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1.  Anti-inflammatory activity and neutrophil reductions mediated by the JAK1/JAK3 inhibitor, CP-690,550, in rat adjuvant-induced arthritis 
The Janus kinase (JAK) family of tyrosine kinases includes JAK1, JAK2, JAK3 and TYK2, and is required for signaling through Type I and Type II cytokine receptors. CP-690,550 is a potent and selective JAK inhibitor currently in clinical trials for rheumatoid arthritis (RA) and other autoimmune disease indications. In RA trials, dose-dependent decreases in neutrophil counts (PBNC) were observed with CP-690,550 treatment. These studies were undertaken to better understand the relationship between JAK selectivity and PBNC decreases observed with CP-690,550 treatment.
Potency and selectivity of CP-690,550 for mouse, rat and human JAKs was evaluated in a panel of in vitro assays. The effect of CP-690,550 on granulopoiesis from progenitor cells was also assessed in vitro using colony forming assays. In vivo the potency of orally administered CP-690,550 on arthritis (paw edema), plasma cytokines, PBNC and bone marrow differentials were evaluated in the rat adjuvant-induced arthritis (AIA) model.
CP-690,550 potently inhibited signaling through JAK1 and JAK3 with 5-100 fold selectivity over JAK2 in cellular assays, despite inhibiting all four JAK isoforms with nM potency in in vitro enzyme assays. Dose-dependent inhibition of paw edema was observed in vivo with CP-690,550 treatment. Plasma cytokines (IL-6 and IL-17), PBNC, and bone marrow myeloid progenitor cells were elevated in the context of AIA disease. At efficacious exposures, CP-690,550 returned all of these parameters to pre-disease levels. The plasma concentration of CP-690,550 at efficacious doses was above the in vitro whole blood IC50 of JAK1 and JAK3 inhibition, but not that of JAK2.
Results from this investigation suggest that CP-690,550 is a potent inhibitor of JAK1 and JAK3 with potentially reduced cellular potency for JAK2. In rat AIA, as in the case of human RA, PBNC were decreased at efficacious exposures of CP-690,550. Inflammatory end points were similarly reduced, as judged by attenuation of paw edema and cytokines IL-6 and IL-17. Plasma concentration at these exposures was consistent with inhibition of JAK1 and JAK3 but not JAK2. Decreases in PBNC following CP-690,550 treatment may thus be related to attenuation of inflammation and are likely not due to suppression of granulopoiesis through JAK2 inhibition.
PMCID: PMC2928212  PMID: 20701804
2.  Modulation of Innate and Adaptive Immune Responses by Tofacitinib (CP-690,550) 
Inhibitors of the JAK family of non-receptor tyrosine kinases have demonstrated clinical efficacy in rheumatoid arthritis and other inflammatory disorders; however, the precise mechanisms by which JAK inhibition improves inflammatory immune responses remain unclear. Here we examined the mode of action of tofacitinib (CP-690,550) on JAK/STAT signaling pathways involved in adaptive and innate immune responses. To determine the extent of inhibition of specific JAK/STAT-dependent pathways, we analyzed cytokine stimulation of mouse and human T cells in vitro. We also investigated the consequences of CP-690,550 treatment on Th cell differentiation of naïve murine CD4+ T cells. CP-690,550 inhibited IL-4-dependent Th2 cell differentiation, and interestingly also interfered with Th17 cell differentiation. Expression of IL-23 receptor and of the Th17 cytokines IL-17A, IL-17F and IL-22 were blocked when naïve Th cells were stimulated with IL-6 and IL-23. In contrast, IL-17A-production was enhanced when Th17 cells were differentiated in the presence of TGF-β. Moreover, CP-690,550 also prevented activation of STAT1, induction of T-bet and subsequent generation of Th1 cells. In a model of established arthritis, CP-690,550 rapidly improved disease by inhibiting production of inflammatory mediators and suppressing STAT1-dependent genes in joint tissue. Furthermore, efficacy in this disease model correlated with inhibition of both JAK1 and JAK3 signaling pathways. CP-690,550 also modulated innate responses to LPS in vivo through a mechanism likely involving inhibition of STAT1 signaling. Thus, CP-690,550 may improve autoimmune diseases and prevent transplant rejection by suppressing the differentiation of pathogenic Th1 and Th17 cells, as well as innate immune cell signaling.
PMCID: PMC3108067  PMID: 21383241
3.  CP690,550 inhibits oncostatin M-induced JAK/STAT signaling pathway in rheumatoid synoviocytes 
Interleukin (IL)-6-type cytokines exert their effects through activation of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling cascade. The JAK/STAT pathways play an important role in rheumatoid arthritis, since JAK inhibitors have exhibited dramatic effects on rheumatoid arthritis (RA) in clinical trials. In this study, we investigated the molecular effects of a small molecule JAK inhibitor, CP690,550 on the JAK/STAT signaling pathways and examined the role of JAK kinases in rheumatoid synovitis.
Fibroblast-like synoviocytes (FLS) were isolated from RA patients and stimulated with recombinant oncostatin M (OSM). The cellular supernatants were analyzed using cytokine protein chips. IL-6 mRNA and protein expression were analyzed by real-time PCR method and ELISA, respectively. Protein phosphorylation of rheumatoid synoviocytes was assessed by Western blot using phospho-specific antibodies.
OSM was found to be a potent inducer of IL-6 in FLS. OSM stimulation elicited rapid phosphorylation of STATs suggesting activation of the JAK/STAT pathway in FLS. CP690,550 pretreatment completely abrogated the OSM-induced production of IL-6, as well as OSM-induced JAK/STAT, and activation of mitogen-activated kinases (MAPKs) in FLS.
These findings suggest that IL-6-type cytokines contribute to rheumatoid synovitis through activation of the JAK/STAT pathway in rheumatoid synoviocytes. Inhibition of these pro-inflammatory signaling pathways by CP690,550 could be important in the treatment of RA.
PMCID: PMC3218881  PMID: 21548952
4.  NSC114792, a novel small molecule identified through structure-based computational database screening, selectively inhibits JAK3 
Molecular Cancer  2010;9:36.
Human or animals lacking either JAK3 or the common gamma chain (γc) expression display severe combined immunodeficiency disease, indicating the crucial role of JAK3 in T-cell development and the homeostasis of the immune system. JAK3 has also been suggested to contribute to the pathogenesis of tumorigenesis. Recent studies identified activating JAK3 mutations in patients with various hematopoietic malignancies, including acute megakaryoblastic leukemia. Importantly, functional analyses of some of those JAK3 mutations have been shown to cause lethal hematopoietic malignancies in animal models. These observations make JAK3 an ideal therapeutic target for the treatment of various human diseases. To identify novel small molecule inhibitors of JAK3, we performed structure-based virtual screen using the 3D structure of JAK3 kinase domain and the NCI diversity set of compounds.
We identified NSC114792 as a lead compound. This compound directly blocked the catalytic activity of JAK3 but not that of other JAK family members in vitro. In addition, treatment of 32D/IL-2Rβ cells with the compound led to a block in IL-2-dependent activation of JAK3/STAT5 but not IL-3-dependent activation of JAK2/STAT5. Consistent with the specificity of NSC114792 for JAK3, it selectively inhibited persistently-activated JAK3, but failed to affect the activity of other JAK family members and other oncogenic kinases in various cancer cell lines. Finally, we showed that NSC114792 decreases cell viability by inducing apoptosis through down-regulating anti-apoptotic gene expression only in cancer cells harboring persistently-active JAK3.
NSC114792 is a lead compound that selectively inhibits JAK3 activity. Therefore, our study suggests that this small molecule inhibitor of JAK3 can be used as a starting point to develop a new class of drugs targeting JAK3 activity, and may have therapeutic potential in various diseases that are caused by aberrant JAK3 activity.
PMCID: PMC2830973  PMID: 20149240
5.  Division of labor by dual feedback regulators controls JAK2/STAT5 signaling over broad ligand range 
Quantitative analysis of time-resolved data in primary erythroid progenitor cells reveals that a dual negative transcriptional feedback mechanism underlies the ability of STAT5 to respond to the broad spectrum of physiologically relevant Epo concentrations.
A mathematical dual feedback model of the Epo-induced JAK2/STAT5 signaling pathway was calibrated with extensive time-resolved quantitative data sets from immunoblotting, mass spectrometry and qRT–PCR experiments in primary erythroid progenitor cells.We show that the amount of nuclear phosphorylated STAT5 integrated for 60 min post Epo stimulation directly correlates with the fraction of surviving cells 24 h later.CIS and SOCS3 were identified as the most relevant transcriptional feedback regulators of JAK2/STAT5 signaling in primary erythroid progenitor cells. Applying the model, we revealed that CIS-mediated inhibitory effects are most important at low ligand concentrations, whereas SOCS3 inhibition is more effective at high ligand doses.The distinct modes of inhibition of CIS and SOCS3 at various Epo concentrations provide a strategy for achieving control of JAK2/STAT5 signaling over the entire range of physiological Epo concentrations.
Cells interpret information encoded by extracellular stimuli through the activation of intracellular signaling networks and translate this information into cellular decisions. A prime example for a system that is exposed to extremely variable ligand concentrations is the erythroid lineage. The key regulator Erythropoietin (Epo) facilitates continuous renewal of erythrocytes at low basal levels but also secures compensation in case of, e.g., blood loss through an up to 1000-fold increase in hormone concentration. The Epo receptor (EpoR) is expressed on erythroid progenitor cells at the colony forming unit erythroid (CFU-E) stage. Stimulation of these cells with Epo leads to rapid but transient activation of receptor and JAK2 phosphorylation followed by phosphorylation of the latent transcription factor STAT5. Although STAT5 is known to be an essential regulator of survival and differentiation of erythroid progenitor cells, a quantitative link between the dynamic properties of STAT5 signaling and survival decisions remained unknown. STAT5-mediated responses in CFU-E cells are modulated by multiple attenuation mechanisms that operate on different time scales. Fast-acting mechanisms such as depletion of Epo by rapid receptor turnover and recruitment of the phosphatase SHP-1 control the initial signal amplitude at the receptor level. Transcriptional feedback regulators such as suppressor of cytokine signaling (SOCS) family members CIS and SOCS3 operate at a slower time scale. Despite the ample knowledge of the individual components involved, only little is known about the specific contributions of these regulators in controlling dynamic properties of STAT5 in response to a broad range of input signals. Therefore, dynamic pathway modeling is required to understand the complex regulatory network of feedback regulators.
To address these questions, we established a dual negative feedback model of JAK2/STAT5 signaling in primary erythroid progenitor cells isolated from mouse fetal livers. We provide a large data set of JAK2/STAT5 signaling dynamics employing quantitative immunoblotting, mass spectrometry and quantitative RT–PCR measured under different perturbation conditions to calibrate our model (Figure 3). The structure of our model was constructed to comprise the minimal number of parameters necessary to explain the data. Thereby, we aimed at a model with fully identifiable parameters that are essential to obtain high predictive power. Parameter identifiability was analyzed by the profile likelihood approach. Applying this method, we could establish a dual negative feedback model of JAK2-STAT5 signaling with structurally and in most cases practically identifiable parameters.
A major bottle-neck in combining signal transduction events with cellular phenotypes is the discrepancy in the time scale and stimuli concentrations that are applied in the different experiments. The sensitivity of biochemical assays to determine phosphorylation events within minutes or hours after stimulation is usually lower than the threshold of sensitivity in assays to determine the physiological response after one or more days. Facilitated by the model, we were able to compute the integrated response of JAK2/STAT5 signaling components for experimentally unaddressable Epo concentrations. Our results demonstrate that the integrated response of pSTAT5 in the nucleus accurately correlates with the experimentally determined survival of CFU-E cells. This provides a quantitative link of the dependency of primary CFU-E cells on pSTAT5 activation dynamics. By correlation analysis, we could identify the early signaling phase (⩽1 h) of STAT5 to be the most predictive for the fraction of surviving cells, which was determined ∼24 h later. Thus, we hypothesize that as a general principle in apoptotic decisions, ligand concentrations translated into kinetic-encoded information of early signaling events downstream of receptors can be predictive for survival decisions 24 h later.
After the first hour of stimulation, it is important to constrain signaling to a residual steady-state level. Constitutive phosphorylation of the JAK2/STAT5 pathway has a crucial role in the onset of polycythemia vera (PV), a disease associated with Epo-independent erythroid differentiation. The two identified transcriptional feedback proteins, CIS and SOCS3, are responsible for adjusting the phosphorylation level of STAT5 after 1 h of stimulation. Since the Epo input signal can vary over a broad range of ligand concentrations, we asked how CIS and SOCS3 can facilitate control of STAT5 long-term phosphorylation levels over the entire physiological relevant hormone concentrations. By using model simulations, we revealed that the two feedbacks are most effective at different Epo concentration ranges. Predicted by our mathematical model, the major role of CIS in modulating STAT5 phosphorylation levels is at low, basal Epo concentrations, whereas SOCS3 is essential to control the STAT5 phosphorylation levels at high Epo doses (Figure 6). As a potential molecular mechanism of this dose-dependent inhibitory effect, we could identify the quantity of pJAK2 relative to pEpoR that increases with higher Epo concentrations. Since SOCS3 can inhibit JAK2 directly via its KIR domain to attenuate downstream STAT5 activation, SOCS3 becomes more effective with the relative increase of JAK2 activation. Hence, CIS and SOCS3 act in a concerted manner to ensure tight regulation of STAT5 responses over the broad physiological range of Epo concentrations.
In summary, our mathematical approach provided new insights into the specific function of feedback regulation in STAT5-mediated life or death decisions of primary erythroid cells. We dissected the roles of the transcriptionally induced proteins CIS and SOCS3 that operate as dual feedback with divided function thereby facilitating the control of STAT5 activation levels over the entire range of physiological Epo concentrations. The detailed understanding of the molecular processes and control distribution of Epo-induced JAK/STAT signaling can be further applied to gain insights into alterations promoting malignant hematopoietic diseases.
Cellular signal transduction is governed by multiple feedback mechanisms to elicit robust cellular decisions. The specific contributions of individual feedback regulators, however, remain unclear. Based on extensive time-resolved data sets in primary erythroid progenitor cells, we established a dynamic pathway model to dissect the roles of the two transcriptional negative feedback regulators of the suppressor of cytokine signaling (SOCS) family, CIS and SOCS3, in JAK2/STAT5 signaling. Facilitated by the model, we calculated the STAT5 response for experimentally unobservable Epo concentrations and provide a quantitative link between cell survival and the integrated response of STAT5 in the nucleus. Model predictions show that the two feedbacks CIS and SOCS3 are most effective at different ligand concentration ranges due to their distinct inhibitory mechanisms. This divided function of dual feedback regulation enables control of STAT5 responses for Epo concentrations that can vary 1000-fold in vivo. Our modeling approach reveals dose-dependent feedback control as key property to regulate STAT5-mediated survival decisions over a broad range of ligand concentrations.
PMCID: PMC3159971  PMID: 21772264
apoptosis; erythropoietin; mathematical modeling; negative feedback; SOCS
6.  Inhibitory effects of the JAK inhibitor CP690,550 on human CD4+ T lymphocyte cytokine production 
BMC Immunology  2011;12:51.
The new JAK3 inhibitor, CP690,550, has shown efficacy in the treatment of rheumatoid arthritis. The present study was undertaken to assess the effects of CP690,550 on cytokine production and cellular signaling in human CD4+ T cells.
CD4+ T cells produced IL-2, IL-4, IL-17, IL-22 and IFN-γ in following stimulation with a CD3 antibody. At the optimal concentration, CP690,550 almost completely inhibited the production of IL-4, IL-17, IL-22 and IFN-γ from these activated CD4+ T cells, but only had marginal effects on IL-2 production. Moreover CP690,550 inhibited anti-CD3-induced phosphorylation of STAT1, STAT3, STAT4, STAT5, and STAT6, but not the TCR-associated phosphorylation of ZAP-70.
Therefore, CP690,550-mediated modification of the JAK/STAT pathway may be a new immunosuppressive strategy in the treatment of autoimmune diseases.
PMCID: PMC3179939  PMID: 21884580
CP690; 550; cytokines; Janus kinase; signal transducers and activators of transcription; T lymphocytes
7.  Proliferation and Survival Signaling from Both Jak2-V617F and Lyn Involving GSK3 and mTOR/p70S6K/4EBP1 in PVTL-1 Cell Line Newly Established from Acute Myeloid Leukemia Transformed from Polycythemia Vera 
PLoS ONE  2014;9(1):e84746.
The gain of function mutation JAK2-V617F is very frequently found in myeloproliferative neoplasms (MPNs) and is strongly implicated in pathogenesis of these and other hematological malignancies. Here we report establishment of a new leukemia cell line, PVTL-1, homozygous for JAK2-V617F from a 73-year-old female patient with acute myeloid leukemia (AML) transformed from MPN. PVTL-1 is positive for CD7, CD13, CD33, CD34, CD117, HLA-DR, and MPO, and has complex karyotypic abnormalities, 44,XX,-5q,-7,-8,add(11)(p11.2),add(11)(q23),−16,+21,−22,+mar1. Sequence analysis of JAK2 revealed only the mutated allele coding for Jak2-V617F. Proliferation of PVTL-1 was inhibited and apoptosis was induced by the pan-Jak inhibitor Jak inhibitor-1 (JakI-1) or dasatinib, which inhibits the Src family kinases as well as BCR/ABL. Consistently, the Src family kinase Lyn was constitutively activated with phosphorylation of Y396 in the activation loop, which was inhibited by dasatinib but not by JakI-1. Further analyses with JakI-1 and dasatinib indicated that Jak2-V617F phosphorylated STAT5 and SHP2 while Lyn phosphorylated SHP1, SHP2, Gab-2, c-Cbl, and CrkL to induce the SHP2/Gab2 and c-Cbl/CrkL complex formation. In addition, JakI-1 and dasatinib inactivated the mTOR/p70S6K/4EBP1 pathway and reduced the inhibitory phosphorylation of GSK3 in PVTL-1 cells, which correlated with their effects on proliferation and survival of these cells. Furthermore, inhibition of GSK3 by its inhibitor SB216763 mitigated apoptosis induced by dasatinib but not by JakI-1. Together, these data suggest that apoptosis may be suppressed in PVTL-1 cells through inactivation of GSK3 by Lyn as well as Jak2-V617F and additionally through activation of STAT5 by Jak2-V617F. It is also speculated that activation of the mTOR/p70S6K/4EBP1 pathway may mediate proliferation signaling from Jak2-V617F and Lyn. PVTL-1 cells may provide a valuable model system to elucidate the molecular mechanisms involved in evolution of Jak2-V617F-expressing MPN to AML and to develop novel therapies against this intractable condition.
PMCID: PMC3880321  PMID: 24404189
8.  Brevilin A, a Novel Natural Product, Inhibits Janus Kinase Activity and Blocks STAT3 Signaling in Cancer Cells 
PLoS ONE  2013;8(5):e63697.
Signal abnormalities in human cells usually cause unexpected consequences for individual health. We focus on these kinds of events involved in JAK-STAT signal pathways, especially the ones triggered by aberrant activated STAT3, an oncoprotein which participates in essential processes of cell survival, growth and proliferation in many types of tumors, as well as immune diseases. By establishing a STAT3 signal based high-throughput drug screening system in human lung cancer A549 cells, we have screened a library from natural products which contained purified compounds from medicinal herbs. One compound, named Brevilin A, exhibited both strong STAT3 signal inhibition and STAT3 signal dependent cell growth inhibition. Further investigations revealed that Brevilin A not only inhibits STAT3 signaling but also STAT1 signaling for cytokines induced phosphorylation of STAT3 and STAT1 as well as the expression of their target genes. In addition, we found Brevilin A could attenuate the JAKs activity by blocking the JAKs tyrosine kinase domain JH1. The levels of cytokine induced phosphorylation of STATs and other substrates were dramatically reduced by treatment of Brevilin A. The roles of Brevilin A targeting on JAKs activity indicate that Brevilin A may not only be used as a STAT3 inhibitor but also a compound blocking other JAK-STAT hyperactivation. Thus, these findings provided a strong impetus for the development of selective JAK-STAT inhibitors and therapeutic drugs in order to improve survival of patients with hyperactivated JAKs and STATs.
PMCID: PMC3660600  PMID: 23704931
9.  Bim and Mcl-1 exert key roles in regulating JAK2V617F cell survival 
BMC Cancer  2011;11:24.
The JAK2V617F 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 JAK2V617F 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 JAK2V617F 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 JAK2V617F mutant cell viability and sensitized the cells to JAK2 inhibition.
We conclude that Bim and Mcl-1 have key opposing roles in regulating JAK2V617F 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 for the treatment of chronic myeloproliferative neoplasms.
PMCID: PMC3037340  PMID: 21247487
10.  MS-1020 is a novel small molecule that selectively inhibits JAK3 activity 
British journal of haematology  2009;148(1):132-143.
To identify JAK/STAT signaling inhibitors, we performed a cell-based high throughput screening using a plant extract library and identified Nb-(α-hydroxynaphthoyl)serotonin called MS-1020 as a novel JAK3 inhibitor. MS-1020 potently inhibited persistently-active STAT3 in a cell type-specific manner. Upon further examination, we found that MS-1020 selectively blocks constitutively-active JAK3. MS-1020 consistently suppressed IL-2-induced JAK3/STAT5 signaling but not prolactin-induced JAK2/STAT5 signaling. Furthermore, MS-1020 affected cell viability only in cancer cells harboring persistently-active JAK3/STATs, and in vitro kinase assays showed MS-1020 binds directly with JAK3, blocking its catalytic activity. Therefore, our study suggested that this reagent selectively inhibits JAK3 and subsequently leads to a block in STAT signaling. Finally, we showed MS-1020 decreases cell survival by inducing apoptosis via down-regulating anti-apoptotic gene expression. Our study suggests that MS-1020 may have therapeutic potential in the treatment of cancers harboring aberrant JAK3 signaling.
PMCID: PMC3034357  PMID: 19793252
JAK/STAT; cancer; small molecule inhibitor; plant extracts; cell-based high throughput screening; Drosophila
11.  Regulation of Inflammatory Responses in Tumor Necrosis Factor - Activated and Rheumatoid Arthritis Synovial Macrophages by Janus Kinase Inhibitors 
Arthritis and rheumatism  2012;64(12):3856-3866.
Inhibitors of the Janus kinases (JAKs) have been developed as anti-inflammatory and immunosuppressive agents and are currently undergoing testing in clinical trials. The JAK inhibitors CP-690,550 (tofacitinib) and INCB018424 (ruxolitinib) have demonstrated clinical efficacy in rheumatoid arthritis (RA). However, the mechanisms that mediate the beneficial actions of these compounds are not known. In this study, we examined the effects of both JAK inhibitors on inflammatory and tumor necrosis factor (TNF) responses in human macrophages (MΦs).
In vitro studies were performed with peripheral blood MΦs from healthy donors treated with TNF and synovial fluid MΦs from patients with RA. Levels of activated signal transducer and activator of transcription (STAT) proteins and other transcription factors were detected by Western blot, and gene expression was measured by real-time polymerase chain reaction. In vivo effects of JAK inhibitors were evaluated in the K/BxN serum-transfer model of arthritis.
JAK inhibitors suppressed activation and expression of STAT1 and downstream inflammatory target genes in TNF-stimulated and RA synovial macrophages. In addition, JAK inhibitors decreased nuclear localization of NF-κB subunits in TNF-stimulated and RA synovial macrophages. CP-690,550 significantly decreased IL6 expression in synovial MΦs. JAK inhibitors augmented nuclear levels of NFATc1 and cJun, followed by increased formation of osteoclast-like cells. CP-690,550 strongly suppressed K/BxN arthritis that is dependent on macrophages but not on lymphocytes.
Our findings demonstrate that JAK inhibitors suppress macrophage activation and attenuate TNF responses, and suggest that suppression of cytokine/chemokine production and innate immunity contributes to the therapeutic efficacy of JAK inhibitors.
PMCID: PMC3510320  PMID: 22941906
macrophages; TNF; STAT1; rheumatoid arthritis; JAK inhibitors
12.  MPLW515L Is a Novel Somatic Activating Mutation in Myelofibrosis with Myeloid Metaplasia  
PLoS Medicine  2006;3(7):e270.
The JAK2V617F allele has recently been identified in patients with polycythemia vera (PV), essential thrombocytosis (ET), and myelofibrosis with myeloid metaplasia (MF). Subsequent analysis has shown that constitutive activation of the JAK-STAT signal transduction pathway is an important pathogenetic event in these patients, and that enzymatic inhibition of JAK2V617F may be of therapeutic benefit in this context. However, a significant proportion of patients with ET or MF are JAK2V617F-negative. We hypothesized that activation of the JAK-STAT pathway might also occur as a consequence of activating mutations in certain hematopoietic-specific cytokine receptors, including the erythropoietin receptor (EPOR), the thrombopoietin receptor (MPL), or the granulocyte-colony stimulating factor receptor (GCSFR).
Methods and Findings
DNA sequence analysis of the exons encoding the transmembrane and juxtamembrane domains of EPOR, MPL, and GCSFR, and comparison with germline DNA derived from buccal swabs, identified a somatic activating mutation in the transmembrane domain of MPL (W515L) in 9% (4/45) of JAKV617F-negative MF. Expression of MPLW515L in 32D, UT7, or Ba/F3 cells conferred cytokine-independent growth and thrombopoietin hypersensitivity, and resulted in constitutive phosphorylation of JAK2, STAT3, STAT5, AKT, and ERK. Furthermore, a small molecule JAK kinase inhibitor inhibited MPLW515L-mediated proliferation and JAK-STAT signaling in vitro. In a murine bone marrow transplant assay, expression of MPLW515L, but not wild-type MPL, resulted in a fully penetrant myeloproliferative disorder characterized by marked thrombocytosis (Plt count 1.9–4.0 × 10 12/L), marked splenomegaly due to extramedullary hematopoiesis, and increased reticulin fibrosis.
Activation of JAK-STAT signaling via MPLW515L is an important pathogenetic event in patients with JAK2V617F-negative MF. The bone marrow transplant model of MPLW515L-mediated myeloproliferative disorders (MPD) exhibits certain features of human MF, including extramedullary hematopoiesis, splenomegaly, and megakaryocytic proliferation. Further analysis of positive and negative regulators of the JAK-STAT pathway is warranted in JAK2V617F-negative MPD.
Editors' Summary
Myelofibrosis with myeloid metaplasia (MF) is one of a group of chronic blood disorders, known as chronic myeloproliferative disorders. These disorders sometimes turn into acute leukemia. The main abnormality in myelofibrosis is for the bone marrow to become filled with fibrous (scar) tissue (hence the name myelofibrosis), which stops it from producing normal blood cells efficiently. In addition, the white blood cells that remain are abnormal (that is, metaplastic). The clinical effect of these abnormalities are that patients are anemic (they have low numbers of red cells), are more likely to get infections because of the abnormal white cells which cannot fight infections normally, and may bleed more easily because of a lack of the platelets that help the blood to clot. Scientists who study this disorder believe that the disease starts from just one abnormal cell, which divides to replace all the other cells—that is, all the abnormal cells are part of one clone.
Why Was This Study Done?
In two similar diseases, polycythemia vera (in which the bone marrow produces too many red blood cells) and essential thrombocytosis (in which the bone marrow produces too many platelets), and in some patients with MF, scientists have found genetic changes which seem to trigger these diseases. However, there are some patients with MF in which no abnormal gene has been found. The scientists here wanted to look at other genes to see if they could find any changes that might trigger MF.
What Did the Researchers Do and Find?
They decoded the DNA sequence of three genes that are known to be involved in how blood cells develop for 45 patients with MF. They looked at DNA from white blood cells, and also from normal cheek cells for comparison. They found that in four of the 45 patients the DNA in the bone marrow, but not the cheek, carried a mutation in a gene for the thrombopoietin receptor (also called MPL). This gene is necessary for the cells that make platelets to grow correctly. The mutation was not present in any samples from patients with diseases related to MF, nor in 270 normal samples. The mutation that was identified was at position 515 in the MPL gene sequence, hence the name MPLW515L—the W and the L are the shorthand way of indicating exactly which change occurred. The change meant that the gene became abnormally active. The researchers tested the effect of the abnormal gene by putting it into cells grown in culture in the laboratory; they found that it made the cells grow more than was normal. In addition, when cells with the abnormal gene were put into mice, the mice developed a blood disorder similar to that seen in humans with MF.
What Do These Findings Mean?
It seems likely that the genetic change that has been identified here is responsible for the MF that develops in some patients. The MPL gene is known to be part of a pathway of genes that control how certain blood cells develop. However, it is not yet clear exactly how the genetic change found here causes the blood cells to grow abnormally, or how it causes the other clinical effects of MF. Further work will also need to be done to see if it is possible to develop drugs that can act on this gene mutation, or on the other genes that it affects so as to return the cells to normal.
Additional Information.
Please access these Web sites via the online version of this summary at
• MedlinePlus, a Web site of the US National Library of Health, has pages of information on myelofibrosis and related diseases
• The National Cancer Institute, which funds research into many cancers, has information for patients on myelofibrosis, including information on clinical trials
• The MPD Foundation has information for patients with myelofibrosis and related diseases
Activation of JAK-STAT signaling via a mutation - MPLW515L- in the thrombopoietin receptor seems to have a role in the pathogenesis of some patients with myelofibrosis.
PMCID: PMC1502153  PMID: 16834459
13.  Z3, a Novel Jak2 Tyrosine Kinase Small Molecule Inhibitor that Suppresses Jak2-mediated Pathologic Cell Growth 
Molecular cancer therapeutics  2008;7(8):2308-2318.
Jak2 tyrosine kinase is essential for animal development and hyper-kinetic Jak2 function has been linked to a host of human diseases. Control of this pathway using Jak2-specific inhibitors would therefore potentially serve as a useful research tool and/or therapeutic agent. Here, we used a high throughput program called DOCK, to predict the ability of 20,000 small molecules to interact with a structural pocket adjacent to the ATP binding site of murine Jak2. One small molecule, 2-methyl-1-phenyl-4-pyridin-2-yl-2-(2-pyridin-2-ylethyl)butan-1-one (herein designated as Z3) bound to Jak2 with a favorable energy score. Z3 inhibited Jak2-V617F and Jak2-WT autophosphorylation in a dose-dependent manner, but was not cytotoxic to cells at concentrations that inhibited kinase activity. Z3 selectively inhibited Jak2 kinase function with no effect of Tyk2 or c-Src kinase function. Z3 significantly inhibited proliferation of the Jak2-V617F-expressing, human erythroleukemia cell line, HEL 92.1.7. The Z3-mediated reduction in cell proliferation correlated with reduced Jak2 and STAT3 tyrosine phosphorylation levels as well as marked cell cycle arrest. Finally, Z3 inhibited the growth of hematopoietic progenitor cells isolated from the bone marrow of an essential thrombocythemia patient harboring the Jak2-V617F mutation and a polycythemia vera patient carrying a Jak2-F537I mutation. Collectively, the data suggest that Z3 is a novel specific inhibitor of Jak2 tyrosine kinase.
PMCID: PMC2579271  PMID: 18723478
Small Molecule Inhibitor; Jak2; Myeloproliferative Disorders
14.  Preclinical characterization of atiprimod, a novel JAK2 AND JAK3 inhibitor 
Investigational new drugs  2010;29(5):818-826.
We herein report on the activity of the JAK2/JAK3 small molecule inhibitor atiprimod on mouse FDCP-EpoR cells carrying either wild-type (JAK2WT) or mutant (JAK2V617F) JAK2, human acute megakaryoblastic leukemia cells carrying JAK2V617F (SET-2 cell line), and human acute megakaryocytic leukemia carrying mutated JAK3 (CMK cells). Atiprimod inhibited more efficaciously the proliferation of FDCP-EpoR JAK2V617F (IC50 0.42 µM) and SET-2 cells (IC50 0.53 µM) than that of CMK (IC50 0.79 µM) or FDCP-EpoR JAK2WT cells (IC50 0.69 µM). This activity was accompanied by inhibition of the phosphorylation of JAK2 and downstream signaling proteins STAT3, STAT5, and AKT in a dose- and time-dependent manner. Atiprimod-induced cell growth inhibition of JAK2V617F–positive cells was coupled with induction of apoptosis, as evidenced by heightened mitochondrial membrane potential and caspase-3 activity, as well as PARP cleavage, increased turnover of the anti-apoptotic X-linked mammalian inhibitor of apoptosis (XIAP) protein, and inhibition of the pro-apoptotic protein BCL-2 in a time- and dose-dependent manner. Furthermore, atiprimod was more effective at inhibiting the proliferation of peripheral blood hematopoietic progenitors obtained from patients with JAK2V617F –positive polycythemia vera than at inhibiting hematopoietic progenitors from normal individuals (p=0.001). The effect on primary expanded erythroid progenitors was paralleled by a decrease in JAK2V617F mutant allele burden in single microaspirated BFU-E and CFU-GM colonies. Taken together, our data supports the clinical testing of atiprimod in patients with hematologic malignancies driven by constitutive activation of JAK2 or JAK3 kinases.
PMCID: PMC4170651  PMID: 20372971
Atiprimod; JAK inhibitor; JAK2 mutation; FDCP cells; Megakaryoblastic cells; Polycythemia vera; Myeloproliferative neoplasia
15.  Transforming and Tumorigenic Activity of JAK2 by Fusion to BCR: Molecular Mechanisms of Action of a Novel BCR-JAK2 Tyrosine-Kinase 
PLoS ONE  2012;7(2):e32451.
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.
PMCID: PMC3288102  PMID: 22384256
16.  Differential Binding to and Regulation of JAK2 by the SH2 Domain and N-Terminal Region of SH2-Bβ 
Molecular and Cellular Biology  2000;20(9):3168-3177.
SH2-Bβ has been shown to bind via its SH2 (Src homology 2) domain to tyrosyl-phosphorylated JAK2 and strongly activate JAK2. In this study, we demonstrate the existence of an additional binding site(s) for JAK2 within the N-terminal region of SH2-Bβ (amino acids 1 to 555) and the ability of this region of SH2-B to inhibit JAK2. Four lines of evidence support the existence of this additional binding site(s). In a glutathione S-transferase pull-down assay, wild-type SH2-Bβ and SH2-Bβ(R555E) with a defective SH2 domain bind to both tyrosyl-phosphorylated JAK2 from growth hormone (GH)-treated cells and non-tyrosyl-phosphorylated JAK2 from control cells, whereas the SH2 domain of SH2-Bβ binds only to tyrosyl-phosphorylated JAK2 from GH-treated cells. Similarly, JAK2 is present in αSH2-B immunoprecipitates in the absence and presence of GH, with GH substantially increasing the coprecipitation of JAK2 with SH2-B. When coexpressed in COS cells, SH2-Bβ coimmunoprecipitates not only wild-type, tyrosyl-phosphorylated JAK2 but also kinase-inactive, non-tyrosyl-phosphorylated JAK2(K882E), although to a lesser extent. ΔC555 (amino acids 1 to 555 of SH2-Bβ) that lacks most of the SH2 domain binds similarly to wild-type JAK2 and kinase-inactive JAK2(K882E). Experiments using a series of N- and C-terminally truncated SH2-Bβ constructs indicate that the pleckstrin homology (PH) domain (amino acids 269 to 410) and amino acids 410 to 555 are necessary for maximal binding of SH2-Bβ to inactive JAK2, but neither region alone is sufficient for maximal binding. The SH2 domain of SH2-Bβ is necessary and sufficient for the stimulatory effect of SH2-Bβ on JAK2 and JAK2-mediated tyrosyl phosphorylation of Stat5B. In contrast, ΔC555 lacking the SH2 domain, and to a lesser extent the PH domain alone, inhibits JAK2. ΔC555 also blocks JAK2-mediated tyrosyl phosphorylation of Stat5B in COS cells and GH-stimulated nuclear accumulation of Stat5B in 3T3-F442A cells. These data indicate that in addition to the SH2 domain, SH2-Bβ has one or more lower-affinity binding sites for JAK2 within amino acids 269 to 555. The interaction via this site(s) in SH2-B with inactive JAK2 seems likely to increase the local concentration of SH2-Bβ around JAK2, thereby facilitating binding of the SH2 domain to ligand-activated JAK2. This would result in a more rapid and robust cellular response to hormones and cytokines that activate JAK2. This interaction between inactive JAK2 and SH2-B may also help prevent abnormal activation of JAK2.
PMCID: PMC85611  PMID: 10757801
17.  The Jak2 Small Molecule Inhibitor, G6, Reduces the Tumorigenic Potential of T98G Glioblastoma Cells In Vitro and In Vivo 
PLoS ONE  2014;9(8):e105568.
Glioblastoma multiforme (GBM) is the most common and the most aggressive form of primary brain tumor. Jak2 is a non-receptor tyrosine kinase that is involved in proliferative signaling through its association with various cell surface receptors. Hyperactive Jak2 signaling has been implicated in numerous hematological disorders as well as in various solid tumors including GBM. Our lab has developed a Jak2 small molecule inhibitor known as G6. It exhibits potent efficacy in vitro and in several in vivo models of Jak2-mediated hematological disease. Here, we hypothesized that G6 would inhibit the pathogenic growth of GBM cells expressing hyperactive Jak2. To test this, we screened several GBM cell lines and found that T98G cells express readily detectable levels of active Jak2. We found that G6 treatment of these cells reduced the phosphorylation of Jak2 and STAT3, in a dose-dependent manner. In addition, G6 treatment reduced the migratory potential, invasive potential, clonogenic growth potential, and overall viability of these cells. The effect of G6 was due to its direct suppression of Jak2 function and not via off-target kinases, as these effects were recapitulated in T98G cells that received Jak2 specific shRNA. G6 also significantly increased the levels of caspase-dependent apoptosis in T98G cells, when compared to cells that were treated with vehicle control. Lastly, when T98G cells were injected into nude mice, G6 treatment significantly reduced tumor volume and this was concomitant with significantly decreased levels of phospho-Jak2 and phospho-STAT3 within the tumors themselves. Furthermore, tumors harvested from mice that received G6 had significantly less vimentin protein levels when compared to tumors from mice that received vehicle control solution. Overall, these combined in vitro and in vivo results indicate that G6 may be a viable therapeutic option against GBM exhibiting hyperactivation of Jak2.
PMCID: PMC4146502  PMID: 25162558
18.  Combined Targeting of JAK2 and Bcl-2/Bcl-xL to Cure Mutant JAK2-Driven Malignancies and Overcome Acquired Resistance to JAK2 Inhibitors 
Cell Reports  2013;5(4):1047-1059.
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.
Graphical Abstract
•Bcl-2 and Bcl-xL are the key survival factors downstream of oncogenic JAK2•Combined targeting of JAK2 and Bcl-2/Bcl-xL is highly efficacious in vivo•Combination therapy is well tolerated in preclinical models of JAK2-driven ALL•Combination therapy can overcome and circumvent resistance to JAK2 inhibitors
Better therapeutic options are required for mutant JAK2-driven hemopoietic malignancies to overcome intrinsic and acquired therapy resistance. Johnstone and colleagues functionally dissect signaling pathways downstream of hyperactive JAK2 and define the STAT5-Bcl-2/Bcl-xL axis as crucial for tumor cell survival and development of acquired JAK inhibitor resistance. Combined inhibition of this oncogenic JAK2 signaling network at two critical points—JAK2 and Bcl-2/Bcl-xL—proved highly efficacious in vivo and was able to circumvent and overcome acquired resistance to single-agent JAK inhibitors.
PMCID: PMC3898474  PMID: 24268771
19.  Random Mutagenesis Reveals Residues of JAK2 Critical in Evading Inhibition by a Tyrosine Kinase Inhibitor 
PLoS ONE  2012;7(8):e43437.
The non-receptor tyrosine kinase JAK2 is implicated in a group of myeloproliferative neoplasms including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. JAK2-selective inhibitors are currently being evaluated in clinical trials. Data from drug-resistant chronic myeloid leukemia patients demonstrate that treatment with a small-molecule inhibitor generates resistance via mutation or amplification of BCR-ABL. We hypothesize that treatment with small molecule inhibitors of JAK2 will similarly generate inhibitor-resistant mutants in JAK2.
In order to identify inhibitor-resistant JAK2 mutations a priori, we utilized TEL-JAK2 to conduct an in vitro random mutagenesis screen for JAK2 alleles resistant to JAK Inhibitor-I. Isolated mutations were evaluated for their ability to sustain cellular growth, stimulate downstream signaling pathways, and phosphorylate a novel JAK2 substrate in the presence of inhibitor.
Mutations were found exclusively in the kinase domain of JAK2. The panel of mutations conferred resistance to high concentrations of inhibitor accompanied by sustained activation of the Stat5, Erk1/2, and Akt pathways. Using a JAK2 substrate, enhanced catalytic activity of the mutant JAK2 kinase was observed in inhibitor concentrations 200-fold higher than is inhibitory to the wild-type protein. When testing the panel of mutations in the context of the Jak2 V617F allele, we observed that a subset of mutations conferred resistance to inhibitor, validating the use of TEL-JAK2 in the initial screen. These results demonstrate that small-molecule inhibitors select for JAK2 inhibitor-resistant alleles, and the design of next-generation JAK2 inhibitors should consider the location of mutations arising in inhibitor-resistant screens.
PMCID: PMC3420867  PMID: 22916261
20.  t(8;9)(p22;p24)/PCM1-JAK2 Activates SOCS2 and SOCS3 via STAT5 
PLoS ONE  2013;8(1):e53767.
Fusions of the tyrosine kinase domain of JAK2 with multiple partners occur in leukemia/lymphoma where they reportedly promote JAK2-oligomerization and autonomous signalling, Affected entities are promising candidates for therapy with JAK2 signalling inhibitors. While JAK2-translocations occur in myeloid, B-cell and T-cell lymphoid neoplasms, our findings suggest their incidence among the last group is low. Here we describe the genomic, transcriptional and signalling characteristics of PCM1-JAK2 formed by t(8;9)(p22;p24) in a trio of cell lines established at indolent (MAC-1) and aggressive (MAC-2A/2B) phases of a cutaneous T-cell lymphoma (CTCL). To investigate signalling, PCM1-JAK2 was subjected to lentiviral knockdown which inhibited 7 top upregulated genes in t(8;9) cells, notably SOCS2/3. SOCS3, but not SOCS2, was also upregulated in a chronic eosinophilic leukemia bearing PCM1-JAK2, highlighting its role as a central signalling target of JAK2 translocation neoplasia. Conversely, expression of GATA3, a key T-cell developmental gene silenced in aggressive lymphoma cells, was partially restored by PCM1-JAK2 knockdown. Treatment with a selective JAK2 inhibitor (TG101348) to which MAC-1/2A/2B cells were conspicuously sensitive confirmed knockdown results and highlighted JAK2 as the active moiety. PCM1-JAK2 signalling required pSTAT5, supporting a general paradigm of STAT5 activation by JAK2 alterations in lymphoid malignancies. MAC-1/2A/2B - the first JAK2–translocation leukemia/lymphoma cell lines described - display conspicuous JAK/STAT signalling accompanied by T-cell developmental and autoimmune disease gene expression signatures, confirming their fitness as CTCL disease models. Our data support further investigation of SOCS2/3 as signalling effectors, prognostic indicators and potential therapeutic targets in cancers with JAK2 rearrangements.
PMCID: PMC3553112  PMID: 23372669
21.  A46, a Benzothiophene Derived Compound, Suppresses Jak2-Mediated Pathologic Cell Growth 
Experimental hematology  2011;40(1):22-34.
Hyperkinetic Jak2 tyrosine kinase signaling has been implicated in several hematological disorders including the myeloproliferative neoplasms (MPNs). Effective Jak2 inhibitors can thus have significant therapeutic potential. Here, using structure based virtual screening, we identified a benzothiophene derived Jak2 inhibitor named A46. We hypothesized that this compound would inhibit Jak2-V617F mediated pathologic cell growth. To test this, A46 was analyzed for its ability to i) inhibit recombinant Jak2 protein catalysis ii) suppress Jak2-mediated pathogenic cell growth in vitro iii) inhibit the aberrant ex vivo growth of Jak2-V617F expressing primary human bone marrow cells and iv) inhibit Jak2-mediated pathogenesis in vivo. To this end, we found that A46 selectively inhibited Jak2-V617F protein when compared to wild type Jak2 protein. The drug also selectively inhibited the proliferation of Jak2-V617F expressing cells in both a time- and dose-dependent manner and this correlated with decreased Jak2 and STAT5 phosphorylation within treated cells. The Jak2-V617F cell growth inhibition correlated with an induction of cell cycle arrest and promotion of apoptosis. A46 also inhibited the pathologic growth of primary Jak2-V617F expressing bone marrow cells, ex vivo. Lastly, using a mouse model of Jak2-V617F mediated MPN, A46 significantly reduced the splenomegaly and megakaryocytic hyperplasia in the spleens of treated mice and the levels of IL-6 in the plasma. Collectively, our data demonstrate that the benzothiophene based compound, A46, suppresses Jak2-mediated pathogenesis, thereby making it a potential candidate drug against Jak2-mediated disorders.
PMCID: PMC3237899  PMID: 22019628
Jak2 kinase; V617F; Small Molecule Inhibitor; Benzothiophene; Myeloproliferative Neoplasms
22.  Adipose Tissue-Derived Stem Cells Secrete CXCL5 Cytokine with Neurotrophic Effects on Cavernous Nerve Regeneration 
The journal of sexual medicine  2010;8(2):437-446.
Previously we reported that paracrine actions likely mediated the therapeutic effects of adipose tissue-derived stem cells (ADSC) on a rat model of cavernous nerve (CN) injury.
To identify potential neurotrophic factors in ADSC’s secretion, test the most promising one, and identify the molecular mechanism of its neurotrophic action.
Rat major pelvic ganglia (MPG) were cultured in conditioned media of ADSC and penile smooth muscle cells (PSMC). Cytokine expression in these two media was probed with a cytokine antibody array. CXCL5 cytokine was quantified in these two media by enzyme-linked immunosorbent assay (ELISA). Activation of JAK/STAT by CXCL5 was tested in neuroblastoma cell lines BE(2)C and SH-SY5Y as well as in Schwann cell line RT4-D6P2T by western blot. Involvement of CXCL5 and JAK/STAT in ADSC-conditioned medium’s neurotrophic effects was confirmed with anti-CXCL5 antibody and JAK inhibitor AG490, respectively.
Main Outcome Measures
Neurotrophic effects of ADSC and PSMC-conditioned media were quantified by measuring neurite length in MPG cultures. Secretion of CXCL5 in these two media was quantified by ELISA. Activation of JAK/STAT by CXCL5 was quantified by densitometry on western blots for STAT1 and STAT3 phosphorylation.
MPG neurite length was significantly longer in ADSC than in PSMC-conditioned medium. CXCL5 was secreted 8 times higher in ADSC than in PSMC-conditioned medium. Anti-CXCL5 antibody blocked the neurotrophic effects of ADSC-conditioned medium. CXCL5 activated JAK/STAT concentration-dependently from 0 to 50 ng/ml in RT4-D6P2T Schwann cells. At 50 ng/ml, CXCL5 activated JAK/STAT time-dependently, peaking at 45 min. AG490 blocked these activities as well as the neurotrophic effects of ADSC-conditioned medium.
CXCL5 was secreted by ADSC at a high level, promoted MPG neurite growth, and activated JAK/STAT in Schwann cells. CXCL5 may contribute to ADSC’s therapeutic efficacy on CN injury-induced ED.
PMCID: PMC3176296  PMID: 21114767
Adipose tissue-derived stem cells; CXCL5 cytokine; cavernous nerve regeneration; erectile dysfunction; JAK/STAT
23.  Biochemical and functional analyses of gp130 mutants unveil JAK1 as a novel therapeutic target in human inflammatory hepatocellular adenoma 
Oncoimmunology  2014;2(12):e27090.
Inflammatory hepatocellular adenomas (IHCAs) are benign liver lesions that can be characterized histologically by the presence of an inflammatory infiltrate and at the molecular level by the overexpression of acute phase inflammatory response genes. Recurrent somatic mutations of the interleukin-6 (IL-6) signal transducer (IL6ST) locus, encoding the critical component of the IL-6 signal transduction machinery gp130, are present in 60% of IHCAs and in a subset (2%) of hepatocellular carcinoma (HCCs). By screening of 256 human hepatic adenoma specimens (the largest genetic analysis of IL6ST performed to date in this setting), we identified 24 distinct somatic IL6ST mutations among 66 mutant adenomas. The functional analysis of nine different gp130 mutants expressed in hepatic cancer cell lines consistently revealed the constitutive and IL-6-independent activation of the JAK/STAT signaling pathway. We further demonstrated that the signaling activity of mutant gp130 in IHCA remains responsive to suppressor of cytokine signaling 3 (SOCS3), a physiological gp130 inhibitor. Specifically, cells expressing a double mutant variant of gp130 with a disrupted SOCS3-binding site at residue 759 (Y186/Y759F) displayed a hyperactivation of signal transducer and activator of transcription 3 (STAT3) as compared with cells expressing the endogenous IHCA-associated Y186 gp130 mutant. Notably, we identified that constitutive signaling via gp130 in IHCA requires the Janus kinase family member JAK1, but not JAK2 or tyrosine kinase 2. In support of this notion, AG490, a tyrosine kinase inhibitor that selectively blocks JAK2, had no effect on gp130 activity. In stark contrast, we showed that ruxolitinib, a JAK1/JAK2-selective tyrosine kinase inhibitor used to treat patients with myelofibrosis, dramatically impaired JAK1-STAT signaling downstream of all IHCA-associated gp130 mutants. In conclusion, our findings provide a rationale for the use of JAK1 inhibitors for the treatment of HCAs expressing mutant gp130 as well as a subset of HCCs that bear similar mutations.
PMCID: PMC3913689  PMID: 24501689
IL6ST; JAK1; ruxolitinib; STAT3; targeted therapy
24.  Resistance of Cancer Cells to Targeted Therapies Through the Activation of Compensating Signaling Loops 
The emergence of low molecular weight kinase inhibitors as “targeted” drugs has led to remarkable advances in the treatment of cancer patients. The clinical benefits of these tumor therapies, however, vary widely in patient populations and with duration of treatment. Intrinsic and acquired resistance against such drugs limits their efficacy. In addition to the well studied mechanisms of resistance based upon drug transport and metabolism, genetic alterations in drug target structures and the activation of compensatory cell signaling have received recent attention. Adaptive responses can be triggered which counteract the initial dependence of tumor cells upon a particular signaling molecule and allow only a transient inhibition of tumor cell growth. These compensating signaling mechanisms are often based upon the relief of repression of regulatory feedback loops. They might involve cell autonomous, intracellular events or they can be mediated via the secretion of growth factor receptor ligands into the tumor microenvironment and signal induction in an auto- or paracrine fashion. The transcription factors Stat3 and Stat5 mediate the biological functions of cytokines, interleukins and growth factors and can be considered as endpoints of multiple signaling pathways. In normal cells this activation is transient and the Stat molecules return to their non-phosphorylated state within a short time period. In tumor cells the balance between activating and de-activating signals is disturbed resulting in the persistent activation of Stat3 or Stat5. The constant activation of Stat3 induces the expression of target genes, which cause the proliferation and survival of cancer cells, as well as their migration and invasive behavior. Activating components of the Jak-Stat pathway have been recognized as potentially valuable drug targets and important principles of compensatory signaling circuit induction during targeted drug treatment have been discovered in the context of kinase inhibition studies in HNSCC cells [1]. The treatment of HNSCC with a specific inhibitor of c-Src, initially resulted in reduced Stat3 and Stat5 activation and subsequently an arrest of cell proliferation and increased apoptosis. However, the inhibition of c-Src only caused a persistent inhibition of Stat5, whereas the inhibition of Stat3 was only transient. The activation of Stat3 was restored within a short time period in the presence of the c-Src inhibitor. This process is mediated through the suppression of P-Stat5 activity and the decrease in the expression of the Stat5 dependent target gene SOCS2, a negative regulator of Jak2. Jak2 activity is enhanced upon SOCS2 downregulation and causes the reactivation of Stat3. A similar observation has been made upon inhibition of Bmx, bone marrow kinase x-linked, activated in the murine glioma cell lines Tu-2449 and Tu-9648. Its inhibition resulted in a transient decrease of P-Stat3 and the induction of a compensatory Stat3 activation mechanism, possibly through the relief of negative feedback inhibition and Jak2 activation. These observations indicate that the inhibition of a single tyrosine kinase might not be sufficient to induce lasting therapeutic effects in cancer patients. Compensatory kinases and pathways might become activated and maintain the growth and survival of tumor cells. The definition of these escape pathways and their preemptive inhibition will suggest effective new combination therapies for cancer.
PMCID: PMC4095943  PMID: 25045345
Drug resistance; signaling redundancy and compensation; targeted tumor therapy.
25.  A Novel Missense (M206K) STAT3 Mutation in Diffuse Large B Cell Lymphoma Deregulates STAT3 Signaling 
PLoS ONE  2013;8(7):e67851.
Persistent STAT3 activation has been found in activated B-cell like diffuse large B cell tumors (DLBCL). To investigate whether genetic mutations play a role in aberrant STAT3 signaling in DLBCL, we bi-directionally sequenced all 24 exons of the STAT3 gene in DLBCL tumors (n = 40). We identified 2 novel point mutations in 2 separate (2/40; 5%) patients at exon 7 and 24. Point mutation 2552G>A was a silent mutation in the stop codon. Another heterozygous mutation 857T>A encoded a methionine substitution by lysine at codon 206 (M206K) in the coiled-coil domain of STAT3. We performed site directed mutagenesis to mutate wild type (WT) STAT3α and STAT3β at codon 206 and constructed stable cell lines by lentiviral transfection of STAT3αWT, STAT3αM206K, STAT3βWT and STAT3βM206K plasmids. The mutation was found to increase STAT3 phosphorylation in STAT3α mutant cell lines with no effect on the STAT3β mutant cell line. Transcriptional activation was also increased in the STAT3α mutant cells compared with STAT3α WT cells as detected by a luciferase reporter assay. Moreover, STAT3αM206K mutant cells were resistant to JAK2 pathway inhibition compared to STAT3α WT cells. These results indicate that missense mutations in STAT3 increase signaling through the JAK/STAT pathway. JAK2 inhibitors may be useful in the patient with this STAT3 mutation as well as those with pathway activation by other mechanisms.
PMCID: PMC3701620  PMID: 23861822

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