dependent upon D 1 receptor activation. This appears to be largely an action at the terminals of the striatonigral GABA projection neurons, which may be enhanced by a partial inhibition of dendritic [ 3 H]dopamine release. (Copyright (c) 1997 Elsevier Science B.V., Amsterdam. All rights reserved.)

The JAK inhibitor tofacitinib suppresses synovial JAK1-STAT signalling in rheumatoid arthritis

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Boyle, D L; Soma, K; Hodge, J; Kavanaugh, A; Mandel, D; Mease, P; Shurmur, R; Singhal, A K; Wei, N; Rosengren, S; Kaplan, I; Krishnaswami, S; Luo, Z; Bradley, J; Firestein, G S

2015-01-01

Objective Tofacitinib is an oral Janus kinase (JAK) inhibitor for the treatment of rheumatoid arthritis (RA). The pathways affected by tofacitinib and the effects on gene expression in situ are unknown. Therefore, tofacitinib effects on synovial pathobiology were investigated. Methods A randomised, double-blind, phase II serial synovial biopsy study (A3921073; NCT00976599) in patients with RA with an inadequate methotrexate response. Patients on background methotrexate received tofacitinib 10 mg twice daily or placebo for 28 days. Synovial biopsies were performed on Days -7 and 28 and analysed by immunoassay or quantitative PCR. Clinical response was determined by disease activity score and European League Against Rheumatism (EULAR) response on Day 28 in A3921073, and at Month 3 in a long-term extension study (A3921024; NCT00413699). Results Tofacitinib exposure led to EULAR moderate to good responses (11/14 patients), while placebo was ineffective (1/14 patients) on Day 28. Tofacitinib treatment significantly reduced synovial mRNA expression of matrix metalloproteinase (MMP)-1 and MMP-3 (pTofacitinib significantly decreased plasma CXCL10 (pTofacitinib reduces metalloproteinase and interferon-regulated gene expression in rheumatoid synovium, and clinical improvement correlates with reductions in STAT1 and STAT3 phosphorylation. JAK1-mediated interferon and interleukin-6 signalling likely play a key role in the synovial response. Trial registration number NCT00976599. PMID:25398374

JAK2V617F mutation is associated with special alleles in essential thrombocythemia.

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Hsiao, Hui-Hua; Liu, Yi-Chang; Tsai, Hui-Jen; Lee, Ching-Ping; Hsu, Jui-Feng; Lin, Sheng-Fung

2011-03-01

Janus kinase 2 mutation (JAK2V617F) has been identified in myeloproliferative neoplasms. Furthermore, special single nucleoside polymorphisms (SNPs) have been found to be associated with the JAK2V617F mutation. Therefore, the associations among JAK2V617F and special SNPs and the allelic location between them were investigated in patients with essential thrombocythemia (ET). A total of 61 patients with ET and 106 healthy individuals were enrolled. The PCR-RFLP method was applied to investigate the pattern of three SNPs, rs10974944, rs12343867, and rs12340895. Allele-specific PCR was used to examine the allelic location between rs10974944 and JAK2V617F. Among the patients with ET, 34 (55.7%, 34/61) were JAK2V617F positive (heterozygous) while the other 27 (44.3%, 27/61) were negative, and there were no MPLW515L/K mutations noted. The pattern of special SNPs in JAK2V617F(+) was significantly different from that in normal individuals (p <0.05), while there was no difference between JAK2V617F(-) patients and normal individuals. Allele-specific PCR showed high association of a cis-location between the special G-allele of rs10974944 and JAK2V617F(+). Based on this small numbered study, the results show the association between special SNPs and JAK2V617F mutation and a cis-location between the special G-allelic form of rs10974944 and the JAK2V617F mutation. These data highlight a close relationship between them in patients with ET.

Loss of function JAK1 mutations occur at high frequency in cancers with microsatellite instability and are suggestive of immune evasion.

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Albacker, Lee A; Wu, Jeremy; Smith, Peter; Warmuth, Markus; Stephens, Philip J; Zhu, Ping; Yu, Lihua; Chmielecki, Juliann

2017-01-01

Immune evasion is a well-recognized hallmark of cancer and recent studies with immunotherapy agents have suggested that tumors with increased numbers of neoantigens elicit greater immune responses. We hypothesized that the immune system presents a common selective pressure on high mutation burden tumors and therefore immune evasion mutations would be enriched in high mutation burden tumors. The JAK family of kinases is required for the signaling of a host of immune modulators in tumor, stromal, and immune cells. Therefore, we analyzed alterations in this family for the hypothesized signature of an immune evasion mutation. Here, we searched a database of 61,704 unique solid tumors for alterations in the JAK family kinases (JAK1/2/3, TYK2). We used The Cancer Genome Atlas and Cancer Cell Line Encyclopedia data to confirm and extend our findings by analyzing gene expression patterns. Recurrent frameshift mutations in JAK1 were associated with high mutation burden and microsatellite instability. These mutations occurred in multiple tumor types including endometrial, colorectal, stomach, and prostate carcinomas. Analyzing gene expression signatures in endometrial and stomach adenocarcinomas revealed that tumors with a JAK1 frameshift exhibited reduced expression of interferon response signatures and multiple anti-tumor immune signatures. Importantly, endometrial cancer cell lines exhibited similar gene expression changes that were expected to be tumor cell intrinsic (e.g. interferon response) but not those expected to be tumor cell extrinsic (e.g. NK cells). From these data, we derive two primary conclusions: 1) JAK1 frameshifts are loss of function alterations that represent a potential pan-cancer adaptation to immune responses against tumors with microsatellite instability; 2) The mechanism by which JAK1 loss of function contributes to tumor immune evasion is likely associated with loss of the JAK1-mediated interferon response.

Autophosphorylation of JAK2 on tyrosines 221 and 570 regulates its activity.

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Argetsinger, Lawrence S; Kouadio, Jean-Louis K; Steen, Hanno; Stensballe, Allan; Jensen, Ole N; Carter-Su, Christin

2004-06-01

The tyrosine kinase JAK2 is a key signaling protein for at least 20 receptors in the cytokine/hematopoietin receptor superfamily and is a component of signaling by insulin receptor and several G-protein-coupled receptors. However, there is only limited knowledge of the physical structure of JAK2 or which of the 49 tyrosines in JAK2 are autophosphorylated. In this study, mass spectrometry and two-dimensional peptide mapping were used to determine that tyrosines 221, 570, and 1007 in JAK2 are autophosphorylated. Phosphorylation of tyrosine 570 is particularly robust. In response to growth hormone, JAK2 was rapidly and transiently phosphorylated at tyrosines 221 and 570, returning to basal levels by 60 min. Analysis of the sequences surrounding tyrosines 221 and 570 in JAK2 and tyrosines in other proteins that are phosphorylated in response to ligands that activate JAK2 suggests that the YXX[L/I/V] motif is one of the motifs recognized by JAK2. Experiments using JAK2 with tyrosines 221 and 570 mutated to phenylalanine suggest that tyrosines 221 and 570 in JAK2 may serve as regulatory sites in JAK2, with phosphorylation of tyrosine 221 increasing kinase activity and phosphorylation of tyrosine 570 decreasing kinase activity and thereby contributing to rapid termination of ligand activation of JAK2.

Role of JAK-STAT signaling in the pathogenesis of myeloproliferative disorders.

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Levine, Ross L; Wernig, Gerlinde

2006-01-01

The identification of JAK2V617F mutations in polycythemia vera (PV), essential thrombocytosis (ET), and myelofibrosis (MF) represents an important advance in our understanding of these myeloproliferative disorders (MPD). Most, if not all, patients with PV and a significant number of patients with ET and MF are JAK2V617F positive, and the mutation likely arises in the hematopoietic stem cell compartment. JAK2V617F is a constitutively active tyrosine kinase that is able to activate JAK-STAT signaling most efficiently when co-expressed with the erythropoietin receptor (EPOR), the thrombopoietin receptor (MPL), or the granulocyte colony-stimulating factor receptor (GCSFR). Data from murine models supports the central role of JAK2V617F in the pathogenesis of MPD, as expression of JAK2V617F in a bone marrow transplantation assay results in polycythemia and myelofibrosis in recipient mice. Activation of JAK-STAT signaling by JAK2V617F in some, but not all MPD patients with ET and MF led to the identification of the constitutively active MPLW515L allele in ET and MF. Small molecule inhibitors of JAK-STAT signaling are currently being developed, which offer potential for molecularly targeted therapy for patients with PV, ET, and MF. Despite these advances, many questions remain regarding the role of a single disease allele in three phenotypically distinct MPD, the potential clinical efficacy of JAK2 inhibitors, and the identity of oncogenic alleles in JAK2V617F/MPLW515-negative MPD.

Autophosphorylation of JAK2 on tyrosines 221 and 570 regulates its activity

DEFF Research Database (Denmark)

Argetsinger, Lawrence S; Kouadio, Jean-Louis K; Steen, Hanno

2004-01-01

or which of the 49 tyrosines in JAK2 are autophosphorylated. In this study, mass spectrometry and two-dimensional peptide mapping were used to determine that tyrosines 221, 570, and 1007 in JAK2 are autophosphorylated. Phosphorylation of tyrosine 570 is particularly robust. In response to growth hormone......, JAK2 was rapidly and transiently phosphorylated at tyrosines 221 and 570, returning to basal levels by 60 min. Analysis of the sequences surrounding tyrosines 221 and 570 in JAK2 and tyrosines in other proteins that are phosphorylated in response to ligands that activate JAK2 suggests that the YXX......[L/I/V] motif is one of the motifs recognized by JAK2. Experiments using JAK2 with tyrosines 221 and 570 mutated to phenylalanine suggest that tyrosines 221 and 570 in JAK2 may serve as regulatory sites in JAK2, with phosphorylation of tyrosine 221 increasing kinase activity and phosphorylation of tyrosine 570...

Melting curve analysis after T allele enrichment (MelcaTle as a highly sensitive and reliable method for detecting the JAK2V617F mutation.

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Soji Morishita

Full Text Available Detection of the JAK2V617F mutation is essential for diagnosing patients with classical myeloproliferative neoplasms (MPNs. However, detection of the low-frequency JAK2V617F mutation is a challenging task due to the necessity of discriminating between true-positive and false-positive results. Here, we have developed a highly sensitive and accurate assay for the detection of JAK2V617F and named it melting curve analysis after T allele enrichment (MelcaTle. MelcaTle comprises three steps: 1 two cycles of JAK2V617F allele enrichment by PCR amplification followed by BsaXI digestion, 2 selective amplification of the JAK2V617F allele in the presence of a bridged nucleic acid (BNA probe, and 3 a melting curve assay using a BODIPY-FL-labeled oligonucleotide. Using this assay, we successfully detected nearly a single copy of the JAK2V617F allele, without false-positive signals, using 10 ng of genomic DNA standard. Furthermore, MelcaTle showed no positive signals in 90 assays screening healthy individuals for JAK2V617F. When applying MelcaTle to 27 patients who were initially classified as JAK2V617F-positive on the basis of allele-specific PCR analysis and were thus suspected as having MPNs, we found that two of the patients were actually JAK2V617F-negative. A more careful clinical data analysis revealed that these two patients had developed transient erythrocytosis of unknown etiology but not polycythemia vera, a subtype of MPNs. These findings indicate that the newly developed MelcaTle assay should markedly improve the diagnosis of JAK2V617F-positive MPNs.

IL-6 Promotes FSH-Induced VEGF Expression Through JAK/STAT3 Signaling Pathway in Bovine Granulosa Cells

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Meng Yang

2017-11-01

Full Text Available Background/Aims: Vascular endothelial growth factor (VEGF has been demonstrated to play a pivotal role in the regulation of angiogenesis in ovarian follicular development, particularly during the preovulatory period. Although numerous studies have shown that interleukin-6 (IL-6 is one of the major inducing factors that regulate the expression of VEGF in non-ovarian cells, whether it involved in regulating the expression of VEGF in normal ovarian granulosa cells is still unknown. The aim of this study was to elucidate the mechanisms underlying the effect of IL-6 on FSH-induced VEGF expression in bovine granulosa cells derived from large follicles. Methods: VEGF mRNA expression in granulosa cells after IL-6 with/without inhibitors treatment was analyzed by RT-qPCR. Phosphorylation levels of ERK1/2 and STAT3 proteins induced by IL-6 were analyzed by western blotting. The protein levels produced by granulosa cells were detected by ELISA. Results: High concentration of IL-6 (10ng/ml can significantly up-regulate FSH-induced VEGF gene and protein expression levels in granulosa cells, and also promote the VEGF upstream regulators HIF-1α and COX2 mRNA expression. VEGF expression levels were significantly decreased after specifically blocking HIF-1α and COX2 by using inhibitors. The up-regulation effect of IL-6 on FSH-induced VEGF expression in granulosa cells mainly through activating the JAK/STAT3 signaling pathway, which can be impaired by JAK inhibitors. Conclusion: IL-6 can promote FSH-induced VEGF expression in granulosa cells, which is mainly achieved by increasing the expression of HIF-1α and COX2.This promoting effect is mediated by activating the JAK/STAT3 pathway. Moreover, there may be a synergistic relationship between FSH and IL-6 in the regulation of VEGF expression.

High density lipoprotein (HDL)-associated sphingosine 1-phosphate (S1P) inhibits macrophage apoptosis by stimulating STAT3 activity and survivin expression.

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Feuerborn, Renata; Becker, Susen; Potì, Francesco; Nagel, Petra; Brodde, Martin; Schmidt, Harmut; Christoffersen, Christina; Ceglarek, Uta; Burkhardt, Ralph; Nofer, Jerzy-Roch

2017-02-01

Macrophage apoptosis is critically involved in atherosclerosis. We here examined the effect of anti-atherogenic high density lipoprotein (HDL) and its component sphingosine-1-phosphate (S1P) on apoptosis in RAW264.7 murine macrophages. Mitochondrial or endoplasmic reticulum-dependent apoptosis was induced by exposure of macrophages to etoposide or thapsigargin/fukoidan, respectively. Cell death induced by these compounds was inhibited by S1P as inferred from reduced annexin V binding, TUNEL staining, and caspase 3, 9 and 12 activities. S1P induced expression of the inhibitor of apoptosis protein (IAP) family proteins cIAP1, cIAP2 and survivin, but only the inhibitor of survivin expression YM155 and not the cIAP1/2 blocker GDC0152 reversed the inhibitory effect of S1P on apoptosis. Moreover, S1P activated signal transducer and activator of transcription 3 (STAT3) and Janus kinase 2 (JAK2) and the stimulatory effect of S1P on survivin expression and inhibitory effects on apoptosis were attenuated by STAT3 or JAK2 inhibitors, S3I-201 or AG490, respectively. The effects of S1P on STAT3 activation, survivin expression and macrophage apoptosis were emulated by HDL, HDL lipids, and apolipoprotein (apo) M-containing HDL, but not by apoA-I or HDL deprived of S1P or apoM. In addition, JTE013 and CAY10444, S1P receptor 2 and 3 antagonists, respectively, compromised the S1P and HDL capacities to stimulate STAT3 activation and survivin expression, and to inhibit apoptosis. HDL-associated S1P inhibits macrophage apoptosis by stimulating STAT3 activity and survivin expression. The suppression of macrophage apoptosis may represent a novel mechanism utilized by HDL to exert its anti-atherogenic effects. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Tofacitinib induces G1 cell-cycle arrest and inhibits tumor growth in Epstein-Barr virus-associated T and natural killer cell lymphoma cells.

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Ando, Shotaro; Kawada, Jun-Ichi; Watanabe, Takahiro; Suzuki, Michio; Sato, Yoshitaka; Torii, Yuka; Asai, Masato; Goshima, Fumi; Murata, Takayuki; Shimizu, Norio; Ito, Yoshinori; Kimura, Hiroshi

2016-11-22

Epstein-Barr virus (EBV) infects not only B cells, but also T cells and natural killer (NK) cells, and is associated with T or NK cell lymphoma. These lymphoid malignancies are refractory to conventional chemotherapy. We examined the activation of the JAK3/STAT5 pathway in EBV-positive and -negative B, T and NK cell lines and in cell samples from patients with EBV-associated T cell lymphoma. We then evaluated the antitumor effects of the selective JAK3 inhibitor, tofacitinib, against these cell lines in vitro and in a murine xenograft model. We found that all EBV-positive T and NK cell lines and patient samples tested displayed activation of the JAK3/STAT5 pathway. Treatment of these cell lines with tofacitinib reduced the levels of phospho-STAT5, suppressed proliferation, induced G1 cell-cycle arrest and decreased EBV LMP1 and EBNA1 expression. An EBV-negative NK cell line was also sensitive to tofacitinib, whereas an EBV-infected NK cell line was more sensitive to tofacitinib than its parental line. Tofacitinib significantly inhibited the growth of established tumors in NOG mice. These findings suggest that tofacitinib may represent a useful therapeutic agent for patients with EBV-associated T and NK cell lymphoma.

Impact of JAK2V617F Mutation Burden on Disease Phenotype in Chinese Patients with JAK2V617F-positive Polycythemia Vera (PV) and Essential thrombocythemia (ET).

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Zhao, Shixiang; Zhang, Xiang; Xu, Yang; Feng, Yufeng; Sheng, Wenhong; Cen, Jiannong; Wu, Depei; Han, Yue

2016-01-01

Most patients with polycythemia vera (PV) and half of essential thrombocythemia (ET) possess an activating JAK2V617F mutation. The objective of this study was to better define the effect of JAK2V617F mutant allele burden on clinical phenotypes in Chinese patients, especially thrombosis. By real-time polymerase chain reaction (RT-PCR), the JAK2V617F mutation burden was detected in 170 JAK2V617F-positive patients, including 54 PV and 116 ET. The results showed that JAK2V617F allele burden was higher in PV than in ET (PET (68.5% VS 26.7%) (PET patients showed increased JAK2V617F allele burden in the group with higher hemoglobin (HGB above 150 g/L) (PET. In PV patients, JAK2V617F mutation burden had influence on WBC counts. And the clinical characteristics of ET patients, such as WBC counts, hemoglobin level, splenomegaly and thrombosis, were influenced by JAK2V617F mutation burden. Male, high hemoglobin (HGB above 150 g/L), and increased JAK2V617F mutation burden (JAK2V617F allele burden ≥ 16.5%) were risks of thrombosis (PET patients by Logistic Regression.

Selective inhibition of distracting input.

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Noonan, MaryAnn P; Crittenden, Ben M; Jensen, Ole; Stokes, Mark G

2017-10-16

We review a series of studies exploring distractor suppression. It is often assumed that preparatory distractor suppression is controlled via top-down mechanisms of attention akin to those that prepare brain areas for target enhancement. Here, we consider two alternative mechanisms: secondary inhibition and expectation suppression within a predictive coding framework. We draw on behavioural studies, evidence from neuroimaging and some animal studies. We conclude that there is very limited evidence for selective top-down control of preparatory inhibition. By contrast, we argue that distractor suppression often relies secondary inhibition of non-target items (relatively non-selective inhibition) and on statistical regularities of the environment, learned through direct experience. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

The JAK/STAT pathway in obesity and diabetes.

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Gurzov, Esteban N; Stanley, William J; Pappas, Evan G; Thomas, Helen E; Gough, Daniel J

2016-08-01

Diabetes mellitus are complex, multi-organ metabolic pathologies characterized by hyperglycemia. Emerging evidence shows that the highly conserved and potent JAK/STAT signaling pathway is required for normal homeostasis, and, when dysregulated, contributes to the development of obesity and diabetes. In this review, we analyze the role of JAK/STAT activation in the brain, liver, muscle, fat and pancreas, and how this affects the course of the disease. We also consider the therapeutic implications of targeting the JAK/STAT pathway in treatment of obesity and diabetes. © 2016 Federation of European Biochemical Societies.

Selective mutism and temperament: the silence and behavioral inhibition to the unfamiliar.

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Gensthaler, Angelika; Khalaf, Sally; Ligges, Marc; Kaess, Michael; Freitag, Christine M; Schwenck, Christina

2016-10-01

Behavioral inhibition (BI) is a suspected precursor of selective mutism. However, investigations on early behavioral inhibition of children with selective mutism are lacking. Children aged 3-18 with lifetime selective mutism (n = 109), social phobia (n = 61), internalizing behavior (n = 46) and healthy controls (n = 118) were assessed using the parent-rated Retrospective Infant Behavioral Inhibition (RIBI) questionnaire. Analyses showed that children with lifetime selective mutism and social phobia were more inhibited as infants and toddlers than children of the internalizing and healthy control groups, who displayed similar low levels of behavioral inhibition. Moreover, behavioral inhibition was higher in infants with lifetime selective mutism than in participants with social phobia according to the Total BI score (p = 0.012) and the Shyness subscale (p selective mutism. Results yield first evidence of the recently hypothesized temperamental origin of selective mutism. Children at risk should be screened for this debilitating child psychiatric condition.

Cytogenetics, JAK2 and MPL mutations in polycythemia vera, primary myelofibrosis and essential thrombocythemia

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Leonardo Caires dos Santos

2011-12-01

Full Text Available BACKGROUND: The detection of molecular and cytogenetic alterations is important for the diagnosis, prognosis and classification of myeloproliferative neoplasms. OBJECTIVE: The aim of this study was to detect the following mutations: JAK2 V617F, JAK2 exon 12 and MPL W515K/L, besides chromosomal abnormalities. Furthermore, molecular and cytogenetic alterations were correlated with the leukocyte and platelet counts, hemoglobin levels and age in all patients and with the degree of fibrosis in primary myelofibrosis cases. METHODS: Twenty cases of polycythemia vera, 17 of essential thrombocythemia and 21 of primary myelofibrosis were selected in the Hematology Department of the Universidade Federal de São Paulo (UNIFESP between February 2008 and December 2009. The JAK2 V617F, JAK2 exon 12 mutations, MPL W515K and MPL W515L mutations were investigated by real-time PCR and direct sequencing. G-band karyotyping and fluorescence in situ hybridization were used to detect chromosomal abnormalities. RESULTS: Chromosomal abnormalities were observed only in polycythemia vera (11.8% and primary myelofibrosis cases (17.6%, without correlation to clinical data. Chromosomal abnormalities were not detected by fluorescence in situ hybridization. The JAK2 V617F mutation was observed in polycythemia vera (90%, primary myelofibrosis (42.8% and essential thrombocythemia (47%. Patients with JAK2 V617F-negative polycythemia vera had lower platelet and leukocyte counts compared to V617F-positive polycythemia vera (p-value = 0.0001 and p-value = 0.023, respectively. JAK2 V617F-positive and MPL W515L-positive primary myelofibrosis cases had a higher degree of fibrosis than V617F-negative cases (p-value = 0.022. JAK2 exon 12 mutations were not detected in polycythemia vera patients. The MPL W515L mutation was observed in one case of primary myelofibrosis and in one of essential thrombocythemia. The MPL W515K mutation was not found in patients with essential thrombocythemia

Cytogenetics, JAK2 and MPL mutations in polycythemia vera, primary myelofibrosis and essential thrombocythemia.

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Dos Santos, Leonardo Caires; Ribeiro, Juliana Corrêa da Costa; Silva, Neusa Pereira; Cerutti, Janete; da Silva, Maria Regina Regis; Chauffaille, Maria de Lourdes Lopes Ferrari

2011-01-01

The detection of molecular and cytogenetic alterations is important for the diagnosis, prognosis and classification of myeloproliferative neoplasms. THE AIM OF THIS STUDY WAS TO DETECT THE FOLLOWING MUTATIONS: JAK2 V617F, JAK2 exon 12 and MPL W515K/L, besides chromosomal abnormalities. Furthermore, molecular and cytogenetic alterations were correlated with the leukocyte and platelet counts, hemoglobin levels and age in all patients and with the degree of fibrosis in primary myelofibrosis cases. Twenty cases of polycythemia vera, 17 of essential thrombocythemia and 21 of primary myelofibrosis were selected in the Hematology Department of the Universidade Federal de São Paulo (UNIFESP) between February 2008 and December 2009. The JAK2 V617F, JAK2 exon 12 mutations, MPL W515K and MPL W515L mutations were investigated by real-time PCR and direct sequencing. G-band karyotyping and fluorescence in situ hybridization were used to detect chromosomal abnormalities. Chromosomal abnormalities were observed only in polycythemia vera (11.8%) and primary myelofibrosis cases (17.6%), without correlation to clinical data. Chromosomal abnormalities were not detected by fluorescence in situ hybridization. The JAK2 V617F mutation was observed in polycythemia vera (90%), primary myelofibrosis (42.8%) and essential thrombocythemia (47%). Patients with JAK2 V617F-negative polycythemia vera had lower platelet and leukocyte counts compared to V617F-positive polycythemia vera (p-value = 0.0001 and p-value = 0.023, respectively). JAK2 V617F-positive and MPL W515L-positive primary myelofibrosis cases had a higher degree of fibrosis than V617F-negative cases (p-value = 0.022). JAK2 exon 12 mutations were not detected in polycythemia vera patients. The MPL W515L mutation was observed in one case of primary myelofibrosis and in one of essential thrombocythemia. The MPL W515K mutation was not found in patients with essential thrombocythemia or primary myelofibrosis. The MPL W515L

JAK2V617F expression in mice amplifies early hematopoietic cells and gives them a competitive advantage that is hampered by IFNα.

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Hasan, Salma; Lacout, Catherine; Marty, Caroline; Cuingnet, Marie; Solary, Eric; Vainchenker, William; Villeval, Jean-Luc

2013-08-22

The acquired gain-of-function V617F mutation in the Janus Kinase 2 (JAK2(V617F)) is the main mutation involved in BCR/ABL-negative myeloproliferative neoplasms (MPNs), but its effect on hematopoietic stem cells as a driver of disease emergence has been questioned. Therefore, we reinvestigated the role of endogenous expression of JAK2(V617F) on early steps of hematopoiesis as well as the effect of interferon-α (IFNα), which may target the JAK2(V617F) clone in humans by using knock-in mice with conditional expression of JAK2(V617F) in hematopoietic cells. These mice develop a MPN mimicking polycythemia vera with large amplification of myeloid mature and precursor cells, displaying erythroid endogenous growth and progressing to myelofibrosis. Interestingly, early hematopoietic compartments [Lin-, LSK, and SLAM (LSK/CD48-/CD150+)] increased with the age. Competitive repopulation assays demonstrated disease appearance and progressive overgrowth of myeloid, Lin-, LSK, and SLAM cells, but not lymphocytes, from a low number of engrafted JAK2(V617F) SLAM cells. Finally, IFNα treatment prevented disease development by specifically inhibiting JAK2(V617F) cells at an early stage of differentiation and eradicating disease-initiating cells. This study shows that JAK2(V617F) in mice amplifies not only late but also early hematopoietic cells, giving them a proliferative advantage through high cell cycling and low apoptosis that may sustain MPN emergence but is lost upon IFNα treatment.

Coexistance of JAK2V617F mutation and BCR/ABL translocation in one patient

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Murat Albayrak

2010-09-01

Full Text Available Dear Editor,The myeloproliferative disorders (MPDs constitute a subcategory of chronic myeloid disorders and include chronic myeloid leukemia (CML, essential thrombocytemia (ET, polycythemia vera (PV and myelofibrosis (MF. In 1960, the discovery of the Philadelphia chromosome (Ph became a cornerstone in CML treatment and led to the development of moleculary targeted therapy. Recently, an acquired mutation in the Janus kinase 2 (JAK2 gene has been discovered in nearly all patents with PV and approximately half of the patients with primary MF and ET. Subsequently, the mutation has been demonstrated in atypical MPDs (chronic neutrophilic leukemia, unclassified, de novo myelodysplastic syndrome or acute myeloid leukemia.1 It has been hoped that targeted inhibition of JAK2V617F should achieve similar disease control as thyrosine kinases has produced in CML.

Hepatic JAK2 protects against atherosclerosis through circulating IGF-1.

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Sivasubramaniyam, Tharini; Schroer, Stephanie A; Li, Angela; Luk, Cynthia T; Shi, Sally Yu; Besla, Rickvinder; Dodington, David W; Metherel, Adam H; Kitson, Alex P; Brunt, Jara J; Lopes, Joshua; Wagner, Kay-Uwe; Bazinet, Richard P; Bendeck, Michelle P; Robbins, Clinton S; Woo, Minna

2017-07-20

Atherosclerosis is considered both a metabolic and inflammatory disease; however, the specific tissue and signaling molecules that instigate and propagate this disease remain unclear. The liver is a central site of inflammation and lipid metabolism that is critical for atherosclerosis, and JAK2 is a key mediator of inflammation and, more recently, of hepatic lipid metabolism. However, precise effects of hepatic Jak2 on atherosclerosis remain unknown. We show here that hepatic Jak2 deficiency in atherosclerosis-prone mouse models exhibited accelerated atherosclerosis with increased plaque macrophages and decreased plaque smooth muscle cell content. JAK2's essential role in growth hormone signalling in liver that resulted in reduced IGF-1 with hepatic Jak2 deficiency played a causal role in exacerbating atherosclerosis. As such, restoring IGF-1 either pharmacologically or genetically attenuated atherosclerotic burden. Together, our data show hepatic Jak2 to play a protective role in atherogenesis through actions mediated by circulating IGF-1 and, to our knowledge, provide a novel liver-centric mechanism in atheroprotection.

Curcumin blocks interleukin (IL)-2 signaling in T-lymphocytes by inhibiting IL-2 synthesis, CD25 expression, and IL-2 receptor signaling

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Forward, Nicholas A.; Conrad, David M.; Power Coombs, Melanie R.; Doucette, Carolyn D.; Furlong, Suzanne J.; Lin, Tong-Jun; Hoskin, David W.

2011-01-01

Highlights: → Curcumin inhibits CD4 + T-lymphocyte proliferation. → Curcumin inhibits interleukin-2 (IL-2) synthesis and CD25 expression by CD4 + T-lymphocytes. → Curcumin interferes with IL-2 receptor signaling by inhibiting JAK3 and STAT5 phosphorylation. → IL-2-dependent regulatory T-lymphocyte function and Foxp3 expression is downregulated by curcumin. -- Abstract: Curcumin (diferulomethane) is the principal curcuminoid in the spice tumeric and a potent inhibitor of activation-induced T-lymphocyte proliferation; however, the molecular basis of this immunosuppressive effect has not been well studied. Here we show that micromolar concentrations of curcumin inhibited DNA synthesis by mouse CD4 + T-lymphocytes, as well as interleukin-2 (IL-2) and CD25 (α chain of the high affinity IL-2 receptor) expression in response to antibody-mediated cross-linking of CD3 and CD28. Curcumin acted downstream of protein kinase C activation and intracellular Ca 2+ release to inhibit IκB phosphorylation, which is required for nuclear translocation of the transcription factor NFκB. In addition, IL-2-dependent DNA synthesis by mouse CTLL-2 cells, but not constitutive CD25 expression, was impaired in the presence of curcumin, which demonstrated an inhibitory effect on IL-2 receptor (IL-2R) signaling. IL-2-induced phosphorylation of STAT5A and JAK3, but not JAK1, was diminished in the presence of curcumin, indicating inhibition of critical proximal events in IL-2R signaling. In line with the inhibitory action of curcumin on IL-2R signaling, pretreatment of CD4 + CD25 + regulatory T-cells with curcumin downregulated suppressor function, as well as forkhead box p3 (Foxp3) expression. We conclude that curcumin inhibits IL-2 signaling by reducing available IL-2 and high affinity IL-2R, as well as interfering with IL-2R signaling.

Clinical effect of driver mutations of JAK2, CALR, or MPL in primary myelofibrosis.

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Rumi, Elisa; Pietra, Daniela; Pascutto, Cristiana; Guglielmelli, Paola; Martínez-Trillos, Alejandra; Casetti, Ilaria; Colomer, Dolors; Pieri, Lisa; Pratcorona, Marta; Rotunno, Giada; Sant'Antonio, Emanuela; Bellini, Marta; Cavalloni, Chiara; Mannarelli, Carmela; Milanesi, Chiara; Boveri, Emanuela; Ferretti, Virginia; Astori, Cesare; Rosti, Vittorio; Cervantes, Francisco; Barosi, Giovanni; Vannucchi, Alessandro M; Cazzola, Mario

2014-08-14

We studied the impact of driver mutations of JAK2, CALR, (calreticulin gene) or MPL on clinical course, leukemic transformation, and survival of patients with primary myelofibrosis (PMF). Of the 617 subjects studied, 399 (64.7%) carried JAK2 (V617F), 140 (22.7%) had a CALR exon 9 indel, 25 (4.0%) carried an MPL (W515) mutation, and 53 (8.6%) had nonmutated JAK2, CALR, and MPL (so-called triple-negative PMF). Patients with CALR mutation had a lower risk of developing anemia, thrombocytopenia, and marked leukocytosis compared with other subtypes. They also had a lower risk of thrombosis compared with patients carrying JAK2 (V617F). At the opposite, triple-negative patients had higher incidence of leukemic transformation compared with either CALR-mutant or JAK2-mutant patients. Median overall survival was 17.7 years in CALR-mutant, 9.2 years in JAK2-mutant, 9.1 years in MPL-mutant, and 3.2 years in triple-negative patients. In multivariate analysis corrected for age, CALR-mutant patients had better overall survival than either JAK2-mutant or triple-negative patients. The impact of genetic lesions on survival was independent of current prognostic scoring systems. These observations indicate that driver mutations define distinct disease entities within PMF. Accounting for them is not only relevant to clinical decision-making, but should also be considered in designing clinical trials. © 2014 by The American Society of Hematology.

Deficiency of PTP1B Attenuates Hypothalamic Inflammation via Activation of the JAK2-STAT3 Pathway in Microglia.

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Tsunekawa, Taku; Banno, Ryoichi; Mizoguchi, Akira; Sugiyama, Mariko; Tominaga, Takashi; Onoue, Takeshi; Hagiwara, Daisuke; Ito, Yoshihiro; Iwama, Shintaro; Goto, Motomitsu; Suga, Hidetaka; Sugimura, Yoshihisa; Arima, Hiroshi

2017-02-01

Protein tyrosine phosphatase 1B (PTP1B) regulates leptin signaling in hypothalamic neurons via the JAK2-STAT3 pathway. PTP1B has also been implicated in the regulation of inflammation in the periphery. However, the role of PTP1B in hypothalamic inflammation, which is induced by a high-fat diet (HFD), remains to be elucidated. Here, we showed that STAT3 phosphorylation (p-STAT3) was increased in microglia in the hypothalamic arcuate nucleus of PTP1B knock-out mice (KO) on a HFD, accompanied by decreased Tnf and increased Il10 mRNA expression in the hypothalamus compared to wild-type mice (WT). In hypothalamic organotypic cultures, incubation with TNFα led to increased p-STAT3, accompanied by decreased Tnf and increased Il10 mRNA expression, in KO compared to WT. Incubation with p-STAT3 inhibitors or microglial depletion eliminated the differences in inflammation between genotypes. These data indicate an important role of JAK2-STAT3 signaling negatively regulated by PTP1B in microglia, which attenuates hypothalamic inflammation under HFD conditions. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Deficiency of PTP1B Attenuates Hypothalamic Inflammation via Activation of the JAK2-STAT3 Pathway in Microglia

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Taku Tsunekawa

2017-02-01

Full Text Available Protein tyrosine phosphatase 1B (PTP1B regulates leptin signaling in hypothalamic neurons via the JAK2-STAT3 pathway. PTP1B has also been implicated in the regulation of inflammation in the periphery. However, the role of PTP1B in hypothalamic inflammation, which is induced by a high-fat diet (HFD, remains to be elucidated. Here, we showed that STAT3 phosphorylation (p-STAT3 was increased in microglia in the hypothalamic arcuate nucleus of PTP1B knock-out mice (KO on a HFD, accompanied by decreased Tnf and increased Il10 mRNA expression in the hypothalamus compared to wild-type mice (WT. In hypothalamic organotypic cultures, incubation with TNFα led to increased p-STAT3, accompanied by decreased Tnf and increased Il10 mRNA expression, in KO compared to WT. Incubation with p-STAT3 inhibitors or microglial depletion eliminated the differences in inflammation between genotypes. These data indicate an important role of JAK2-STAT3 signaling negatively regulated by PTP1B in microglia, which attenuates hypothalamic inflammation under HFD conditions.

Characterization and Prognosis Significance of JAK2 (V617F), MPL, and CALR Mutations in Philadelphia-Negative Myeloproliferative Neoplasms

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Singdong, Roongrudee; Siriboonpiputtana, Teerapong; Chareonsirisuthigul, Takol; Kongruang, Adcharee; Limsuwanachot, Nittaya; Sirirat, Tanasan; Chuncharunee, Suporn; Rerkamnuaychoke, Budsaba

2016-10-01

Background: The discovery of somatic acquired mutations of JAK2 (V617F) in Philadelphia-negative myeloproliferative neoplasms (Ph-negative MPNs) including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) has not only improved rational disease classification and prognostication but also brings new understanding insight into the pathogenesis of diseases. Dosage effects of the JAK2 (V617F) allelic burden in Ph-negative MPNs may partially influence clinical presentation, disease progression, and treatment outcome. Material and Methods: Pyrosequencing was performed to detect JAK2 (V617F) and MPL (W515K/L) and capillary electrophoresis to identify CALR exon 9.0 mutations in 100.0 samples of Ph-negative MPNs (38.0 PV, 55 ET, 4 PMF, and 3 MPN-U). Results: The results showed somatic mutations of JAK2 (V617F) in 94.7% of PV, 74.5% of ET, 25.0% of PMF, and all MPN-U. A high proportion of JAK2 (V617F) mutant allele burden (mutational load > 50.0%) was predominantly observed in PV when compared with ET. Although a high level of JAK2 (V617F) allele burden was strongly associated with high WBC counts in both PV and ET, several hematological parameters (hemoglobin, hematocrit, and platelet count) were independent of JAK2 (V617F) mutational load. MPL (W515K/L) mutations could not be detected whereas CALR exon 9.0 mutations were identified in 35.7% of patients with JAK2 negative ET and 33.3% with JAK2 negative PMF. Conclusions: The JAK2 (V617F) allele burden may be involved in progression of MPNs. Furthermore, a high level of JAK2 (V617F) mutant allele appears strongly associated with leukocytosis in both PV and ET. Creative Commons Attribution License

The dipeptide Pro-Asp promotes IGF-1 secretion and expression in hepatocytes by enhancing JAK2/STAT5 signaling pathway.

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Wang, Songbo; Wang, Guoqing; Zhang, Mengyuan; Zhuang, Lu; Wan, Xiaojuan; Xu, Jingren; Wang, Lina; Zhu, Xiaotong; Gao, Ping; Xi, Qianyun; Zhang, Yongliang; Shu, Gang; Jiang, Qingyan

2016-11-15

It has been implicated that IGF-1 secretion can be regulated by dietary protein. However, whether the dipeptides, one of digested products of dietary protein, have influence on IGF-1 secretion remain largely unknown. Our study aimed to investigate the effects of the dipeptide Pro-Asp on IGF-1 secretion and expression in hepatocytes and to explore the possible underlying mechanisms. Our findings demonstrated that Pro-Asp promoted the secretion and gene expression of IGF-1 in HepG2 cells and primary porcine hepatocytes. Meanwhile, Pro-Asp activated the ERK and Akt signaling pathways, downstream of IGF-1. In addition, Pro-Asp enhanced GH-mediated JAK2/STAT5 signaling pathway, while inhibition of JAK2/STAT5 blocked the promotive effect of Pro-Asp on IGF-1 secretion and expression. Moreover, acute injection of Pro-Asp stimulated IGF-1 expression and activated JAK2/STAT5 signaling pathway in mice liver. Together, these results suggested that the dipeptide Pro-Asp promoted IGF-1 secretion and expression in hepatocytes by enhancing GH-mediated JAK2/STAT5 signaling pathway. Copyright © 2016. Published by Elsevier Ireland Ltd.

CXCL12 suppresses cisplatin-induced apoptosis through activation of JAK2/STAT3 signaling in human non-small-cell lung cancer cells

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Wang M

2017-06-01

Full Text Available Meng Wang,1 Tie Lin,2 Yicun Wang,3 Song Gao,4 Zhaoyang Yang,1 Xuan Hong,1 Gongyan Chen1 1Department of Respiratory Medicine, Harbin Medical University Cancer Hospital, 2Department of Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, 3Jilin Provincial Key Laboratory on Molecular and Chemical Genetics, The Second Hospital of Jilin University, Changchun, 4Department of Clinical Oncology, Shengjing Hospital, China Medical University, Shenyang, People’s Republic of China Aims: Poor efficacy of chemotherapy drugs in non-small-cell lung cancer (NSCLC is the key reason for the failure of treatment, but the mechanism of this remains largely unknown. Stromal cell-derived factor 1-alpha (SDF-1α/CXCL12 is a small chemotactic cytokine protein that plays an important role in tumor progression. In this study, we investigated the anti-apoptotic mechanism of the CXCL12/CXCR4 axis in response to cisplatin, a commonly used chemotherapeutic drug, in human lung adenocarcinoma A549 cells.Methods: CXCL12 blocks cisplatin-induced apoptosis in A549, and the results were shown by propidium iodide/annexin V staining in vitro. The mechanism of CXCL12 stimulating phosphorylation of STAT3 through CXCR4/JAK2 was demonstrated by immunofluorescence and Western blotting. The expression of CXCL12 and p-STAT3 in clinical specimens was examined by immunohistochemistry.Results: CXCL12 significantly decreased the ratio of apoptotic cells and stimulation of phospho-signal transducer and activator of transcription (p-STAT-3 in a time-dependent manner through interaction with CXCR4. Among the signaling molecules downstream of CXCR4, the JAK2/STAT3 pathway plays a predominant role in the anti-apoptotic effect of CXCL12. Analysis of clinical specimens revealed that increased CXCL12 and p-STAT3 expression correlates with enhanced lung cancer progression.Conclusion: These data suggest that CXCR4 contributes to CXCL12-mediated anti-apoptosis by activating JAK2

Enu mutagenesis identifies a novel platelet phenotype in a loss-of-function Jak2 allele.

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Nicole M Anderson

Full Text Available Utilizing ENU mutagenesis, we identified a mutant mouse with elevated platelets. Genetic mapping localized the mutation to an interval on chromosome 19 that encodes the Jak2 tyrosine kinase. We identified a A3056T mutation resulting in a premature stop codon within exon 19 of Jak2 (Jak2(K915X, resulting in a protein truncation and functionally inactive enzyme. This novel platelet phenotype was also observed in mice bearing a hemizygous targeted disruption of the Jak2 locus (Jak2(+/-. Timed pregnancy experiments revealed that Jak2(K915X/K915X and Jak2(-/- displayed embryonic lethality; however, Jak2(K915X/K915X embryos were viable an additional two days compared to Jak2(-/- embryos. Our data suggest that perturbing JAK2 activation may have unexpected consequences in elevation of platelet number and correspondingly, important implications for treatment of hematological disorders with constitutive Jak2 activity.

MSM enhances GH signaling via the Jak2/STAT5b pathway in osteoblast-like cells and osteoblast differentiation through the activation of STAT5b in MSCs.

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Youn Hee Joung

Full Text Available Methylsulfonylmethane (MSM is a naturally occurring sulfur compound with well-known anti-oxidant properties and anti-inflammatory activities. But, its effects on bone are unknown. Growth hormone (GH is regulator of bone growth and bone metabolism. GH activates several signaling pathways such as the Janus kinase (Jak/signal transducers and activators of transcription (STAT pathway, thereby regulating expression of genes including insulin-like growth factor (IGF-1. GH exerts effects both directly and via IGF-1, which signals by activating the IGF-1 receptor (IGF-1R. In this study, we investigated the effects of MSM on the GH signaling via the Jak/STAT pathway in osteoblasts and the differentiation of primary bone marrow mesenchymal stem cells (MSCs. MSM was not toxic to osteoblastic cells and MSCs. MSM increased the expression of GH-related proteins including IGF-1R, p-IGF-1R, STAT5b, p-STAT5b, and Jak2 in osteoblastic cells and MSCs. MSM increased IGF-1R and GHR mRNA expression in osteoblastic cells. The expression of MSM-induced IGF-1R and GHR was inhibited by AG490, a Jak2 kinase inhibitor. MSM induced binding of STAT5 to the IGF-1R and increased IGF-1 and IGF-1R promoter activities. Analysis of cell extracts by immunoprecipitation and Western blot showed that MSM enhanced GH-induced activation of Jak2/STAT5b. We found that MSM and GH, separately or in combination, activated GH signaling via the Jak2/STAT5b pathway in UMR-106 cells. Using siRNA analysis, we found that STAT5b plays an essential role in GH signaling activation in C3H10T1/2 cells. Osteogenic marker genes (ALP, ON, OCN, BSP, OSX, and Runx2 were activated by MSM, and siRNA-mediated STAT5b knockdown inhibited MSM-induced expression of osteogenic markers. Furthermore, MSM increased ALP activity and the mineralization of MSCs. Taken together, these results indicated that MSM can promote osteogenic differentiation of MSCs through activation of STAT5b.

Novel fused oxazepino-indoles (FOIs) attenuate liver carcinogenesis via IL-6/JAK2/STAT3 signaling blockade as evidenced through data-based mathematical modeling.

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Singh, Ashok K; Bhadauria, Archana Singh; Kumar, Umesh; Raj, Vinit; Maurya, Vimal; Kumar, Dinesh; Maity, Biswanath; Prakash, Anand; De, Arnab; Samanta, Amalesh; Saha, Sudipta

2018-05-15

To potentiate the well-documented tumor protecting ability of paullones, literatures demand for rational modifications in paullone ring structure and exploration of a precise mechanism underlying their antitumor effects. Thus, recently we synthesized novel paullone-like scaffold, 5H-benzo [2, 3][1,4]oxazepino[5,6-b]indoles, where compounds 13a and 14a attenuated the growth of liver cancer specific Hep-G2 cells in vitro and formed stable binding complex with IL-6. Henceforth, we hypothesized that this action is probably due to the blockade of IL-6 mediated JAK2/STAT3 signaling cascade. A preclinical study was conducted using NDEA-induced HCC rat model by oral administration of FOIs at 10 mg/kg dose for 15 days. The molecular insights were confirmed through ELISA, qRT-PCR, western blot analyses. The study was further confirmed by data-based mathematical modeling using the quantitative data obtained from western blot analysis. 1 H NMR based metabolomics study was also performed to unveil metabolite discriminations among various studied groups. We identified that the HCC condition was produced due to the IL-6 induced activation of JAK2 and STAT3 which, in turn, was due to enhanced phosphorylation of JAK2 and STAT3. The treatment with FOIs led to the significant blockade of the IL-6 mediated JAK2/STAT3 signaling pathway. Besides, FOIs showed their potential ability in restoring perturbed metabolites linked to HCC. In particular, the anticancer efficacy of compound 13a was comparable or somewhat better than marketed chemotherapeutics, 5-flurouracil. These findings altogether opened up possibilities of developing fused oxazepino-indoles (FOIs) as new candidate molecule for plausible alternative of paullones to treat liver cancer. Copyright © 2018 Elsevier Inc. All rights reserved.

IL-10 Promotes Neurite Outgrowth and Synapse Formation in Cultured Cortical Neurons after the Oxygen-Glucose Deprivation via JAK1/STAT3 Pathway.

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Chen, Hongbin; Lin, Wei; Zhang, Yixian; Lin, Longzai; Chen, Jianhao; Zeng, Yongping; Zheng, Mouwei; Zhuang, Zezhong; Du, Houwei; Chen, Ronghua; Liu, Nan

2016-07-26

As a classic immunoregulatory and anti-inflammatory cytokine, interleukin-10 (IL-10) provides neuroprotection in cerebral ischemia in vivo or oxygen-glucose deprivation (OGD)-induced injury in vitro. However, it remains blurred whether IL-10 promotes neurite outgrowth and synapse formation in cultured primary cortical neurons after OGD injury. In order to evaluate its effect on neuronal apoptosis, neurite outgrowth and synapse formation, we administered IL-10 or IL-10 neutralizing antibody (IL-10NA) to cultured rat primary cortical neurons after OGD injury. We found that IL-10 treatment activated the Janus kinase 1 (JAK1)/signal transducers and activators of transcription 3 (STAT3) signaling pathway. Moreover, IL-10 attenuated OGD-induced neuronal apoptosis by down-regulating the Bax expression and up-regulating the Bcl-2 expression, facilitated neurite outgrowth by increasing the expression of Netrin-1, and promoted synapse formation in cultured primary cortical neurons after OGD injury. These effects were partly abolished by JAK1 inhibitor GLPG0634. Contrarily, IL-10NA produced opposite effects on the cultured cortical neurons after OGD injury. Taken together, our findings suggest that IL-10 not only attenuates neuronal apoptosis, but also promotes neurite outgrowth and synapse formation via the JAK1/STAT3 signaling pathway in cultured primary cortical neurons after OGD injury.

A bipolar clamp mechanism for activation of Jak-family protein tyrosine kinases.

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Dipak Barua

2009-04-01

Full Text Available Most cell surface receptors for growth factors and cytokines dimerize in order to mediate signal transduction. For many such receptors, the Janus kinase (Jak family of non-receptor protein tyrosine kinases are recruited in pairs and juxtaposed by dimerized receptor complexes in order to activate one another by trans-phosphorylation. An alternative mechanism for Jak trans-phosphorylation has been proposed in which the phosphorylated kinase interacts with the Src homology 2 (SH2 domain of SH2-B, a unique adaptor protein with the capacity to homo-dimerize. Building on a rule-based kinetic modeling approach that considers the concerted nature and combinatorial complexity of modular protein domain interactions, we examine these mechanisms in detail, focusing on the growth hormone (GH receptor/Jak2/SH2-Bbeta system. The modeling results suggest that, whereas Jak2-(SH2-Bbeta(2-Jak2 heterotetramers are scarcely expected to affect Jak2 phosphorylation, SH2-Bbeta and dimerized receptors synergistically promote Jak2 trans-activation in the context of intracellular signaling. Analysis of the results revealed a unique mechanism whereby SH2-B and receptor dimers constitute a bipolar 'clamp' that stabilizes the active configuration of two Jak2 molecules in the same macro-complex.

CALR, JAK2 and MPL mutation status in Argentinean patients with BCR-ABL1- negative myeloproliferative neoplasms.

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Ojeda, Mara Jorgelina; Bragós, Irma Margarita; Calvo, Karina Lucrecia; Williams, Gladis Marcela; Carbonell, María Magdalena; Pratti, Arianna Flavia

2018-05-01

To establish the frequency of JAK2, MPL and CALR mutations in Argentinean patients with BCR-ABL1-negative  myeloproliferative neoplasms (MPN) and to compare their clinical and haematological features. Mutations of JAK2V617F, JAK2 exon 12, MPL W515L/K and CALR were analysed in 439 Argentinean patients with BCR-ABL1-negative MPN, including 176 polycythemia vera (PV), 214 essential thrombocythemia (ET) and 49 primary myelofibrosis (PMF). In 94.9% of PV, 85.5% ET and 85.2% PMF, we found mutations in JAK2, MPL or CALR. 74.9% carried JAK2V617F, 12.3% CALR mutations, 2.1% MPL mutations and 10.7% were triple negative. In ET, nine types of CALR mutations were identified, four of which were novel. PMF patients were limited to types 1 and 2, type 2 being more frequent. In ET, patients with CALR mutation were younger and had higher platelet counts than those with JAK2V617F and triple negative. In addition, JAK2V617F patients had high leucocyte and haemoglobin values compared with CALR-mutated and triple-negative patients. In PMF, patients with mutant CALR were associated with higher platelet counts. Our study underscores the importance of JAK2, MPL and CALR genotyping for accurate diagnosis of patients with BCR-ABL1-negative MPN.

Mimetics of Suppressor of cytokine signalling 3: novel potential therapeutics in triple breast cancer.

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La Manna, Sara; Lee, Eunmi; Ouzounova, Maria; Di Natale, Concetta; Novellino, Ettore; Merlino, Antonello; Korkaya, Hasan; Marasco, Daniela

2018-05-11

Suppressor of cytokine signaling (SOCS) family of proteins plays critical role in the regulation of immune responses controlling JAK/STAT mediated inflammatory cytokines. Among the members, SOCS1 and SOCS3 contain a kinase inhibitory region (KIR) and SOCS3 binds to JAK/STAT/gp130 complex by inhibiting the downstream signaling and suppressing inflammatory cytokines. Loss or reduced levels of SOCS3 have been linked to cancer-associated inflammation and suppressive immunity leading to enhanced tumour growth and metastasis. In line with these reports, we previously demonstrated that proteolytic degradation of SOCS3 in triple negative breast cancer (TNBC) subtype drives the expression of inflammatory cytokines. Therefore, we postulated that SOCS3 mimetics might suppress the inflammatory cytokine production in TNBC subtype and inhibit tumor growth and metastasis. Here we designed and characterized five linear peptides derived from the N-terminal region of SOCS3 encompassing regions that interface with the JAK2/gp130 complex by using the Circular Dichroism and Surface Plasmon Resonance spectroscopies. The KIRESS peptide resulted the sequence containing the most part of the hot-spots required for binding to JAK2 and was further investigated in vivo in mouse xenografts of MDA-MB-231-luci tumours as models of human TNBC subtype. Expectedly, this peptide showed a significant inhibition of primary tumour growth and pulmonary metastasis. Our studies suggest that SOCS3 peptidomimetics may possess a therapeutic potential in aggressive cancers, such as TNBC subtype, with activated inflammatory cytokines. This article is protected by copyright. All rights reserved. © 2018 UICC.

Janus Kinase 2 (JAK2) Dissociates Hepatosteatosis from Hepatocellular Carcinoma in Mice.

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Shi, Sally Yu; Luk, Cynthia T; Schroer, Stephanie A; Kim, Min Jeong; Dodington, David W; Sivasubramaniyam, Tharini; Lin, Lauren; Cai, Erica P; Lu, Shun-Yan; Wagner, Kay-Uwe; Bazinet, Richard P; Woo, Minna

2017-03-03

Hepatocellular carcinoma is an end-stage complication of non-alcoholic fatty liver disease (NAFLD). Inflammation plays a critical role in the progression of non-alcoholic fatty liver disease and the development of hepatocellular carcinoma. However, whether steatosis per se promotes liver cancer, and the molecular mechanisms that control the progression in this disease spectrum remain largely elusive. The Janus kinase signal transducers and activators of transcription (JAK-STAT) pathway mediates signal transduction by numerous cytokines that regulate inflammation and may contribute to hepatocarcinogenesis. Mice with hepatocyte-specific deletion of JAK2 (L-JAK2 KO) develop extensive fatty liver spontaneously. We show here that this simple steatosis was insufficient to drive carcinogenesis. In fact, L-JAK2 KO mice were markedly protected from chemically induced tumor formation. Using the methionine choline-deficient dietary model to induce steatohepatitis, we found that steatohepatitis development was completely arrested in L-JAK2 KO mice despite the presence of steatosis, suggesting that JAK2 is the critical factor required for inflammatory progression in the liver. In line with this, L-JAK2 KO mice exhibited attenuated inflammation after chemical carcinogen challenge. This was associated with increased hepatocyte apoptosis without elevated compensatory proliferation, thus thwarting expansion of transformed hepatocytes. Taken together, our findings identify an indispensable role of JAK2 in hepatocarcinogenesis through regulating critical inflammatory pathways. Targeting the JAK-STAT pathway may provide a novel therapeutic option for the treatment of hepatocellular carcinoma. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Inhibition of STAT3 activity delays obesity-induced thyroid carcinogenesis in a mouse model

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Park, Jeong Won; Han, Cho Rong; Zhao, Li; Willingham, Mark C.; Cheng, Sheue-yann

2015-01-01

Compelling epidemiologic studies indicate that obesity is a risk factor for many human cancers, including thyroid cancer. In recent decades, the incidence of thyroid cancer has dramatically increased along with a marked rise in obesity prevalence. We previously demonstrated that a high fat diet (HFD) effectively induced the obese phenotype in a mouse model of thyroid cancer (ThrbPV/PVPten+/− mice). Moreover, HFD activates the STAT3 signal pathway to promote more aggressive tumor phenotypes. The aim of the present study was to evaluate the effect of S3I-201, a specific inhibitor of STAT3 activity, on HFD-induced aggressive cancer progression in the mouse model of thyroid cancer. Wild type and ThrbPV/PVPten+/− mice were treated with HFD together with S3I-201 or vehicle-only as controls. We assessed the effects of S3I-201 on HFD-induced thyroid cancer progression, the leptin-JAK2-STAT3 signaling pathway, and key regulators of epithelial-mesenchymal transition. S3I-201 effectively inhibited HFD-induced aberrant activation of STAT3 and its downstream targets to markedly inhibit thyroid tumor growth and to prolong survival. Decreased protein levels of cyclins D1 and B1, cyclin dependent kinase (CDK) 4, CDK 6, and phosphorylated retinoblastoma protein led to the inhibition of tumor cell proliferation in S3I-201-treated ThrbPV/PVPten+/− mice. Reduced occurrence of vascular invasion and blocking of anaplasia and lung metastasis in thyroid tumors of S3I-201-treated ThrbPV/PVPten+/− mice were mediated via decreased expression of vimentin and matrix metalloproteinases, two key effectors of epithelial-mesenchymal transition. The present findings suggest that inhibition of the STAT3 activity would be a novel treatment strategy for obesity-induced thyroid cancer. PMID:26552408

RECQL5 Suppresses Oncogenic JAK2-Induced Replication Stress and Genomic Instability

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Edwin Chen

2015-12-01

Full Text Available JAK2V617F is the most common oncogenic lesion in patients with myeloproliferative neoplasms (MPNs. Despite the ability of JAK2V617F to instigate DNA damage in vitro, MPNs are nevertheless characterized by genomic stability. In this study, we address this paradox by identifying the DNA helicase RECQL5 as a suppressor of genomic instability in MPNs. We report increased RECQL5 expression in JAK2V617F-expressing cells and demonstrate that RECQL5 is required to counteract JAK2V617F-induced replication stress. Moreover, RECQL5 depletion sensitizes JAK2V617F mutant cells to hydroxyurea (HU, a pharmacological inducer of replication stress and the most common treatment for MPNs. Using single-fiber chromosome combing, we show that RECQL5 depletion in JAK2V617F mutant cells impairs replication dynamics following HU treatment, resulting in increased double-stranded breaks and apoptosis. Cumulatively, these findings identify RECQL5 as a critical regulator of genome stability in MPNs and demonstrate that replication stress-associated cytotoxicity can be amplified specifically in JAK2V617F mutant cells through RECQL5-targeted synthetic lethality.

JAK2 mutation in a patient with CLL with coexistent myeloproliferative neoplasm (MPN).

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Kodali, Srinivas; Chen, Chi; Rathnasabapathy, Chenthilmurugan; Wang, Jen Chin

2009-12-01

JAK2 mutation has not been described in patients with chronic lymphocytic leukemia (CLL). We found JAK2 mutation in a patient with CLL and coexisting myeloproliferative neoplasm (MPN). In this patient, we demonstrated the presence of the JAK2 mutation in CD34(+) progenitor cells, myeloid lineage cells, megakaryocytes, B lymphocytes but not in T lymphocytes. This case represents the first case report of JAK2 mutation in CLL and may also suggest that, JAK2 mutation most likely represents a secondary event from primary gene mutations involving the primitive stem cells which give rise to MPN and CLL. Furthermore, in this case, we believe that we are the first to demonstrate that JAK2 mutation in myeloid and B lymphoid cells but not T lymphocytes in a case of coexisting CLL and MPN.

Autophosphorylation of JAK2 on Tyrosines 221 and 570 Regulates Its Activity

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Argetsinger, Lawrence S.; Kouadio, Jean-Louis K.; Steen, Hanno; Stensballe, Allan; Jensen, Ole N.; Carter-Su, Christin

2004-01-01

The tyrosine kinase JAK2 is a key signaling protein for at least 20 receptors in the cytokine/hematopoietin receptor superfamily and is a component of signaling by insulin receptor and several G-protein-coupled receptors. However, there is only limited knowledge of the physical structure of JAK2 or which of the 49 tyrosines in JAK2 are autophosphorylated. In this study, mass spectrometry and two-dimensional peptide mapping were used to determine that tyrosines 221, 570, and 1007 in JAK2 are a...

A phase 2 study of ruxolitinib, an oral JAK1 and JAK2 Inhibitor, in patients with advanced polycythemia vera who are refractory or intolerant to hydroxyurea.

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Verstovsek, Srdan; Passamonti, Francesco; Rambaldi, Alessandro; Barosi, Giovanni; Rosen, Peter J; Rumi, Elisa; Gattoni, Elisabetta; Pieri, Lisa; Guglielmelli, Paola; Elena, Chiara; He, Shui; Contel, Nancy; Mookerjee, Bijoyesh; Sandor, Victor; Cazzola, Mario; Kantarjian, Hagop M; Barbui, Tiziano; Vannucchi, Alessandro M

2014-02-15

Polycythemia vera (PV) is a myeloproliferative neoplasm associated with somatic gain-of-function mutations of Janus kinase-2 (JAK2). Therapeutic options are limited in patients with advanced disease. Ruxolitinib, an oral JAK1/JAK2 inhibitor, is active in preclinical models of PV. The long-term efficacy and safety of ruxolitinib in patients with advanced PV who are refractory or intolerant to hydroxyurea were studied in a phase 2 trial. Response was assessed using modified European LeukemiaNet criteria, which included a reduction in hematocrit to cell and platelet counts, and reduction in PV-associated symptoms. Thirty-four patients received ruxolitinib for a median of 152 weeks (range, 31 weeks-177 weeks) or 35.0 months (range, 7.1 months-40.7 months). Hematocrit night sweats, and bone pain were observed within 4 weeks of the initiation of therapy and maintained with continued treatment. Ruxolitinib treatment also reduced elevated levels of inflammatory cytokines and granulocyte activation. Thrombocytopenia and anemia were the most common adverse events.Thrombocytopenia of grade 3 or anemia of grade 3 (according to National Cancer Institute Common Terminology Criteria for Adverse Events,version 3.0) occurred in 3 patients each (9%) (1 patient had both) and were managed with dose modification. Ruxolitinib was generally well tolerated and provided rapid and durable clinical benefits in patients with advanced PV who were refractory or intolerant to hydroxyurea.

Curcumin blocks interleukin (IL)-2 signaling in T-lymphocytes by inhibiting IL-2 synthesis, CD25 expression, and IL-2 receptor signaling

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Forward, Nicholas A.; Conrad, David M. [Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia (Canada); Power Coombs, Melanie R.; Doucette, Carolyn D. [Department of Pathology, Dalhousie University, Halifax, Nova Scotia (Canada); Furlong, Suzanne J. [Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia (Canada); Lin, Tong-Jun [Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia (Canada); Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia (Canada); Hoskin, David W., E-mail: d.w.hoskin@dal.ca [Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia (Canada); Department of Pathology, Dalhousie University, Halifax, Nova Scotia (Canada); Department of Surgery, Dalhousie University, Halifax, Nova Scotia (Canada)

2011-04-22

Highlights: {yields} Curcumin inhibits CD4{sup +} T-lymphocyte proliferation. {yields} Curcumin inhibits interleukin-2 (IL-2) synthesis and CD25 expression by CD4{sup +} T-lymphocytes. {yields} Curcumin interferes with IL-2 receptor signaling by inhibiting JAK3 and STAT5 phosphorylation. {yields} IL-2-dependent regulatory T-lymphocyte function and Foxp3 expression is downregulated by curcumin. -- Abstract: Curcumin (diferulomethane) is the principal curcuminoid in the spice tumeric and a potent inhibitor of activation-induced T-lymphocyte proliferation; however, the molecular basis of this immunosuppressive effect has not been well studied. Here we show that micromolar concentrations of curcumin inhibited DNA synthesis by mouse CD4{sup +} T-lymphocytes, as well as interleukin-2 (IL-2) and CD25 ({alpha} chain of the high affinity IL-2 receptor) expression in response to antibody-mediated cross-linking of CD3 and CD28. Curcumin acted downstream of protein kinase C activation and intracellular Ca{sup 2+} release to inhibit I{kappa}B phosphorylation, which is required for nuclear translocation of the transcription factor NF{kappa}B. In addition, IL-2-dependent DNA synthesis by mouse CTLL-2 cells, but not constitutive CD25 expression, was impaired in the presence of curcumin, which demonstrated an inhibitory effect on IL-2 receptor (IL-2R) signaling. IL-2-induced phosphorylation of STAT5A and JAK3, but not JAK1, was diminished in the presence of curcumin, indicating inhibition of critical proximal events in IL-2R signaling. In line with the inhibitory action of curcumin on IL-2R signaling, pretreatment of CD4{sup +}CD25{sup +} regulatory T-cells with curcumin downregulated suppressor function, as well as forkhead box p3 (Foxp3) expression. We conclude that curcumin inhibits IL-2 signaling by reducing available IL-2 and high affinity IL-2R, as well as interfering with IL-2R signaling.

The JAK2 GGCC (46/1 Haplotype in Myeloproliferative Neoplasms: Causal or Random?

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Luisa Anelli

2018-04-01

Full Text Available The germline JAK2 haplotype known as “GGCC or 46/1 haplotype” (haplotypeGGCC_46/1 consists of a combination of single nucleotide polymorphisms (SNPs mapping in a region of about 250 kb, extending from the JAK2 intron 10 to the Insulin-like 4 (INLS4 gene. Four main SNPs (rs3780367, rs10974944, rs12343867, and rs1159782 generating a “GGCC” combination are more frequently indicated to represent the JAK2 haplotype. These SNPs are inherited together and are frequently associated with the onset of myeloproliferative neoplasms (MPN positive for both JAK2 V617 and exon 12 mutations. The association between the JAK2 haplotypeGGCC_46/1 and mutations in other genes, such as thrombopoietin receptor (MPL and calreticulin (CALR, or the association with triple negative MPN, is still controversial. This review provides an overview of the frequency and the role of the JAK2 haplotypeGGCC_46/1 in the pathogenesis of different myeloid neoplasms and describes the hypothetical mechanisms at the basis of the association with JAK2 gene mutations. Moreover, possible clinical implications are discussed, as different papers reported contrasting data about the correlation between the JAK2 haplotypeGGCC_46/1 and blood cell count, survival, or disease progression.

JAK2, MPL, and CALR mutations in Chinese Han patients with essential thrombocythemia.

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Wang, Jing; Zhang, Biao; Chen, Bing; Zhou, Rong-Fu; Zhang, Qi-Guo; Li, Juan; Yang, Yong-Gong; Zhou, Min; Shao, Xiao-Yan; Xu, Yong; Xu, Xi-Hui; Ouyang, Jian; Xu, Jingyan; Ye, Qing

2017-04-01

Mutations in Janus kinase 2 (JAK2), myeloproliferative leukemia (MPL), and CALR are highly relevant to Philadelphia chromosome (Ph)-negative myeloproliferative neoplasms. Assessing the prevalence of molecular mutations in Chinese Han patients with essential thrombocythemia (ET), and correlating their mutational profile with disease characteristics/phenotype. Of the 110 subjects studied, 62 carried the JAK2 V617F mutation, 21 had CALR mutations, one carried an MPL (W515) mutation, and 28 had non-mutated JAK2, CALR, and MPL (so-called triple-negative ET). Mutations in JAK2 exon 12 were not detected in any patient. Two ET patients had both CALR and JAK2 V617F mutations. Comparing the hematological parameters of the patients with JAK2 mutations with those of the patients with CALR mutations showed that the ET patients with CALR mutations were younger (p = 0.045) and had higher platelet counts (p = 0.043). Genotyping for CALR could be a useful diagnostic tool for JAK2/MPL-negative ET, since the data suggest that CALR is much more prevalent than MPL, therefore testing for CALR should be considered in patients who are JAK2 negative as its frequency is almost 20 times that of MPL mutation.

Distinct patterns of DNA damage response and apoptosis correlate with Jak/Stat and PI3kinase response profiles in human acute myelogenous leukemia.

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David B Rosen

Full Text Available BACKGROUND: Single cell network profiling (SCNP utilizing flow cytometry measures alterations in intracellular signaling responses. Here SCNP was used to characterize Acute Myeloid Leukemia (AML disease subtypes based on survival, DNA damage response and apoptosis pathways. METHODOLOGY AND PRINCIPAL FINDINGS: Thirty four diagnostic non-M3 AML samples from patients with known clinical outcome were treated with a panel of myeloid growth factors and cytokines, as well as with apoptosis-inducing agents. Analysis of induced Jak/Stat and PI3K pathway responses in blasts from individual patient samples identified subgroups with distinct signaling profiles that were not seen in the absence of a modulator. In vitro exposure of patient samples to etoposide, a DNA damaging agent, revealed three distinct "DNA damage response (DDR/apoptosis" profiles: 1 AML blasts with a defective DDR and failure to undergo apoptosis; 2 AML blasts with proficient DDR and failure to undergo apoptosis; 3 AML blasts with proficiency in both DDR and apoptosis pathways. Notably, AML samples from clinical responders fell within the "DDR/apoptosis" proficient profile and, as well, had low PI3K and Jak/Stat signaling responses. In contrast, samples from clinical non responders had variable signaling profiles often with in vitro apoptotic failure and elevated PI3K pathway activity. Individual patient samples often harbored multiple, distinct, leukemia-associated cell populations identifiable by their surface marker expression, functional performance of signaling pathway in the face of cytokine or growth factor stimulation, as well as their response to apoptosis-inducing agents. CONCLUSIONS AND SIGNIFICANCE: Characterizing and tracking changes in intracellular pathway profiles in cell subpopulations both at baseline and under therapeutic pressure will likely have important clinical applications, potentially informing the selection of beneficial targeted agents, used either alone or in

Response of the JAK-STAT signaling pathway to oxygen deprivation in the red eared slider turtle, Trachemys scripta elegans.

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Bansal, Saumya; Biggar, Kyle K; Krivoruchko, Anastasia; Storey, Kenneth B

2016-11-15

The red-eared slider turtle, Trachemys scripta elegans, is a model organism commonly used to study the environmental stress of anoxia. It exhibits multiple biochemical adaptations to ensure its survival during the winter months where quantities of oxygen are largely depleted. We proposed that JAK-STAT signaling would display stress responsive regulation to mediate the survival of the red-eared slider turtle, Trachemys scripta elegans, during anoxic stress. Importantly, the JAK-STAT signaling pathway is involved in transmitting extracellular signals to the nucleus resulting in the expression of select genes that aid cell survival and growth. Immunoblotting was used to compare the relative phosphorylation levels of JAK proteins, STAT proteins, and two of its inhibitors, SOCS and PIAS, in response to anoxia. A clear activation of the JAK-STAT pathway was observed in the liver tissue while no significant changes were found in the skeletal muscle. To further support our findings we also found an increase in mRNA transcripts of downstream targets of STATs, namely bcl-xL and bcl-2, using PCR analysis in the liver tissues. These findings suggest an important role for the JAK-STAT pathway in exhibiting natural anoxia tolerance by the red-eared slider turtle. Copyright © 2016 Elsevier B.V. All rights reserved.

The association of JAK2V617F mutation and leukocytosis with thrombotic events in essential thrombocythemia.

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Hsiao, Hui-Hua; Yang, Ming-Yu; Liu, Yi-Chang; Lee, Ching-Ping; Yang, Wen-Chi; Liu, Ta-Chih; Chang, Chao-Sung; Lin, Sheng-Fung

2007-11-01

The Janus kinase 2 mutation, JAK2 (V617F), and megakaryocytic mutations, MPL (W515L/K), have been identified and correlated with a subtype of essential thrombocythemia (ET) patients. We investigated the frequency of mutations in ET patients and analyzed the relationship with their clinical features. Fifty-three ET patients were enrolled in the study. The amplification refractory mutation system was applied for the mutation survey of the JAK2V617F, while the polymerase chain reaction with sequencing was used for the mutation survey of MPLW515L/K. Thirty-five (66%) patients harboring the JAK2 (V617F) mutation, including 3 homozygous and 32 heterozygous changes, but no MPLW515L/K mutation, were found. During follow-up, 17 (32.1%) patients suffered from documented thrombotic events, with 15 having JAK2V617F mutations. Statistical analysis showed that patients with the JAK2 mutation had significantly higher leukocytes, hemoglobin level, and thrombotic event (p = 0.043, p = 0.001, and p = 0.029, respectively). Thrombotic events were also significantly correlated with leukocytosis and older age. The JAK2V617F mutation was noted in a certain population of ET patients and correlated with leukocytosis, high hemoglobin level, and thrombosis. Therefore, detection of the JAK2V617F mutation can affect not only the diagnosis, but also the management of ET patients.

JAK2 V617F-dependent upregulation of PU.1 expression in the peripheral blood of myeloproliferative neoplasm patients.

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Tamotsu Irino

Full Text Available Myeloproliferative neoplasms (MPN are multiple disease entities characterized by clonal expansion of one or more of the myeloid lineages (i.e. granulocytic, erythroid, megakaryocytic and mast cell. JAK2 mutations, such as the common V617F substitution and the less common exon 12 mutations, are frequently detected in such tumor cells and have been incorporated into the diagnostic criteria published by the World Health Organization since 2008. However, the mechanism by which these mutations contribute to MPN development is poorly understood. We examined gene expression profiles of MPN patients focusing on genes in the JAK-STAT signaling pathway using low-density real-time PCR arrays. We identified the following 2 upregulated genes in MPN patients: a known target of the JAK-STAT axis, SOCS3, and a potentially novel target, SPI1, encoding PU.1. Induction of PU.1 expression by JAK2 V617F in JAK2-wildtype K562 cells and its downregulation by JAK2 siRNA transfection in JAK2 V617F-positive HEL cells supported this possibility. We also found that the ABL1 kinase inhibitor imatinib was very effective in suppressing PU.1 expression in BCR-ABL1-positive K562 cells but not in HEL cells. This suggests that PU.1 expression is regulated by both JAK2 and ABL1. The contribution of the two kinases in driving PU.1 expression was dominant for JAK2 and ABL1 in HEL and K562 cells, respectively. Therefore, PU.1 may be a common transcription factor upregulated in MPN. PU.1 is a transcription factor required for myeloid differentiation and is implicated in erythroid leukemia. Therefore, expression of PU.1 downstream of activated JAK2 may explain why JAK2 mutations are frequently observed in MPN patients.

Whole-exome sequencing identifies novel MPL and JAK2 mutations in triple-negative myeloproliferative neoplasms.

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Milosevic Feenstra, Jelena D; Nivarthi, Harini; Gisslinger, Heinz; Leroy, Emilie; Rumi, Elisa; Chachoua, Ilyas; Bagienski, Klaudia; Kubesova, Blanka; Pietra, Daniela; Gisslinger, Bettina; Milanesi, Chiara; Jäger, Roland; Chen, Doris; Berg, Tiina; Schalling, Martin; Schuster, Michael; Bock, Christoph; Constantinescu, Stefan N; Cazzola, Mario; Kralovics, Robert

2016-01-21

Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are chronic diseases characterized by clonal hematopoiesis and hyperproliferation of terminally differentiated myeloid cells. The disease is driven by somatic mutations in exon 9 of CALR or exon 10 of MPL or JAK2-V617F in >90% of the cases, whereas the remaining cases are termed "triple negative." We aimed to identify the disease-causing mutations in the triple-negative cases of ET and PMF by applying whole-exome sequencing (WES) on paired tumor and control samples from 8 patients. We found evidence of clonal hematopoiesis in 5 of 8 studied cases based on clonality analysis and presence of somatic genetic aberrations. WES identified somatic mutations in 3 of 8 cases. We did not detect any novel recurrent somatic mutations. In 3 patients with clonal hematopoiesis analyzed by WES, we identified a somatic MPL-S204P, a germline MPL-V285E mutation, and a germline JAK2-G571S variant. We performed Sanger sequencing of the entire coding region of MPL in 62, and of JAK2 in 49 additional triple-negative cases of ET or PMF. New somatic (T119I, S204F, E230G, Y591D) and 1 germline (R321W) MPL mutation were detected. All of the identified MPL mutations were gain-of-function when analyzed in functional assays. JAK2 variants were identified in 5 of 57 triple-negative cases analyzed by WES and Sanger sequencing combined. We could demonstrate that JAK2-V625F and JAK2-F556V are gain-of-function mutations. Our results suggest that triple-negative cases of ET and PMF do not represent a homogenous disease entity. Cases with polyclonal hematopoiesis might represent hereditary disorders. © 2016 by The American Society of Hematology.

Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2

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Nangalia, J.; Massie, C.E.; Baxter, E.J.; Nice, F.L.; Gundem, G.; Wedge, D.C.; Avezov, E.; Li, J.; Kollmann, K.; Kent, D.G.; Aziz, A.; Godfrey, A.L.; Hinton, J.; Martincorena, I.; Loo, P. Van; Jones, A.V.; Guglielmelli, P.; Tarpey, P.; Harding, H.P.; Fitzpatrick, J.D.; Goudie, C.T.; Ortmann, C.A.; Loughran, S.J.; Raine, K.; Jones, D.R.; Butler, A.P.; Teague, J.W.; O'Meara, S.; McLaren, S.; Bianchi, M.; Silber, Y.; Dimitropoulou, D.; Bloxham, D.; Mudie, L.; Maddison, M.; Robinson, B.; Keohane, C.; Maclean, C.; Hill, K.; Orchard, K.; Tauro, S.; Du, M.Q.; Greaves, M.; Bowen, D.; Huntly, B.J.; Harrison, C.N.; Cross, N.C.; Ron, D.; Vannucchi, A.M.; Papaemmanuil, E.; Campbell, P.J.; Green, A.R.

2013-01-01

BACKGROUND: Somatic mutations in the Janus kinase 2 gene (JAK2) occur in many myeloproliferative neoplasms, but the molecular pathogenesis of myeloproliferative neoplasms with nonmutated JAK2 is obscure, and the diagnosis of these neoplasms remains a challenge. METHODS: We performed exome sequencing

Prognostic significance of ASXL1, JAK2V617F mutations and JAK2V617F allele burden in Philadelphia-negative myeloproliferative neoplasms

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Yonal-Hindilerden I

2015-06-01

Full Text Available Ipek Yonal-Hindilerden, Aynur Daglar-Aday, Basak Akadam-Teker, Ceylan Yilmaz, Meliha Nalcaci, Akif Selim Yavuz, Deniz SarginDivision of Hematology, Department of Internal Medicine, Istanbul Medical Faculty, Istanbul University, Fatih-Istanbul, Turkey Background: Despite insights into the genetic basis of Philadelphia-negative myeloproliferative neoplasms (Ph-negative MPNs, a significant proportion of essential thrombocythemia (ET and primary myelofibrosis (PMF patients present with no known MPN disease alleles. There were no previous studies investigating the impact of ASXL1 mutations in Ph-negative MPNs in Turkey. In the current study, we investigated the prognostic significance of ASXL1 mutations in Turkish MPN patients. We also aimed to determine the prognostic significance of JAK2V617F allele burden and the relationship of JAK2V617F mutation with ASXL1 mutations in Ph-negative MPNs. Methods: About 184 patients from a single center diagnosed with Ph-negative MPNs were screened for ASXL1, JAK2V617F mutations, and JAK2V617F allele burden: 107 ET and 77 PMF. Results: A total of 29 ASXL1 mutations were detected in 24.7% of PMF and 8.4% of ET patients. ASXL1-mutated ET patients showed a trend toward an increase in the incidence of cerebrovascular events and higher total leukocyte counts. ASXL1-mutation in PMF was associated with older age and a higher prevalence of bleeding complications. In univariate analysis, overall survival (OS was significantly reduced in ASXL1-mutated PMF patients. In multivariate analysis, Dynamic International Prognostic Scoring System-plus high-risk category and ASXL1 mutation status were independently associated with shorter survival in PMF. In PMF, mutational status and allele burden of JAK2V617F showed no difference in terms of OS and leukemia-free survival. Conclusion: We conclude that ASXL1 mutations are molecular predictors of short OS in PMF. Keywords: Philadelphia-negative myeloproliferative neoplasms (Ph

Clinical features of Japanese polycythemia vera and essential thrombocythemia patients harboring CALR, JAK2V617F, JAK2Ex12del, and MPLW515L/K mutations.

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Okabe, Masahiro; Yamaguchi, Hiroki; Usuki, Kensuke; Kobayashi, Yutaka; Kawata, Eri; Kuroda, Junya; Kimura, Shinya; Tajika, Kenji; Gomi, Seiji; Arima, Nobuyoshi; Mori, Sinichiro; Ito, Shigeki; Koizumi, Masayuki; Ito, Yoshikazu; Wakita, Satoshi; Arai, Kunihito; Kitano, Tomoaki; Kosaka, Fumiko; Dan, Kazuo; Inokuchi, Koiti

2016-01-01

The risk of complication of polycythemia vera (PV) and essential thrombocythemia (ET) by thrombosis in Japanese patients is clearly lower than in western populations, suggesting that genetic background such as race may influence the clinical features. This study aimed to clarify the relationship between genetic mutations and haplotypes and clinical features in Japanese patients with PV and ET. Clinical features were assessed prospectively among 74 PV and 303 ET patients. There were no clinical differences, including JAK2V617F allele burden, between PV patients harboring the various genetic mutations. However, CALR mutation-positive ET patients had a significantly lower WBC count, Hb value, Ht value, and neutrophil alkaline phosphatase score (NAP), and significantly more platelets, relative to JAK2V617F-positive ET patients and ET patients with no mutations. Compared to normal controls, the frequency of the JAK246/1 haplotype was significantly higher among patients with JAK2V617F, JAK2Ex12del, or MPL mutations, whereas no significant difference was found among CALR mutation-positive patients. CALR mutation-positive patients had a lower incidence of thrombosis relative to JAK2V617F-positive patients. Our findings suggest that JAK2V617F-positive ET patients and CALR mutation-positive patients have different mechanisms of occurrence and clinical features of ET, suggesting the potential need for therapy stratification in the future. Copyright © 2015 Elsevier Ltd. All rights reserved.

Therapeutic activity of multiple common γ-chain cytokine inhibition in acute and chronic GVHD.

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Hechinger, Anne-Kathrin; Smith, Benjamin A H; Flynn, Ryan; Hanke, Kathrin; McDonald-Hyman, Cameron; Taylor, Patricia A; Pfeifer, Dietmar; Hackanson, Björn; Leonhardt, Franziska; Prinz, Gabriele; Dierbach, Heide; Schmitt-Graeff, Annette; Kovarik, Jiri; Blazar, Bruce R; Zeiser, Robert

2015-01-15

The common γ chain (CD132) is a subunit of the interleukin (IL) receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Because levels of several of these cytokines were shown to be increased in the serum of patients developing acute and chronic graft-versus-host disease (GVHD), we reasoned that inhibition of CD132 could have a profound effect on GVHD. We observed that anti-CD132 monoclonal antibody (mAb) reduced acute GVHD potently with respect to survival, production of tumor necrosis factor, interferon-γ, and IL-6, and GVHD histopathology. Anti-CD132 mAb afforded protection from GVHD partly via inhibition of granzyme B production in CD8 T cells, whereas exposure of CD8 T cells to IL-2, IL-7, IL-15, and IL-21 increased granzyme B production. Also, T cells exposed to anti-CD132 mAb displayed a more naive phenotype in microarray-based analyses and showed reduced Janus kinase 3 (JAK3) phosphorylation upon activation. Consistent with a role of JAK3 in GVHD, Jak3(-/-) T cells caused less severe GVHD. Additionally, anti-CD132 mAb treatment of established chronic GVHD reversed liver and lung fibrosis, and pulmonary dysfunction characteristic of bronchiolitis obliterans. We conclude that acute GVHD and chronic GVHD, caused by T cells activated by common γ-chain cytokines, each represent therapeutic targets for anti-CD132 mAb immunomodulation. © 2015 by The American Society of Hematology.

Efficacy of ALK5 inhibition in myelofibrosis

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Zhao, Wanke; Ho, Wanting Tina; Han, Ying; Murdun, Cem; Mailloux, Adam W.; Zhang, Ling; Wang, Xuefeng; Budhathoki, Anjali; Pradhan, Kith; Rapaport, Franck; Wang, Huaquan; Shao, Zonghong; Ren, Xiubao; Steidl, Ulrich; Levine, Ross L.; Zhao, Zhizhuang Joe; Verma, Amit; Epling-Burnette, Pearlie K.

2017-01-01

Myelofibrosis (MF) is a bone marrow disorder characterized by clonal myeloproliferation, aberrant cytokine production, extramedullary hematopoiesis, and bone marrow fibrosis. Although somatic mutations in JAK2, MPL, and CALR have been identified in the pathogenesis of these diseases, inhibitors of the Jak2 pathway have not demonstrated efficacy in ameliorating MF in patients. TGF-β family members are profibrotic cytokines and we observed significant TGF-β1 isoform overexpression in a large cohort of primary MF patient samples. Significant overexpression of TGF-β1 was also observed in murine clonal MPLW515L megakaryocytic cells. TGF-β1 stimulated the deposition of excessive collagen by mesenchymal stromal cells (MSCs) by activating the TGF-β receptor I kinase (ALK5)/Smad3 pathway. MSCs derived from MPLW515L mice demonstrated sustained overproduction of both collagen I and collagen III, effects that were abrogated by ALK5 inhibition in vitro and in vivo. Importantly, use of galunisertib, a clinically active ALK5 inhibitor, significantly improved MF in both MPLW515L and JAK2V617F mouse models. These data demonstrate the role of malignant hematopoietic stem cell (HSC)/TGF-β/MSC axis in the pathogenesis of MF, and provide a preclinical rationale for ALK5 blockade as a therapeutic strategy in MF. PMID:28405618

The Prevalence of JAK2, MPL, and CALR Mutations in Chinese Patients With BCR-ABL1-Negative Myeloproliferative Neoplasms.

Science.gov (United States)

Lin, Yani; Liu, Enbin; Sun, Qi; Ma, Jiao; Li, QingHua; Cao, Zeng; Wang, Jun; Jia, Yujiao; Zhang, Hongju; Song, Zhen; Ai, Xiaofei; Shi, Lihui; Feng, Xiaofang; Li, Chenwei; Wang, Jianxiang; Ru, Kun

2015-07-01

To evaluate the mutation frequency of JAK2 V617F, JAK2 exon 12, MPL exon 10, and CALR exon 9 and the value of the combined tests in the diagnosis of BCR-ABL1-negative myeloproliferative neoplasms (MPNs). In the current study, mutations of JAK2 V617F, JAK2 exon 12, MPL exon 10, and CALR exon 9 were analyzed in 929 Chinese patients with BCR-ABL1-negative MPN, including 234 cases of polycythemia vera (PV), 428 ETs, 187 PMFs, and 80 unclassifiable MPNs (MPN-Us). Our result showed that the positive rate of any of four mutations in patients with PV, ET, PMF, and MPN-U was 89.3%, 83.4%, 87.2%, and 77.5%, respectively, which significantly improved the diagnostic rate, especially in ET and PMF. Meanwhile, we also found that the patients without any of four mutations were younger than those with one or more mutations. Unexpectedly, the coexistence of JAK2 V617F and CALR exon 9 was identified in six (0.6%) patients, and JAK2 V617F and MPL exon 10 were present simultaneously in two (0.2%) patients. In addition, we also identified several novel mutation types in CALR exon 9. The combined genetic tests of JAK2 V617F, JAK2 exon 12, MPL exon 10, and CALR exon 9 help improve the diagnostic rate for BCR-ABL1-negative MPN. Copyright© by the American Society for Clinical Pathology.

Fluoxetine-induced inhibition of synaptosomal [3H]5-HT release: Possible Ca2+-channel inhibition

International Nuclear Information System (INIS)

Stauderman, K.A.; Gandhi, V.C.; Jones, D.J.

1992-01-01

Fluoxetine, a selective 5-Ht uptake inhibitor, inhibited 15 mM K + -induced [ 3 H]5-HT release from rat spinal cord and cortical synaptosomes at concentrations > 0.5 uM. This effect reflected a property shared by another selective 5-HT uptake inhibitor paroxetine but not by less selective uptake inhibitors such as amitriptyline, desipramine, imipramine or nortriptyline. Inhibition of release by fluoxetine was inversely related to both the concentration of K + used to depolarize the synaptosomes and the concentration of external Ca 2+ . Experiments aimed at determining a mechanism of action revealed that fluoxetine did not inhibit voltage-independent release of [ 3 H]5-HT release induced by the Ca 2+ -ionophore A 23187 or Ca 2+ -independent release induced by fenfluramine. Moreover the 5-HT autoreceptor antagonist methiothepin did not reverse the inhibitory actions of fluoxetine on K + -induced release. Further studies examined the effects of fluoxetine on voltage-dependent Ca 2+ channels and Ca 2+ entry

JAK2 and MPL gene mutations in V617F-negative myeloproliferative neoplasms.

NARCIS (Netherlands)

Siemiatkowska, A.M.; Bieniaszewska, M.; Hellmann, A.; Limon, J.

2010-01-01

We report three novel mutations in JAK2 exons 12, 19 and 25 in V617F-negative patients with polycythemia vera, essential thrombocythemia and idiopathic myelofibrosis. Scanning of JAK2 exons 12-25 and MPL exon 10 revealed the presence of JAK2 alterations in six and MPL W515L/K mutations in five of 34

Treatment and management of myelofibrosis in the era of JAK inhibitors

Directory of Open Access Journals (Sweden)

Keohane C

2013-08-01

Full Text Available Clodagh Keohane, Deepti H Radia, Claire N HarrisonDepartment of Haematology, Guy's and St Thomas' NHS Foundation Trust, London, UKAbstract: Myelofibrosis (MF can present as a primary disorder or evolve from polycythemia vera (PV or essential thrombocythemia (ET to post-PV MF or post-ET MF, respectively. MF is characterized by bone marrow fibrosis, splenomegaly, leukoerythroblastosis, extramedullary hematopoiesis, and a collection of debilitating symptoms. Until recently, the therapeutic options for patients with MF consisted of allogeneic hematopoietic stem cell transplant (alloHSCT, the use of cytoreductive agents (ie, hydroxyurea, splenectomy and splenic irradiation for treatment of splenomegaly, and management of anemia with transfusions, erythropoiesis-stimulating agents (ESAs, androgens, and immunomodulatory agents. However, with increased understanding of the pathogenesis of MF resulting from dysregulated Janus kinase (JAK signaling, new targeted JAK inhibitor therapies, such as ruxolitinib, are now available. The purpose of this article is to review the clinical features of MF, discuss the use and future of JAK inhibitors, reassess when and how to use conventional MF treatments in the context of JAK inhibitors, and provide a perspective on the future of MF treatment.Keywords: myelofibrosis, ruxolitinib, JAK inhibitor

Distractor Inhibition: Principles of Operation during Selective Attention

Science.gov (United States)

Wyatt, Natalie; Machado, Liana

2013-01-01

Research suggests that although target amplification acts as the main determinant of the efficacy of selective attention, distractor inhibition contributes under some circumstances. Here we aimed to gain insight into the operating principles that regulate the use of distractor inhibition during selective attention. The results suggest that, in…

Selective inhibition of Biotin Protein Ligase from Staphylococcus aureus*

Science.gov (United States)

Soares da Costa, Tatiana P.; Tieu, William; Yap, Min Y.; Pendini, Nicole R.; Polyak, Steven W.; Sejer Pedersen, Daniel; Morona, Renato; Turnidge, John D.; Wallace, John C.; Wilce, Matthew C. J.; Booker, Grant W.; Abell, Andrew D.

2012-01-01

There is a well documented need to replenish the antibiotic pipeline with new agents to combat the rise of drug resistant bacteria. One strategy to combat resistance is to discover new chemical classes immune to current resistance mechanisms that inhibit essential metabolic enzymes. Many of the obvious drug targets that have no homologous isozyme in the human host have now been investigated. Bacterial drug targets that have a closely related human homologue represent a new frontier in antibiotic discovery. However, to avoid potential toxicity to the host, these inhibitors must have very high selectivity for the bacterial enzyme over the human homolog. We have demonstrated that the essential enzyme biotin protein ligase (BPL) from the clinically important pathogen Staphylococcus aureus could be selectively inhibited. Linking biotin to adenosine via a 1,2,3 triazole yielded the first BPL inhibitor selective for S. aureus BPL over the human equivalent. The synthesis of new biotin 1,2,3-triazole analogues using click chemistry yielded our most potent structure (Ki 90 nm) with a >1100-fold selectivity for the S. aureus BPL over the human homologue. X-ray crystallography confirmed the mechanism of inhibitor binding. Importantly, the inhibitor showed cytotoxicity against S. aureus but not cultured mammalian cells. The biotin 1,2,3-triazole provides a novel pharmacophore for future medicinal chemistry programs to develop this new antibiotic class. PMID:22437830

Selective inhibition of biotin protein ligase from Staphylococcus aureus.

Science.gov (United States)

Soares da Costa, Tatiana P; Tieu, William; Yap, Min Y; Pendini, Nicole R; Polyak, Steven W; Sejer Pedersen, Daniel; Morona, Renato; Turnidge, John D; Wallace, John C; Wilce, Matthew C J; Booker, Grant W; Abell, Andrew D

2012-05-18

There is a well documented need to replenish the antibiotic pipeline with new agents to combat the rise of drug resistant bacteria. One strategy to combat resistance is to discover new chemical classes immune to current resistance mechanisms that inhibit essential metabolic enzymes. Many of the obvious drug targets that have no homologous isozyme in the human host have now been investigated. Bacterial drug targets that have a closely related human homologue represent a new frontier in antibiotic discovery. However, to avoid potential toxicity to the host, these inhibitors must have very high selectivity for the bacterial enzyme over the human homolog. We have demonstrated that the essential enzyme biotin protein ligase (BPL) from the clinically important pathogen Staphylococcus aureus could be selectively inhibited. Linking biotin to adenosine via a 1,2,3 triazole yielded the first BPL inhibitor selective for S. aureus BPL over the human equivalent. The synthesis of new biotin 1,2,3-triazole analogues using click chemistry yielded our most potent structure (K(i) 90 nM) with a >1100-fold selectivity for the S. aureus BPL over the human homologue. X-ray crystallography confirmed the mechanism of inhibitor binding. Importantly, the inhibitor showed cytotoxicity against S. aureus but not cultured mammalian cells. The biotin 1,2,3-triazole provides a novel pharmacophore for future medicinal chemistry programs to develop this new antibiotic class.

AKT is a therapeutic target in myeloproliferative neoplasms.

Science.gov (United States)

Khan, I; Huang, Z; Wen, Q; Stankiewicz, M J; Gilles, L; Goldenson, B; Schultz, R; Diebold, L; Gurbuxani, S; Finke, C M; Lasho, T L; Koppikar, P; Pardanani, A; Stein, B; Altman, J K; Levine, R L; Tefferi, A; Crispino, J D

2013-09-01

The majority of patients with BCR-ABL1-negative myeloproliferative neoplasms (MPN) harbor mutations in JAK2 or MPL, which lead to constitutive activation of the JAK/STAT, PI3K and ERK signaling pathways. JAK inhibitors by themselves are inadequate in producing selective clonal suppression in MPN and are associated with hematopoietic toxicities. MK-2206 is a potent allosteric AKT inhibitor that was well tolerated, including no evidence of myelosuppression, in a phase I study of solid tumors. Herein, we show that inhibition of PI3K/AKT signaling by MK-2206 affected the growth of both JAK2V617F- or MPLW515L-expressing cells via reduced phosphorylation of AKT and inhibition of its downstream signaling molecules. Moreover, we demonstrate that MK-2206 synergizes with ruxolitinib in suppressing the growth of JAK2V617F-mutant SET2 cells. Importantly, MK-2206 suppressed colony formation from hematopoietic progenitor cells in patients with primary myelofibrosis and alleviated hepatosplenomegaly and reduced megakaryocyte burden in the bone marrows, livers and spleens of mice with MPLW515L-induced MPN. Together, these findings establish AKT as a rational therapeutic target in the MPNs.

How Do Parameters of Motor Response Influence Selective Inhibition? Evidence from the Stop-Signal Paradigm

Directory of Open Access Journals (Sweden)

Chien Hui Tang

2011-05-01

Full Text Available The ability to selectively inhibit the execution of an action while performing other ones is crucial in humans' multitasking daily life. The current study aims to compare selective inhibition for choice reaction involving two effectors or response directions. We adopted a variation of the stop-signal paradigm to examine how selective inhibition is modulated by the way potential motor responses are combined and inhibited. Experiment 1 investigated selective inhibition under different combinations of effectors, namely “index and middle fingers” versus “hand and foot”. The results showed SSRT of the index finger was longer when the other response option was the foot than the middle finger. Experiment 2 examined how selective inhibition differs between selective stopping of effectors and movement directions, and that for most of the situations SSRT is longer for stopping a response based on its direction than effector. After equating complexity of response mapping between direction and effector conditions in Experiment 2, Experiment 3 still showed that SSRT differs between selecting direction or effectors. To summarize, SSRT varies depending on the way response effectors are paired and selectively stopped. Selective inhibition is thus likely not amodal and may involve different inhibitory mechanisms depending on parameters specifying the motor response.

Presence of calreticulin mutations in JAK2-negative polycythemia vera.

Science.gov (United States)

Broséus, Julien; Park, Ji-Hye; Carillo, Serge; Hermouet, Sylvie; Girodon, François

2014-12-18

Calreticulin (CALR) mutations have been reported in Janus kinase 2 (JAK2)- and myeloproliferative leukemia (MPL)-negative essential thrombocythemia and primary myelofibrosis. In contrast, no CALR mutations have ever been reported in the context of polycythemia vera (PV). Here, we describe 2 JAK2(V617F)-JAK2(exon12)-negative PV patients who presented with a CALR mutation in peripheral granulocytes at the time of diagnosis. In both cases, the CALR mutation was a 52-bp deletion. Single burst-forming units-erythroid (BFU-E) from 1 patient were grown in vitro and genotyped: the same CALR del 52-bp mutation was noted in 31 of the 37 colonies examined; 30 of 31 BFU-E were heterozygous for CALR del 52 bp, and 1 of 31 BFU-E was homozygous for CALR del 52 bp. In summary, although unknown mutations leading to PV cannot be ruled out, our results suggest that CALR mutations can be associated with JAK2-negative PV. © 2014 by The American Society of Hematology.

Functions of the Drosophila JAK-STAT pathway

Science.gov (United States)

Amoyel, Marc; Bach, Erika A.

2012-01-01

JAK-STAT signaling has been proposed to act in numerous stem cells in a variety of organisms. Here we provide an overview of its roles in three well characterized stem cell populations in Drosophila, in the intestine, lymph gland and testis. In flies, there is a single JAK and a single STAT, which has made the genetic dissection of pathway function considerably easier and facilitated the analysis of communication between stem cells, their niches and offspring. Studies in flies have revealed roles for this pathway as diverse as regulating bona fide intrinsic self-renewal, integrating response to environmental cues that control quiescence and promoting mitogenic responses to stress. PMID:24058767

Treatment and management of myelofibrosis in the era of JAK inhibitors.

Science.gov (United States)

Keohane, Clodagh; Radia, Deepti H; Harrison, Claire N

2013-01-01

Myelofibrosis (MF) can present as a primary disorder or evolve from polycythemia vera (PV) or essential thrombocythemia (ET) to post-PV MF or post-ET MF, respectively. MF is characterized by bone marrow fibrosis, splenomegaly, leukoerythroblastosis, extramedullary hematopoiesis, and a collection of debilitating symptoms. Until recently, the therapeutic options for patients with MF consisted of allogeneic hematopoietic stem cell transplant (alloHSCT), the use of cytoreductive agents (ie, hydroxyurea), splenectomy and splenic irradiation for treatment of splenomegaly, and management of anemia with transfusions, erythropoiesis-stimulating agents (ESAs), androgens, and immunomodulatory agents. However, with increased understanding of the pathogenesis of MF resulting from dysregulated Janus kinase (JAK) signaling, new targeted JAK inhibitor therapies, such as ruxolitinib, are now available. The purpose of this article is to review the clinical features of MF, discuss the use and future of JAK inhibitors, reassess when and how to use conventional MF treatments in the context of JAK inhibitors, and provide a perspective on the future of MF treatment.

Domains of the growth hormone receptor required for association and activation of JAK2 tyrosine kinase

DEFF Research Database (Denmark)

VanderKuur, J A; Wang, X; Zhang, L

1994-01-01

Growth hormone (GH) has recently been shown to activate the GH receptor (GHR)-associated tyrosine kinase JAK2. In the present study, regions of the GHR required for JAK2 association with GHR were identified. GH-dependent JAK2 association with GHR was detected in Chinese hamster ovary (CHO) cells...... and RIN-5AH cells, the ability of JAK2 to associate with the mutated GHR was found to correlate with GH-dependent activation of JAK2, tyrosyl phosphorylation of GHR (in the case of GHR1-638 and GHR1-454), and the ability of the GHR to copurify with tyrosine kinase activity. In CHO cells expressing mutated......, and that tyrosines in the N-terminal half of the cytoplasmic domain of the GHR are phosphorylated by JAK2. The finding that a specific interaction with the C-terminal half of GHR appears to be necessary for p97 phosphorylation indicates that while JAK2 activation may be necessary for a full biological response to GH...

The PLA2R1-JAK2 pathway upregulates ERRα and its mitochondrial program to exert tumor-suppressive action.

Science.gov (United States)

Griveau, A; Devailly, G; Eberst, L; Navaratnam, N; Le Calvé, B; Ferrand, M; Faull, P; Augert, A; Dante, R; Vanacker, J M; Vindrieux, D; Bernard, D

2016-09-22

Little is known about the biological role of the phospholipase A2 receptor (PLA2R1) transmembrane protein. In recent years, PLA2R1 has been shown to have an important role in regulating tumor-suppressive responses via JAK2 activation, but the underlying mechanisms are largely undeciphered. In this study, we observed that PLA2R1 increases the mitochondrial content, judged by increased levels of numerous mitochondrial proteins, of the mitochondrial structural component cardiolipin, of the mitochondrial DNA content, and of the mitochondrial DNA replication and transcription factor TFAM. This effect of PLA2R1 relies on a transcriptional program controlled by the estrogen-related receptor alpha1 (ERRα) mitochondrial master regulator. Expression of ERRα and of its nucleus-encoded mitochondrial targets is upregulated upon PLA2R1 ectopic expression, and this effect is mediated by JAK2. Conversely, downregulation of PLA2R1 decreases the level of ERRα and of its nucleus-encoded mitochondrial targets. Finally, blocking the ERRα-controlled mitochondrial program largely inhibits the PLA2R1-induced tumor-suppressive response. Together, our data document ERRα and its mitochondrial program as downstream effectors of the PLA2R1-JAK2 pathway leading to oncosuppression.

Stat5 Exerts Distinct, Vital Functions in the Cytoplasm and Nucleus of Bcr-Abl+ K562 and Jak2(V617F)+ HEL Leukemia Cells

International Nuclear Information System (INIS)

Weber, Axel; Borghouts, Corina; Brendel, Christian; Moriggl, Richard; Delis, Natalia; Brill, Boris; Vafaizadeh, Vida; Groner, Bernd

2015-01-01

Signal transducers and activators of transcription (Stats) play central roles in the conversion of extracellular signals, e.g., cytokines, hormones and growth factors, into tissue and cell type specific gene expression patterns. In normal cells, their signaling potential is strictly limited in extent and duration. The persistent activation of Stat3 or Stat5 is found in many human tumor cells and contributes to their growth and survival. Stat5 activation plays a pivotal role in nearly all hematological malignancies and occurs downstream of oncogenic kinases, e.g., Bcr-Abl in chronic myeloid leukemias (CML) and Jak2(V617F) in other myeloproliferative diseases (MPD). We defined the mechanisms through which Stat5 affects growth and survival of K562 cells, representative of Bcr-Abl positive CML, and HEL cells, representative for Jak2(V617F) positive acute erythroid leukemia. In our experiments we suppressed the protein expression levels of Stat5a and Stat5b through shRNA mediated downregulation and demonstrated the dependence of cell survival on the presence of Stat5. Alternatively, we interfered with the functional capacities of the Stat5 protein through the interaction with a Stat5 specific peptide ligand. This ligand is a Stat5 specific peptide aptamer construct which comprises a 12mer peptide integrated into a modified thioredoxin scaffold, S5-DBD-PA. The peptide sequence specifically recognizes the DNA binding domain (DBD) of Stat5. Complex formation of S5-DBD-PA with Stat5 causes a strong reduction of P-Stat5 in the nuclear fraction of Bcr-Abl-transformed K562 cells and a suppression of Stat5 target genes. Distinct Stat5 mediated survival mechanisms were detected in K562 and Jak2(V617F)-transformed HEL cells. Stat5 is activated in the nuclear and cytosolic compartments of K562 cells and the S5-DBD-PA inhibitor most likely affects the viability of Bcr-Abl + K562 cells through the inhibition of canonical Stat5 induced target gene transcription. In HEL cells, Stat5

Targeted sequencing identifies associations between IL7R-JAK mutations and epigenetic modulators in T-cell acute lymphoblastic leukemia

Science.gov (United States)

Vicente, Carmen; Schwab, Claire; Broux, Michaël; Geerdens, Ellen; Degryse, Sandrine; Demeyer, Sofie; Lahortiga, Idoya; Elliott, Alannah; Chilton, Lucy; La Starza, Roberta; Mecucci, Cristina; Vandenberghe, Peter; Goulden, Nicholas; Vora, Ajay; Moorman, Anthony V.; Soulier, Jean; Harrison, Christine J.; Clappier, Emmanuelle; Cools, Jan

2015-01-01

T-cell acute lymphoblastic leukemia is caused by the accumulation of multiple oncogenic lesions, including chromosomal rearrangements and mutations. To determine the frequency and co-occurrence of mutations in T-cell acute lymphoblastic leukemia, we performed targeted re-sequencing of 115 genes across 155 diagnostic samples (44 adult and 111 childhood cases). NOTCH1 and CDKN2A/B were mutated/deleted in more than half of the cases, while an additional 37 genes were mutated/deleted in 4% to 20% of cases. We found that IL7R-JAK pathway genes were mutated in 27.7% of cases, with JAK3 mutations being the most frequent event in this group. Copy number variations were also detected, including deletions of CREBBP or CTCF and duplication of MYB. FLT3 mutations were rare, but a novel extracellular mutation in FLT3 was detected and confirmed to be transforming. Furthermore, we identified complex patterns of pairwise associations, including a significant association between mutations in IL7R-JAK genes and epigenetic regulators (WT1, PRC2, PHF6). Our analyses showed that IL7R-JAK genetic lesions did not confer adverse prognosis in T-cell acute lymphoblastic leukemia cases enrolled in the UK ALL2003 trial. Overall, these results identify interconnections between the T-cell acute lymphoblastic leukemia genome and disease biology, and suggest a potential clinical application for JAK inhibitors in a significant proportion of patients with T-cell acute lymphoblastic leukemia. PMID:26206799

Activation of Stat-3 is involved in the induction of apoptosis after ligation of major histocompatibility complex class I molecules on human Jurkat T cells

DEFF Research Database (Denmark)

Skov, S; Nielsen, M; Bregenholt, S

1998-01-01

Activation of Janus tyrosine kinases (Jak) and Signal transducers and activators of transcription (Stat) after ligation of major histocompatibility complex class I (MHC-I) was explored in Jurkat T cells. Cross-linking of MHC-I mediated tyrosine phosphorylation of Tyk2, but not Jak1, Jak2, and Jak3......-probe derived from the interferon-gamma activated site (GAS) in the c-fos promoter, a common DNA sequence for Stat protein binding. An association between hSIE and Stat-3 after MHC-I ligation was directly demonstrated by precipitating Stat-3 from nuclear extracts with biotinylated hSIE probe and avidin......-coupled agarose. To investigate the function of the activated Stat-3, Jurkat T cells were transiently transfected with a Stat-3 isoform lacking the transactivating domain. This dominant-negative acting Stat-3 isoform significantly inhibited apoptosis induced by ligation of MHC-I. In conclusion, our data suggest...

Cucurbitacin E reduces obesity and related metabolic dysfunction in mice by targeting JAK-STAT5 signaling pathway.

Science.gov (United States)

Murtaza, Munazza; Khan, Gulnaz; Aftab, Meha Fatima; Afridi, Shabbir Khan; Ghaffar, Safina; Ahmed, Ayaz; Hafizur, Rahman M; Waraich, Rizwana Sanaullah

2017-01-01

Several members of cucurbitaceae family have been reported to regulate growth of cancer by interfering with STAT3 signaling. In the present study, we investigated the unique role and molecular mechanism of cucurbitacins (Cucs) in reducing symptoms of metabolic syndrome in mice. Cucurbitacin E (CuE) was found to reduce adipogenesis in murine adipocytes. CuE treatment diminished hypertrophy of adipocytes, visceral obesity and lipogenesis gene expression in diet induced mice model of metabolic syndrome (MetS). CuE also ameliorated adipose tissue dysfunction by reducing hyperleptinemia and TNF-alpha levels and enhancing hypoadiponectinemia. Results show that CuE mediated these effects by attenuating Jenus kinase- Signal transducer and activator of transcription 5 (JAK- STAT5) signaling in visceral fat tissue. As a result, CuE treatment also reduced PPAR gamma expression. Glucose uptake enhanced in adipocytes after stimulation with CuE and insulin resistance diminished in mice treated with CuE, as reflected by reduced glucose intolerance and glucose stimulated insulin secretion. CuE restored insulin sensitivity indirectly by inhibiting JAK phosphorylation and improving AMPK activity. Consequently, insulin signaling was up-regulated in mice muscle. As CuE positively regulated adipose tissue function and suppressed visceral obesity, dyslipedemia, hyperglycemia and insulin resistance in mice model of MetS, we suggest that CuE can be used as novel approach to treat metabolic diseases.

Significance of combined detection of JAK2V617F, MPL and CALR gene mutations in patients with essential thrombocythemia.

Science.gov (United States)

Ji, Liying; Qian, Mengyao; Wu, Nana; Wu, Jianmin

2017-03-01

The aim of this study was to analyze the mutation rate of JAK2V617F, MPLW515L/K and CALR genes in adult patients with essential thrombocythemia (ET) and the accuracy of the combined detection by the receiver operating curve. Three hundred and forty-two cases with high-platelets (≥300×10 9 /l) were consecutively selected. The patients were analyzed for routine blood examination, bone marrow biopsy and genetic testing. One hundred and fifty-four cases (45.03%) were diagnosed with ET and 188 cases of secondary thrombocythemia according to the hematopoietic and lymphoid tissue tumor classification standards of 2008. It was found that the mutant type of three genes showed three bands, whereas only one band for wild-type. The JAK2V617F and MPL mutations did not cause a change in the open reading frame and the CALR mutation resulted in its change. The mutation rate of JAK2V617F and CALR in ET group was significantly higher than that in the secondary thrombocythemia group (p<0.05). The positive mutation rate of MPL was only 4.55%. JAK2V617F-positive mutation alone was used to diagnose with ET. The area under the curve (AUC) was 0.721. The sensitivity was 72.4%, the specificity was 79.5% and the cut-off value was 0.25. When CALR-positive mutation alone was used to diagnose ET, the AUC, sensitivity, specificity and cut-off value were 0.664, 68.4, 82.4 and 0.09%, respectively. JAK2V617F combined with CALR mutation were used for diagnosis of ET. The AUC was 0.862, the sensitivity was 85.9%, the specificity was 87.8%, and the cut-off values were 0.21 and 0.07. In conclusion, the positive mutation rate of JAK2V617F and CALR in ET was higher, and the sensitivity, specificity and accuracy of the diagnosis of ET were significantly improved using the detection of JAK2V617F and CALR.

Determining the role of inflammation in the selection of JAK2 mutant cells in myeloproliferative neoplasms.

Science.gov (United States)

Zhang, Jie; Fleischman, Angela G; Wodarz, Dominik; Komarova, Natalia L

2017-07-21

Myeloproliferative neoplasm (MPN) is a hematologic malignancy characterized by the clonal outgrowth of hematopoietic cells with a somatically acquired mutation most commonly in JAK2 (JAK2 V617F ). This mutation endows upon myeloid progenitors cytokine independent growth and consequently leads to excessive production of myeloid lineage cells. It has been previously suggested that inflammation may play a role in the clonal evolution of JAK2 V617F mutants. In particular, it is possible that one or more cellular kinetic parameters of hematopoietic stem cells (HSCs) are affected by inflammation, such as division or death rates of cells, and the probability of HSC differentiation. This suggests a mechanism that can steer the outcome of the cellular competition in favor of the mutants, initiating the disease. In this paper we create a number of mathematical evolutionary models, from very abstract to more concrete, that describe cellular competition in the context of inflammation. It is possible to build a model axiomatically, where only very general assumptions are imposed on the modeling components and no arbitrary (and generally unknown) functional forms are used, and still generate a set of testable predictions. In particular, we show that, if HSC death is negligible, the evolutionary advantage of mutant cells can only be conferred by an increase in differentiation probability of HSCs in the presence of inflammation, and if death plays a significant role in the dynamics, an additional mechanism may be an increase of HSC's division-to-death ratio in the presence of inflammation. Further, we show that in the presence of inflammation, the wild type cell population is predicted to shrink under inflammation (even in the absence of mutants). Finally, it turns out that if only the differentiation probability is affected by the inflammation, then the resulting steady state population of wild type cells will contain a relatively smaller percentage of HSCs under inflammation. If

Vascular events in Korean patients with myeloproliferative neoplasms and their relationship to JAK2 mutation.

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Bang, Soo-Mee; Lee, Jong-Seok; Ahn, Jeong Yeal; Lee, Jae Hoon; Hyun, Myung Soo; Kim, Bong Seog; Park, Moo Rim; Chi, Hyun-Sook; Kim, Ho Young; Kim, Hyo Jung; Lee, Moon Hee; Kim, Hwak; Won, Jong Ho; Yoon, Hwi Joong; Oh, Do-Yeun; Nam, Eun-Mi; Bae, Sung Hwa; Kim, Byoung-Kook

2009-03-01

Evaluation of the Janus kinase 2 (JAK2) V617F mutation has been widely used for the diagnosis of myeloproliferative neoplasms (MPN). However, its prognostic relevance to clinical outcome is not completely understood. We investigated the association of JAK2 V617F with vascular events in Korean patients with myeloproliferative neoplasms (MPN). We studied 283 patients from 15 centers, who were diagnosed with MPN. The JAK2 V617F status was evaluated by allele-specific polymerase chain reaction (PCR) and sequencing. The patients' diagnoses were essential thrombocythemia (ET n = 146), polycythemia vera (PV n = 120), primary myelofibrosis (n = 12), and unclassifiable MPN (MPNu n = 5). JAK2 V617F was detected in 89 (61%) patients with ET, 103 (86%) with PV, four (33%) with myelofibrosis, and four (80%) with MPNu. A higher number of leukocytes, haemoglobin levels and BM cellularity as well as an older age, lower platelet counts, and diagnosis of PV were significantly correlated with JAK2 V617F. Eighty-three and 43 episodes of thrombosis and bleeding occurred in 100 patients each before and after the diagnosis. Vascular events more frequently occurred in 37% of patients with JAK2 V617F than in 29% of those without the mutation (p = 0.045). Among 175 patients whose samples were available for sequencing, 28 patients with homozygous JAK2 V617F had vascular events more frequently (57%) than those who were heterozygotes (39%) or had the wild type (27%) (p = 0.03). The multivariate analysis showed that a JAK2 homozygous mutation, hypercholesterolemia and older age were independent risk factors for a vascular event. The results of this study showed that Korean patients with MPN had a similar JAK2 mutation rate and frequency of vascular events when compared to Western patients. The presence of V617F was significantly related to vascular events. Therefore, initial evaluation for the JAK2 mutation and careful monitoring for vascular events should be performed in MPN patients.

The role of JAK1/2 inhibitors in the treatment of chronic myeloproliferative neoplasms.

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Keohane, Clodagh; Mesa, Ruben; Harrison, Claire

2013-01-01

In 2005, the description of the JAK2V617F mutation for the first time provided a molecular key to enable more rapid diagnosis and target for novel therapeutics in the myeloproliferative neoplasms. In 2007, the first-in-class agent INC18424, ruxolitinib, JAKafi, or JAKAVI was first tested in patients with intermediate-risk 2 or high-risk myelofibrosis regardless of whether they possessed the JAK2V617F mutation. Patients treated with this agent had major reduction in splenomegaly as well as impressive reduction, and in some cases resolution, of symptoms. This study was followed by the two Controlled Myelofibrosis Study with Oral JAK Inhibitor Therapy (COMFORT) trials (the first-ever phase III trials in myelofibrosis), which confirmed results in these aspects were superior to either placebo or standard care, and updated results show a survival advantage with this therapy. This paper discusses these results and data from other JAK inhibitors while speculating on the future of these therapies. It also reflects on the fact that the true targets and agents' mode of action are uncertain. Unlike targeted therapy for chronic myeloid leukemia (CML), these agents do not deliver molecular remission, and it is not clear whether their predominant benefit is mediated via JAK2, JAK1, or both. Nonetheless, the advent of the JAK inhibitor is a welcome advance and has made a dramatic improvement to the therapeutic landscape of these conditions.

Molecular genetic tests for JAK2V617F, Exon12_JAK2 and MPLW515K/L are highly informative in the evaluation of patients suspected to have BCR-ABL1-negative myeloproliferative neoplasms.

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dos Santos, Marcos Tadeu; Mitne-Neto, Miguel; Miyashiro, Kozue; Chauffaille, Maria de Lourdes L Ferrari; Rizzatti, Edgar Gil

2014-02-01

Polycythaemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (MF), are the most common myeloproliferative neoplasms (MPN) in patients without the BCR-ABL1 gene rearrangement. They are caused by clonal expansion of haematopoietic stem cells and share, as a diagnostic criterion, the identification of JAK2V617F mutation. Classically, when other clinical criteria are present, a JAK2V617F negative case requires the analysis of Exon12_JAK2 for the diagnosis of PV, and of MPL515K/L mutations for the diagnosis of ET and MF. Here, we evaluated 78 samples from Brazilian patients suspected to have MPN, without stratification for PV, ET or MF. We found that 28 (35.9%) are JAK2V617F carriers; from the 50 remaining samples, one (2%) showed an Exon12_JAK2 mutation, and another (2%) was positive for MPLW515L mutation. In summary, the investigation of JAK2V617F, Exon12_JAK2 and MPLW515K/L was relevant for the diagnosis of 38.4% of patients suspected to have BCR-ABL1-negative MPN, suggesting that molecular genetic tests are useful for a quick and unequivocal diagnosis of MPN.

Molecular basis of cyclooxygenase enzymes (COXs) selective inhibition

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Limongelli, Vittorio; Bonomi, Massimiliano; Marinelli, Luciana; Gervasio, Francesco Luigi; Cavalli, Andrea; Novellino, Ettore; Parrinello, Michele

2010-01-01

The widely used nonsteroidal anti-inflammatory drugs block the cyclooxygenase enzymes (COXs) and are clinically used for the treatment of inflammation, pain, and cancers. A selective inhibition of the different isoforms, particularly COX-2, is desirable, and consequently a deeper understanding of the molecular basis of selective inhibition is of great demand. Using an advanced computational technique we have simulated the full dissociation process of a highly potent and selective inhibitor, SC-558, in both COX-1 and COX-2. We have found a previously unreported alternative binding mode in COX-2 explaining the time-dependent inhibition exhibited by this class of inhibitors and consequently their long residence time inside this isoform. Our metadynamics-based approach allows us to illuminate the highly dynamical character of the ligand/protein recognition process, thus explaining a wealth of experimental data and paving the way to an innovative strategy for designing new COX inhibitors with tuned selectivity. PMID:20215464

Treatment and management of myelofibrosis in the era of JAK inhibitors [Corrigendum

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Keohane C

2013-10-01

Full Text Available Keohane C, Radia DH, Harrison CN. Biologics: Targets and Therapy. 2013;7:189–198. On page 193 note that the paragraph beginning "Pacritinib (SB1518; Cell Technology, Inc, Mountain View, CA, USA is a JAK2 and FLT3 inhibitor currently being evaluated at a dose of 400 mg daily in a Phase II study (N=34 that included patients with low platelet counts (<50 × 109/L." should have been "Pacritinib (SB1518; Cell Therapeutics, Inc, Seattle, WA, USA is a JAK2 and FLT3 inhibitor which was evaluated at a dose of 400 mg daily in a Phase II study (N=34 that included patients with low platelet counts (<50 × 109/L." On page 193 in the same paragraph note that "Post-Essential Thrombocythemia Myelofibrosis: PERSIST." should have been "Post-Essential Thrombocythemia Myelofibrosis-1: PERSIST-1."Read the original article

Prevalence and clinical correlates of JAK2 mutations in Down syndrome acute lymphoblastic leukemia

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Gaikwad, Amos; Rye, Cassia L.; Devidas, Meenakshi; Heerema, Nyla A.; Carroll, Andrew J.; Izraeli, Shai; Plon, Sharon E.; Basso, Giuseppe; Pession, Andrea; Rabin, Karen R.

2009-01-01

Summary Recurrent, prognostically significant chromosomal abnormalities occur in approximately 75% of pediatric acute lymphoblastic leukemia (ALL), but only infrequently in children with Down syndrome (DS) and ALL. Recently, novel somatic activating mutations in Janus kinase 2 (JAK2) were reported in 18% of DS ALL. Here we report identification and clinical correlates of JAK2 mutations in an independent cohort. JAK2 activating mutations occurred in 10 of 53 DS ALL cases (18.9%). Mutations were overrepresented in males (p<0.03), occurred once in association with high hyperdiploidy, and were not significantly correlated with age, initial white blood count, or event-free survival. Our results confirm significance of JAK-STAT pathway activation in DS ALL. PMID:19120350

[The quantitative testing of V617F mutation in gen JAK2 using pyrosequencing technique].

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Dunaeva, E A; Mironov, K O; Dribnokhodova, T E; Subbotina, E E; Bashmakova; Ol'hovskiĭ, I A; Shipulin, G A

2014-11-01

The somatic mutation V617F in gen JAK2 is a frequent cause of chronic myeloprolific diseases not conditioned by BCR/ABL mutation. The quantitative testing of relative percentage of mutant allele can be used in establishing severity of disease and its prognosis and in prescription of remedy inhibiting activity of JAK2. To quantitatively test mutation the pyrosequencing technique was applied. The developed technique permits detecting and quantitatively, testing percentage of mutation fraction since 7%. The "gray zone" is presented by samples with percentage of mutant allele from 4% to 7%. The dependence of expected percentage of mutant fraction in analyzed sample from observed value of signal is described by equation of line with regression coefficients y = - 0.97, x = -1.32 and at that measurement uncertainty consists ± 0.7. The developed technique is approved officially on clinical material from 192 patients with main forms of myeloprolific diseases not conditioned by BCR/ABL mutation. It was detected 64 samples with mautant fraction percentage from 13% to 91%. The developed technique permits implementing monitoring of therapy of myeloprolific diseases and facilitates to optimize tactics of treatment.

Stat5 Exerts Distinct, Vital Functions in the Cytoplasm and Nucleus of Bcr-Abl{sup +} K562 and Jak2(V617F){sup +} HEL Leukemia Cells

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Weber, Axel [Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main 60596 (Germany); Borghouts, Corina [Ganymed Pharmaceuticals AG, Mainz 55131 (Germany); Brendel, Christian [Boston Children’s Hospital, Division of Hematology/Oncology, Boston, MA 02115 (United States); Moriggl, Richard [Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna 1090 (Austria); Delis, Natalia; Brill, Boris; Vafaizadeh, Vida; Groner, Bernd, E-mail: Groner@em.uni-frankfurt.de [Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main 60596 (Germany)

2015-03-19

Signal transducers and activators of transcription (Stats) play central roles in the conversion of extracellular signals, e.g., cytokines, hormones and growth factors, into tissue and cell type specific gene expression patterns. In normal cells, their signaling potential is strictly limited in extent and duration. The persistent activation of Stat3 or Stat5 is found in many human tumor cells and contributes to their growth and survival. Stat5 activation plays a pivotal role in nearly all hematological malignancies and occurs downstream of oncogenic kinases, e.g., Bcr-Abl in chronic myeloid leukemias (CML) and Jak2(V617F) in other myeloproliferative diseases (MPD). We defined the mechanisms through which Stat5 affects growth and survival of K562 cells, representative of Bcr-Abl positive CML, and HEL cells, representative for Jak2(V617F) positive acute erythroid leukemia. In our experiments we suppressed the protein expression levels of Stat5a and Stat5b through shRNA mediated downregulation and demonstrated the dependence of cell survival on the presence of Stat5. Alternatively, we interfered with the functional capacities of the Stat5 protein through the interaction with a Stat5 specific peptide ligand. This ligand is a Stat5 specific peptide aptamer construct which comprises a 12mer peptide integrated into a modified thioredoxin scaffold, S5-DBD-PA. The peptide sequence specifically recognizes the DNA binding domain (DBD) of Stat5. Complex formation of S5-DBD-PA with Stat5 causes a strong reduction of P-Stat5 in the nuclear fraction of Bcr-Abl-transformed K562 cells and a suppression of Stat5 target genes. Distinct Stat5 mediated survival mechanisms were detected in K562 and Jak2(V617F)-transformed HEL cells. Stat5 is activated in the nuclear and cytosolic compartments of K562 cells and the S5-DBD-PA inhibitor most likely affects the viability of Bcr-Abl{sup +} K562 cells through the inhibition of canonical Stat5 induced target gene transcription. In HEL cells

Impact of JAK2V617F Mutational Status on Phenotypic Features in Essential Thrombocythemia and Primary Myelofibrosis

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İpek Yönal

2016-05-01

Full Text Available Objective: The JAK2V617F mutation is present in the majority of patients with essential thrombocythemia (ET and primary myelofibrosis (PMF. The impact of this mutation on disease phenotype in ET and PMF is still a matter of discussion. This study aims to determine whether there are differences in clinical presentation and disease outcome between ET and PMF patients with and without the JAK2V617F mutation. Materials and Methods: In this single-center study, a total of 184 consecutive Philadelphia-negative chronic myeloproliferative neoplasms, 107 cases of ET and 77 cases of PMF, were genotyped for JAK2V617F mutation using the JAK2 Ipsogen MutaScreen assay, which involves allele-specific polymerase chain reaction. Results: ET patients positive for JAK2V617F mutation had higher hemoglobin (Hb and hematocrit (Hct levels, lower platelet counts, and more prevalent splenomegaly at diagnosis compared to patients negative for the JAK2V617F mutation, but rates of major thrombotic events, arterial thrombosis, and venous thrombosis were comparable between the groups. At presentation, PMF patients with JAK2V617F mutation had significantly higher Hb and Hct levels and leukocyte counts than patients without the mutation. Similar to the findings of ET patients, thromboembolic rates were similar in PMF patients with and without theJAK2V617F mutation. For ET and PMF patients, no difference was observed in rates of death with respect to JAK2V617F mutational status. Moreover, leukemic transformation rate was not different in our PMF patients with and without JAK2V617F mutation. Conclusion: We conclude that JAK2V617F-mutated ET patients express a polycythemia vera-like phenotype and JAK2V617F mutation in PMF patients is associated with a more pronounced myeloproliferative phenotype.

The thrombopoietin receptor, MPL, is critical for development of a JAK2V617F-induced myeloproliferative neoplasm.

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Sangkhae, Veena; Etheridge, S Leah; Kaushansky, Kenneth; Hitchcock, Ian S

2014-12-18

The most frequent contributing factor in Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) is the acquisition of a V617F mutation in Janus kinase 2 (JAK2) in hematopoietic stem cells (HSCs). Recent evidence has demonstrated that to drive MPN transformation, JAK2V617F needs to directly associate with a functional homodimeric type I cytokine receptor, suggesting that, although acquiring JAK2V617F may promote disease, there are additional cellular components necessary for MPN development. Here we show that loss of the thrombopoietin (TPO) receptor (MPL) significantly ameliorates MPN development in JAK2V617F(+) transgenic mice, whereas loss of TPO only mildly affects the disease phenotype. Specifically, compared with JAK2V617F(+) mice, JAK2V617F(+)Mpl(-/-) mice exhibited reduced thrombocythemia, neutrophilia, splenomegaly, and neoplastic stem cell pool. The importance of MPL is highlighted as JAK2V617FMpl(+/-) mice displayed a significantly reduced MPN phenotype, indicating that Mpl level may have a substantial effect on MPN development and severity. Splenomegaly and the increased neoplastic stem cell pool were retained in JAK2V617F(+)Tpo(-/-) mice, although thrombocytosis was reduced compared with JAK2V617F(+) mice. These results demonstrate that Mpl expression, but not Tpo, is fundamental in the development of JAK2V617F(+) MPNs, highlighting an entirely novel target for therapeutic intervention. © 2014 by The American Society of Hematology.

Inhibition of STAT3 signaling and induction of SHP1 mediate antiangiogenic and antitumor activities of ergosterol peroxide in U266 multiple myeloma cells

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Rhee, Yun-Hee; Jeong, Soo-Jin; Lee, Hyo-Jeong; Lee, Hyo-Jung; Koh, Wonil; Jung, Ji Hoon; Kim, Sun-Hee; Sung-Hoon, Kim

2012-01-01

Ergosterol peroxide (EP) derived from edible mushroom has been shown to exert anti-tumor activity in several cancer cells. In the present study, anti-angiogenic activity of EP was investigated with the underlying molecular mechanisms in human multiple myeloma U266 cells. Despite weak cytotoxicity against U266 cells, EP suppressed phosphorylation, DNA binding activity and nuclear translocalization of signal transducer and activator of transcription 3 (STAT3) in U266 cells at nontoxic concentrations. Also, EP inhibited phosphorylation of the upstream kinases Janus kinase 2 (JAK2) and Src in a time-dependent manner. Furthermore, EP increased the expression of protein tyrosine phosphatase SHP-1 at protein and mRNA levels, and conversely silencing of the SHP-1 gene clearly blocked EP-mediated STAT3 inactivation. In addition, EP significantly decreased vascular endothelial growth factor (VEGF), one of STAT3 target genes at cellular and protein levels as well as disrupted in vitro tube formation assay. Moreover, EP significantly suppressed the growth of U266 cells inoculated in female BALB/c athymic nude mice and immunohistochemistry revealed that EP effectively reduced the expression of STAT3 and CD34 in tumor sections compared to untreated control. These findings suggest that EP can exert antitumor activity in multiple myeloma U266 cells partly with antiangiogenic activity targeting JAK2/STAT3 signaling pathway as a potent cancer preventive agent for treatment of multiple myeloma cells

Combination therapy with interferon and JAK1-2 inhibitor is feasible

DEFF Research Database (Denmark)

Bjørn, M E; de Stricker, K; Kjær, L

2014-01-01

We report a 55 year old woman with post-ET PV for 12 years, who experienced resolution of severe constitutional symptoms within 3 days, a marked reduction in splenomegaly and a rapid decline in the JAK2V617F allele burden during combination therapy with interferon-alpha2a and ruxolitinib. Within ...

5-hydroxy-2-methyl-1,4-naphthoquinone, a vitamin K3 analogue, suppresses STAT3 activation pathway through induction of protein tyrosine phosphatase, SHP-1: potential role in chemosensitization.

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Sandur, Santosh K; Pandey, Manoj K; Sung, Bokyung; Aggarwal, Bharat B

2010-01-01

The activation of signal transducers and activators of transcription 3 (STAT3) has been linked with carcinogenesis through survival, proliferation, and angiogenesis of tumor cells. Agents that can suppress STAT3 activation have potential not only for prevention but also for treatment of cancer. In the present report, we investigated whether 5-hydroxy-2-methyl-1,4-naphthoquinone (plumbagin), an analogue of vitamin K, and isolated from chitrak (Plumbago zeylanica), an Ayurvedic medicinal plant, can modulate the STAT3 pathway. We found that plumbagin inhibited both constitutive and interleukin 6-inducible STAT3 phosphorylation in multiple myeloma (MM) cells and this correlated with the inhibition of c-Src, Janus-activated kinase (JAK)1, and JAK2 activation. Vanadate, however, reversed the plumbagin-induced downregulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase. Indeed, we found that plumbagin induced the expression of the protein tyrosine phosphatase, SHP-1, and silencing of the SHP-1 abolished the effect of plumbagin. This agent also downregulated the expression of STAT3-regulated cyclin D1, Bcl-xL, and vascular endothelial growth factor; activated caspase-3; induced poly (ADP ribose) polymerase cleavage; and increased the sub-G(1) population of MM cells. Consistent with these results, overexpression of constitutive active STAT3 significantly reduced the plumbagin-induced apoptosis. When compared with AG490, a rationally designed STAT3/JAK2 inhibitor, plumbagin was found more potent in suppressing the proliferation of cells. Plumbagin also significantly potentiated the apoptotic effects of thalidomide and bortezomib in MM cells. Overall, these results suggest that the plumbagin inhibits STAT3 activation pathway through the induction of SHP-1 and this may mediate the sensitization of STAT3 overexpressing cancers to chemotherapeutic agents.

Jak to bylo s fúzí - část čtvrtá

Czech Academy of Sciences Publication Activity Database

Řípa, Milan

Leden (2016) Institutional support: RVO:61389021 Keywords : Fusion * Argentina * Ronald Richter * Proyecto Huemu Subject RIV: BL - Plasma and Gas Discharge Physics http://www.3pol.cz/cz/rubriky/jaderna-fyzika-a-energetika/1771-jak-to-bylo-s-fuzi-cast-ctvrta

The JAK2V617 mutation induces constitutive activation and agonist hypersensitivity in basophils from patients with polycythemia vera

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Pieri, Lisa; Bogani, Costanza; Guglielmelli, Paola; Zingariello, Maria; Rana, Rosa Alba; Bartalucci, Niccolò; Bosi, Alberto; Vannucchi, Alessandro M.

2009-01-01

Background The JAK2V617F mutation has been associated with constitutive and enhanced activation of neutrophils, while no information is available concerning other leukocyte subtypes. Design and Methods We evaluated correlations between JAK2V617F mutation and the count of circulating basophils, the number of activated CD63+ basophils, their response in vitro to agonists as well as the effects of a JAK2 inhibitor. Results We found that basophil count was increased in patients with JAK2V617F -positive myeloproliferative neoplasms, particularly in those with polycythemia vera, and was correlated with the V617F burden. The burden of V617F allele was similar in neutrophils and basophils from patients with polycythemia vera, while total JAK2 mRNA content was remarkably greater in the basophils; however, the content of JAK2 protein in basophils was not increased. The number of CD63+ basophils was higher in patients with polycythemia vera than in healthy subjects or patients with essential thrombocythemia or primary myelofibrosis and was correlated with the V617F burden. Ultrastructurally, basophils from patients with polycythemia vera contained an increased number of granules, most of which were empty suggesting cell degranulation in vivo. Ex vivo experiments revealed that basophils from patients with polycythemia vera were hypersensitive to the priming effect of interleukin-3 and to f-MLP-induced activation; pre-treatment with a JAK2 inhibitor reduced polycythemia vera basophil activation. Finally, we found that the number of circulating CD63+ basophils was significantly greater in patients suffering from aquagenic pruritus, who also showed a higher V617F allele burden. Conclusions These data indicate that the number of constitutively activated and hypersensitive circulating basophils is increased in polycythemia vera, underscoring a role of JAK2V617F in these cells’ abnormal function and, putatively, in the pathogenesis of pruritus. PMID:19608683

Heterozygous and homozygous JAK2(V617F states modeled by induced pluripotent stem cells from myeloproliferative neoplasm patients.

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Joseph Saliba

Full Text Available JAK2(V617F is the predominant mutation in myeloproliferative neoplasms (MPN. Modeling MPN in a human context might be helpful for the screening of molecules targeting JAK2 and its intracellular signaling. We describe here the derivation of induced pluripotent stem (iPS cell lines from 2 polycythemia vera patients carrying a heterozygous and a homozygous mutated JAK2(V617F, respectively. In the patient with homozygous JAK2(V617F, additional ASXL1 mutation and chromosome 20 allowed partial delineation of the clonal architecture and assignation of the cellular origin of the derived iPS cell lines. The marked difference in the response to erythropoietin (EPO between homozygous and heterozygous cell lines correlated with the constitutive activation level of signaling pathways. Strikingly, heterozygous iPS cells showed thrombopoietin (TPO-independent formation of megakaryocytic colonies, but not EPO-independent erythroid colony formation. JAK2, PI3K and HSP90 inhibitors were able to block spontaneous and EPO-induced growth of erythroid colonies from GPA(+CD41(+ cells derived from iPS cells. Altogether, this study brings the proof of concept that iPS can be used for studying MPN pathogenesis, clonal architecture, and drug efficacy.

Selective Inhibition and Naming Performance in Semantic Blocking, Picture-Word Interference, and Color-Word Stroop Tasks

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Shao, Zeshu; Roelofs, Ardi; Martin, Randi C.; Meyer, Antje S.

2015-01-01

In 2 studies, we examined whether explicit distractors are necessary and sufficient to evoke selective inhibition in 3 naming tasks: the semantic blocking, picture-word interference, and color-word Stroop task. Delta plots were used to quantify the size of the interference effects as a function of reaction time (RT). Selective inhibition was…

Virtual screening and optimization of Type II inhibitors of JAK2 from a natural product library.

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Ma, Dik-Lung; Chan, Daniel Shiu-Hin; Wei, Guo; Zhong, Hai-Jing; Yang, Hui; Leung, Lai To; Gullen, Elizabeth A; Chiu, Pauline; Cheng, Yung-Chi; Leung, Chung-Hang

2014-11-21

Amentoflavone has been identified as a JAK2 inhibitor by structure-based virtual screening of a natural product library. In silico optimization using the DOLPHIN model yielded analogues with enhanced potency against JAK2 activity and HCV activity in cellulo. Molecular modeling and kinetic experiments suggested that the analogues may function as Type II inhibitors of JAK2.

Corydalis hendersonii Hemsl. protects against myocardial injury by attenuating inflammation and fibrosis via NF-κB and JAK2-STAT3 signaling pathways.

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Bai, Ruifeng; Yin, Xu; Feng, Xiao; Cao, Yuan; Wu, Yan; Zhu, Zhixiang; Li, Chun; Tu, Pengfei; Chai, Xingyun

2017-07-31

group; attenuated the increase levels of CK-MB and LDH in serum; reduced expressions of AngII, TNF-α, IL-6 and IL-1ß in plasma, MMP-2 and MMP-9 expressions in the cardiac tissue homogenate; and down-regulated myocardial expressions of p-p65, p-IκBα, p-JAK2, p-STAT3, MMP-2, and MMP-9 in AMI mice. Also, an obvious reduction in inflammatory cell infiltration in the myocardial infarct was found in all CH treated groups. Besides, CH also inhibited platelet aggregation induced by THR, ADP, and AA. CH extract exerted a protective effect against myocardial ischemic injury via inhibition of inflammation, myocardial fibrosis, and platelet aggregation. This study demonstrates such protection for the first time and provides a basis for development of CH-based drugs for treatment of ischemic heart disease in clinical settings. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Frequency of JAK2 V617F mutation in patients with Philadelphia positive Chronic Myeloid Leukemia in Pakistan.

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Tabassum, Najia; Saboor, Mohammed; Ghani, Rubina; Moinuddin, Moinuddin

2014-01-01

Co-existence of myeloproliferative disorders (MPD) and Janus associated kinase 2 mutation (JAK2 V617F) is a well-established fact. Only few case reports are available showing presence of JAK2 V617F mutation in chronic myeloid leukemia (CML). Purpose of this study was to determine the frequency of JAK2 V617F mutation in Philadelphia Chromosome positive (Ph (+)) CML patients in Pakistan. The study was conducted from August 2009 to July 2010 at Civil Hospital and Baqai Institute of Hematology (BIH) Karachi. Blood samples from 25 patients with CML were collected. Multiplex reverse transcription polymerase chain reaction (RT-PCR) was performed for Breakpoint Cluster Region - Abelson (BCR-ABL) rearrangement. Conventional PCR was performed for JAK2 V617F mutation on BCR-ABL positive samples. All 25 samples showed BCR-ABL rearrangement. Out of these 11 samples (44%) had JAK2 V617F mutation; the remaining 14 (56%) cases showed JAK2 617V wild type. It is concluded that the co-existence of Ph (+)CML and JAK2 V617F mutation is possible.

JAK2V617F mutation in chronic myeloid leukemia predicts early disease progression

International Nuclear Information System (INIS)

Pahore, Z.A.A.; Shamsi, T.S.; Taj, M.; Farzana, T.; Ansari, S.H.; Nadeem, M.; Ahmad, M.; Naz, A.

2011-01-01

Objective: To determine the association of JAK2V617F mutation along with BCR-ABL translocation or Philadelphia chromosome in chronic myeloid leukemia with early disease progression to advanced stages (accelerated phase or blast crisis) and poor outcome. Study Design: Case series. Place and Duration of Study: National Institute of Blood Diseases and Bone Marrow Transplantation, Karachi, from February 2008 to August 2009. Methodology: All the newly diagnosed cases of BCR-ABL or Philadelphia positive CML were tested for JAK2V617F mutation by Nested PCR. Demographic data, spleen size, hemoglobin levels, white blood cell and platelet counts were recorded. Independent sample t-test was used for age, haemoglobin level and spleen size. Fisher's exact test was applied to compare disease progression in JAK2V617F mutation positive and negative cases. Results: Out of 45 newly diagnosed cases of CML, 40 were in chronic phase, 01 in accelerated phase and 04 in blast crisis. JAK2V617F mutation was detected in 12 (26.7%) patients; 09 (22.5%) in chronic phase, none in accelerated phase and 03 (75%) in blast crisis. During a mean follow-up of 8 months, 03 patients in chronic phase transformed in blast crisis and 02 into accelerated phase. Overall 08 out of 11 (73%) JAK2V617F positive patients either had advanced disease or showed disease progression. Only 2 of 20 (10%) available patients, negative for the mutation, showed disease progression by transforming into blast crisis (p < 0.001). No statistically significant difference was seen in the age, spleen size, haemoglobin levels, white blood cells and platelets counts in JAK2V617F positive patients. Conclusion: JAK2V617F mutation was detected in 26.7% cases of chronic myeloid leukemia. A significant proportion of them showed early disease progression. (author)

Improved diagnosis of the transition to JAK2 (V⁶¹⁷F homozygosity: the key feature for predicting the evolution of myeloproliferative neoplasms.

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Mariana Selena Gonzalez

Full Text Available Most cases of BCR-ABL1-negative myeloproliferative neoplasms (MPNs, essential thrombocythemia, polycythemia vera and primary myelofibrosis are associated with JAK2 (V617F mutations. The outcomes of these cases are critically influenced by the transition from JAK2 (V617F heterozygosity to homozygosity. Therefore, a technique providing an unbiased assessment of the critical allele burden, 50% JAK2 (V617F, is highly desirable. In this study, we present an approach to assess the JAK2 (V617F burden from genomic DNA (gDNA and complementary DNA (cDNA using one-plus-one template references for allele-specific quantitative-real-time-PCR (qPCR. Plasmidic gDNA and cDNA constructs encompassing one PCR template for JAK2 (V617F spaced from one template for JAK2(Wild Type were constructed by multiple fusion PCR amplifications. Repeated assessments of the 50% JAK2(V617F burden within the dynamic range of serial dilutions of gDNA and cDNA constructs resulted in 52.53 ± 4.2% and 51.46 ± 4.21%, respectively. The mutation-positive cutoff was estimated to be 3.65% (mean +2 standard deviation using 20 samples from a healthy population. This qPCR approach was compared with the qualitative ARMS-PCR technique and with two standard methods based on qPCR, and highly significant correlations were obtained in all cases. qPCR assays were performed on paired gDNA/cDNA samples from 20 MPN patients, and the JAK2 (V617F expression showed a significant correlation with the allele burden. Our data demonstrate that the qPCR method using one-plus-one template references provides an improved assessment of the clinically relevant transition of JAK2 (V617F from heterozygosity to homozygosity.

JAK2 V617F, MPL, and CALR mutations in essential thrombocythaemia and major thrombotic complications: a single-institute retrospective analysis.

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Pósfai, Éva; Marton, Imelda; Király, Péter Attila; Kotosz, Balázs; Kiss-László, Zsuzsanna; Széll, Márta; Borbényi, Zita

2015-07-01

Thrombo-haemorrhagic events are the main cause of morbidity and mortality in essential thrombocythemia. The aim of this study was to estimate the incidence of thrombotic events and the impact of the JAK2V617F, MPL (W515L, W515K, W515R, W515A and S505N) and CALR (type-1, type-2) mutations on 101 essential thrombocythaemia patients (72 females and 29 males with a mean age of 61 years) diagnosed in a Southern Hungarian regional academic centre. The incidence of major thrombosis was 13.86 %. Sixty percent of the patients carried the JAK2V617F mutation. The MPL mutations were analysed by sequencing and the W515L was the only one we could identify with an incidence of 3.96 %. Type-2 CALR mutation could be identified in 3 cases among the patients who had JAK2/MPL-unmutated ET. Statistical analyses revealed that the JAK2V617F mutation was associated with significantly increased levels of platelet (p = 0.042), haemoglobin (p = 0.000), red blood cell (p = 0.000) and haematocrit (p = 0.000) and hepatomegaly (p = 0.045) at diagnosis compared to JAK2V617F negative counterparts, however there was no significant association between the JAK2V617F mutation status (relative risk: 1.297, 95 % CI 0.395-4.258; p = 0.668) and subsequent thrombotic complications. The impact of JAK2V617F, MPL W515L and CALR mutations on the clinical findings at the diagnosis of ET was obvious, but their statistically significant role in the prediction of thrombotic events could not be proven in this study. Our results indirectly support the concept that, besides the quantitative and qualitative changes in the platelets, the mechanisms leading to thrombosis are more complex and multifactorial.

Frequency and clinical features of the JAK2 V617F mutation in pediatric patients with sporadic essential thrombocythemia.

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Nakatani, Takuya; Imamura, Toshihiko; Ishida, Hiroyuki; Wakaizumi, Katsuji; Yamamoto, Tohru; Otabe, Osamu; Ishigami, Tsuyoshi; Adachi, Souichi; Morimoto, Akira

2008-12-01

Pediatric essential thrombocythemia (ET) is a rare and heterogenous disease entity. While several recent studies have focused on the role of the JAK2 V617F mutation in pediatric ET, the frequency of pediatric ET cases with this mutation and the associated clinical features remain unclear. We examined six childhood cases who had been diagnosed with ET according to WHO criteria (onset age: 0.2-14 years) for the presence of the JAK2 V617F mutation, MPLW515L mutation and JAK2 exon 12 mutations. Two sensitive PCR-based methods were used for the JAK2 V617F genotyping. We also examined the expression of polycythemia rubra vera-1 (PRV-1), which is a diagnostic marker for clonal ET. We found that three of the six cases had the JAK2 V617F mutation and that all six cases expressed PRV-1 in their peripheral granulocytes. Neither MPL W515L mutation nor JAK2 exon 12 mutations was detected in the patients without JAK2 V617F mutation. The two patients who developed thrombocythemia during infancy were JAK2 V617F-negative. These findings suggest that the JAK2 V617F mutation is not rare in childhood sporadic ET cases, and that these cases might be older and myeloproliferative features.

Areca nut components stimulate ADAM17, IL-1α, PGE2 and 8-isoprostane production in oral keratinocyte: role of reactive oxygen species, EGF and JAK signaling.

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Chang, Mei-Chi; Chan, Chiu-Po; Chen, Yi-Jane; Hsien, Hsiang-Chi; Chang, Ya-Ching; Yeung, Sin-Yuet; Jeng, Po-Yuan; Cheng, Ru-Hsiu; Hahn, Liang-Jiunn; Jeng, Jiiang-Huei

2016-03-29

Betel quid (BQ) chewing is an etiologic factor of oral submucous fibrosis (OSF) and oral cancer. There are 600 million BQ chewers worldwide. The mechanisms for the toxic and inflammatory responses of BQ are unclear. In this study, both areca nut (AN) extract (ANE) and arecoline stimulated epidermal growth factor (EGF) and interleukin-1α (IL-1α) production of gingival keratinocytes (GKs), whereas only ANE can stimulate a disintegrin and metalloproteinase 17 (ADAM17), prostaglandin E2 (PGE2) and 8-isoprostane production. ANE-induced EGF production was inhibited by catalase. Addition of anti-EGF neutralizing antibody attenuated ANE-induced cyclooxygenase-2 (COX-2), mature ADAM9 expression and PGE2 and 8-isoprostane production. ANE-induced IL-1α production was inhibited by catalase, anti-EGF antibody, PD153035 (EGF receptor antagonist) and U0126 (MEK inhibitor) but not by α-naphthoflavone (cytochrome p450-1A1 inhibitor). ANE-induced ADAM17 production was inhibited by pp2 (Src inhibitor), U0126, α-naphthoflavone and aspirin. AG490 (JAK inhibitor) prevented ANE-stimulated ADAM17, IL-1α, PGE2 production, COX-2 expression, ADAM9 maturation, and the ANE-induced decline in keratin 5 and 14, but showed little effect on cdc2 expression and EGF production. Moreover, ANE-induced 8-isoprostane production by GKs was inhibited by catalase, anti-EGF antibody, AG490, pp2, U0126, α-naphthoflavone, Zinc protoporphyrin (ZnPP) and aspirin. These results indicate that AN components may involve in BQ-induced oral cancer by induction of reactive oxygen species, EGF/EGFR, IL-1α, ADAMs, JAK, Src, MEK/ERK, CYP1A1, and COX signaling pathways, and the aberration of cell cycle and differentiation. Various blockers against ROS, EGF, IL-1α, ADAM, JAK, Src, MEK, CYP1A1, and COX can be used for prevention or treatment of BQ chewing-related diseases.

Efficacy of the JAK2 inhibitor INCB16562 in a murine model of MPLW515L-induced thrombocytosis and myelofibrosis

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Koppikar, Priya; Abdel-Wahab, Omar; Hedvat, Cyrus; Marubayashi, Sachie; Patel, Jay; Goel, Aviva; Kucine, Nicole; Gardner, Jeffrey R.; Combs, Andrew P.; Vaddi, Kris; Haley, Patrick J.; Burn, Timothy C.; Rupar, Mark; Bromberg, Jacqueline F.; Heaney, Mark L.

2010-01-01

The discovery of JAK2 and MPL mutations in patients with myeloproliferative neoplasms (MPNs) provided important insight into the genetic basis of these disorders and led to the development of JAK2 kinase inhibitors for MPN therapy. Although recent studies have shown that JAK2 kinase inhibitors demonstrate efficacy in a JAK2V617F murine bone marrow transplantation model, the effects of JAK2 inhibitors on MPLW515L-mediated myeloproliferation have not been investigated. In this report, we descri...

[Clinical significance of JAK2、CALR and MPL gene mutations in 1 648 Philadelphia chromosome negative myeloproliferative neoplasms patients from a single center].

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Li, M Y; Chao, H Y; Sun, A N; Qiu, H Y; Jin, Z M; Tang, X W; Han, Y; Fu, C C; Chen, S N; Wu, D P

2017-04-14

Objective: To explore the prevalences of JAK2, CALR and MPL gene mutations and the mutation types in patients with Philadelphia chromosome negative myeloproliferative neoplasms (MPNs) , and to compare their clinical characteristics of different mutation types with each other and mutation negative group. Methods: The mutations of JAK2 V617F, JAK2 gene at exon 12, CALR gene at exon 9 and MPL gene at exon 10 in 1 648 Ph negative MPNs patients were detected by direct sequencing. Results: ① The JAK2V617F mutation was found in 471 (92.7%) of 508 PV patients, 819 (78.1%) of 1 049 ET patients and 74 (81.3%) of 91 PMF patients respectively, with the total mutation rate as 82.8% (1 364/1 648) . The JAK2 exon12 mutation was found in 9 (1.7%) of 508 PV patients, none was found in ET or PMF patients, with the total mutation rate as 0.5% (9/1 648) . The CALR mutation was found in 132 (12.6%) of 1 049 ET patients and 11 (12.1%) of 91 PMF patients respectively, with the total mutation rate as 8.7% (143/1 648) ; the MPL mutation was found in 9 (0.9%) of 1 049 ET patients and 1 (1.1%) of 91 PMF patients respectively, with the total mutation rate as 0.6% (10/1 648) . The co-occurrence of any two types of driver gene mutations was not detected by direct sequencing. ②The median onset age of patients with JAK2V617F[61 (15-95) y] was significant higher than of with JAK2 exon12 mutation[49 (33-62) y] or without mutations[42 (3-78) y] ( P MPL mutation[59 (22-71) y] ( P >0.05) . Patients with JAK2V617F had higher white blood cell count and hemoglobin level ( P MPL mutation ( P =0.013) . The platelet count of patients with CALR mutation was significantly higher than of with JAK2V617F[966 (400-2 069) ×10(9)/L vs 800 (198-3 730) ×10(9)/L, P MPL gene mutation revealed normal karyotype. Conclusions: Driver gene mutations detection could ensure the diagnosis and prognosis judgment of MPN more reliable, different subtypes of MPNs had different profiles of driver gene mutations, the latter

Marburg virus evades interferon responses by a mechanism distinct from ebola virus.

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Charalampos Valmas

2010-01-01

Full Text Available Previous studies have demonstrated that Marburg viruses (MARV and Ebola viruses (EBOV inhibit interferon (IFN-alpha/beta signaling but utilize different mechanisms. EBOV inhibits IFN signaling via its VP24 protein which blocks the nuclear accumulation of tyrosine phosphorylated STAT1. In contrast, MARV infection inhibits IFNalpha/beta induced tyrosine phosphorylation of STAT1 and STAT2. MARV infection is now demonstrated to inhibit not only IFNalpha/beta but also IFNgamma-induced STAT phosphorylation and to inhibit the IFNalpha/beta and IFNgamma-induced tyrosine phosphorylation of upstream Janus (Jak family kinases. Surprisingly, the MARV matrix protein VP40, not the MARV VP24 protein, has been identified to antagonize Jak and STAT tyrosine phosphorylation, to inhibit IFNalpha/beta or IFNgamma-induced gene expression and to inhibit the induction of an antiviral state by IFNalpha/beta. Global loss of STAT and Jak tyrosine phosphorylation in response to both IFNalpha/beta and IFNgamma is reminiscent of the phenotype seen in Jak1-null cells. Consistent with this model, MARV infection and MARV VP40 expression also inhibit the Jak1-dependent, IL-6-induced tyrosine phosphorylation of STAT1 and STAT3. Finally, expression of MARV VP40 is able to prevent the tyrosine phosphorylation of Jak1, STAT1, STAT2 or STAT3 which occurs following over-expression of the Jak1 kinase. In contrast, MARV VP40 does not detectably inhibit the tyrosine phosphorylation of STAT2 or Tyk2 when Tyk2 is over-expressed. Mutation of the VP40 late domain, essential for efficient VP40 budding, has no detectable impact on inhibition of IFN signaling. This study shows that MARV inhibits IFN signaling by a mechanism different from that employed by the related EBOV. It identifies a novel function for the MARV VP40 protein and suggests that MARV may globally inhibit Jak1-dependent cytokine signaling.

STAT3 inhibitor WP1066 as a novel therapeutic agent for bCCI neuropathic pain rats.

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Xue, Zhao-Jing; Shen, Le; Wang, Zhi-Yao; Hui, Shang-Yi; Huang, Yu-Guang; Ma, Chao

2014-10-02

Activation of signal transducer and activator of transcription-3 (STAT3) is suggested to be critically involved in the development of chronic pain, but the complex regulation of STAT3-dependent pathway and the functional significance of inhibiting this pathway during the development of neuropathic pain remain elusive. To evaluate the contribution of the JAK2/STAT3 pathway to neuropathic pain and the potentiality of this pathway as a novel therapeutic target, we examined the effects of the STAT3 inhibitor WP1066 by intrathecal administration in a rat model of bilateral chronic constriction injury (bCCI). The pain behavior tests were performed before the surgery and on postoperative day 3, 7, 14 and 21. L4-L6 dorsal spinal cord were harvested at each time point. Both RT-PCR and Western blot were performed to evaluate the activation of JAK2/STAT3 pathway. To observe the influence of WP1066 on neuropathic pain and its molecular mechanism, WP1066 (10 μl, 10 mmol/L in DMSO) or the same capacity of DMSO as the control were applied through the intrathecal tube on the day before bCCI surgery, and on the postoperative day 3 and 5. Behavioral tests were performed to observe the therapeutic effect on mechanical, thermal and cold hyperalgesia. L4-L6 dorsal spinal cord was harvested on postoperative day fourteen, followed by RT-PCR and Western blot evaluation of the JAK2/STAT3 pathway activation. The mechanical, thermal and cold hyperalgesia of the bCCI rats were significantly decreased when compared with the Sham or the Naïve group at each postoperative time point (PbCCI rats, accompanied by SOCS3 mRNA with a similar tendency. Western blot analysis showed that JAK2 and phosphorylated STAT3 increased significantly since 3 days after bCCI. JAK2 peaked on postoperative day 14 while phosphorylated STAT3 peaked on postoperative day 7 and gradually decreased thereafter and SOCS3׳s peak level on postoperative day 3. When WP1066 were administered intrathecally, the pain behaviors of

Influenza A virus inhibits type I IFN signaling via NF-kappaB-dependent induction of SOCS-3 expression.

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Eva-K Pauli

2008-11-01

Full Text Available The type I interferon (IFN system is a first line of defense against viral infections. Viruses have developed various mechanisms to counteract this response. So far, the interferon antagonistic activity of influenza A viruses was mainly observed on the level of IFNbeta gene induction via action of the viral non-structural protein 1 (NS1. Here we present data indicating that influenza A viruses not only suppress IFNbeta gene induction but also inhibit type I IFN signaling through a mechanism involving induction of the suppressor of cytokine signaling-3 (SOCS-3 protein. Our study was based on the observation that in cells that were infected with influenza A virus and subsequently stimulated with IFNalpha/beta, phosphorylation of the signal transducer and activator of transcription protein 1 (STAT1 was strongly reduced. This impaired STAT1 activation was not due to the action of viral proteins but rather appeared to be induced by accumulation of viral 5' triphosphate RNA in the cell. SOCS proteins are potent endogenous inhibitors of Janus kinase (JAK/STAT signaling. Closer examination revealed that SOCS-3 but not SOCS-1 mRNA levels increase in an RNA- and nuclear factor kappa B (NF-kappaB-dependent but type I IFN-independent manner early in the viral replication cycle. This direct viral induction of SOCS-3 mRNA and protein expression appears to be relevant for suppression of the antiviral response since in SOCS-3 deficient cells a sustained phosphorylation of STAT1 correlated with elevated expression of type I IFN-dependent genes. As a consequence, progeny virus titers were reduced in SOCS-3 deficient cells or in cells were SOCS-3 expression was knocked-down by siRNA. These data provide the first evidence that influenza A viruses suppress type I IFN signaling on the level of JAK/STAT activation. The inhibitory effect is at least in part due to the induction of SOCS-3 gene expression, which results in an impaired antiviral response.

Children of Few Words: Relations Among Selective Mutism, Behavioral Inhibition, and (Social) Anxiety Symptoms in 3- to 6-Year-Olds.

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Muris, Peter; Hendriks, Eline; Bot, Suili

2016-02-01

Children with selective mutism (SM) fail to speak in specific public situations (e.g., school), despite speaking normally in other situations (e.g., at home). The current study explored the phenomenon of SM in a sample of 57 non-clinical children aged 3-6 years. Children performed two speech tasks to assess their absolute amount of spoken words, while their parents completed questionnaires for measuring children's levels of SM, social anxiety and non-social anxiety symptoms as well as the temperament characteristic of behavioral inhibition. The results indicated that high levels of parent-reported SM were primarily associated with high levels of social anxiety symptoms. The number of spoken words was negatively related to behavioral inhibition: children with a more inhibited temperament used fewer words during the speech tasks. Future research is necessary to test whether the temperament characteristic of behavioral inhibition prompts children to speak less in novel social situations, and whether it is mainly social anxiety that turns this taciturnity into the psychopathology of SM.

Kefir peptides prevent high-fructose corn syrup-induced non-alcoholic fatty liver disease in a murine model by modulation of inflammation and the JAK2 signaling pathway.

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Chen, H L; Tsai, T C; Tsai, Y C; Liao, J W; Yen, C C; Chen, C M

2016-12-12

In recent years, people have changed their eating habits, and high-fructose-containing bubble tea has become very popular. High-fructose intake has been suggested to be a key factor that induces non-alcoholic fatty liver disease (NAFLD). Kefir, a fermented milk product composed of microbial symbionts, has demonstrated numerous biological activities, including antibacterial, antioxidant and immunostimulating effects. The present study aims to evaluate the effects of kefir peptides on high-fructose-induced hepatic steatosis and the possible molecular mechanism. An animal model of 30% high-fructose-induced NAFLD in C57BL/6J mice was established. The experiment is divided into the following six groups: (1) normal: H 2 O drinking water; (2) mock: H 2 O+30% fructose; (3) KL: low-dose kefir peptides (50 mg kg -1 )+30% fructose; (4) KM: medium-dose kefir peptides (100 mg kg -1 )+30% fructose; (5) KH: high-dose kefir peptides (150 mg kg -1 )+30% fructose; and (6) CFM: commercial fermented milk (100 mg kg -1 )+30% fructose. The results show that kefir peptides improve fatty liver syndrome by decreasing body weight, serum alanine aminotransferase, triglycerides, insulin and hepatic triglycerides, cholesterol, and free fatty acids as well as the inflammatory cytokines (TNF-α, IL-6 and IL-1β) that had been elevated in fructose-induced NAFLD mice. In addition, kefir peptides markedly increased phosphorylation of AMPK to downregulate its targeted enzymes, ACC (acetyl-CoA carboxylase) and SREBP-1c (sterol regulatory element-binding protein 1), and inhibited de novo lipogenesis. Furthermore, kefir peptides activated JAK2 to stimulate STAT3 phosphorylation, which can translocate to the nucleus, and upregulated several genes, including the CPT1 (carnitine palmitoyltransferase-1) involved in fatty acid oxidation. Our data have demonstrated that kefir peptides can improve the symptoms of NAFLD, including body weight, energy intake, inflammatory reaction and the

Kefir peptides prevent high-fructose corn syrup-induced non-alcoholic fatty liver disease in a murine model by modulation of inflammation and the JAK2 signaling pathway

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Chen, H L; Tsai, T C; Tsai, Y C; Liao, J W; Yen, C C; Chen, C M

2016-01-01

Objective: In recent years, people have changed their eating habits, and high-fructose-containing bubble tea has become very popular. High-fructose intake has been suggested to be a key factor that induces non-alcoholic fatty liver disease (NAFLD). Kefir, a fermented milk product composed of microbial symbionts, has demonstrated numerous biological activities, including antibacterial, antioxidant and immunostimulating effects. The present study aims to evaluate the effects of kefir peptides on high-fructose-induced hepatic steatosis and the possible molecular mechanism. Results: An animal model of 30% high-fructose-induced NAFLD in C57BL/6J mice was established. The experiment is divided into the following six groups: (1) normal: H2O drinking water; (2) mock: H2O+30% fructose; (3) KL: low-dose kefir peptides (50 mg kg−1)+30% fructose; (4) KM: medium-dose kefir peptides (100 mg kg−1)+30% fructose; (5) KH: high-dose kefir peptides (150 mg kg−1)+30% fructose; and (6) CFM: commercial fermented milk (100 mg kg−1)+30% fructose. The results show that kefir peptides improve fatty liver syndrome by decreasing body weight, serum alanine aminotransferase, triglycerides, insulin and hepatic triglycerides, cholesterol, and free fatty acids as well as the inflammatory cytokines (TNF-α, IL-6 and IL-1β) that had been elevated in fructose-induced NAFLD mice. In addition, kefir peptides markedly increased phosphorylation of AMPK to downregulate its targeted enzymes, ACC (acetyl-CoA carboxylase) and SREBP-1c (sterol regulatory element-binding protein 1), and inhibited de novo lipogenesis. Furthermore, kefir peptides activated JAK2 to stimulate STAT3 phosphorylation, which can translocate to the nucleus, and upregulated several genes, including the CPT1 (carnitine palmitoyltransferase-1) involved in fatty acid oxidation. Conclusion: Our data have demonstrated that kefir peptides can improve the symptoms of NAFLD, including body weight, energy intake

Driver mutations (JAK2V617F, MPLW515L/K or CALR), pentraxin-3 and C-reactive protein in essential thrombocythemia and polycythemia vera

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Lussana, Federico; Carobbio, Alessandra; Salmoiraghi, Silvia; Guglielmelli, Paola; Vannucchi, Alessandro Maria; Bottazzi, Barbara; Leone, Roberto; Mantovani, Alberto; Barbui, Tiziano; Rambaldi, Alessandro

2017-01-01

Abstract Background The driver mutations JAK2V617F, MPLW515L/K and CALR influence disease phenotype of myeloproliferative neoplasms (MPNs) and might sustain a condition of chronic inflammation. Pentraxin 3 (PTX3) and high-sensitivity C-reactive protein (hs-CRP) are inflammatory biomarkers potentially useful for refining prognostic classification of MPNs. Methods We evaluated 305 with essential thrombocythemia (ET) and 172 polycythemia vera (PV) patients diagnosed according to the 2016 WHO cri...

Coexistence of gain-of-function JAK2 germ line mutations with JAK2(V617F) in polycythemia vera

Czech Academy of Sciences Publication Activity Database

Láníková, Lucie; Babošová, Oľga; Swierczek, S.; Wang, L.; Wheeler, D.A.; Divoky, V.; Kořínek, Vladimír; Prchal, J.T.

2016-01-01

Roč. 128, č. 18 (2016), s. 2266-2270 ISSN 0006-4971 R&D Projects: GA ČR GJ15-18046Y; GA MŠk LO1419 Institutional support: RVO:68378050 Keywords : JAK2 mutations * polycythemia vera Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 13.164, year: 2016

The C allele of JAK2 rs4495487 is an additional candidate locus that contributes to myeloproliferative neoplasm predisposition in the Japanese population

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Ohyashiki Junko H

2012-01-01

Full Text Available Abstract Background Polycythemia vera (PV, essential thrombocythemia (ET, and primary myelofibrosis (PMF are myeloproliferative neoplasms (MPNs characterized in most cases by a unique somatic mutation, JAK2 V617F. Recent studies revealed that JAK2 V617F occurs more frequently in a specific JAK2 haplotype, named JAK2 46/1 or GGCC haplotype, which is tagged by rs10974944 (C/G and/or rs12343867 (T/C. This study examined the impact of single nucleotide polymorphisms (SNPs of the JAK2 locus on MPNs in a Japanese population. Methods We sequenced 24 JAK2 SNPs in Japanese patients with PV. We then genotyped 138 MPN patients (33 PV, 96 ET, and 9 PMF with known JAK2 mutational status and 107 controls for a novel SNP, in addition to two SNPs known to be part of the 46/1 haplotype (rs10974944 and rs12343867. Associations with risk of MPN were estimated by odds ratios and their 95% confidence intervals using logistic regression. Results A novel locus, rs4495487 (T/C, with a mutated T allele was significantly associated with PV. Similar to rs10974944 and rs12343867, rs4495487 in the JAK2 locus is significantly associated with JAK2-positive MPN. Based on the results of SNP analysis of the three JAK2 locus, we defined the "GCC genotype" as having at least one minor allele in each SNP (G allele in rs10974944, C allele in rs4495487, and C allele in rs12343867. The GCC genotype was associated with increased risk of both JAK2 V617F-positive and JAK2 V617F-negative MPN. In ET patients, leukocyte count and hemoglobin were significantly associated with JAK2 V617F, rather than the GCC genotype. In contrast, none of the JAK2 V617F-negative ET patients without the GCC genotype had thrombosis, and splenomegaly was frequently seen in this subset of ET patients. PV patients without the GCC genotype were significantly associated with high platelet count. Conclusions Our results indicate that the C allele of JAK2 rs4495487, in addition to the 46/1 haplotype, contributes

Detection of CALR and MPL Mutations in Low Allelic Burden JAK2 V617F Essential Thrombocythemia.

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Usseglio, Fabrice; Beaufils, Nathalie; Calleja, Anne; Raynaud, Sophie; Gabert, Jean

2017-01-01

Myeloproliferative neoplasms are clonal hematopoietic stem cell disorders characterized by aberrant proliferation and an increased tendency toward leukemic transformation. The genes JAK2, MPL, and CALR are frequently altered in these syndromes, and their mutations are often a strong argument for diagnosis. We analyzed the mutational profiles of these three genes in a cohort of 164 suspected myeloproliferative neoplasms. JAK2 V617F mutation was detected by real-time PCR, whereas high-resolution melting analysis followed by Sanger sequencing were used for searching for mutations in JAK2 exon 12, CALR, and MPL. JAK2 V617F mutation was associated with CALR (n = 4) and MPL (n = 4) mutations in 8 of 103 essential thrombocytosis patients. These cases were harboring a JAK2 V617F allelic burden of MPL genes in myeloproliferative neoplasms and suggest that CALR and MPL should be analyzed not only in JAK2-negative patients but also in low V617F mutation patients. Follow-up of these double-mutation cases will be important for determining whether this group of patients presents particular evolution or complications. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Reduced expression of Jak-1 and Tyk-2 proteins leads to interferon resistance in Hepatitis C virus replicon

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Luftig Ronald

2007-09-01

Full Text Available Abstract Background Alpha interferon in combination with ribavirin is the standard therapy for hepatitis C virus infection. Unfortunately, a significant number of patients fail to eradicate their infection with this regimen. The mechanisms of IFN-resistance are unclear. The aim of this study was to determine the contribution of host cell factors to the mechanisms of interferon resistance using replicon cell lines. Results HCV replicons with high and low activation of the IFN-promoter were cultured for a prolonged period of time in the presence of interferon-alpha (IFN-alpha2b. Stable replicon cell lines with resistant phenotype were isolated and characterized by their ability to continue viral replication in the presence of IFN-alpha. Interferon resistant cell colonies developed only in replicons having lower activation of the IFN promoter and no resistant colonies arose from replicons that exhibit higher activation of the IFN promoter. Individual cell clones were isolated and nine IFN resistant cell lines were established. HCV RNA and protein levels in these cells were not altered by IFN- alpha2b. Reduced signaling and IFN-resistant phenotype was found in all Huh-7 cell lines even after eliminating HCV, suggesting that cellular factors are involved. Resistant phenotype in the replicons is not due to lack of interferon receptor expression. All the cell lines show defect in the JAK-STAT signaling and phosphorylation of STAT 1 and STAT 2 proteins were strongly inhibited due to reduced expression of Tyk2 and Jak-1 protein. Conclusion This in vitro study provides evidence that altered expression of the Jak-Stat signaling proteins can cause IFN resistance using HCV replicon cell clones.

High prevalence of arterial thrombosis in JAK2 mutated essential thrombocythaemia: independence of the V617F allele burden

DEFF Research Database (Denmark)

Larsen, Thomas Stauffer; Pallisgaard, Niels; Møller, Michael Boe

2008-01-01

Approximately half of the patients with essential thrombocythaemia (ET) harbor the JAK2 V617F mutation. Despite a phenotypic mimicry of JAK2 V617F positive ET and polycythaemia vera (PV), the data on thromboembolic risk and correlation to JAK2 mutation status are ambiguous. On a strictly WHO defi...

Efficacy of vorinostat in a murine model of polycythemia vera

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Akada, Hajime; Akada, Saeko; Gajra, Ajeet; Bair, Alicia; Graziano, Stephen; Hutchison, Robert E.

2012-01-01

The discovery of the JAK2V617F mutation in most patients with Ph-negative myeloproliferative neoplasms has led to the development of JAK2 kinase inhibitors. However, JAK2 inhibitor therapy has shown limited efficacy and dose-limiting hematopoietic toxicities in clinical trials. In the present study, we describe the effects of vorinostat, a small-molecule inhibitor of histone deacetylase, against cells expressing JAK2V617F and in an animal model of polycythemia vera (PV). We found that vorinostat markedly inhibited proliferation and induced apoptosis in cells expressing JAK2V617F. In addition, vorinostat significantly inhibited JAK2V617F-expressing mouse and human PV hematopoietic progenitors. Biochemical analyses revealed significant inhibition of phosphorylation of JAK2, Stat5, Stat3, Akt, and Erk1/2 in vorinostat-treated, JAK2V617F-expressing human erythroleukemia (HEL) cells. Expression of JAK2V617F and several other genes, including GATA1, KLF1, FOG1, SCL, C/EPBα, PU.1, and NF-E2, was significantly down-regulated, whereas the expression of SOCS1 and SOCS3 was up-regulated by vorinostat treatment. More importantly, we observed that vorinostat treatment normalized the peripheral blood counts and markedly reduced splenomegaly in Jak2V617F knock-in mice compared with placebo treatment. Vorinostat treatment also decreased the mutant allele burden in mice. Our results suggest that vorinostat may have therapeutic potential for the treatment of PV and other JAK2V617F-associated myeloproliferative neoplasms. PMID:22408262

The Role of Janus Kinase 3 in the Regulation of Na+/K+ ATPase under Energy Depletion

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Zohreh Hosseinzadeh

2015-05-01

Full Text Available Background/Aims: Janus kinase-3 (JAK3 is activated during energy depletion. Energy-consuming pumps include the Na+/K+-ATPase. The present study explored whether JAK3 regulates Na+/K+-ATPase in dendritic cells (DCs. Methods: Ouabain (100 µM-sensitive (Iouabain and K+-induced (Ipump outward currents were determined by utilizing whole cell patch-clamp, Na+/K+-ATPase α1-subunit mRNA levels by RT-PCR, Na+/K+-ATPase protein abundance by flow cytometry or immunofluorescence, and cellular ATP by luciferase-assay in DCs from bone marrow of JAK3-knockout (jak3-/- or wild-type mice (jak3+/+. Ipump was further determined by voltage clamp in Xenopus oocytes expressing JAK3, active A568VJAK3 or inactive K851AJAK3. Results: Na+/K+-ATPase α1-subunit mRNA and protein levels, as well as Ipump and Iouabain were significantly higher in jak3-/-DCs than in jak3+/+DCs. Energy depletion by 4h pre-treatment with 2,4-dinitro-phenol significantly decreased Ipump in jak3+/+ DCs but not in jak3-/-DCs. Cellular ATP was significantly lower in jak3-/-DCs than in jak3+/+DCs and decreased in both genotypes by 2,4-dinitro-phenol, an effect significantly more pronounced in jak3-/-DCs than in jak3+/+DCs and strongly blunted by ouabain in both jak3+/+ and jak3-/-DCs. Ipump and Iouabain in oocytes were decreased by expression of JAK3 and of A568VJAK3 but not of K851AJAK3. JAK3 inhibitor WHI-P154 (4-[(3'-bromo-4'-hydroxyphenylamino]-6,7-dimethoxyquinazoline, 22 μM enhanced Ipump and Iouabain in JAK3 expressing oocytes. The difference between A568VJAK3 and K851AJAK3 expressing oocytes was virtually abrogated by actinomycin D (50 nM. Conclusions: JAK3 down-regulates Na+/K+-ATPase activity, an effect involving gene expression and profoundly curtailing ATP consumption.

Development and inter-laboratory validation of unlabeled probe melting curve analysis for detection of JAK2 V617F mutation in polycythemia vera.

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Wu, Zhiyuan; Yuan, Hong; Zhang, Xinju; Liu, Weiwei; Xu, Jinhua; Zhang, Wei; Guan, Ming

2011-01-01

JAK2 V617F, a somatic point mutation that leads to constitutive JAK2 phosphorylation and kinase activation, has been incorporated into the WHO classification and diagnostic criteria of myeloid neoplasms. Although various approaches such as restriction fragment length polymorphism, amplification refractory mutation system and real-time PCR have been developed for its detection, a generic rapid closed-tube method, which can be utilized on routine genetic testing instruments with stability and cost-efficiency, has not been described. Asymmetric PCR for detection of JAK2 V617F with a 3'-blocked unlabeled probe, saturate dye and subsequent melting curve analysis was performed on a Rotor-Gene® Q real-time cycler to establish the methodology. We compared this method to the existing amplification refractory mutation systems and direct sequencing. Hereafter, the broad applicability of this unlabeled probe melting method was also validated on three diverse real-time systems (Roche LightCycler® 480, Applied Biosystems ABI® 7500 and Eppendorf Mastercycler® ep realplex) in two different laboratories. The unlabeled probe melting analysis could genotype JAK2 V617F mutation explicitly with a 3% mutation load detecting sensitivity. At level of 5% mutation load, the intra- and inter-assay CVs of probe-DNA heteroduplex (mutation/wild type) covered 3.14%/3.55% and 1.72%/1.29% respectively. The method could equally discriminate mutant from wild type samples on the other three real-time instruments. With a high detecting sensitivity, unlabeled probe melting curve analysis is more applicable to disclose JAK2 V617F mutation than conventional methodologies. Verified with the favorable inter- and intra-assay reproducibility, unlabeled probe melting analysis provided a generic mutation detecting alternative for real-time instruments.

Development and inter-laboratory validation of unlabeled probe melting curve analysis for detection of JAK2 V617F mutation in polycythemia vera.

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Zhiyuan Wu

Full Text Available BACKGROUND: JAK2 V617F, a somatic point mutation that leads to constitutive JAK2 phosphorylation and kinase activation, has been incorporated into the WHO classification and diagnostic criteria of myeloid neoplasms. Although various approaches such as restriction fragment length polymorphism, amplification refractory mutation system and real-time PCR have been developed for its detection, a generic rapid closed-tube method, which can be utilized on routine genetic testing instruments with stability and cost-efficiency, has not been described. METHODOLOGY/PRINCIPAL FINDINGS: Asymmetric PCR for detection of JAK2 V617F with a 3'-blocked unlabeled probe, saturate dye and subsequent melting curve analysis was performed on a Rotor-Gene® Q real-time cycler to establish the methodology. We compared this method to the existing amplification refractory mutation systems and direct sequencing. Hereafter, the broad applicability of this unlabeled probe melting method was also validated on three diverse real-time systems (Roche LightCycler® 480, Applied Biosystems ABI® 7500 and Eppendorf Mastercycler® ep realplex in two different laboratories. The unlabeled probe melting analysis could genotype JAK2 V617F mutation explicitly with a 3% mutation load detecting sensitivity. At level of 5% mutation load, the intra- and inter-assay CVs of probe-DNA heteroduplex (mutation/wild type covered 3.14%/3.55% and 1.72%/1.29% respectively. The method could equally discriminate mutant from wild type samples on the other three real-time instruments. CONCLUSIONS: With a high detecting sensitivity, unlabeled probe melting curve analysis is more applicable to disclose JAK2 V617F mutation than conventional methodologies. Verified with the favorable inter- and intra-assay reproducibility, unlabeled probe melting analysis provided a generic mutation detecting alternative for real-time instruments.

Model Based Targeting of IL-6-Induced Inflammatory Responses in Cultured Primary Hepatocytes to Improve Application of the JAK Inhibitor Ruxolitinib.

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Sobotta, Svantje; Raue, Andreas; Huang, Xiaoyun; Vanlier, Joep; Jünger, Anja; Bohl, Sebastian; Albrecht, Ute; Hahnel, Maximilian J; Wolf, Stephanie; Mueller, Nikola S; D'Alessandro, Lorenza A; Mueller-Bohl, Stephanie; Boehm, Martin E; Lucarelli, Philippe; Bonefas, Sandra; Damm, Georg; Seehofer, Daniel; Lehmann, Wolf D; Rose-John, Stefan; van der Hoeven, Frank; Gretz, Norbert; Theis, Fabian J; Ehlting, Christian; Bode, Johannes G; Timmer, Jens; Schilling, Marcel; Klingmüller, Ursula

2017-01-01

IL-6 is a central mediator of the immediate induction of hepatic acute phase proteins (APP) in the liver during infection and after injury, but increased IL-6 activity has been associated with multiple pathological conditions. In hepatocytes, IL-6 activates JAK1-STAT3 signaling that induces the negative feedback regulator SOCS3 and expression of APPs. While different inhibitors of IL-6-induced JAK1-STAT3-signaling have been developed, understanding their precise impact on signaling dynamics requires a systems biology approach. Here we present a mathematical model of IL-6-induced JAK1-STAT3 signaling that quantitatively links physiological IL-6 concentrations to the dynamics of IL-6-induced signal transduction and expression of target genes in hepatocytes. The mathematical model consists of coupled ordinary differential equations (ODE) and the model parameters were estimated by a maximum likelihood approach, whereas identifiability of the dynamic model parameters was ensured by the Profile Likelihood. Using model simulations coupled with experimental validation we could optimize the long-term impact of the JAK-inhibitor Ruxolitinib, a therapeutic compound that is quickly metabolized. Model-predicted doses and timing of treatments helps to improve the reduction of inflammatory APP gene expression in primary mouse hepatocytes close to levels observed during regenerative conditions. The concept of improved efficacy of the inhibitor through multiple treatments at optimized time intervals was confirmed in primary human hepatocytes. Thus, combining quantitative data generation with mathematical modeling suggests that repetitive treatment with Ruxolitinib is required to effectively target excessive inflammatory responses without exceeding doses recommended by the clinical guidelines.

Identification of an imidazopyridine scaffold to generate potent and selective TYK2 inhibitors that demonstrate activity in an in vivo psoriasis model.

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Liang, Jun; Van Abbema, Anne; Balazs, Mercedesz; Barrett, Kathy; Berezhkovsky, Leo; Blair, Wade S; Chang, Christine; Delarosa, Donnie; DeVoss, Jason; Driscoll, Jim; Eigenbrot, Charles; Goodacre, Simon; Ghilardi, Nico; MacLeod, Calum; Johnson, Adam; Bir Kohli, Pawan; Lai, Yingjie; Lin, Zhonghua; Mantik, Priscilla; Menghrajani, Kapil; Nguyen, Hieu; Peng, Ivan; Sambrone, Amy; Shia, Steven; Smith, Jan; Sohn, Sue; Tsui, Vickie; Ultsch, Mark; Williams, Karen; Wu, Lawren C; Yang, Wenqian; Zhang, Birong; Magnuson, Steven

2017-09-15

Herein we report identification of an imidazopyridine class of potent and selective TYK2 inhibitors, exemplified by prototype 6, through constraint of the rotatable amide bond connecting the pyridine and aryl rings of compound 1. Further optimization led to generation of compound 30 that potently inhibits the TYK2 enzyme and the IL-23 pathway in cells, exhibits selectivity against cellular JAK2 activity, and has good pharmacokinetic properties. In mice, compound 30 demonstrated dose-dependent reduction of IL-17 production in a PK/PD model as well as in an imiquimod-induced psoriasis model. In this efficacy model, the IL-17 decrease was accompanied by a reduction of ear thickness indicating the potential of TYK2 inhibition as a therapeutic approach for psoriasis patients. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mechanisms of JAK/STAT pathway negative regulation by the short coreceptor Eye Transformer/Latran.

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Fisher, Katherine H; Stec, Wojciech; Brown, Stephen; Zeidler, Martin P

2016-02-01

Transmembrane receptors interact with extracellular ligands to transduce intracellular signaling cascades, modulate target gene expression, and regulate processes such as proliferation, apoptosis, differentiation, and homeostasis. As a consequence, aberrant signaling events often underlie human disease. Whereas the vertebrate JAK/STAT signaling cascade is transduced via multiple receptor combinations, the Drosophila pathway has only one full-length signaling receptor, Domeless (Dome), and a single negatively acting receptor, Eye Transformer/Latran (Et/Lat). Here we investigate the molecular mechanisms underlying Et/Lat activity. We demonstrate that Et/Lat negatively regulates the JAK/STAT pathway activity and can bind to Dome, thus reducing Dome:Dome homodimerization by creating signaling-incompetent Dome:Et/Lat heterodimers. Surprisingly, we find that Et/Lat is able to bind to both JAK and STAT92E but, despite the presence of putative cytokine-binding motifs, does not detectably interact with pathway ligands. We find that Et/Lat is trafficked through the endocytic machinery for lysosomal degradation but at a much slower rate than Dome, a difference that may enhance its ability to sequester Dome into signaling-incompetent complexes. Our data offer new insights into the molecular mechanism and regulation of Et/Lat in Drosophila that may inform our understanding of how short receptors function in other organisms. © 2016 Fisher et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Evolution of JAK-STAT pathway components: mechanisms and role in immune system development.

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Clifford Liongue

Full Text Available BACKGROUND: Lying downstream of a myriad of cytokine receptors, the Janus kinase (JAK-Signal transducer and activator of transcription (STAT pathway is pivotal for the development and function of the immune system, with additional important roles in other biological systems. To gain further insight into immune system evolution, we have performed a comprehensive bioinformatic analysis of the JAK-STAT pathway components, including the key negative regulators of this pathway, the SH2-domain containing tyrosine phosphatase (SHP, Protein inhibitors against Stats (PIAS, and Suppressor of cytokine signaling (SOCS proteins across a diverse range of organisms. RESULTS: Our analysis has demonstrated significant expansion of JAK-STAT pathway components co-incident with the emergence of adaptive immunity, with whole genome duplication being the principal mechanism for generating this additional diversity. In contrast, expansion of upstream cytokine receptors appears to be a pivotal driver for the differential diversification of specific pathway components. CONCLUSION: Diversification of JAK-STAT pathway components during early vertebrate development occurred concurrently with a major expansion of upstream cytokine receptors and two rounds of whole genome duplications. This produced an intricate cell-cell communication system that has made a significant contribution to the evolution of the immune system, particularly the emergence of adaptive immunity.

SOCS2 and SOCS3 expression in ulcerative colitis and their correlation with inflammatory response and immune response

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Le Huang1

2017-05-01

Full Text Available Objective: To study the correlation of SOCS2 and SOCS3 expression in ulcerative colitis tissue with inflammatory response and immune response. Methods: Ulcerative colitis lesions and normal mucosa from colonoscopic biopsy in Central Hospital of Zibo Mining Refco Group Ltd between May 2014 and July 2016 were selected and enrolled in UC group and control group respectively. RNA was extracted to determine mRNA expression of SOCS2 and SOCS3 as well as inflammatory response JAKs/STATs pathway molecules; protein was extracted to determine the contents of immune response cytokines. Results: SOCS2 mRNA expression in intestinal mucosa of UC group was not significantly different from that of control group, and SOCS3 mRNA expression was significantly lower than that of control group; JAK1, JAK2, JAK3, STAT1, STAT3 and STAT5 mRNA expression as well as IFN-γ and IL-17 protein contents in intestinal mucosa of UC group were significantly higher than those of control group while IL-4 and IL-10 protein contents were significantly lower than those of control group; JAK1, JAK2, JAK3, STAT1, STAT3 and STAT5 mRNA expression as well as IFN-γ and IL-17 protein contents in UC group of intestinal mucosa with low SOCS3 expression were significantly higher than those of intestinal mucosa with high SOCS3 expression while IL-4 and IL-10 protein contents were significantly lower than those of intestinal mucosa with high SOCS3 expression. Conclusion: Low expression of SOCS3 in ulcerative colitis can aggravate the inflammatory reaction and cause the imbalance of Th1/Th2 and Th17/Treg immune response.

Jak to bylo s fúzí – část první

Czech Academy of Sciences Publication Activity Database

Řípa, Milan

2015-01-01

Roč. 8, červen (2015) Institutional support: RVO:61389021 Keywords : Albert Einstein * Ernest Rutherford * Arthur Eddinghton * Francis W. Aston * Fritz Houtermans * Robert Atkinson * George Gamow * Hans Bethe * Fusion * Sun * mass defect * proton-proton cyclus Subject RIV: BL - Plasma and Gas Discharge Physics http://www.3pol.cz/cz/rubriky/jaderna-fyzika-a-energetika/1705-jak-to-bylo-s-fuzi-cast-prvni

Regulation of spatial selectivity by crossover inhibition.

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Cafaro, Jon; Rieke, Fred

2013-04-10

Signals throughout the nervous system diverge into parallel excitatory and inhibitory pathways that later converge on downstream neurons to control their spike output. Converging excitatory and inhibitory synaptic inputs can exhibit a variety of temporal relationships. A common motif is feedforward inhibition, in which an increase (decrease) in excitatory input precedes a corresponding increase (decrease) in inhibitory input. The delay of inhibitory input relative to excitatory input originates from an extra synapse in the circuit shaping inhibitory input. Another common motif is push-pull or "crossover" inhibition, in which increases (decreases) in excitatory input occur together with decreases (increases) in inhibitory input. Primate On midget ganglion cells receive primarily feedforward inhibition and On parasol cells receive primarily crossover inhibition; this difference provides an opportunity to study how each motif shapes the light responses of cell types that play a key role in visual perception. For full-field stimuli, feedforward inhibition abbreviated and attenuated responses of On midget cells, while crossover inhibition, though plentiful, had surprisingly little impact on the responses of On parasol cells. Spatially structured stimuli, however, could cause excitatory and inhibitory inputs to On parasol cells to increase together, adopting a temporal relation very much like that for feedforward inhibition. In this case, inhibitory inputs substantially abbreviated a cell's spike output. Thus inhibitory input shapes the temporal stimulus selectivity of both midget and parasol ganglion cells, but its impact on responses of parasol cells depends strongly on the spatial structure of the light inputs.

Mangiferin ameliorates Porphyromonas gingivalis-induced experimental periodontitis by inhibiting phosphorylation of nuclear factor-κB and Janus kinase 1-signal transducer and activator of transcription signaling pathways.

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Li, H; Wang, Q; Ding, Y; Bao, C; Li, W

2017-02-01

Mangiferin is a natural polyphenol compound with anti-inflammatory properties. However, there have been few reports on the effect of mangiferin on periodontitis. Here, we investigated the anti-inflammatory effects of this compound on experimental periodontitis and the underlying mechanisms. Mice were inoculated with Porphyromonas gingivalis to induce periodontitis, and treated with mangiferin orally (50 mg/kg bodyweight, once a day) for 8 wk. Then, the alveolar bone loss was examined using a scanning electronic microscope. Expression of tumor necrosis factor-α (TNF-α) and the phosphorylation levels of nuclear factor-κB (NF-κB) and Janus kinase 1-signal transducer and activator of adhesion (JAK1-STAT) pathways in the gingival epithelium were detected using western blot analysis and immunohistochemical staining. The results showed that mice with periodontitis exhibited greater alveolar bone loss, stronger expression of TNF-α and higher phosphorylation levels of NF-κB and JAK1-STAT1/3 pathways in gingival epithelia, compared with control mice with no periodontitis. Moreover, treatment with mangiferin could significantly inhibit alveolar bone loss, TNF-α production and phosphorylation of NF-κB and JAK1-STAT1/3 pathways in gingival epithelia. Mangiferin has anti-inflammatory effects on periodontitis, which is associated with its ability to down-regulate the phosphorylation of NF-κB and JAK1-STAT1/3 pathways in gingival epithelia. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Model Based Targeting of IL-6-Induced Inflammatory Responses in Cultured Primary Hepatocytes to Improve Application of the JAK Inhibitor Ruxolitinib

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Svantje Sobotta

2017-10-01

Full Text Available IL-6 is a central mediator of the immediate induction of hepatic acute phase proteins (APP in the liver during infection and after injury, but increased IL-6 activity has been associated with multiple pathological conditions. In hepatocytes, IL-6 activates JAK1-STAT3 signaling that induces the negative feedback regulator SOCS3 and expression of APPs. While different inhibitors of IL-6-induced JAK1-STAT3-signaling have been developed, understanding their precise impact on signaling dynamics requires a systems biology approach. Here we present a mathematical model of IL-6-induced JAK1-STAT3 signaling that quantitatively links physiological IL-6 concentrations to the dynamics of IL-6-induced signal transduction and expression of target genes in hepatocytes. The mathematical model consists of coupled ordinary differential equations (ODE and the model parameters were estimated by a maximum likelihood approach, whereas identifiability of the dynamic model parameters was ensured by the Profile Likelihood. Using model simulations coupled with experimental validation we could optimize the long-term impact of the JAK-inhibitor Ruxolitinib, a therapeutic compound that is quickly metabolized. Model-predicted doses and timing of treatments helps to improve the reduction of inflammatory APP gene expression in primary mouse hepatocytes close to levels observed during regenerative conditions. The concept of improved efficacy of the inhibitor through multiple treatments at optimized time intervals was confirmed in primary human hepatocytes. Thus, combining quantitative data generation with mathematical modeling suggests that repetitive treatment with Ruxolitinib is required to effectively target excessive inflammatory responses without exceeding doses recommended by the clinical guidelines.

Discovery and antiplatelet activity of a selective PI3Kβ inhibitor (MIPS-9922).

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Zheng, Zhaohua; Pinson, Jo-Anne; Mountford, Simon J; Orive, Stephanie; Schoenwaelder, Simone M; Shackleford, David; Powell, Andrew; Nelson, Erin M; Hamilton, Justin R; Jackson, Shaun P; Jennings, Ian G; Thompson, Philip E

2016-10-21

A series of amino-substituted triazines were developed and examined for PI3Kβ inhibition and anti-platelet function. Structural adaptations of a morpholine ring of the prototype pan-PI3K inhibitor ZSTK474 yielded PI3Kβ selective compounds, where the selectivity largely derives from an interaction with the non-conserved Asp862 residue, as shown by site directed mutagenesis. The most PI3Kβ selective inhibitor from the series was studied in detail through a series of in vitro and in vivo functional studies. MIPS-9922, 10 potently inhibited ADP-induced washed platelet aggregation. It also inhibited integrin αIIbβ3 activation and αIIbβ3 dependent platelet adhesion to immobilized vWF under high shear. It prevented arterial thrombus formation in the in vivo electrolytic mouse model of thrombosis without inducing prolonged bleeding or excess blood loss. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

[Prognostic value of JAK2, MPL and CALR mutations in Chinese patients with primary myelofibrosis].

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Xu, Z F; Li, B; Liu, J Q; Li, Y; Ai, X F; Zhang, P H; Qin, T J; Zhang, Y; Wang, J Y; Xu, J Q; Zhang, H L; Fang, L W; Pan, L J; Hu, N B; Qu, S Q; Xiao, Z J

2016-07-01

To evaluate the prognostic value of JAK2, MPL and CALR mutations in Chinese patients with primary myelofibrosis (PMF). Four hundred and two Chinese patients with PMF were retrospectively analyzed. The Kaplan-Meier method, the Log-rank test, the likelihood ratio test and the Cox proportional hazards regression model were used to evaluate the prognostic scoring system. This cohort of patients included 209 males and 193 females with a median age of 55 years (range: 15- 89). JAK2V617F mutations were detected in 189 subjects (47.0% ), MPLW515 mutations in 13 (3.2%) and CALR mutations in 81 (20.1%) [There were 30 (37.0%) type-1, 48 (59.3%) type-2 and 3 (3.7%) less common CALR mutations], respectively. 119 subjects (29.6%) had no detectable mutation in JAK2, MPL or CALR. Univariate analysis indicated that patients with CALR type-2 mutations or no detectable mutations had inferior survival compared to those with JAK2, MPL or CALR type- 1 or other less common CALR mutations (the median survival was 74vs 168 months, respectively [HR 2.990 (95% CI 1.935-4.619),P<0.001]. Therefore, patients were categorized into the high-risk with CALR type- 2 mutations or no detectable driver mutations and the low- risk without aforementioned mutations status. The DIPSS-Chinese molecular prognostic model was proposed by adopting mutation categories and DIPSS-Chinese risk group. The median survival of patients classified in low risk (132 subjects, 32.8% ), intermediate- 1 risk (143 subjects, 35.6%), intermediate- 2 risk (106 subjects, 26.4%) and high risk (21 subjects, 5.2%) were not reached, 156 (95% CI 117- 194), 60 (95% CI 28- 91) and 22 (95% CI 10- 33) months, respectively, and there was a statistically significant difference in overall survival among the four risk groups (P<0.001). There was significantly higher predictive power for survival according to the DIPSS-Chinese molecular prognostic model compared with the DIPSS-Chinese model (P=0.005, -2 log-likelihood ratios of 855.6 and 869

Multimodal Microvascular Imaging Reveals that Selective Inhibition of Class I PI3K Is Sufficient to Induce an Antivascular Response

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Deepak Sampath

2013-07-01

Full Text Available The phosphatidylinositol 3-kinase (PI3K pathway is a central mediator of vascular endothelial growth factor (VEGF-driven angiogenesis. The discovery of small molecule inhibitors that selectively target PI3K or PI3K and mammalian target of rapamycin (mTOR provides an opportunity to pharmacologically determine the contribution of these key signaling nodes in VEGF-A-driven tumor angiogenesis in vivo. This study used an array of microvascular imaging techniques to monitor the antivascular effects of selective class I PI3K, mTOR, or dual PI3K/ mTOR inhibitors in colorectal and prostate cancer xenograft models. Micro-computed tomography (micro-CT angiography, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI, vessel size index (VSI MRI, and DCE ultrasound (DCE-U/S were employed to quantitatively evaluate the vascular (structural and physiological response to these inhibitors. GDC-0980, a dual PI3K/mTOR inhibitor, was found to reduce micro-CT angiography vascular density, while VSI MRI demonstrated a significant reduction in vessel density and an increase in mean vessel size, consistent with a loss of small functional vessels and a substantial antivascular response. DCE-MRI showed that GDC-0980 produces a strong functional response by decreasing the vascular permeability/perfusion-related parameter, Ktrans. Interestingly, comparable antivascular effects were observed for both GDC-980 and GNE-490 (a selective class I PI3K inhibitor. In addition, mTOR-selective inhibitors did not affect vascular density, suggesting that PI3K inhibition is sufficient to generate structural changes, characteristic of a robust antivascular response. This study supports the use of noninvasive microvascular imaging techniques (DCE-MRI, VSI MRI, DCE-U/S as pharmacodynamic assays to quantitatively measure the activity of PI3K and dual PI3K/mTOR inhibitors in vivo.

CLINICAL AND LABORATORY FEATURES OF ESSENTIAL THROMBOCYTOSIS AND PRIMARY MYELOFIBROSIS DEPENDING ON JAK2 AND CALR1 MUTATION STATUS

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E. G. lisina

2017-01-01

Full Text Available Introduction. JAK2V617F mutation is detected in approximately 50 % of patients with essential thrombocytosis (ET and primary myelofibrosis (PMF. In 2013 most of the JAK2 negative patients showed mutations in the CALR gene. Diagnostic value of JAK2 and CALR mutations is high, but their prognostic significance is not sufficiently clear. Data on impact of JAK2 and CALR mutational status on thrombotic complications in ET and myelofibrosis patients are contradictory.The aim of the study was to identify clinical and laboratory features in patients with ET and PMF in accordance with the mutational status of JAK2V617F and CALR gene.Materials and methods. Patients treated in Almazov National Medical Research Center (St. Petersburg, Chuvash Republican Clinical Hospital (Cheboksary, Irkutsk Regional Clinical Hospital (Irkutsk,  Kirov Research Institute of Hematology and Blood Transfusion (Kirov was included in the retrospective study. CALR mutation (1 and 2 types, MPL W515L/K and JAK2V617F mutation were detected in peripheral blood cells.Results. We identified that 21 % (n = 16 of ET patients had thrombotic complications, and they occurred more often among JAK2V617F positive patients (p <0.05. The median of hemoglobin level in PMF was the lowest in the group of triple negative patients. The level of leukocytes in PMF was higher in the group of triple negative patients than in the group with mutated CALR (p = 0.014.Conclusion. JAK2V617F mutation in ET patients was associated with a high risk of thrombosis. Patients with CALR mutations may have a favorable prognosis regarding to thrombotic complications. Some laboratory features of CALR mutations in ET and PMF patients have been revealed.

JAK inhibition induces silencing of T Helper cytokine secretion and a profound reduction in T regulatory cells.

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Keohane, Clodagh; Kordasti, Shahram; Seidl, Thomas; Perez Abellan, Pilar; Thomas, Nicholas S B; Harrison, Claire N; McLornan, Donal P; Mufti, Ghulam J

2015-10-01

CD4(+) T cells maintain cancer surveillance and immune tolerance. Chronic inflammation has been proposed as a driver of clonal evolution in myeloproliferative neoplasms (MPN), suggesting that T cells play an important role in their pathogenesis. Treatment with JAK inhibitors (JAKi) results in improvements in MPN-associated constitutional symptoms as well as reductions in splenomegaly. However, effects of JAKi on T cells in MPN are not well established and the baseline immune signature remains unclear. We investigated the frequency and function of CD4(+) T cell subsets in 50 MPN patients at baseline as well as during treatment with either ruxolitinib or fedratinib in a subset. We show that CD4(+)  CD127(low)  CD25(high)  FOXP3(+) T regulatory cells are reduced in MPN patients compared to healthy controls and that this decrease is even more pronounced following JAKi therapy. Moreover, we show that after 6 months of treatment the number of T helper (Th)-17 cells increased. We also describe a functional 'silencing' of T helper cells both in vivo and in vitro and a blockade of pro-inflammatory cytokines from these cells. This profound effect of JAKi on T cell function may underlay augmented rates of atypical infections that have been reported with use of these drugs. © 2015 John Wiley & Sons Ltd.

[JAK2 V617F and exon 12 genetic variations in Korean patients with BCR/ABL1-negative myeloproliferative neoplasms].

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Kim, Jeong Tae; Cho, Yong Gon; Choi, Sam Im; Lee, Young Jin; Kim, Hye Ran; Jang, Sook Jin; Moon, Dae Soo; Park, Young Jin; Park, Geon

2010-12-01

JAK2 genetic variations have been described in a high proportion of patients with BCR/ABL1-negative myeloproliferative neoplasms (MPN). This study was designed to analyze the frequencies of JAK2 V617F and exon 12 variations, and their correlations with clinical characteristics of Korean patients with BCR/ABL1-negative MPN. We examined a total of 154 patients with BCR/ABL1-negative MPN that included 24, 26, 89, and 15 patients with polycythemia vera (PV), primary myelofibrosis (PMF), essential thrombocythemia (ET), and unclassified myeloproliferative neoplasms (MPNU), respectively. We performed allele-specific PCR to detect V617F in all BCR/ABL1-negative patients, and performed direct sequencing to detect exon 12 variations in 47 V617F-negative MPN patients. JAK2 c.1641+179_183del5 variation was detected by restriction fragment length polymorphism assay in 176 healthy subjects. JAK2 V617F was detected in 91 patients (59.1%): PV (91.6%), PMF (46.2%), ET (52.8%), and MPNU (66.7%). In V617F-negative MPN patients, no mutations were found in exon 12. The c.1641+179_183del5 was detected in 68.1% of V617F-negative MPN patients and 45.4% of healthy subjects (P=0.008). JAK2 V617F was closely correlated with age and leukocytosis in BCR/ABL1-negative MPN patients (P<0.05). However, c.1641+179_183del5 was not related to age, sex, or complete blood cell count parameters in V617F-negative MPN patients and healthy subjects. The c.1641+179_183del5 was associated with an increased odds ratio for MPN (odds ratio, 2.6; 95% confidences interval, 1.3-5.1; P=0.007). Frequencies of V617F are similar to reported results. JAK2 exon 12 mutations may be rare and c.1641+179_183del5 may influence the occurrence of MPN in Korean patients with V6 17F-negative MPN.

LPS-induced release of IL-6 from glia modulates production of IL-1beta in a JAK2-dependent manner

LENUS (Irish Health Repository)

Minogue, Aedín M

2012-06-14

AbstractBackgroundCompelling evidence has implicated neuroinflammation in the pathogenesis of a number of neurodegenerative conditions. Chronic activation of both astrocytes and microglia leads to excessive secretion of proinflammatory molecules such as TNFα, IL-6 and IL-1β with potentially deleterious consequences for neuronal viability. Many signaling pathways involving the mitogen-activated protein kinases (MAPKs), nuclear factor κB (NFκB) complex and the Janus kinases (JAKs)\\/signal transducers and activators of transcription (STAT)-1 have been implicated in the secretion of proinflammatory cytokines from glia. We sought to identify signaling kinases responsible for cytokine production and to delineate the complex interactions which govern time-related responses to lipopolysaccharide (LPS).MethodsWe examined the time-related changes in certain signaling events and the release of proinflammatory cytokines from LPS-stimulated co-cultures of astrocytes and microglia isolated from neonatal rats.ResultsTNFα was detected in the supernatant approximately 1 to 2 hours after LPS treatment while IL-1β and IL-6 were detected after 2 to 3 and 4 to 6 hours, respectively. Interestingly, activation of NFκB signaling preceded release of all cytokines while phosphorylation of STAT1 was evident only after 2 hours, indicating that activation of JAK\\/STAT may be important in the up-regulation of IL-6 production. Additionally, incubation of glia with TNFα induced both phosphorylation of JAK2 and STAT1 and the interaction of JAK2 with the TNFα receptor (TNFR1). Co-treatment of glia with LPS and recombinant IL-6 protein attenuated the LPS-induced release of both TNFα and IL-1β while potentiating the effect of LPS on suppressor of cytokine signaling (SOCS)3 expression and IL-10 release.ConclusionsThese data indicate that TNFα may regulate IL-6 production through activation of JAK\\/STAT signaling and that the subsequent production of IL-6 may impact on the release of

Extending Jak2V617F and MplW515 mutation analysis to single hematopoietic colonies and B and T lymphocytes.

Science.gov (United States)

Pardanani, Animesh; Lasho, Terra L; Finke, Christy; Mesa, Ruben A; Hogan, William J; Ketterling, Rhett P; Gilliland, Dwight Gary; Tefferi, Ayalew

2007-09-01

JAK2V617F and MPLW515L/K are myeloproliferative disorder (MPD)-associated mutations. We genotyped 552 individual hematopoietic colonies obtained by CD34+ cell culture from 16 affected patients (13 JAK2V617F and 3 MPLW515L/K) to determine (a) the proportion of colonies harboring a particular mutation in the presence or absence of cytokines, (b) the lineage distribution of endogenous colonies for each mutation, and (c) the differences (if any) in the pattern of mutation among the various MPDs, as established by genotyping of individual colonies. Genotyping analysis revealed cohabitation of mutation-negative and mutation-positive endogenous colonies in polycythemia vera as well as other MPDs. Culture of progenitor cells harboring MPLW515L/K yielded virtually no endogenous erythroid colonies in contrast to JAK2V617F-harboring progenitor cells. The mutation pattern (i.e., relative distribution of homozygous, heterozygous, or wild-type colonies) was not a distinguishing feature among the MPDs, and MPLW515 mutations were detected in B and/or T lymphocytes in all three patients tested. These observations suggest that clonal myelopoiesis antedates acquisition of JAK2V617F or MPLW515L/K mutations and that the latter is acquired in a lympho-myeloid progenitor cell.

Activation of the protein tyrosine phosphatase SHP2 via the interleukin-6 signal transducing receptor protein gp130 requires tyrosine kinase Jak1 and limits acute-phase protein expression.

Science.gov (United States)

Schaper, F; Gendo, C; Eck, M; Schmitz, J; Grimm, C; Anhuf, D; Kerr, I M; Heinrich, P C

1998-11-01

Stimulation of the interleukin-6 (IL-6) signalling pathway occurs via the IL-6 receptor-glycoprotein 130 (IL-6R-gp130) receptor complex and results in the regulation of acute-phase protein genes in liver cells. Ligand binding to the receptor complex leads to tyrosine phosphorylation and activation of Janus kinases (Jak), phosphorylation of the signal transducing subunit gp130, followed by recruitment and phosphorylation of the signal transducer and activator of transcription factors STAT3 and STAT1 and the src homology domain (SH2)-containing protein tyrosine phosphatase (SHP2). The tyrosine phosphorylated STAT factors dissociate from the receptor, dimerize and translocate to the nucleus where they bind to enhancer sequences of IL-6 target genes. Phosphorylated SHP2 is able to bind growth factor receptor bound protein (grb2) and thus might link the Jak/STAT pathway to the ras/raf/mitogen-activated protein kinase pathway. Here we present data on the dose-dependence, kinetics and kinase requirements for SHP2 phosphorylation after the activation of the signal transducer, gp130, of the IL-6-type family receptor complex. When human fibrosarcoma cell lines deficient in Jak1, Jak2 or tyrosine kinase 2 (Tyk2) were stimulated with IL-6-soluble IL-6R complexes it was found that only in Jak1-, but not in Jak 2- or Tyk2-deficient cells, SHP2 activation was greatly impaired. It is concluded that Jak1 is required for the tyrosine phosphorylation of SHP2. This phosphorylation depends on Tyr-759 in the cytoplasmatic domain of gp130, since a Tyr-759-->Phe exchange abrogates SHP2 activation and in turn leads to elevated and prolonged STAT3 and STAT1 activation as well as enhanced acute-phase protein gene induction. Therefore, SHP2 plays an important role in acute-phase gene regulation.

The mutation profile of JAK2, MPL and CALR in Mexican patients with Philadelphia chromosome-negative myeloproliferative neoplasms.

Science.gov (United States)

Labastida-Mercado, Nancy; Galindo-Becerra, Samantha; Garcés-Eisele, Javier; Colunga-Pedraza, Perla; Guzman-Olvera, Valeria; Reyes-Nuñez, Virginia; Ruiz-Delgado, Guillermo J; Ruiz-Argüelles, Guillermo J

2015-03-01

By using molecular markers, it is possible to gain information on both the classification and etiopathogenesis of chronic myeloproliferative neoplasias (MPN). In a group of 27 Mexican mestizo patients with MPNs, we studied seven molecular markers: the BCR/ABL1 fusion gene, the JAK2 V617F mutation, the JAK2 exon 12 mutations, the MPL W515L mutation, the MPL W515K mutation, and the calreticulin (CALR) exon 9 deletion or insertion. Patients with the BCR/ABL1 fusion gene were excluded. We studied 14 patients with essential thrombocythemia (ET), eight with polycythemia vera (PV), four with primary myelofibrosis (MF), and one with undifferentiated MPN. We found twelve individuals with the JAK2 V617F mutation; five of them had been clinically classified as PV, five as ET, and one as MF. One patient with the MPL W515L was identified with a clinical picture of ET. Five patients with the CALR mutation were identified, four ET and one MF. No individuals with either the MPL W515K mutation or the JAK2 exon 12 mutations were identified. The most consistent relationship was that between PV and the JAK2 V617F mutation (p=.01). Despite its small size, the study shows much less prevalence of JAK2 mutation in PV, ET and MF, which does not match international data. Copyright © 2015 King Faisal Specialist Hospital & Research Centre. Published by Elsevier B.V. All rights reserved.

EXEL-8232, a small-molecule JAK2 inhibitor, effectively treats thrombocytosis and extramedullary hematopoiesis in a murine model of myeloproliferative neoplasm induced by MPLW515L.

Science.gov (United States)

Wernig, G; Kharas, M G; Mullally, A; Leeman, D S; Okabe, R; George, T; Clary, D O; Gilliland, D G

2012-04-01

About 10% of patients with essential thrombocythemia (ET) or myelofibrosis (MF) that lack mutations in JAK2 harbor an activating mutation in the thrombopoietin receptor, MPLW515L. Distinct from the JAK2V617F retroviral transplant model, the MPLW515L model recapitulates many features of ET and MF, including severe fibrosis and thrombocytosis. We have tested EXEL-8232, an experimental potent JAK2 inhibitor, for efficacy in suppression of thrombocytosis in vivo and for its ability to attenuate MPLW515L myeloproliferative disease. EXEL-8232 was administered for 28 days q12 h by oral gavage at doses of 30 or 100 mg/kg, prospectively. Animals treated with EXEL-8232 at 100 mg/kg had normalized high platelet counts, eliminated extramedullary hematopoiesis in the spleen and eliminated bone marrow fibrosis, whereas the wild-type controls did not develop thrombocytopenia. Consistent with a clinical response in this model, we validated surrogate end points for response to treatment, including a reduction of endogenous colony growth and signaling inhibition in immature erythroid and myeloid primary cells both in vitro and upon treatment in vivo. We conclude that EXEL-8232 has efficacy in treatment of thrombocytosis in vivo in a murine model of ET and MF, and may be of therapeutic benefit for patients with MPL-mutant MPN.

Van gen naar ziekte; JAK2 en polycythaemia vera

NARCIS (Netherlands)

Koene, H. R.; Biemond, B. J.; van der Schoot, C. E.

2007-01-01

The identification of a point mutation in the JAK2 gene in most patients with polycythaemia vera (PV) has led to increased insight into the pathogenesis of the disease. The mutation causes cytokine-independent growth and proliferation of haematopoietic precursor cells, leading to erythrocytosis. The

The use of naturally occurring selectively isolated bacteria for inhibiting paraffin deposition

International Nuclear Information System (INIS)

Lazar, I.; Voicu, A.; Dobrota, S.; Petrisor, I.G.; Stefanescu, M.; Sandulescu, L.; Nicolescu, C.; Mucenica, D.

1999-01-01

One of the most severe problems at any oil fields producing paraffinic oils is that of paraffin depositions. Romania which has a long experience in oil production is also faced with this problem in many oil fields. The microbial treatment, based on the activity of naturally occurring, selectively isolated bacteria, is already proved as an effective alternative to conventional methods to prevent and remove paraffin damage. Using such kind of bacterial products, exciting results for inhibiting paraffin depositions have been obtained. In this paper results concerning the naturally occurring bacteria selectively isolated from hydrocarbon polluted sites as well as from paraffinic oils, semi-solid and solid paraffin depositions are presented. After a laboratory screening, 15 bacterial strains (BS 1-15), three bacterial consortia (BC 1-3) and a Special Bacterial Consortium (SBC1) were selected. For the selection of bacterial consortia, the classical enrichment culture method has been used. The Special Bacterial Consortium resulted from a mixture of BS 1-15 and BC 1-3 following the steps of the classical enrichment culture method. The BS 1-15, BC 1-3 and SBC1 have been tested for their performances in producing biosurfactants and biosolvents as well as for hydrocarbon utilisation. The SBC1 has been tested for its ability in degradation of hydrocarbons contained in several types of paraffinic or non-paraffinic oils, and then for inhibiting paraffin deposition on a 'flow equipment' using two types of paraffinic oils. The SBC1 has been also tested for degradation of hydrocarbons contained in semi-solid and solid paraffin depositions. The results obtained could support further applications to prevent and control paraffin depositions

The use of naturally occurring selectively isolated bacteria for inhibiting paraffin deposition

Energy Technology Data Exchange (ETDEWEB)

Lazar, I.; Voicu, A.; Dobrota, S.; Petrisor, I.G.; Stefanescu, M.; Sandulescu, L. [Institute of Biology of the Romanian Academy, Spl. Independentei 296, Bucharest (Romania); Nicolescu, C.; Mucenica, D. [PETROSTAR Ploiesti, Bdul Bucuresti 35, Ploiesti (Romania)

1999-01-01

One of the most severe problems at any oil fields producing paraffinic oils is that of paraffin depositions. Romania which has a long experience in oil production is also faced with this problem in many oil fields. The microbial treatment, based on the activity of naturally occurring, selectively isolated bacteria, is already proved as an effective alternative to conventional methods to prevent and remove paraffin damage. Using such kind of bacterial products, exciting results for inhibiting paraffin depositions have been obtained. In this paper results concerning the naturally occurring bacteria selectively isolated from hydrocarbon polluted sites as well as from paraffinic oils, semi-solid and solid paraffin depositions are presented. After a laboratory screening, 15 bacterial strains (BS 1-15), three bacterial consortia (BC 1-3) and a Special Bacterial Consortium (SBC1) were selected. For the selection of bacterial consortia, the classical enrichment culture method has been used. The Special Bacterial Consortium resulted from a mixture of BS 1-15 and BC 1-3 following the steps of the classical enrichment culture method. The BS 1-15, BC 1-3 and SBC1 have been tested for their performances in producing biosurfactants and biosolvents as well as for hydrocarbon utilisation. The SBC1 has been tested for its ability in degradation of hydrocarbons contained in several types of paraffinic or non-paraffinic oils, and then for inhibiting paraffin deposition on a `flow equipment` using two types of paraffinic oils. The SBC1 has been also tested for degradation of hydrocarbons contained in semi-solid and solid paraffin depositions. The results obtained could support further applications to prevent and control paraffin depositions

WHO-defined 'myelodysplastic syndrome with isolated del(5q)' in 88 consecutive patients: survival data, leukemic transformation rates and prevalence of JAK2, MPL and IDH mutations.

Science.gov (United States)

Patnaik, M M; Lasho, T L; Finke, C M; Gangat, N; Caramazza, D; Holtan, S G; Pardanani, A; Knudson, R A; Ketterling, R P; Chen, D; Hoyer, J D; Hanson, C A; Tefferi, A

2010-07-01

The 2008 World Health Organization (WHO) criteria were used to identify 88 consecutive Mayo Clinic patients with 'myelodysplastic syndrome with isolated del(5q)' (median age 74 years; 60 females). In all, 60 (68%) patients were followed up to the time of their death. Overall median survival was 66 months; leukemic transformation was documented in five (5.7%) cases. Multivariable analysis identified age >or=70 years (P=0.01), transfusion need at diagnosis (P=0.04) and dysgranulopoiesis (P=0.02) as independent predictors of shortened survival; the presence of zero (low risk), one (intermediate risk) or >or=2 (high risk) risk factors corresponded to median survivals of 102, 52 and 27 months, respectively. Janus kinase 2 (JAK2), thrombopoietin receptor (MPL), isocitrate dehydrogenase 1 (IDH1) and IDH2 mutational analysis was performed on archived bone marrows in 78 patients; JAK2V617F and MPLW515L mutations were shown in five (6.4%) and three (3.8%) patients, respectively, and did not seem to affect phenotype or prognosis. IDH mutations were not detected. Survival was not affected by serum ferritin and there were no instances of death directly related to iron overload. The current study is unique in its strict adherence to WHO criteria for selecting study patients and providing information on long-term survival, practical prognostic factors, baseline risk of leukemic transformation and the prevalence of JAK2, MPL and IDH mutations.

Microfluidic generated EGF-gradients induce chemokinesis of transplantable retinal progenitor cells via the JAK/STAT and PI3kinase signaling pathways.

Directory of Open Access Journals (Sweden)

Uchenna J Unachukwu

Full Text Available A growing number of studies are evaluating retinal progenitor cell (RPC transplantation as an approach to repair retinal degeneration and restore visual function. To advance cell-replacement strategies for a practical retinal therapy, it is important to define the molecular and biochemical mechanisms guiding RPC motility. We have analyzed RPC expression of the epidermal growth factor receptor (EGFR and evaluated whether exposure to epidermal growth factor (EGF can coordinate motogenic activity in vitro. Using Boyden chamber analysis as an initial high-throughput screen, we determined that RPC motility was optimally stimulated by EGF concentrations in the range of 20-400 ng/ml, with decreased stimulation at higher concentrations, suggesting concentration-dependence of EGF-induced motility. Using bioinformatics analysis of the EGF ligand in a retina-specific gene network pathway, we predicted a chemotactic function for EGF involving the MAPK and JAK-STAT intracellular signaling pathways. Based on targeted inhibition studies, we show that ligand binding, phosphorylation of EGFR and activation of the intracellular STAT3 and PI3kinase signaling pathways are necessary to drive RPC motility. Using engineered microfluidic devices to generate quantifiable steady-state gradients of EGF coupled with live-cell tracking, we analyzed the dynamics of individual RPC motility. Microfluidic analysis, including center of mass and maximum accumulated distance, revealed that EGF induced motility is chemokinetic with optimal activity observed in response to low concentration gradients. Our combined results show that EGFR expressing RPCs exhibit enhanced chemokinetic motility in the presence of low nanomole levels of EGF. These findings may serve to inform further studies evaluating the extent to which EGFR activity, in response to endogenous ligand, drives motility and migration of RPCs in retinal transplantation paradigms.

Reversal of CD8 T-Cell–Mediated Mucocutaneous Graft-Versus-Host-Like Disease by the JAK Inhibitor Tofacitinib

Science.gov (United States)

Okiyama, Naoko; Furumoto, Yasuko; Villarroel, Vadim A; Linton, Jay T; Tsai, Wanxia L; Gutermuth, Jan; Ghoreschi, Kamran; Gadina, Massimo; O'Shea, John J; Katz, Stephen I

2014-01-01

The utility of allogeneic hematopoietic stem cell transplantation is limited by graft-versus-host disease (GVHD), a significant cause of morbidity and mortality. Patients with GVHD exhibit cutaneous manifestations with histological features of interface dermatitis followed by scleroderma-like changes. JAK inhibitors represent a class of immunomodulatory drugs that inhibit signaling by multiple cytokines. Herein we report the effects of tofacitinib in a murine model of GVHD. Oral administration of tofacitinib prevented GVHD-like disease manifested by weight loss and mucocutaneous lesions. More importantly, tofacitinib was also effective in reversing established disease. Tofacitinib diminished the expansion and activation of murine CD8 T cells in this model, and had similar effects on IL-2-stimulated human CD8 T cells. Tofacitinib also inhibited the expression of IFN-γ-inducible chemoattractants by keratinocytes, and IFN-γ-inducible cell death of keratinocytes. Tofacitinib may be an effective drug for treatment against CD8 T-cell–mediated mucocutaneous diseases in patients with GVHD. PMID:24213371

HIV-1 Promotes the Degradation of Components of the Type 1 IFN JAK/STAT Pathway and Blocks Anti-viral ISG Induction.

Science.gov (United States)

Gargan, Siobhan; Ahmed, Suaad; Mahony, Rebecca; Bannan, Ciaran; Napoletano, Silvia; O'Farrelly, Cliona; Borrow, Persephone; Bergin, Colm; Stevenson, Nigel J

2018-04-01

Anti-retroviral therapy successfully suppresses HIV-1 infection, but fails to provide a cure. During infection Type 1 IFNs normally play an essential role in viral clearance, but in vivo IFN-α only has a modest impact on HIV-1 infection, suggesting its possible targeting by HIV. Here, we report that the HIV protein, Vif, inhibits effective IFN-α signalling via degradation of essential JAK/STAT pathway components. We found that STAT1 and STAT3 are specifically reduced in HEK293T cells expressing Vif and that full length, infectious HIV-1 IIIB strain promotes their degradation in a Vif-dependent manner. HIV-1 IIIB infection of myeloid ThP-1 cells also reduced the IFN-α-mediated induction of the anti-viral gene, ISG15, but not MxA, revealing a functional consequence of this HIV-1-mediated immune evasion strategy. Interestingly, while total STAT levels were not reduced upon in vitro IIIB infection of primary human PBMCs, IFN-α-mediated phosphorylation of STAT1 and STAT3 and ISG induction were starkly reduced, with removal of Vif (IIIBΔVif), partially restoring pSTATs, ISG15 and MxB induction. Similarly, pSTAT1 and pSTAT3 expression and IFN-α-induced ISG15 were reduced in PBMCs from HIV-infected patients, compared to healthy controls. Furthermore, IFN-α pre-treatment of a CEM T lymphoblast cells significantly inhibited HIV infection/replication (measured by cellular p24), only in the absence of Vif (IIIBΔVif), but was unable to suppress full length IIIB infection. When analysing the mechanism by which Vif might target the JAK/STAT pathway, we found Vif interacts with both STAT1 and STAT3, (but not STAT2), and its expression promotes ubiquitination and MG132-sensitive, proteosomal degradation of both proteins. Vif's Elongin-Cullin-SOCS-box binding motif enables the formation of an active E3 ligase complex, which we found to be required for Vif's degradation of STAT1 and STAT3. In fact, the E3 ligase scaffold proteins, Cul5 and Rbx2, were also found to be

Quantitative threefold allele-specific PCR (QuanTAS-PCR) for highly sensitive JAK2 V617F mutant allele detection

International Nuclear Information System (INIS)

Zapparoli, Giada V; Jorissen, Robert N; Hewitt, Chelsee A; McBean, Michelle; Westerman, David A; Dobrovic, Alexander

2013-01-01

The JAK2 V617F mutation is the most frequent somatic change in myeloproliferative neoplasms, making it an important tumour-specific marker for diagnostic purposes and for the detection of minimal residual disease. Sensitive quantitative assays are required for both applications, particularly for the monitoring of minimal residual disease, which requires not only high sensitivity but also very high specificity. We developed a highly sensitive probe-free quantitative mutant-allele detection method, Quantitative Threefold Allele-Specific PCR (QuanTAS-PCR), that is performed in a closed-tube system, thus eliminating the manipulation of PCR products. QuantTAS-PCR uses a threefold approach to ensure allele-specific amplification of the mutant sequence: (i) a mutant allele-specific primer, (ii) a 3′dideoxy blocker to suppress false-positive amplification from the wild-type template and (iii) a PCR specificity enhancer, also to suppress false-positive amplification from the wild-type template. Mutant alleles were quantified relative to exon 9 of JAK2. We showed that the addition of the 3′dideoxy blocker suppressed but did not eliminate false-positive amplification from the wild-type template. However, the addition of the PCR specificity enhancer near eliminated false-positive amplification from the wild-type allele. Further discrimination between true and false positives was enabled by using the quantification cycle (Cq) value of a single mutant template as a cut-off point, thus enabling robust distinction between true and false positives. As 10,000 JAK2 templates were used per replicate, the assay had a sensitivity of 1/10 -4 per replicate. Greater sensitivity could be reached by increasing the number of replicates analysed. Variation in replicates when low mutant-allele templates were present necessitated the use of a statistics-based approach to estimate the load of mutant JAK2 copies. QuanTAS-PCR showed comparable quantitative results when validated against a

Molecular modeling-driven approach for identification of Janus kinase 1 inhibitors through 3D-QSAR, docking and molecular dynamics simulations.

Science.gov (United States)

Itteboina, Ramesh; Ballu, Srilata; Sivan, Sree Kanth; Manga, Vijjulatha

2017-10-01

Janus kinase 1 (JAK 1) belongs to the JAK family of intracellular nonreceptor tyrosine kinase. JAK-signal transducer and activator of transcription (JAK-STAT) pathway mediate signaling by cytokines, which control survival, proliferation and differentiation of a variety of cells. Three-dimensional quantitative structure activity relationship (3 D-QSAR), molecular docking and molecular dynamics (MD) methods was carried out on a dataset of Janus kinase 1(JAK 1) inhibitors. Ligands were constructed and docked into the active site of protein using GLIDE 5.6. Best docked poses were selected after analysis for further 3 D-QSAR analysis using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) methodology. Employing 60 molecules in the training set, 3 D-QSAR models were generate that showed good statistical reliability, which is clearly observed in terms of r 2 ncv and q 2 loo values. The predictive ability of these models was determined using a test set of 25 molecules that gave acceptable predictive correlation (r 2 Pred ) values. The key amino acid residues were identified by means of molecular docking, and the stability and rationality of the derived molecular conformations were also validated by MD simulation. The good consonance between the docking results and CoMFA/CoMSIA contour maps provides helpful clues about the reasonable modification of molecules in order to design more efficient JAK 1 inhibitors. The developed models are expected to provide some directives for further synthesis of highly effective JAK 1 inhibitors.

Mechanism of protein tyrosine phosphatase 1B-mediated inhibition of leptin signalling

DEFF Research Database (Denmark)

Lund, I K; Hansen, J A; Andersen, H S

2005-01-01

Upon leptin binding, the leptin receptor is activated, leading to stimulation of the JAK/STAT signal transduction cascade. The transient character of the tyrosine phosphorylation of JAK2 and STAT3 suggests the involvement of protein tyrosine phosphatases (PTPs) as negative regulators...

WHO-defined ‘myelodysplastic syndrome with isolated del(5q)' in 88 consecutive patients: survival data, leukemic transformation rates and prevalence of JAK2, MPL and IDH mutations

Science.gov (United States)

Patnaik, M M; Lasho, T L; Finke, C M; Gangat, N; Caramazza, D; Holtan, S G; Pardanani, A; Knudson, R A; Ketterling, R P; Chen, D; Hoyer, J D; Hanson, C A; Tefferi, A

2010-01-01

The 2008 World Health Organization (WHO) criteria were used to identify 88 consecutive Mayo Clinic patients with ‘myelodysplastic syndrome with isolated del(5q)' (median age 74 years; 60 females). In all, 60 (68%) patients were followed up to the time of their death. Overall median survival was 66 months; leukemic transformation was documented in five (5.7%) cases. Multivariable analysis identified age ⩾70 years (P=0.01), transfusion need at diagnosis (P=0.04) and dysgranulopoiesis (P=0.02) as independent predictors of shortened survival; the presence of zero (low risk), one (intermediate risk) or ⩾2 (high risk) risk factors corresponded to median survivals of 102, 52 and 27 months, respectively. Janus kinase 2 (JAK2), thrombopoietin receptor (MPL), isocitrate dehydrogenase 1 (IDH1) and IDH2 mutational analysis was performed on archived bone marrows in 78 patients; JAK2V617F and MPLW515L mutations were shown in five (6.4%) and three (3.8%) patients, respectively, and did not seem to affect phenotype or prognosis. IDH mutations were not detected. Survival was not affected by serum ferritin and there were no instances of death directly related to iron overload. The current study is unique in its strict adherence to WHO criteria for selecting study patients and providing information on long-term survival, practical prognostic factors, baseline risk of leukemic transformation and the prevalence of JAK2, MPL and IDH mutations. PMID:20485371

TC-PTP and PTP1B: Regulating JAK-STAT signaling, controlling lymphoid malignancies.

Science.gov (United States)

Pike, Kelly A; Tremblay, Michel L

2016-06-01

Lymphoid malignancies are characterized by an accumulation of genetic lesions that act co-operatively to perturb signaling pathways and alter gene expression programs. The Janus kinases (JAK)-signal transducers and activators of transcription (STATs) pathway is one such pathway that is frequently mutated in leukemia and lymphoma. In response to cytokines and growth factors, a cascade of reversible tyrosine phosphorylation events propagates the JAK-STAT pathway from the cell surface to the nucleus. Activated STAT family members then play a fundamental role in establishing the transcriptional landscape of the cell. In leukemia and lymphoma, somatic mutations have been identified in JAK and STAT family members, as well as, negative regulators of the pathway. Most recently, inactivating mutations in the protein tyrosine phosphatase (PTP) genes PTPN1 (PTP1B) and PTPN2 (TC-PTP) were sequenced in B cell lymphoma and T cell acute lymphoblastic leukemia (T-ALL) respectively. The loss of PTP1B and TC-PTP phosphatase activity is associated with an increase in cytokine sensitivity, elevated JAK-STAT signaling, and changes in gene expression. As inactivation mutations in PTPN1 and PTPN2 are restricted to distinct subsets of leukemia and lymphoma, a future challenge will be to identify in which cellular contexts do they contributing to the initiation or maintenance of leukemogenesis or lymphomagenesis. As well, the molecular mechanisms by which PTP1B and TC-PTP loss co-operates with other genetic aberrations will need to be elucidated to design more effective therapeutic strategies. Copyright © 2016 Elsevier Ltd. All rights reserved.

Possible involvement of the JAK/STAT signaling pathway in N-acetylcysteine-mediated antidepressant-like effects

OpenAIRE

Al-Samhari, Marwa M; Al-Rasheed, Nouf M; Al-Rejaie, Salim; Al-Rasheed, Nawal M; Hasan, Iman H; Mahmoud, Ayman M; Dzimiri, Nduna

2015-01-01

Advances in depression research have targeted inflammation and oxidative stress to develop novel types of treatment. The JAK/STAT signaling pathway plays pivotal roles in immune and inflammatory responses. The present study was designed to investigate the effects of N-acetylcysteine, a putative precursor of the antioxidant glutathione, in an animal model of depression, with an emphasis on the JAK/STAT signaling pathway. Fluoxetine, a classical antidepressant drug was also under investigation....

Possible involvement of the JAK/STAT signaling pathway in N-acetylcysteine-mediated antidepressant-like effects.

Science.gov (United States)

Al-Samhari, Marwa M; Al-Rasheed, Nouf M; Al-Rejaie, Salim; Al-Rasheed, Nawal M; Hasan, Iman H; Mahmoud, Ayman M; Dzimiri, Nduna

2016-03-01

Advances in depression research have targeted inflammation and oxidative stress to develop novel types of treatment. The JAK/STAT signaling pathway plays pivotal roles in immune and inflammatory responses. The present study was designed to investigate the effects of N-acetylcysteine, a putative precursor of the antioxidant glutathione, in an animal model of depression, with an emphasis on the JAK/STAT signaling pathway. Fluoxetine, a classical antidepressant drug was also under investigation. Male Wistar rats were subjected to forced swimming test and given N-acetylcysteine and fluoxetine immediately after the pre-test session, 5 h later and 1 h before the test session of the forced swimming test. N-acetylcysteine decreased immobility time (P acetylcysteine produced significant (P acetylcysteine significantly (P acetylcysteine. Therefore, depression research may target the JAK/STAT signaling pathway to provide a novel effective therapy. © 2015 by the Society for Experimental Biology and Medicine.

ATF3 activates Stat3 phosphorylation through inhibition of p53 expression in skin cancer cells.

Science.gov (United States)

Hao, Zhen-Feng; Ao, Jun-Hong; Zhang, Jie; Su, You-Ming; Yang, Rong-Ya

2013-01-01

ATF3, a member of the ATF/CREB family of transcription factors, has been found to be selectively induced by calcineurin/NFAT inhibition and to enhance keratinocyte tumor formation, although the precise role of ATF3 in human skin cancer and possible mechanisms remain unknown. In this study, clinical analysis of 30 skin cancer patients and 30 normal donors revealed that ATF3 was accumulated in skin cancer tissues. Functional assays demonstrated that ATF3 significantly promoted skin cancer cell proliferation. Mechanically, ATF3 activated Stat3 phosphorylation in skin cancer cell through regulation of p53 expression. Moreover, the promotion effect of ATF3 on skin cancer cell proliferation was dependent on the p53-Stat3 signaling cascade. Together, the results indicate that ATF3 might promote skin cancer cell proliferation and enhance skin keratinocyte tumor development through inhibiting p53 expression and then activating Stat3 phosphorylation.

Selective inhibition of prostaglandin E2 receptors EP2 and EP4 inhibits adhesion of human endometriotic epithelial and stromal cells through suppression of integrin-mediated mechanisms.

Science.gov (United States)

Lee, JeHoon; Banu, Sakhila K; Burghardt, Robert C; Starzinski-Powitz, Anna; Arosh, Joe A

2013-03-01

Endometriosis is a chronic gynecological disease of reproductive age women characterized by the presence of functional endometrial tissues outside the uterine cavity. Interactions between the endometriotic cells and the peritoneal extracellular matrix proteins (ECM) are crucial mechanisms that allow adhesion of the endometriotic cells into peritoneal mesothelia. Prostaglandin E2 (PGE2) plays an important role in the pathogenesis of endometriosis. In previous studies, we have reported that selective inhibition of PGE2 receptors PTGER2 and PTGER4 decreases survival and invasion of human endometriotic epithelial and stromal cells through multiple mechanisms. Results of the present study indicates that selective inhibition of PTGER2- and PTGER4-mediated PGE2 signaling 1) decreases the expression and/or activity of specific integrin receptor subunits Itgb1 (beta1) and Itgb3 (beta3) but not Itgb5 (beta5), Itga1 (alpha1), Itga2 (alpha2), Itga5 (alpha5), and Itgav (alphav); 2) decreases integrin-signaling components focal adhesion kinase or protein kinase 2 (PTK2) and talin proteins; 3) inhibits interactions between Itgb1/Itgb3 subunits, PTK2, and talin and PTGER2/PTGER4 proteins through beta-arrestin-1 and Src kinase protein complex in human endometriotic epithelial cells 12Z and stromal cells 22B; and 4) decreases adhesion of 12Z and 22B cells to ECM collagen I, collagen IV, fibronectin, and vitronectin in a substrate-specific manner. These novel findings provide an important molecular framework for further evaluation of selective inhibition of PTGER2 and PTGER4 as potential nonsteroidal therapy to expand the spectrum of currently available treatment options for endometriosis in child-bearing age women.

Transformation of an Unclassified Myeloproliferative Neoplasm with a Rare BCR-JAK2 Fusion Transcript Resulting from the Translocation (9;22)(p24;q11)

OpenAIRE

A. N. Chamseddine; P. Etancelin; D. Penther; F. Parmentier; C. Kuadjovi; V. Camus; N. Contentin; P. Lenain; C. Bastard; H. Tilly; F. Jardin

2015-01-01

BCR-ABL1 negative myeloproliferative neoplasms (MPNs) are known to contain alterations of the tyrosine kinase JAK2 (located on 9p24) that result in constitutive activation of the encoded protein. JAK2 fusions are reported in acute and chronic leukemias of myeloid and lymphoid phenotypes. Here, we report an unclassified case of MPN (MPN-U) showing a t(9;22)(p24;q11), which generates a BCR-JAK2 fusion gene by fusing the BCR at intron 13 to JAK2 at intron 17 on the derivative chromosome 22. Most...

[Expression of JAK2V617F and MPLW515L/K mutation in 30 suspected cases of early myeloproliferative disorders].

Science.gov (United States)

Fan, Zheng; Zhang, Ri; Shen, Yi-Min; Fei, Hai-Rong; Zhu, Zi-Ling; Cen, Jian-Nong

2008-09-01

To investigate the prevalence of JAK2V617F and MPLW515L/K mutation in patients with slightly elevated platelets (BPC) or hemoglobin (Hb) not meeting the criteria of polycythemia vera (PV) or essential thrombocythemia (ET). Genomic DNA from bone marrow or blood mononuclear cells was screened with allele specific polymerase chain reaction (AS-PCR) for JAK2V617F and MPLW515L/K mutation. The history of thrombosis was assessed retrospectively by patients files. Of 30 patients, 14 (46.7%) were positive for the JAK2V617F mutation, none of them had the MPLW515L/ K. Five of these 14 patients had a history of thrombosis. Follow-up results were available in 22 patients. Among them, 12 patients with JAK2V617F mutation turned out to be MPD in 6-24 months; only 2 out of 10 patients without this mutation evolved to MPD. JAK2V617F mutation could be one of the diagnosis criteria of early MPD. No MPLW515L/K expression was found in early MPD.

Selective inhibition of monoamine oxidase A by purpurin, an anthraquinone.

Science.gov (United States)

Lee, Hyun Woo; Ryu, Hyung Won; Kang, Myung-Gyun; Park, Daeui; Oh, Sei-Ryang; Kim, Hoon

2017-03-01

Monoamine oxidase (MAO) catalyzes the oxidation of monoamines that act as neurotransmitters. During a target-based screening of natural products using two isoforms of recombinant human MAO-A and MAO-B, purpurin (an anthraquinone derivative) was found to potently and selectively inhibit MAO-A, with an IC 50 value of 2.50μM, and not to inhibit MAO-B. Alizarin (also an anthraquinone) inhibited MAO-A less potently with an IC 50 value of 30.1μM. Furthermore, purpurin was a reversible and competitive inhibitor of MAO-A with a K i value of 0.422μM. A comparison of their chemical structures suggested the 4-hydroxy group of purpurin might play an important role in its inhibition of MAO-A. Molecular docking simulation showed that the binding affinity of purpurin for MAO-A (-40.0kcal/mol) was higher than its affinity for MAO-B (-33.9kcal/mol), and that Ile 207 and Gly 443 of MAO-A were key residues for hydrogen bonding with purpurin. The findings of this study suggest purpurin is a potent, selective, reversible inhibitor of MAO-A, and that it be considered a new potential lead compound for development of novel reversible inhibitors of MAO-A (RIMAs). Copyright © 2017 Elsevier Ltd. All rights reserved.

Ruxolitinib combined with vorinostat suppresses tumor growth and alters metabolic phenotype in hematological diseases.

Science.gov (United States)

Civallero, Monica; Cosenza, Maria; Pozzi, Samantha; Sacchi, Stefano

2017-11-28

JAK-2 dysregulation plays an important role as an oncogenic driver, and is thus a promising therapeutic target in hematological malignancies. Ruxolitinib is a pyrrolo[2.3-d]pyrimidine derivative with inhibitory activity against JAK1 and JAK2, moderate activity against TYK2, and minor activity against JAK3. Vorinostat is an HDAC inhibitor that reduces JAK-2 expression, thus affecting JAK-2 mRNA expression and increasing JAK-2 proteasomal deterioration. Here we hypothesized that the combination of ruxolitinib and vorinostat could have synergistic effects against hematological disease. We tested combinations of low doses of ruxolitinib and vorinostat in 12 cell lines, and observed highly synergistic cytotoxic action in six cell lines, which was maintained for up to 120 h in the presence of stromal cells. The sensitivity of the six cell lines may be explained by the broad effects of the drug combination, which can affect various targets. Treatment with the combination of ruxolitinib and vorinostat appeared to induce a possible reversal of the Warburg effect, with associated ROS production, apoptotic events, and growth inhibition. Decreased glucose metabolism may have markedly sensitized the six more susceptible cell lines to combined treatment. Therapeutic inhibition of the JAK/STAT pathway seems to offer substantial anti-tumor benefit, and combined therapy with ruxolitinib and vorinostat may represent a promising novel therapeutic modality for hematological neoplasms.

Self-renewal of single mouse hematopoietic stem cells is reduced by JAK2V617F without compromising progenitor cell expansion.

Science.gov (United States)

Kent, David G; Li, Juan; Tanna, Hinal; Fink, Juergen; Kirschner, Kristina; Pask, Dean C; Silber, Yvonne; Hamilton, Tina L; Sneade, Rachel; Simons, Benjamin D; Green, Anthony R

2013-01-01

Recent descriptions of significant heterogeneity in normal stem cells and cancers have altered our understanding of tumorigenesis, emphasizing the need to understand how single stem cells are subverted to cause tumors. Human myeloproliferative neoplasms (MPNs) are thought to reflect transformation of a hematopoietic stem cell (HSC) and the majority harbor an acquired V617F mutation in the JAK2 tyrosine kinase, making them a paradigm for studying the early stages of tumor establishment and progression. The consequences of activating tyrosine kinase mutations for stem and progenitor cell behavior are unclear. In this article, we identify a distinct cellular mechanism operative in stem cells. By using conditional knock-in mice, we show that the HSC defect resulting from expression of heterozygous human JAK2V617F is both quantitative (reduced HSC numbers) and qualitative (lineage biases and reduced self-renewal per HSC). The defect is intrinsic to individual HSCs and their progeny are skewed toward proliferation and differentiation as evidenced by single cell and transplantation assays. Aged JAK2V617F show a more pronounced defect as assessed by transplantation, but mice that transform reacquire competitive self-renewal ability. Quantitative analysis of HSC-derived clones was used to model the fate choices of normal and JAK2-mutant HSCs and indicates that JAK2V617F reduces self-renewal of individual HSCs but leaves progenitor expansion intact. This conclusion is supported by paired daughter cell analyses, which indicate that JAK2-mutant HSCs more often give rise to two differentiated daughter cells. Together these data suggest that acquisition of JAK2V617F alone is insufficient for clonal expansion and disease progression and causes eventual HSC exhaustion. Moreover, our results show that clonal expansion of progenitor cells provides a window in which collaborating mutations can accumulate to drive disease progression. Characterizing the mechanism(s) of JAK2V617F

Thromboembolism with Janus Kinase (JAK) Inhibitors for Rheumatoid Arthritis: How Real is the Risk?

Science.gov (United States)

Scott, Ian C; Hider, Samantha L; Scott, David L

2018-03-02

Two different Janus kinase (JAK) inhibitors-baricitinib and tofacitinib-are effective and licensed in active rheumatoid arthritis (RA). There have been recent concerns about potential thromboembolic risks with these drugs. Concerns about baricitinib focus on clinical trial findings. Using all publically available data, we estimate thromboembolic risks are approximately five events per 1000 patient years with 4 mg baricitinib daily. Concerns about tofacitinib have been raised by analyses of the Federal Drug Administration Adverse Event Reporting System (FAERs). These show some evidence of increased risks of pulmonary thrombosis, though not pulmonary embolism or venous thrombosis. Observational studies suggest in the general population and non-RA controls there are one to four thromboembolic events per 1000 patient years. In RA, thromboembolic risks increase to three to seven per 1000 patient years. The impact of biologics and disease-modifying anti-rheumatic drugs (DMARDs) on disease risk appears minimal, and the number of thromboembolic events is between four and eight per 1000 patient years. In the short term, full details of thromboembolic events in trials of JAK inhibitors need to be published. As the numbers of thromboembolic events will be small and patients enrolled in trials are not representative of all RA patients who may receive JAK inhibitors, this information is unlikely to provide definitive answers. Consequently, in the longer term, large observational studies are needed to accurately quantify thromboembolic risks attributable to JAK inhibitors and other drugs used to treat RA, and differentiate these from risks attributable to RA itself and its comorbidities.

ATF3 inhibits adipocyte differentiation of 3T3-L1 cells

International Nuclear Information System (INIS)

Jang, Min Kyung; Kim, Cho Hee; Seong, Je Kyung; Jung, Myeong Ho

2012-01-01

Highlights: ► Overexpression of ATF3 inhibits adipocyte differentiation in 3T3-L1 cells. ► Overexpression of ATF3 represses C/EBPα expression. ► ATF3 directly binds to mouse C/EBPα promoter spanning from −1928 to −1907. ► ATF3 may play a role in hypoxia-mediated inhibition of adipocyte differentiation. -- Abstract: ATF3 is a stress-adaptive gene that regulates proliferation or apoptosis under stress conditions. However, the role of ATF3 is unknown in adipocyte cells. Therefore, in this study, we investigated the functional role of ATF3 in adipocytes. Both lentivirus-mediated overexpression of ATF3 and stably-overexpressed ATF3 inhibited adipocyte differentiation in 3T3-L1 cells, as revealed by decreased lipid staining with oil red staining and reduction in adipogenic genes. Thapsigargin treatment and overexpression of ATF3 decreased C/EBPα transcript and repressed the activity of the 3.6-kb mouse C/EBPα promoter, demonstrating that ATF3 downregulates C/EBPα expression. Transfection studies using mutant constructs containing 5′-deletions in the C/EBPα promoter revealed that a putative ATF/CRE element, GGATGTCA, is located between −1921 and −1914. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that ATF3 directly binds to mouse C/EBPα promoter spanning from −1928 to −1907. Both chemical hypoxia-mimetics or physical hypoxia led to reduce the C/EBPα mRNA and repress the promoter activity of the C/EBPα gene, whereas increase ATF3 mRNA, suggesting that ATF3 may contribute to the inhibition of adipocyte differentiation in hypoxia through downregulation of C/EBPα expression. Collectively, these results demonstrate that ATF3 represses the C/EBPα gene, resulting in inhibition of adipocyte differentiation, and thus plays a role in hypoxia-mediated inhibition of adipocyte differentiation.

Inhibition of CYP1 by berberine, palmatine, and jatrorrhizine: Selectivity, kinetic characterization, and molecular modeling

International Nuclear Information System (INIS)

Lo, Sheng-Nan; Chang, Yu-Ping; Tsai, Keng-Chang; Chang, Chia-Yu; Wu, Tian-Shung; Ueng, Yune-Fang

2013-01-01

Cytochrome P450 (P450, CYP) 1 family plays a primary role in the detoxification and bioactivation of polycyclic aromatic hydrocarbons. Human CYP1A1, CYP1A2, and CYP1B1 exhibit differential substrate specificity and tissue distribution. Berberine, palmatine, and jatrorrhizine are protoberberine alkaloids present in several medicinal herbs, such as Coptis chinensis (Huang-Lian) and goldenseal. These protoberberines inhibited CYP1A1.1- and CYP1B1.1-catalyzed 7-ethoxyresorufin O-deethylation (EROD) activities, whereas CYP1A2.1 activity was barely affected. Kinetic analysis revealed that berberine noncompetitively inhibited EROD activities of CYP1A1.1 and CYP1B1.1, whereas palmatine and jatrorrhizine caused either competitive or mixed type of inhibition. Among protoberberines, berberine caused the most potent and selective inhibitory effect on CYP1B1.1 with the least K i value of 44 ± 16 nM. Berberine also potently inhibited CYP1B1.1 activities toward 7-ethoxycoumarin and 7-methoxyresorufin, whereas the inhibition of benzo(a)pyrene hydroxylation activity was less pronounced. Berberine inhibited the polymorphic variants, CYP1B1.3 (V432L) and CYP1B1.4 (N453S), with IC 50 values comparable to that for CYP1B1.1 inhibition. Berberine-mediated inhibition was abolished by a mutation of Asn228 to Thr in CYP1B1.1, whereas the inhibition was enhanced by a reversal mutation of Thr223 to Asn in CYP1A2.1. This result in conjugation with the molecular modeling revealed the crucial role of hydrogen-bonding interaction of Asn228 on CYP1B1.1 with the methoxy moiety of berberine. These findings demonstrate that berberine causes a selective CYP1B1-inhibition, in which Asn228 appears to be crucial. The inhibitory effects of berberine on CYP1B1 activities toward structurally diverse substrates can be different. - Highlights: • Berberine preferentially inhibited CYP1B1 activity. • Berberine caused similar inhibitory effects on CYP1B1.1, CYP1B1.3 and CYP1B1.4. • Asn228 in CYP1B1 was an

Inhibition of CYP1 by berberine, palmatine, and jatrorrhizine: Selectivity, kinetic characterization, and molecular modeling

Energy Technology Data Exchange (ETDEWEB)

Lo, Sheng-Nan [National Research Institute of Chinese Medicine, Taipei 112, Taiwan, ROC (China); Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan, ROC (China); Chang, Yu-Ping; Tsai, Keng-Chang [National Research Institute of Chinese Medicine, Taipei 112, Taiwan, ROC (China); Chang, Chia-Yu [National Research Institute of Chinese Medicine, Taipei 112, Taiwan, ROC (China); Institute of Medical Sciences, Taipei Medical University, Taipei 101, Taiwan, ROC (China); Wu, Tian-Shung [Department of Chemistry, National Chung-Kung University, Tainan 701, Taiwan, ROC (China); Ueng, Yune-Fang, E-mail: ueng@nricm.edu.tw [National Research Institute of Chinese Medicine, Taipei 112, Taiwan, ROC (China); Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan, ROC (China); Institute of Medical Sciences, Taipei Medical University, Taipei 101, Taiwan, ROC (China)

2013-11-01

Cytochrome P450 (P450, CYP) 1 family plays a primary role in the detoxification and bioactivation of polycyclic aromatic hydrocarbons. Human CYP1A1, CYP1A2, and CYP1B1 exhibit differential substrate specificity and tissue distribution. Berberine, palmatine, and jatrorrhizine are protoberberine alkaloids present in several medicinal herbs, such as Coptis chinensis (Huang-Lian) and goldenseal. These protoberberines inhibited CYP1A1.1- and CYP1B1.1-catalyzed 7-ethoxyresorufin O-deethylation (EROD) activities, whereas CYP1A2.1 activity was barely affected. Kinetic analysis revealed that berberine noncompetitively inhibited EROD activities of CYP1A1.1 and CYP1B1.1, whereas palmatine and jatrorrhizine caused either competitive or mixed type of inhibition. Among protoberberines, berberine caused the most potent and selective inhibitory effect on CYP1B1.1 with the least K{sub i} value of 44 ± 16 nM. Berberine also potently inhibited CYP1B1.1 activities toward 7-ethoxycoumarin and 7-methoxyresorufin, whereas the inhibition of benzo(a)pyrene hydroxylation activity was less pronounced. Berberine inhibited the polymorphic variants, CYP1B1.3 (V432L) and CYP1B1.4 (N453S), with IC{sub 50} values comparable to that for CYP1B1.1 inhibition. Berberine-mediated inhibition was abolished by a mutation of Asn228 to Thr in CYP1B1.1, whereas the inhibition was enhanced by a reversal mutation of Thr223 to Asn in CYP1A2.1. This result in conjugation with the molecular modeling revealed the crucial role of hydrogen-bonding interaction of Asn228 on CYP1B1.1 with the methoxy moiety of berberine. These findings demonstrate that berberine causes a selective CYP1B1-inhibition, in which Asn228 appears to be crucial. The inhibitory effects of berberine on CYP1B1 activities toward structurally diverse substrates can be different. - Highlights: • Berberine preferentially inhibited CYP1B1 activity. • Berberine caused similar inhibitory effects on CYP1B1.1, CYP1B1.3 and CYP1B1.4. • Asn228 in CYP

Jak se z úspěchu stal neúspěch

Czech Academy of Sciences Publication Activity Database

Řípa, Milan

-, říjen (2015) Institutional support: RVO:61389021 Keywords : John Cockcroft * ZETA * Peter Thonemann * Harwell Subject RIV: BL - Plasma and Gas Discharge Physics http://www.vedaprozivot.cz/sd/novinky/hlavni-stranka/151007-jak-se-z-uspechu-stal-neuspech.html

JAK kinases are required for the bacterial RNA and poly I:C induced tyrosine phosphorylation of PKR

Science.gov (United States)

Bleiblo, Farag; Michael, Paul; Brabant, Danielle; Ramana, Chilakamarti V; Tai, TC; Saleh, Mazen; Parrillo, Joseph E; Kumar, Anand; Kumar, Aseem

2013-01-01

Discriminating the molecular patterns associated with RNA is central to innate immunity. The protein kinase PKR is a cytosolic sensor involved in the recognition of viral dsRNA and triggering interferon-induced signaling. Here, we identified bacterial RNA as a novel distinct pattern recognized by PKR. We show that the tyrosine phosphorylation of PKR induced by either bacterial RNA or poly I:C is impaired in mutant cells lacking TYK2, JAK1, or JAK2 kinases. PKR was found to be a direct substrate for the activated JAKs. Our results indicated that the double-stranded structures of bacterial RNA are required to fully activate PKR. These results suggest that bacterial RNA signaling is analogous in some respects to that of viral RNA and interferons and may have implications in bacterial immunity. PMID:23236554

Gene expression profiling of loss of TET2 and/or JAK2V617F mutant hematopoietic stem cells from mouse models of myeloproliferative neoplasms

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Takuro Kameda

2015-06-01

Full Text Available Myeloproliferative neoplasms (MPNs are clinically characterized by the chronic overproduction of differentiated peripheral blood cells and the gradual expansion of malignant intramedullary/extramedullary hematopoiesis. In MPNs mutations in JAK2 MPL or CALR are detected mutually exclusive in more than 90% of cases [1,2]. Mutations in them lead to the abnormal activation of JAK/STAT signaling and the autonomous growth of differentiated cells therefore they are considered as “driver” gene mutations. In addition to the above driver gene mutations mutations in epigenetic regulators such as TET2 DNMT3A ASXL1 EZH2 or IDH1/2 are detected in about 5%–30% of cases respectively [3]. Mutations in TET2 DNMT3A EZH2 or IDH1/2 commonly confer the increased self-renewal capacity on normal hematopoietic stem cells (HSCs but they do not lead to the autonomous growth of differentiated cells and only exhibit subtle clinical phenotypes [4,6–8,5]. It was unclear how mutations in such epigenetic regulators influenced abnormal HSCs with driver gene mutations how they influenced the disease phenotype or whether a single driver gene mutation was sufficient for the initiation of human MPNs. Therefore we focused on JAK2V617F and loss of TET2—the former as a representative of driver gene mutations and the latter as a representative of mutations in epigenetic regulators—and examined the influence of single or double mutations on HSCs (Lineage−Sca-1+c-Kit+ cells (LSKs by functional analyses and microarray whole-genome expression analyses [9]. Gene expression profiling showed that the HSC fingerprint genes [10] was statistically equally enriched in TET2-knockdown-LSKs but negatively enriched in JAK2V617F–LSKs compared to that in wild-type-LSKs. Double-mutant-LSKs showed the same tendency as JAK2V617F–LSKs in terms of their HSC fingerprint genes but the expression of individual genes differed between the two groups. Among 245 HSC fingerprint genes 100 were more

Association of TNF polymorphisms with JAK2 (V617F) myeloproliferative neoplasms in Brazilian patients.

Science.gov (United States)

Macedo, Luciana Conci; de Cesare Quintero, Fernanda; Pagliari-E-Silva, Sara; Pagnano, Katia Borgia Barbosa; Rodrigues, Camila; de Alencar, Josiane Bazzo; Sell, Ana Maria; Visentainer, Jeane Eliete Laguila

2016-03-01

The classical chromosome Philadelphia-negative myeloproliferative neoplasms (MPNs) are a group of disorders that share clinical, hematological, and histological features. Proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) are elevated in patients with MPN. The aim of this study was to verify the association between the polymorphisms of TNF gene (-308G/A and -238 G/A) in BCR-ABL-negative MPN in our population. Blood samples obtained from MPN patients were genotyped for the JAK2V617F mutation and both TNF polymorphisms using PCR-RFLP. Thirty three (26.8%) patients with polycythemia vera (PV), 35 (28.7%) essential thrombocythemia (ET), 22 (17.7%) primary myelofibrosis (PMF), and 33 (26.8%) with unclassifiable MPN (MPNu) were included in the study. The JAK2 V617F mutation was detected in 94 (76.42%) patients. Were observed a significant increase on the frequency of the TNF-238 GA genotype in MPN patients compared to controls (OR=2.21, 95% CI=1.02-4.80, P<0.04). The distribution of the genotypes and allelic frequencies of TNF-308 was significantly different among the MPNs, JAK2V617F positive, PV and PMF, and controls. Our data has demonstrated that the polymorphisms on TNF-238 GA, TNF-308 GA were associated to MPN development in this population, triggered by JAK2 V617F mutation. Copyright © 2015. Published by Elsevier Inc.

15-Deoxy-Δ12,14-Prostaglandin J2 regulates leukemia inhibitory factor signaling through JAK-STAT pathway in mouse embryonic stem cells

International Nuclear Information System (INIS)

Rajasingh, Johnson; Bright, John J.

2006-01-01

Embryonic stem (ES) cells are genetically normal, pluripotent cells, capable of self-renewal and differentiation into all cell lineages. While leukemia inhibitory factor (LIF) maintains pluripotency in mouse ES cells, retinoic acid and other nuclear hormones induce neuro-glial differentiation in mouse and human ES cells in culture. Peroxisome-proliferator-activated receptors (PPARs) are ligand-dependent nuclear receptor transcription factors that regulate cell growth and differentiation in many cell types. However, the role of PPARs in the regulation of ES cell growth and differentiation is not known. In this study, we show that LIF induces proliferation and self-renewal of mouse D3-ES cells in culture. However, treatment with 15-Deoxy-Δ 12,14 -Prostaglandin J 2 (15d-PGJ2), a natural ligand for PPARγ, or all-trans retinoic acid (ATRA) results in a dose-dependent decrease in proliferation and self-renewal in D3-ES cells. Immunoprecipitation and Western blot analyses showed that LIF induces tyrosine phosphorylation of JAK1, TYK2 and STAT3 in 30 min and treatment with 15d-PGJ2 or ATRA results in a dose-dependent decrease in LIF-induced phosphorylation of JAK1 and STAT3 in D3-ES cells. However, treatment of D3-ES cells with Ciglitazone or 15d-PGJ2 for 48 h in culture resulted in a dose-dependent increase in PPARγ protein expression. These results suggest that PPARγ agonists regulate LIF signaling through JAK-STAT pathway leading to growth and self-renewal of ES cells

Pathogenetic Role of JAK2 V617F Mutation in Chronic Myeloproliferative Disorders

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Hui-Chi Hsu

2007-03-01

Full Text Available The molecular pathogenesis of chronic myeloproliferative disorders (MPDs is poorly understood. The hematopoietic progenitor cells of patients with polycythemia vera (PV or essential thrombocythemia (ET are characterized by hypersensitiv-ity to hematopoietic growth factors and formation of endogenous erythroid colonies. Recently, 4 groups reported almost simultaneously Janus kinase 2 (JAK2 V617F mutation in more than 80% of PV patients, 30% of patients with ET and in about 50% of patients with idiopathic myelofibrosis. The identification of the JAK2 mutation represents a major advance in the understanding of the molecular pathogenesis of MPDs that will likely permit a new classification and the development of novel therapeutic strategies for these diseases.

DMPD: Crosstalk among Jak-STAT, Toll-like receptor, and ITAM-dependent pathways inmacrophage activation. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 17502339 Crosstalk among Jak-STAT, Toll-like receptor, and ITAM-dependent pathways inmacrophage...May 14. (.png) (.svg) (.html) (.csml) Show Crosstalk among Jak-STAT, Toll-like receptor, and ITAM-dependent pathways inmacrophage...T, Toll-like receptor, and ITAM-dependent pathways inmacrophage activation. Authors Hu X, Chen J, Wang L, Iv

The Process and Strategy for Developing Selective Histone Deacetylase 3 Inhibitors

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Fangyuan Cao

2018-03-01

Full Text Available Histone deacetylases (HDACs are epigenetic drug targets that have gained major scientific attention. Inhibition of these important regulatory enzymes is used to treat cancer, and has the potential to treat a host of other diseases. However, currently marketed HDAC inhibitors lack selectivity for the various HDAC isoenzymes. Several studies have shown that HDAC3, in particular, plays an important role in inflammation and degenerative neurological diseases, but the development of selective HDAC3 inhibitors has been challenging. This review provides an up-to-date overview of selective HDAC3 inhibitors, and aims to support the development of novel HDAC3 inhibitors in the future.

Frequency of janus associated kinase 2 (jak2) mutation in patients of bcr-abl negative myeloproliferative neoplasms

International Nuclear Information System (INIS)

Sadiq, M.A.; Ahmed, S.; Ali, N.

2013-01-01

To determine the frequency of Janus associated kinase 2 mutation in the patients of BCR-ABL negative classical myeloproliferative neoplasms. Study Design: Cross-sectional descriptive study Place and Duration of Study: Molecular Department of Haematology, Armed Forces Institute of Pathology (AFIP), Rawalpindi from Jul 2011 to Jul 2012. Patients and Methods: Ninety three consecutive patients of Polycythaemia vera (PV), Essential thrombocythaemia (ET) and Idiopathic myelofibrosis (IMF) diagnosed by the conventional haematological criteria were included in the study. All patients were screened for G-T point mutation (V617F) in the JAK2 gene on chromosome 9 by an allele specific PCR. Results: Out of the 93 myeloproliferative neoplasm (MPN) patients, 33(35%) had polycythaemia vera, 36(39%) had essential thrombocythaemia and 24(26%) had idiopathic myelofibrosis. JAK2 mutation was seen in 64/93 (69%) patients including 33/33(100%) in PV, 19/36(52.6%) in ET and 12/24(50%) in IMF. Conclusion: Classical myeloproliferative neoplasms are an important group of heamatological disorder in our country. JAK2 gene mutation is seen in significant proportion of these disorders (69%). JAK2 mutation analysis can be used to differentiate between polycythemia vera and secondary polycythemia in most cases with near certainty, where it was found in 100% of the cases. (author)

The selective estrogen receptor modulator raloxifene inhibits neutrophil extracellular trap formation.

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Roxana Flores

2016-12-01

Full Text Available Raloxifene is a selective estrogen receptor modulator typically prescribed for the prevention/treatment of osteoporosis in postmenopausal women. Although raloxifene is known to have anti-inflammatory properties, its effect on human neutrophils, the primary phagocytic leukocytes of the immune system, remain poorly understood. Here, through a screen of pharmacologically active small molecules, we find that raloxifene prevents neutrophil cell death in response to the classical activator phorbol 12-myristate 13-acetate (PMA, a compound known to induce formation of DNA-based neutrophil extracellular traps (NETs. Inhibition of PMA-induced NET production by raloxifene was confirmed using quantitative and imaging-based assays. Human neutrophils from both male and female donors express the nuclear estrogen receptors ERα and ERβ, known targets of raloxifene. Like raloxifene, selective antagonists of these receptors inhibit PMA-induced NET production. Furthermore, raloxifene inhibited PMA-induced ERK phosphorylation but not reactive oxygen species (ROS production, pathways known to be key modulators of NET production. Finally, we found that raloxifene inhibited PMA-induced, NET-based killing of the leading human bacterial pathogen, methicillin-resistant Staphylococcus aureus (MRSA. Our results reveal that raloxifene is a potent modulator of neutrophil function and NET production.

Effects of matrine on JAK-STAT signaling transduction pathways in ...

African Journals Online (AJOL)

The current study aims to investigate the effects of matrine on the JAK-STAT signaling transduction pathways in bleomycin (BLM)-induced pulmonary fibrosis (PF) and to explore its action mechanism. A total of 72 male C57BL/6 mice were randomized into the control, model, and treatment groups. PF models were ...

Scalable gastroscopic video summarization via similar-inhibition dictionary selection.

Science.gov (United States)

Wang, Shuai; Cong, Yang; Cao, Jun; Yang, Yunsheng; Tang, Yandong; Zhao, Huaici; Yu, Haibin

2016-01-01

This paper aims at developing an automated gastroscopic video summarization algorithm to assist clinicians to more effectively go through the abnormal contents of the video. To select the most representative frames from the original video sequence, we formulate the problem of gastroscopic video summarization as a dictionary selection issue. Different from the traditional dictionary selection methods, which take into account only the number and reconstruction ability of selected key frames, our model introduces the similar-inhibition constraint to reinforce the diversity of selected key frames. We calculate the attention cost by merging both gaze and content change into a prior cue to help select the frames with more high-level semantic information. Moreover, we adopt an image quality evaluation process to eliminate the interference of the poor quality images and a segmentation process to reduce the computational complexity. For experiments, we build a new gastroscopic video dataset captured from 30 volunteers with more than 400k images and compare our method with the state-of-the-arts using the content consistency, index consistency and content-index consistency with the ground truth. Compared with all competitors, our method obtains the best results in 23 of 30 videos evaluated based on content consistency, 24 of 30 videos evaluated based on index consistency and all videos evaluated based on content-index consistency. For gastroscopic video summarization, we propose an automated annotation method via similar-inhibition dictionary selection. Our model can achieve better performance compared with other state-of-the-art models and supplies more suitable key frames for diagnosis. The developed algorithm can be automatically adapted to various real applications, such as the training of young clinicians, computer-aided diagnosis or medical report generation. Copyright © 2015 Elsevier B.V. All rights reserved.

Mechanism of inhibition of mouse Slo3 (KCa 5.1) potassium channels by quinine, quinidine and barium.

Science.gov (United States)

Wrighton, David C; Muench, Stephen P; Lippiat, Jonathan D

2015-09-01

The Slo3 (KCa 5.1) channel is a major component of mammalian KSper (sperm potassium conductance) channels and inhibition of these channels by quinine and barium alters sperm motility. The aim of this investigation was to determine the mechanism by which these drugs inhibit Slo3 channels. Mouse (m) Slo3 (KCa 5.1) channels or mutant forms were expressed in Xenopus oocytes and currents recorded with 2-electrode voltage-clamp. Gain-of-function mSlo3 mutations were used to explore the state-dependence of the inhibition. The interaction between quinidine and mSlo3 channels was modelled by in silico docking. Several drugs known to block KSper also affected mSlo3 channels with similar levels of inhibition. The inhibition induced by extracellular barium was prevented by increasing the extracellular potassium concentration. R196Q and F304Y mutations in the mSlo3 voltage sensor and pore, respectively, both increased channel activity. The F304Y mutation did not alter the effects of barium, but increased the potency of inhibition by both quinine and quinidine approximately 10-fold; this effect was not observed with the R196Q mutation. Block of mSlo3 channels by quinine, quinidine and barium is not state-dependent. Barium inhibits mSlo3 outside the cell by interacting with the selectivity filter, whereas quinine and quinidine act from the inside, by binding in a hydrophobic pocket formed by the S6 segment of each subunit. Furthermore, we propose that the Slo3 channel activation gate lies deep within the pore between F304 in the S6 segment and the selectivity filter. © 2015 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.

Inhibition of AMPK catabolic action by GSK3

Science.gov (United States)

Suzuki, Tsukasa; Bridges, Dave; Nakada, Daisuke; Skiniotis, Georgios; Morrison, Sean J.; Lin, Jiandie; Saltiel, Alan R.; Inoki, Ken

2013-01-01

SUMMARY AMP-activated protein kinase (AMPK) regulates cellular energy homeostasis by inhibiting anabolic and activating catabolic processes. While AMPK activation has been extensively studied, mechanisms that inhibit AMPK remain elusive. Here we report that glycogen synthase kinase 3 (GSK3) inhibits AMPK function. GSK3 forms a stable complex with AMPK through interactions with the AMPK β regulatory subunit and phosphorylates the AMPK α catalytic subunit. This phosphorylation enhances the accessibility of the activation loop of the α subunit to phosphatases, thereby inhibiting AMPK kinase activity. Surprisingly, PI3K-Akt signaling, which is a major anabolic signaling and normally inhibits GSK3 activity, promotes GSK3 phosphorylation and inhibition of AMPK, thus revealing how AMPK senses anabolic environments in addition to cellular energy levels. Consistently, disrupting GSK3 function within the AMPK complex sustains higher AMPK activity and cellular catabolic processes even under anabolic conditions, indicating that GSK3 acts as a critical sensor for anabolic signaling to regulate AMPK. PMID:23623684

Hesperetin-7,3'-O-dimethylether selectively inhibits phosphodiesterase 4 and effectively suppresses ovalbumin-induced airway hyperresponsiveness with a high therapeutic ratio

Directory of Open Access Journals (Sweden)

Yang You-Lan

2011-11-01

Full Text Available Abstract Background Hesperetin was reported to selectively inhibit phosphodiesterase 4 (PDE4. While hesperetin-7,3'-O-dimethylether (HDME is a synthetic liposoluble hesperetin. Therefore, we were interested in investigating its selectivity on PDE4 and binding ability on high-affinity rolipram-binding sites (HARBs in vitro, and its effects on ovalbumin-induced airway hyperresponsiveness in vivo, and clarifying its potential for treating asthma and chronic obstructive pulmonary disease (COPD. Methods PDE1~5 activities were measured using a two-step procedure. The binding of HDME on high-affinity rolipram-binding sites was determined by replacing 2 nM [3H]-rolipram. AHR was assessed using the FlexiVent system and barometric plethysmography. Inflammatory cells were counted using a hemocytometer. Cytokines were determined using mouse T helper (Th1/Th2 cytokine CBA kits, and total immunoglobulin (IgE or IgG2a levels were done using ELISA method. Xylazine (10 mg/kg/ketamine (70 mg/kg-induced anesthesia was performed. Results HDME revealed selective phosphodiesterase 4 (PDE4 inhibition with a therapeutic (PDE4H/PDE4L ratio of 35.5 in vitro. In vivo, HDME (3~30 μmol/kg, orally (p.o. dose-dependently and significantly attenuated the airway resistance (RL and increased lung dynamic compliance (Cdyn, and decreased enhanced pause (Penh values induced by methacholine in sensitized and challenged mice. It also significantly suppressed the increases in the numbers of total inflammatory cells, macrophages, lymphocytes, neutrophils, and eosinophils, and levels of cytokines, including interleukin (IL-2, IL-4, IL-5, interferon-γ, and tumor necrosis factor-α in bronchoalveolar lavage fluid (BALF of these mice. In addition, HDME (3~30 μmol/kg, p.o. dose-dependently and significantly suppressed total and ovalbumin-specific immunoglobulin (IgE levels in the BALF and serum, and enhanced IgG2a level in the serum of these mice. Conclusions HDME exerted anti

Bone marrow histomorphology and JAK2 mutation status in essential thrombocythemia

DEFF Research Database (Denmark)

Stauffer Larsen, Thomas; Hasselbalch, Hans Carl; Pallisgaard, Niels

2007-01-01

for evaluation. 14 patients were reclassified as having prefibrotic idiopathic myelofibrosis (IMF), whilst the ET diagnosis was sustained in 19 patients. The individual bone marrow parameters of the reviewed diagnosis showed no correlation with JAK2 V617F mutation status, which was determined by a highly...

Genetic Alterations of the Thrombopoietin/MPL/JAK2 Axis Impacting Megakaryopoiesis.

Science.gov (United States)

Plo, Isabelle; Bellanné-Chantelot, Christine; Mosca, Matthieu; Mazzi, Stefania; Marty, Caroline; Vainchenker, William

2017-01-01

Megakaryopoiesis is an original and complex cell process which leads to the formation of platelets. The homeostatic production of platelets is mainly regulated and controlled by thrombopoietin (TPO) and the TPO receptor (MPL)/JAK2 axis. Therefore, any hereditary or acquired abnormality affecting this signaling axis can result in thrombocytosis or thrombocytopenia. Thrombocytosis can be due to genetic alterations that affect either the intrinsic MPL signaling through gain-of-function (GOF) activity ( MPL, JAK2, CALR ) and loss-of-function (LOF) activity of negative regulators ( CBL, LNK ) or the extrinsic MPL signaling by THPO GOF mutations leading to increased TPO synthesis. Alternatively, thrombocytosis may paradoxically result from mutations of MPL leading to an abnormal MPL trafficking, inducing increased TPO levels by alteration of its clearance. In contrast, thrombocytopenia can also result from LOF THPO or MPL mutations, which cause a complete defect in MPL trafficking to the cell membrane, impaired MPL signaling or stability, defects in the TPO/MPL interaction, or an absence of TPO production.

Tofacitinib attenuates arthritis manifestations and reduces the pathogenic CD4 T cells in adjuvant arthritis rats.

Science.gov (United States)

Gertel, Smadar; Mahagna, Hussein; Karmon, Gidi; Watad, Abdulla; Amital, Howard

2017-11-01

Rheumatoid arthritis (RA) is an autoimmune disease characterized by pronounced inflammation and leukocyte infiltration in affected joints. Tofacitinib is new agent, a selective inhibitor of Janus kinase (JAK) signaling pathways mediated by JAK1 and JAK3 and inhibits the key transcription factors STAT1 and STAT3. We investigated the action mechanisms of tofacitinib in rats with adjuvant-induced-arthritis (AIA). AIA-rats were treated orally with tofacitinib or with methotrexate. Arthritis severity and serum C-reactive protein (CRP) levels were evaluated, splenic cells were examined by flow cytometry and cytokines were analyzed by real-time PCR. Tofacitinib markedly reduced the clinical status of treated rats in comparison to control group. Reduced joints inflammation and down-regulated serum CRP levels reflected the clinical manifestations of the treated rats. Tofacitinib down-regulated significantly the frequency of CD4 + IFN-γ + T cells and reduced IL-1β mRNA expression levels in the spleen of the treated rats. These results show that tofacitinib attenuated arthritis severity, modified splenic populations and cytokine imbalance. Copyright © 2017. Published by Elsevier Inc.

Histone deacetylase 3 inhibition improves glycaemia and insulin secretion in obese diabetic rats

DEFF Research Database (Denmark)

Lundh, Morten; Galbo, Thomas; Poulsen, Steen Seier

2015-01-01

Failure of pancreatic β cells to compensate for insulin resistance is a prerequisite for the development of type 2 diabetes. Sustained elevated circulating levels of free fatty acids and glucose contribute to β-cell failure. Selective inhibition of Histone deacetylase (HDAC)-3 protects pancreatic β...... cells against inflammatory and metabolic insults in vitro. Here we tested the ability of a selective HDAC3 inhibitor, BRD3308, to reduce hyperglycemia and increase insulin secretion in an animal model of type 2 diabetes. At diabetes onset, an ambulatory hyperglycemic clamp was performed. HDAC3......3 as a key therapeutic target for β-cell protection in type 2 diabetes....

Concurrent MPL515 and JAK2V617F mutations in myelofibrosis: chronology of clonal emergence and changes in mutant allele burden over time.

Science.gov (United States)

Lasho, Terra L; Pardanani, Animesh; McClure, Rebecca F; Mesa, Ruben A; Levine, Ross L; Gilliland, D Gary; Tefferi, Ayalew

2006-12-01

MPLW515L/K and JAK2V617F can co-exist in myelofibrosis with myeloid metaplasia (MMM). The chronology of clonal emergence was studied in three such cases using serially stored bone marrow. At diagnosis, a major MPL515 mutant clone was accompanied by a minor JAK2V617F clone in all three instances. At 25 time points over a period of 4-8 years, allele burden fluctuated but remained high for MPLW515L/K and low for JAK2V617F. We conclude that MPLW515L/K and JAK2V617F are both early events in MMM and allele burden, rather than the mere presence of these mutations, might be relevant to phenotypic variation in myeloproliferative disorders.

Segregating Top-Down Selective Attention from Response Inhibition in a Spatial Cueing Go/NoGo Task: An ERP and Source Localization Study.

Science.gov (United States)

Hong, Xiangfei; Wang, Yao; Sun, Junfeng; Li, Chunbo; Tong, Shanbao

2017-08-29

Successfully inhibiting a prepotent response tendency requires the attentional detection of signals which cue response cancellation. Although neuroimaging studies have identified important roles of stimulus-driven processing in the attentional detection, the effects of top-down control were scarcely investigated. In this study, scalp EEG was recorded from thirty-two participants during a modified Go/NoGo task, in which a spatial-cueing approach was implemented to manipulate top-down selective attention. We observed classical event-related potential components, including N2 and P3, in the attended condition of response inhibition. While in the ignored condition of response inhibition, a smaller P3 was observed and N2 was absent. The correlation between P3 and CNV during the foreperiod suggested an inhibitory role of P3 in both conditions. Furthermore, source analysis suggested that P3 generation was mainly localized to the midcingulate cortex, and the attended condition showed increased activation relative to the ignored condition in several regions, including inferior frontal gyrus, middle frontal gyrus, precentral gyrus, insula and uncus, suggesting that these regions were involved in top-down attentional control rather than inhibitory processing. Taken together, by segregating electrophysiological correlates of top-down selective attention from those of response inhibition, our findings provide new insights in understanding the neural mechanisms of response inhibition.

Svět očima zvířat aneb jak ptáci vnímají barvy

Czech Academy of Sciences Publication Activity Database

Šulc, Michal; Honza, Marcel

2014-01-01

Roč. 62, č. 4 (2014), s. 180-183 ISSN 0044-4812 R&D Projects: GA MŠk EE2.3.35.0026 Institutional support: RVO:68081766 Keywords : Birds * Colouration Subject RIV: EG - Zoology http://ziva.avcr.cz/files/ziva/pdf/svet-ocima-zvirat-aneb-jak-ptaci-vnimaji-barvy.pdf

JAK kinases are required for the bacterial RNA and poly I:C induced tyrosine phosphorylation of PKR

OpenAIRE

Bleiblo, Farag; Michael, Paul; Brabant, Danielle; Ramana, Chilakamarti V; Tai, TC; Saleh, Mazen; Parrillo, Joseph E; Kumar, Anand; Kumar, Aseem

2012-01-01

Discriminating the molecular patterns associated with RNA is central to innate immunity. The protein kinase PKR is a cytosolic sensor involved in the recognition of viral dsRNA and triggering interferon-induced signaling. Here, we identified bacterial RNA as a novel distinct pattern recognized by PKR. We show that the tyrosine phosphorylation of PKR induced by either bacterial RNA or poly I:C is impaired in mutant cells lacking TYK2, JAK1, or JAK2 kinases. PKR was found to be a direct substra...

JAK/STAT-1 Signaling Is Required for Reserve Intestinal Stem Cell Activation during Intestinal Regeneration Following Acute Inflammation

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Camilla A. Richmond

2018-01-01

Full Text Available The intestinal epithelium serves as an essential barrier to the outside world and is maintained by functionally distinct populations of rapidly cycling intestinal stem cells (CBC ISCs and slowly cycling, reserve ISCs (r-ISCs. Because disruptions in the epithelial barrier can result from pathological activation of the immune system, we sought to investigate the impact of inflammation on ISC behavior during the regenerative response. In a murine model of αCD3 antibody-induced small-intestinal inflammation, r-ISCs proved highly resistant to injury, while CBC ISCs underwent apoptosis. Moreover, r-ISCs were induced to proliferate and functionally contribute to intestinal regeneration. Further analysis revealed that the inflammatory cytokines interferon gamma and tumor necrosis factor alpha led to r-ISC activation in enteroid culture, which could be blocked by the JAK/STAT inhibitor, tofacitinib. These results highlight an important role for r-ISCs in response to acute intestinal inflammation and show that JAK/STAT-1 signaling is required for the r-ISC regenerative response.

Structure-Based Design of a Novel Series of Potent, Selective Inhibitors of the Class I Phosphatidylinositol 3-Kinases

Energy Technology Data Exchange (ETDEWEB)

Smith, Adrian L.; D’Angelo, Noel D.; Bo, Yunxin Y.; Booker, Shon K.; Cee, Victor J.; Herberich, Brad; Hong, Fang-Tsao; Jackson, Claire L.M.; Lanman, Brian A.; Liu, Longbin; Nishimura, Nobuko; Pettus, Liping H.; Reed, Anthony B.; Tadesse, Seifu; Tamayo, Nuria A.; Wurz, Ryan P.; Yang, Kevin; Andrews, Kristin L.; Whittington, Douglas A.; McCarter, John D.; Miguel, Tisha San; Zalameda, Leeanne; Jiang, Jian; Subramanian, Raju; Mullady, Erin L.; Caenepeel, Sean; Freeman, Daniel J.; Wang, Ling; Zhang, Nancy; Wu, Tian; Hughes, Paul E.; Norman, Mark H. (Amgen)

2012-09-17

A highly selective series of inhibitors of the class I phosphatidylinositol 3-kinases (PI3Ks) has been designed and synthesized. Starting from the dual PI3K/mTOR inhibitor 5, a structure-based approach was used to improve potency and selectivity, resulting in the identification of 54 as a potent inhibitor of the class I PI3Ks with excellent selectivity over mTOR, related phosphatidylinositol kinases, and a broad panel of protein kinases. Compound 54 demonstrated a robust PD-PK relationship inhibiting the PI3K/Akt pathway in vivo in a mouse model, and it potently inhibited tumor growth in a U-87 MG xenograft model with an activated PI3K/Akt pathway.

Phenyl Ring-Substituted Lobelane Analogs: Inhibition of [3H]Dopamine Uptake at the Vesicular Monoamine Transporter-2

OpenAIRE

Nickell, Justin R.; Zheng, Guangrong; Deaciuc, Agripina G.; Crooks, Peter A.; Dwoskin, Linda P.

2011-01-01

Lobeline attenuates the behavioral effects of methamphetamine via inhibition of the vesicular monoamine transporter (VMAT2). To increase selectivity for VMAT2, chemically defunctionalized lobeline analogs, including lobelane, were designed to eliminate nicotinic acetylcholine receptor affinity. The current study evaluated the ability of lobelane analogs to inhibit [3H]dihydrotetrabenazine (DTBZ) binding to VMAT2 and [3H]dopamine (DA) uptake into isolated synaptic vesicles and determined the m...

Laser isotope separation using selective inhibition and encouragement of dimer formation

International Nuclear Information System (INIS)

Kivel, B.

1979-01-01

Method and apparatus for inhibiting dimer formation of molecules of a selected isotope type in a cooled flow of gas to enhance the effectiveness of mass difference isotope separation techniques are described. Molecules in the flow containing atoms of the selected isotope type are selectively excited by infrared radiation in order to inhibit the formation of dimers and larger clusters of such molecules, while the molecules not containing atoms of the selected, excited type are encouraged to form dimers and higher order aggregates by the cooling of the gaseous flow. The molecules with the excited isotope will predominate in monomers and will constitute the enriched product stream, while the aggregated group comprising molecules having the unexcited isotope will predominate in dimers and larger clusters of molecules, forming the tails stream. The difference in diffusion coefficientts between particles of the excited and unexcited isotopes is enhanced by the greater mass differences resulting from aggregation of unexcited particles into dimers and larger clusters. Prior art separation techniques which exploit differences in isotopic diffusion rates will consequently exhibit enhanced enrichment per stage by the utilization of the present invention

Characterization and Prognosis Significance of JAK2 (V617F), MPL, and CALR Mutations in Philadelphia-Negative Myeloproliferative Neoplasms

OpenAIRE

Singdong, Roongrudee; Siriboonpiputtana, Teerapong; Chareonsirisuthigul, Takol; Kongruang, Adcharee; Limsuwanachot, Nittaya; Sirirat, Tanasan; Chuncharunee, Suporn; Rerkamnuaychoke, Budsaba

2016-01-01

Background: The discovery of somatic acquired mutations of JAK2 (V617F) in Philadelphia-negative myeloproliferative neoplasms (Ph-negative MPNs) including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) has not only improved rational disease classification and prognostication but also brings new understanding insight into the pathogenesis of diseases. Dosage effects of the JAK2 (V617F) allelic burden in Ph-negative MPNs may partially influence clinical ...

Role of STAT3 in Transformation and Drug Resistance in CML

International Nuclear Information System (INIS)

Nair, Rajesh R.; Tolentino, Joel H.; Hazlehurst, Lori A.

2012-01-01

Chronic myeloid leukemia (CML) is initially driven by the bcr–abl fusion oncoprotein. The identification of bcr–abl led to the discovery and rapid translation into the clinic of bcr–abl kinase inhibitors. Although, bcr–abl inhibitors are efficacious, experimental evidence indicates that targeting bcr–abl is not sufficient for elimination of minimal residual disease found within the bone marrow (BM). Experimental evidence indicates that the failure to eliminate the leukemic stem cell contributes to persistent minimal residual disease. Thus curative strategies will likely need to focus on strategies where bcr–abl inhibitors are given in combination with agents that specifically target the leukemic stem cell or the leukemic stem cell niche. One potential target to be exploited is the Janus kinase (JAK)/signal transducers and activators of transcription 3 (STAT3) pathway. Recently using STAT3 conditional knock-out mice it was shown that STAT3 is critical for initiating the disease. Interestingly, in the absence of treatment, STAT3 was not shown to be required for maintenance of the disease, suggesting that STAT3 is required only in the tumor initiating stem cell population (Hoelbl et al., 2010). In the context of the BM microenvironment, STAT3 is activated in a bcr–abl independent manner by the cytokine milieu. Activation of JAK/STAT3 was shown to contribute to cell survival even in the event of complete inhibition of bcr–abl activity within the BM compartment. Taken together, these studies suggest that JAK/STAT3 is an attractive therapeutic target for developing strategies for targeting the JAK–STAT3 pathway in combination with bcr–abl kinase inhibitors and may represent a viable strategy for eliminating or reducing minimal residual disease located in the BM in CML.

Role of STAT3 in Transformation and Drug Resistance in CML

Energy Technology Data Exchange (ETDEWEB)

Nair, Rajesh R.; Tolentino, Joel H.; Hazlehurst, Lori A., E-mail: lori.hazlehurst@moffitt.org [Molecular Oncology Program, H. Lee Moffitt Cancer Center, Tampa, FL (United States)

2012-04-10

Chronic myeloid leukemia (CML) is initially driven by the bcr–abl fusion oncoprotein. The identification of bcr–abl led to the discovery and rapid translation into the clinic of bcr–abl kinase inhibitors. Although, bcr–abl inhibitors are efficacious, experimental evidence indicates that targeting bcr–abl is not sufficient for elimination of minimal residual disease found within the bone marrow (BM). Experimental evidence indicates that the failure to eliminate the leukemic stem cell contributes to persistent minimal residual disease. Thus curative strategies will likely need to focus on strategies where bcr–abl inhibitors are given in combination with agents that specifically target the leukemic stem cell or the leukemic stem cell niche. One potential target to be exploited is the Janus kinase (JAK)/signal transducers and activators of transcription 3 (STAT3) pathway. Recently using STAT3 conditional knock-out mice it was shown that STAT3 is critical for initiating the disease. Interestingly, in the absence of treatment, STAT3 was not shown to be required for maintenance of the disease, suggesting that STAT3 is required only in the tumor initiating stem cell population (Hoelbl et al., 2010). In the context of the BM microenvironment, STAT3 is activated in a bcr–abl independent manner by the cytokine milieu. Activation of JAK/STAT3 was shown to contribute to cell survival even in the event of complete inhibition of bcr–abl activity within the BM compartment. Taken together, these studies suggest that JAK/STAT3 is an attractive therapeutic target for developing strategies for targeting the JAK–STAT3 pathway in combination with bcr–abl kinase inhibitors and may represent a viable strategy for eliminating or reducing minimal residual disease located in the BM in CML.

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