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.
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.
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.
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.
CP690; 550; cytokines; Janus kinase; signal transducers and activators of transcription; T lymphocytes
Identification of JAK2V617F in myeloproliferative disorders makes JAK2 an important marker for disease diagnosis and a highly attractive target for therapeutic drug development. This study is intended to identify a sensitive and specific substrate for assays of the JAK2 enzymatic activity.
We expressed a glutathione S-transferase (GST) fusion protein designated GST-JAKS which carries a peptide sequence derived from the autophosphorylation sites of human JAK2. The protein was purified from E. coli cells and was used to analyze to tyrosine kinase activities of purified enzymes and crude cell extracts from cells including mononuclear cells of JAK2V617F -positive polycythemia vera blood. It was also used to perform JAK2 kinase assays to screen inhibitors of JAK2.
GST-JAKS is strongly phosphorylated by activated forms of JAK2 including JAK2V617F and recombinant protein containing its catalytic domain alone. It showed minimal responses to wild type JAK2 and was not phosphorylated by the epidermal growth receptor and the insulin receptor tyrosine kinases. Kinase assays with GST-JAKS provide a sharp contrast between wild type and mutant JAK2V617F and are sensitive enough to detect minute amounts of JAK2V617F found in crude cell extracts. The assays can be scaled up to screen for inhibitors of JAK2 in a dot blot format.
GST-JAKS is sensitive and specific protein substrate for JAK2 assays. It may have clinical applications in diagnosis of diseases related to abnormal JAK2 activity. It is also an excellent substrate for development of large scale assays to screen JAK2 inhibitors.
Myeloproliferative disorders; tyrosine kinases; inhibitor; enzyme assays
Both the core JAK-STAT pathway components and their in vivo roles have been widely conserved between vertebrates and invertebrate models such as Drosophila melanogaster. Misregulation of JAK-STAT pathway activity has also been identified as a key factor in the development of multiple human malignancies. Recently, whole genome RNA interference (RNAi) screens in cultured Drosophila cells have identified both positively and negatively acting JAK-STAT pathway regulators. Here, we describe the analysis of 73 human genes representing homologs of 56 Drosophila genes originally identified by genome-wide RNAi screening as regulators of JAK-STAT signaling. Using assays for human STAT1 and STAT3 protein levels and phosphorylation status, as well as assays measuring the expression of endogenous STAT1 and STAT3 transcriptional targets, we have tested siRNAs targeting these 73 human genes and have identified potential JAK-STAT pathway regulatory roles in 69 (95%) of these. The genes identified represent a wide range of human JAK-STAT pathway regulators and include genes not previously known to modulate this signaling cascade. These results underline the value of model system based approaches for the identification of pathway regulators and have led to the identification of loci whose misregulation may ultimately be implicated in JAK-STAT pathway-mediated human disease.
assay; Drosophila; GBP1; HeLa; phosphorylation; RNAi; screening; SOCS3
Jak2/Stat-mediated prolactin signaling culminates in Stat5a-DNA-binding. However, not all Jak2-dependent genes have Stat5 sites. Western analysis with inhibitors showed Jak2 is a proximal intermediate in prolactin-induced RUSH phosphorylation. Transfection assays with HRE-H9 cells showed the RUSH-binding site mediated the ability of prolactin to augment progesterone-dependent transcription of the RUSH gene. Jak2 inhibitors or targeted RUSH-site mutation blocked the prolactin effect. RUSH co-immunoprecipitated with phospho-Jak2 from nuclear extracts. Jak2 inhibitors abolished the nuclear pool of phospho-RUSH not the nuclear content of RUSH in HRE-H9 cells. Nucleolar-affiliated partners, e.g. nucleolin, were identified by μLC/MS/MS analysis of nuclear proteins that co-immunoprecipitated with RUSH/GST-RING. RUSH did not exclusively co-localize with fibrillarin to the nucleolus. MG-132 (proteasomal inhibitor) failed to block Tyrene CR4-mediated decrease in phospho-RUSH, and did not promote RUSH accumulation in the nucleolus. These studies authenticate prolactin-dependent Jak2 phosphorylation of RUSH, and provide functional implications on the RUSH network of nuclear interactions.
Jak2; prolactin; HLTF; nucleolin; actin; HRE-H9 cells (rabbit endometrium)
Here, we examine the significance that stereochemistry plays within the clinically relevant Janus Kinase 3 (Jak3) inhibitor CP-690,550. A synthesis of all four enantiopure stereoisomers of the drug was carried out and an examination of each compound revealed that only the enantiopure 3R, 4R isomer was capable of blocking Stat5 phosphorylation (Jak3 dependent). Each compound was profiled across a panel of over 350 kinases which revealed a high level of selectivity for the Jak family kinases for these related compounds. Each stereoisomer retained a degree of binding to Jak3 and Jak2 and the 3R, 4S and 3S, 4R stereoisomers were further revealed to have binding affinity for selected members of the STE7 and STE20 subfamily of kinases. Finally, an appraisal of the minimum energy conformation of each stereoisomer and molecular docking at Jak3 was performed in an effort to better understand each compounds selectivity and potency profiles.
Janus Kinase 3; CP-690,550; Kinase inhibition; Chiral drugs
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.
JAK/STAT; cancer; small molecule inhibitor; plant extracts; cell-based high throughput screening; Drosophila
Cytokines in the bone marrow of multiple myeloma patients activate Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways in tumor cells and promote tumor growth, survival, and drug resistance. INCB16562 was developed as a novel, selective, and orally bioavailable small-molecule inhibitor of JAK1 and JAK2 markedly selective over JAK3. The specific cellular activity of the inhibitor was demonstrated by its potent and dose-dependent inhibition of cytokine-dependent JAK/STAT signaling and cell proliferation in the absence of effects on Bcr-Abl-expressing cells. Treatment of myeloma cells with INCB16562 potently inhibited interleukin-6 (IL-6)-induced phosphorylation of STAT3. Moreover, the proliferation and survival of myeloma cells dependent on IL-6 for growth, as well as the IL-6-induced growth of primary bone marrow-derived plasma cells from a multiple myeloma patient, were inhibited by INCB16562. Induction of caspase activation and apoptosis was observed and attributed, at least in part, to the suppression of Mcl-1 expression. Importantly, INCB16562 abrogated the protective effects of recombinant cytokines or bone marrow stromal cells and sensitized myeloma cells to cell death by exposure to dexamethasone, melphalan, or bortezomib. Oral administration of INCB16562 antagonized the growth of myeloma xenografts in mice and enhanced the antitumor activity of relevant agents in combination studies. Taken together, these data suggest that INCB16562 is a potent JAK1/2 inhibitor and that mitigation of JAK/STAT signaling by targeting JAK1 and JAK2 will be beneficial in the treatment of myeloma patients, particularly in combination with other agents.
Signal Transducer and Activator of Transcription 3 (STAT3) is an oncogene, which promotes cell survival, proliferation, motility and progression in cancer cells. Targeting STAT3 signaling may lead to the development of novel therapeutic approaches for human cancers. Here, we examined the effects of epigallocathechin gallate (EGCG) on STAT3 signaling in pancreatic cancer cells, and assessed the therapeutic potential of EGCG with gemcitabine or JAK3 inhibitor CP690550 (Tasocitinib) for the treatment and/or prevention of pancreatic cancer.
Cell viability and apoptosis were measured by XTT assay and TUNEL staining, respectively. Gene and protein expressions were measured by qRT-PCR and Western blot analysis, respectively. The results revealed that EGCG inhibited the expression of phospho and total JAK3 and STAT3, STAT3 transcription and activation, and the expression of STAT3-regulated genes, resulting in the inhibition of cell motility, migration and invasion, and the induction of caspase-3 and PARP cleavage. The inhibition of STAT3 enhanced the inhibitory effects of EGCG on cell motility and viability. Additionally, gemcitabine and CP690550 alone inhibited STAT3 target genes and synergized with EGCG to inhibit cell viability and induce apoptosis in pancreatic cancer cells.
Overall, these results suggest that EGCG suppresses the growth, invasion and migration of pancreatic cancer cells, and induces apoptosis by interfering with the STAT3 signaling pathway. Moreover, EGCG further enhanced the therapeutic potential of gemcitabine and CP690550 against pancreatic cancer.
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.
Janus kinase 2 (JAK2) couples ligand activation of cell surface cytokine receptors to the regulation of cellular functions including cell cycle progression, differentiation and apoptosis. It thereby coordinates biological programs such as development and hematopoiesis. Unscheduled activation of JAK2 by point mutations or chromosomal translocations can induce hyperproliferation and hematological malignancies. Typical signal transduction by the JAK2 tyrosine kinase comprises phosphorylation of STAT transcription factors. In this study, we describe the identification of the cyclin-dependent kinase (CDK) inhibitor p27Kip1 as a novel JAK2 substrate. JAK2 can directly bind and phosphorylate p27Kip1. Both, the JAK2 FERM domain and its kinase domain bind to p27Kip1. JAK2 phosphorylates tyrosine residue 88 (Y88) of p27Kip1. We previously reported that Y88 phosphorylation of p27Kip1 by oncogenic tyrosine kinases impairs p27Kip1-mediated CDK inhibition, and initiates its ubiquitin-dependent proteasomal degradation. Consistently, we now find that active oncogenic JAK2V617F reduces p27Kip1 stability and protein levels in patient-derived cell lines harboring the mutant JAK2V617F allele. Moreover, tyrosine phosphorylation of p27Kip1 is impaired and p27Kip1 expression is restored upon JAK2V617F inactivation by small hairpin RNA-mediated knockdown or by the pyridone-containing tetracycle JAK inhibitor-I, indicating that direct phosphorylation of p27Kip1 can contribute to hyperproliferation of JAK2V617F-transformed cells. Activation of endogenous JAK2 by interleukin-3 (IL-3) induces Y88 phosphorylation of p27Kip1, thus unveiling a novel link between cytokine signaling and cell cycle control in non-transformed cells. Oncogenic tyrosine kinases could use this novel pathway to promote hyperproliferation in tumor cells.
cell cycle control; CDK inhibitors; p27Kip1; tyrosine kinases; JAK2; JAK2V617F
Aberrant activation of the Janus Kinase (JAK)/Signal Activator of Transcription (STAT) pathway has been implicated in glioblastoma (GBM) progression. To develop a therapeutic strategy to inhibit STAT-3 signaling, we have evaluated the effects of AZD1480, a pharmacological inhibitor of JAK1 and JAK2. In this study, the in vitro efficacy of AZD1480 was tested in human and murine glioma cell lines. AZD1480 treatment effectively blocks constitutive and stimulus-induced JAK1, JAK2 and STAT-3 phosphorylation in both human and murine glioma cells, and leads to a decrease in cell proliferation and induction of apoptosis. Furthermore, we utilized human xenograft GBM samples as models for the study of JAK/STAT-3 signaling in vivo, since human GBM samples propagated as xenografts in nude mice retain both the hallmark genetic alterations and the invasive phenotype seen in vivo. In these xenograft tumors, JAK2 and STAT-3 are constitutively active, but levels vary among tumors, which is consistent with the heterogeneity of GBMs. AZD1480 inhibits constitutive and stimulus-induced phosphorylation of JAK2 and STAT-3 in these GBM xenograft tumors in vitro, downstream gene expression and inhibits cell proliferation. Furthermore, AZD1480 suppresses STAT-3 activation in the glioma-initiating cell population in GBM tumors. In vivo, AZD1480 inhibits the growth of subcutaneous tumors and increases survival of mice bearing intracranial GBM tumors by inhibiting STAT-3 activity, indicating that pharmacological inhibition of the JAK/STAT-3 pathway by AZD1480 should be considered for study in the treatment of patients with GBM tumors.
AZD1480; glioma; STAT-3; JAK1; JAK2
Since the discovery of mutant Janus Kinase 2 (JAK2), JAK2 V617F, in a major proportion of myeloproliferative neoplasm (MPN) patients, there has been a flurry of activity in the development of JAK2 inhibitors. Pan-JAK, predominantly JAK2 and off-target JAK2 inhibitors have been developed in the short span of the past 5 years. These compounds have since been tested to varying success in both in vitro and in vivo settings with several proceeding on to advanced clinical trials. Although it was hoped that these inhibitors would be the silver bullet in the manner than imatinib was to chronic myeloid leukemia, it is becoming apparent that this is not the case for various reasons, chief of which is that a significant reduction of the underlying pathogenic clone is not achieved. In fact, the very notion that the target of JAK2 inhibitors (be it pan-JAK or JAK2 specific) is the mutant JAK2 V617F is being challenged with findings from several clinical trials showing a poor correlation between the reduction in JAK2 V617F mutant allele burden and clinical response. In view of this, it is not surprising that several groups are now investigating combinations of JAK2 inhibitors and other agents in MPN. Although much knowledge has been added in this short span of time, it is apparent that our understanding of the role of JAK2 inhibitors in the treatment scheme of MPN is only beginning.
JAK2 inhibitors; JAK2 V617F; myeloproliferative neoplasm
Hsp90 inhibition in B cell acute lymphoblastic leukemia overcomes resistance to JAK2 inhibitors.
Enzymatic inhibitors of Janus kinase 2 (JAK2) are in clinical development for the treatment of myeloproliferative neoplasms (MPNs), B cell acute lymphoblastic leukemia (B-ALL) with rearrangements of the cytokine receptor subunit cytokine receptor–like factor 2 (CRLF2), and other tumors with constitutive JAK2 signaling. In this study, we identify G935R, Y931C, and E864K mutations within the JAK2 kinase domain that confer resistance across a panel of JAK inhibitors, whether present in cis with JAK2 V617F (observed in MPNs) or JAK2 R683G (observed in B-ALL). G935R, Y931C, and E864K do not reduce the sensitivity of JAK2-dependent cells to inhibitors of heat shock protein 90 (HSP90), which promote the degradation of both wild-type and mutant JAK2. HSP90 inhibitors were 100–1,000-fold more potent against CRLF2-rearranged B-ALL cells, which correlated with JAK2 degradation and more extensive blockade of JAK2/STAT5, MAP kinase, and AKT signaling. In addition, the HSP90 inhibitor AUY922 prolonged survival of mice xenografted with primary human CRLF2-rearranged B-ALL further than an enzymatic JAK2 inhibitor. Thus, HSP90 is a promising therapeutic target in JAK2-driven cancers, including those with genetic resistance to JAK enzymatic inhibitors.
Signal Transducer and Activator of Transcription 3 (STAT3) is persistently activated and contributes to malignant progression in various cancers. Janus kinases (JAKs) phosphorylate STAT3 in response to stimulation with cytokines or growth factors. The STAT3 signaling pathway has been validated as a promising target for development of anti-cancer therapeutics. Small-molecule inhibitors of JAK/STAT3 signaling represent potential molecular-targeted cancer therapeutic agents. In this study, we investigated the role of JAK/STAT3 signaling in 6-bromoindirubin-3'-oxime (6BIO) mediated growth inhibition of human melanoma cells and assessed 6BIO as an anticancer drug candidate. We found that 6BIO is a pan-JAK inhibitor that induced apoptosis of human melanoma cells. 6BIO directly inhibited JAK family kinase activity both in vitro and in cancer cells. Apoptosis of human melanoma cells induced by 6BIO was associated with reduced phosphorylation of JAKs and STAT3 in both a dose- and time-dependent manners. Consistent with inhibition of STAT3 signaling, the anti-apoptotic protein Mcl-1 was down-regulated. In contrast to the decreased levels of phosphorylation of JAKs and STAT3, phosphorylation levels of the AKT and MAPK signaling proteins were not inhibited in cells treated with 6BIO. Importantly, 6BIO suppressed tumor growth in vivo with low toxicity in a mouse xenograft model of melanoma. Taken together, these results demonstrate that 6BIO is a novel pan-JAK inhibitor that can selectively inhibit STAT3 signaling and induced tumor cell apoptosis. Our findings support further development of 6BIO as a potential anti-cancer therapeutic agent that targets JAK/STAT3 signaling in tumor cells.
bromoindirubin; JAK inhibitor; STAT3 signaling; apoptosis; melanoma
Persistent activation of Stat3 is oncogenic and is prevalent in a wide variety of human cancers. Chronic cytokine stimulation is associated with Stat3 activation in some tumors, implicating cytokine receptor-associated Jak family kinases. Using Jak2 inhibitors, we demonstrate a central role of Jaks in modulating basal and cytokine-induced Stat3 activation in human solid tumor cell lines. Inhibition of Jak2 activity is associated with abrogation of Stat3 nuclear translocation and tumorigenesis. The Jak2 inhibitor, AZD1480, suppresses the growth of human solid tumor xenografts harboring persistent Stat3 activity. We demonstrate the essential role of Stat3 downstream of Jaks by inhibition of tumor growth using shRNA targeting Stat3. Our data support a key role of Jak kinase activity in Stat3-dependent tumorigenesis.
There is accumulating evidence that dysregulated JAK signaling occurs in a wide variety of cancer types. In particular, mutations in JAK2 can result in the constitutive activation of STAT transcription factors and lead to oncogenic growth. JAK kinases are established Hsp90 client proteins and here we show that the novel small molecule Hsp90 inhibitor ganetespib (formerly STA-9090) exhibits potent in vitro and in vivo activity in a range of solid and hematological tumor cells that are dependent on JAK2 activity for growth and survival. Of note, ganetespib treatment results in sustained depletion of JAK2, including the constitutively active JAK2V617F mutant, with subsequent loss of STAT activity and reduced STAT-target gene expression. In contrast, treatment with the pan-JAK inhibitor P6 results in only transient effects on these processes. Further differentiating these modes of intervention, RNA and protein expression studies show that ganetespib additionally modulates cell cycle regulatory proteins, while P6 does not. The concomitant impact of ganetespib on both cell growth and cell division signaling translates to potent antitumor efficacy in mouse models of xenografts and disseminated JAK/STAT-driven leukemia. Overall, our findings support Hsp90 inhibition as a novel therapeutic approach for combating diseases dependent on JAK/STAT signaling, with the multimodal action of ganetespib demonstrating advantages over JAK-specific inhibitors.
Hematopoiesis is the cumulative result of intricately regulated signaling pathways that are mediated by cytokines and their receptors. Studies conducted over the past 10–15 years have revealed that hematopoietic cytokine receptor signaling is largely mediated by a family of tyrosine kinases termed Janus Kinases (JAKs) and their downstream transcription factors, termed STATs (signal transducers and activators of transcription). Aberrations in these pathways, such as those caused by the recently identified JAK2V617F mutation and translocations of the JAK2 gene, are underlying causes of leukemias and other myeloproliferative disorders. This review discusses the role of JAK/STAT signaling in normal hematopoiesis as well as myeloproliferative and myelodisplastic syndromes. This review also summarizes the status of several small molecule JAK2 inhibitors that are currently at various stages of clinical development. Several of these compounds appear to improve the quality of life of patients with myeloproliferative disorders by palliation of disease-related symptoms. However, to date, these agents do not seem to significantly affect bone marrow fibrosis, alter marrow histopathology, reverse cytopenias, reduce red cell transfusion requirements or significantly reduce allele burden. These results suggest that additional mutational events might be associated with the development of these neoplasms and indicate the need for additional therapeutic approaches as the nature of these additional molecular events is better understood.
JAK; STAT; cytokine receptor; myeloproliferative disorders; JAK2 inhibitors
Hematopoiesis is the cumulative result of intricately regulated signaling pathways that are mediated by cytokines and their receptors. Studies conducted over the past 10 to 15 years have revealed that hematopoietic cytokine receptor signaling is largely mediated by a family of tyrosine kinases termed Janus kinases (JAKs) and their downstream transcription factors, termed STATs (signal transducers and activators of transcription). Aberrations in these pathways, such as those caused by the recently identified JAK2V617F mutation and translocations of the JAK2 gene, are underlying causes of leukemias and other myeloproliferative disorders. This review discusses the role of JAK/STAT signaling in normal hematopoiesis as well as genetic abnormalities associated with myeloproliferative and myelodisplastic syndromes. This review also summarizes the status of several small molecule JAK2 inhibitors that are currently at various stages of clinical development. Several of these compounds appear to improve the quality of life of patients with myeloproliferative disorders by palliation of disease-related symptoms. However, to date, these agents do not seem to significantly affect bone marrow fibrosis, alter marrow histopathology, reverse cytopenias, reduce red cell transfusion requirements, or significantly reduce allele burden. These results suggest the possibility that additional mutational events might be associated with the development of these neoplasms, and indicate the need for combination therapies as the nature and significance of these additional molecular events is better understood.
JAK; STAT; cytokine receptor; myeloproliferative disorders; JAK2 inhibitors
Myeloproliferative neoplasms (MPN) are debilitating stem cell-derived clonal myeloid malignancies. Conventional treatments for the BCR-ABL1-negative MPN including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) have, so far, been unsatisfactory. Following the discovery of dysregulated JAK-STAT signaling in patients with MPN, many efforts have been directed toward the development of molecularly targeted therapies, including inhibitors of JAK1 and JAK2. Ruxolitinib (previously known as INCB018424; Incyte Corporation, Wilmington, Delaware, USA) is a rationally designed potent oral JAK1 and JAK2 inhibitor that has undergone clinical trials in patients with PV, ET, and PMF. Ruxolitinib was approved on November 16, 2011 by the United States Food and Drug Administration for the treatment of intermediate or high-risk myelofibrosis (MF), including patients with PMF, post-PV MF, and post-ET MF. In randomized phase III studies, ruxolitinib treatment resulted in significant and durable reductions in splenomegaly and improvements in disease-related symptoms in patients with MF compared with placebo or best available therapy. The most common adverse events were anemia and thrombocytopenia, which were manageable and rarely led to discontinuation. This review addresses the cellular and molecular biology, and the clinical management of MPN.
Essential thrombocythemia; janus kinase; JAK inhibitor; JAK-STAT; myelofibrosis; myeloproliferative neoplasms; polycythemia vera; primary myelofibrosis; quality of life; ruxolitinib; splenomegaly; symptoms.
IL-6 and downstream JAK-dependent signaling pathways have critical roles in the pathophysiology of multiple myeloma. We investigated the effects of a novel small-molecule JAK inhibitor (AZD1480) on IL-6/JAK signal transduction and its biological consequences on the human myeloma-derived cell lines U266 and Kms.11. At low micromolar concentrations, AZD1480 blocks cell proliferation and induces apoptosis of myeloma cell lines. These biological responses to AZD1480 are associated with concomitant inhibition of phosphorylation of JAK2, STAT3 and MAPK signaling proteins. In addition, there is inhibition of expression of STAT3 target genes, particularly Cyclin D2. Examination of a wider variety of myeloma cells (RPMI 8226, OPM-2, NCI-H929, Kms.18, MM1.S, IM-9) as well as primary myeloma cells showed that AZD1480 has broad efficacy. By contrast, viability of normal PBMCs and CD138+ cells derived from healthy controls was not significantly inhibited. Importantly, AZD1480 induces cell death of Kms.11 cells grown in the presence of HS-5 bone marrow-derived stromal cells and inhibits tumor growth in a Kms.11 xenograft mouse model, accompanied with inhibition of phospho-FGFR3, phospho-JAK2, phospho-STAT3 and Cyclin D2 levels. In sum, AZD1480 blocks proliferation, survival, FGFR3 and JAK/STAT3 signaling in myeloma cells cultured alone or co-cultured with bone marrow stromal cells and in vivo. Thus, AZD1480 represents a potential new therapeutic agent for patients with multiple myeloma.
myeloma; JAK2; STAT3; FGFR3
Macrophages, T and B cells, and neutrophils concentrate mainly into the synovial tissue of rheumatoid arthritis (RA) patients and produce several inflammatory mediators including cytokines. More recently, small molecule inhibitors of signalling mediators which have intracellular targets (mainly in T and B cells) such as the Janus kinase (JAK) family of tyrosine kinases have been introduced in RA treatment. The JAK family consist of four types: JAK1, JAK2, JAK3 and TyK2. In particular, JAK3 is the only JAK family member that associates with just one cytokine receptor, the common gamma chain, which is exclusively used by the receptors for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 critically involved in T and natural killer (NK)-cell development, and B-cell function and proliferation. Tofacitinib is one of the first JAK inhibitors tested and mainly interacts with JAK1 and JAK3. Four phase II (one A and three B dose-ranging) trials in RA patients, lasting from 6 to 24 weeks, achieved significant improvements of American College of Rheumatology 20% improvement criteria (ACR20) and Disease Activity Score in 28 joints using the C-reactive protein level (DAS28-CRP) or erythrocyte sedimentation rate (DAS-ESR; in one study that analysed this), as early as week 2 and sustained at week 24 in two studies. Doses ranged from 1, 3, 5, 10, 15, 20 up to 30 mg and were administered orally twice a day. ACR20 response rates for dosages ≥3 mg were found to be significantly (p ≤ 0.05) greater than those for placebo in all phase II studies. In general, the major adverse effects included liver test elevation, neutropenia, lipid and creatinine elevation and increased incidence of infections. More recently, RA patients randomly assigned to 5 or 10 mg of tofacitinib twice daily, in both 6- and 12-month phase III trials, achieved a significantly higher ACR20 than those receiving placebo. Adverse events occurred more frequently with tofacitinib than with placebo, and included pulmonary tuberculosis and other serious infections. The balance of efficacy and safety of tofacitinib compared with standard of care therapy is bringing this first orally available biological disease-modifying antirheumatic drug (DMARD) a step closer for RA patients.
biological disease-modifying antirheumatic drug (DMARD); Janus kinase inhibitors; kinases; rheumatoid arthritis; therapy for rheumatoid arthritis
Aberrant JAK-STAT signaling is a hallmark of myeloproliferative neoplasms (MPNs). These hyperproliferative disorders are classically associated with activating mutations in tyrosine kinases such as JAK2 and the thrombopoietin (TPO) receptor MPL. Activation of JAK-STAT signaling and responses to JAK2 inhibitors have been observed in MPN patients lacking JAK2 or MPL mutations, suggesting that other regulatory elements in the JAK-STAT pathway are altered. However, the molecular basis for this observation has been unclear. Recently, the role of inhibitory regulators of JAK-STAT signaling in MPN pathogenesis has been increasingly recognized. LNK is an adaptor protein that forms a negative feedback loop by binding to MPL and JAK2 and inhibiting downstream STAT activation. Murine models indicate that loss of LNK function can promote the development of a MPN phenotype. Several recent studies have identified novel LNK mutations in MPNs, thus validating this notion in humans. These findings represent a novel genetic paradigm of loss of negative feedback regulation of JAK-STAT activation in MPNs and have implications for the future development of targeted therapies in MPNs.
essential thrombocythemia; JAK2 V617F; JAK-STAT; LNK; myeloproliferative neoplasms; polycythemia vera; primary myelofibrosis