Oncogene database

3717

JAK2

Janus kinase 2;JAK2;Janus kinase 2

JAK-2|Janus kinase 2 (a protein tyrosine kinase)|tyrosine-protein kinase JAK2

9p24

protein-coding

The leukemia-associated TEL-Jak2 fusion protein possesses a constitutive tyrosine kinase activity and transforming properties in hematopoietic cell lines and animal models. In the murine pro-B Ba/F3 cell line, this fusion constitutively activates the Signal Transducer and Activator of Transcription 5 (Stat5) factors and, as a consequence, induces the sustained expression of various Stat5-target genes including the Cytokine Inducible SH2-containing protein (Cis) gene, which codes for a member of the Suppressor of Cytokine Signaling (Socs) protein family. In TEL-Jak2-transformed Ba/F3 cells, we also observed the upregulation of the Socs1 gene, whose product has been reported to negatively regulate the Jak kinase activity. In transient transfection experiments, Socs1 physically interacts with TEL-Jak2 and interferes with the TEL-Jak2-induced phosphorylation and activation of Stat5 factors, probably through the Socs1-induced proteasome-mediated degradation of the fusion protein. Interestingly, TEL-Jak2-expressing Ba/F3 cells were found to be resistant to the anti-proliferative activities of gamma interferon (IFN-gamma) seemingly as a consequence of Socs1 constitutive expression. These results indicate that the Socs1-dependent cytokine feedback loop, although active, is bypassed by the TEL-Jak2 fusion, but may play a role in the leukemogenic process by altering the cytokine responses of the leukemic cells. Our results also suggest that Socs1 plays a role in shutting down the signaling from the normally activated Jak2 kinase by inducing its proteasome-dependent degradation.

OBJECTIVE: Various TEL-JAK2 fusions have been identified in patients with lymphoblastic and myeloid leukemias that result in constitutive activation of the JAK2 kinase domain. Such fusions can mediate factor-independent growth of hematopoietic cell lines and induction of malignancy in mouse models. MATERIALS AND METHODS: To assess whether zebrafish could be utilized as a suitable model for the study of myeloid oncogenesis, we generated a zebrafish tel-jak2a fusion oncoprotein based on that seen in a case of chronic myeloid leukemia. This was transiently expressed in zebrafish embryos under the control of the spi1 promoter, which is strongly active in myeloid precursors. RESULTS: Visual, histological, and molecular analysis revealed disruption of normal embryonic hematopoiesis, including perturbation of the myeloid and erythroid lineages. CONCLUSION: These results indicate that the zebrafish tel-jak2a oncoprotein is functional, and suggest that this organism will be useful for the experimental study of myeloid malignancy.

BACKGROUND AND AIMS: Obesity is a major risk factor for the development of colon cancer. Secretion of the hormone leptin from adipocytes is increased in obesity, and serum levels are proportional to body fat mass. Serum leptin levels are an independent risk factor for colon cancer. Leptin receptors are expressed in normal, premalignant and malignant colonic epithelia. We have investigated the effects of leptin on proliferation and apoptosis of colonic cancer cells and the early signalling events involved. METHODS: Proliferation of HT-29 colon cancer cells in response to leptin was assessed by 3-[4, 5-dimethylthiazol-2-y-l]-2, 5-diphenyltetrazolium bromide (MTT) assay, and apoptosis was quantified by enzyme-linked immunosorbent assay (ELISA) for intracellular nucleosomes. Signalling pathways involved were determined by using specific inhibitors, quantification of phosphorylated active intermediates and ELISA of active nuclear-translocated transcription factors. RESULTS: Leptin stimulated HT-29 cell proliferation and inhibited both serum-starvation and celecoxib-induced apoptosis. The proliferative and anti-apoptotic effects of leptin were abolished by inhibition of JAK2 with AG490, phosphatidylinositol 3 -kinase (PI3 kinase) with LY294002 and c-Jun NH(2)-terminal kinase (JNK) with SP600125. Stimulation of HT-29 cells with leptin increased phosphorylation of JAK2, Akt and JNK. Activation of JAK2 was upstream of PI3 kinase/Akt but not of JNK. Activation of JAK2 was followed by activation and nuclear translocation of STAT3 and JNK activation led to increased activator protein 1 (AP-1) transcriptional activity. CONCLUSIONS: Leptin stimulates proliferation and inhibits apoptosis in human colon cancer cells and may be an important factor in the increased incidence of colon cancer in obesity. This effect involves JAK2, PI3 kinase and JNK and activation of the oncogenic transcription factors signal transducer and activator of transcription (STAT)3 and AP-1.

Unlike the inherited mutations, which are present in ALL cells, somatic (acquired) mutations occur only in certain cells of the body and, quite often, are oncogenic. Quantification of mutant allele burden (percentage of the mutant allele) is critical for diagnosis, monitoring of therapy, and detection of minimal residual disease. With point mutations, the challenge is to quantify the mutant allele while discriminating from a large excess of the normal allele that differs in a single base-pair. To this end, we report the first bioluminometric assay for quantification of the allele burden and its application to JAK2 V617F somatic point mutation, which is a recently (2005) discovered molecular marker for myeloproliferative neoplasms. The method is performed in microtiter wells and involves a single PCR, for amplification of both alleles, followed by primer extension reactions with allele-specific primers. The products are captured in microtiter wells and detected by oligo(dT)-conjugated photoprotein aequorin. The photoprotein is measured within seconds by simply adding Ca(2+). We have demonstrated that the percent (%) luminescence signal due to the mutant allele is linearly related to the allele burden. As low as 0.85% of mutant allele can be detected and the linearity extends to 100%. The assay is complete within 50 min after the amplification step.

BCR/ABL1-negative chronic myeloproliferative neoplasms (CMPNs) are a heterogeneous group of clonal hematological malignancies. Over recent years, some genetic events in tyrosine kinase (TK) genes have been described as causal events of these diseases. To identify new genetic aberrations underlying these diseases, we used denaturing high performance liquid chromatography and fluorescence in situ hybridization (FISH) to analyze 17 genes from two receptor-TK families (III and IV) and from three cytoplasmic-TK families (Syk, Abl, and Jak) on samples from 44 BCR/ABL1-negative and JAK2(V617F)-negative CMPN patients with different clinical phenotypes. Although screening by FISH did not reveal novel chromosomal aberrations, several sequence changes were detected. None of them were frequent events, but we identified a new potential activating mutation in the FERM domain of JAK2(R340Q). None of the germline JAK2(V617F) single-nucleotide polymorphisms detected differed in distribution between patients and control subjects. In summary, data presented here show that these genes are not frequently mutated or rearranged in CMPNs, suggesting that molecular events causing these disorders must be located in other genes.

The oncogenic JAK2V617F mutation is found in myeloproliferative neoplasms (MPNs) and is believed to be critical for leukemogenesis. Here we show that JAK2V617F requires an intact SH2 domain for constitutive activation of downstream signaling pathways. In addition, there is a strict requirement of cytokine receptor expression for the activation of this oncogene. Further analysis showed that the SH2 domain mutation did not interfere with JAK2 membrane distribution. However, coimmunoprecipitated experiments revealed a role for the SH2 domain in the aggregation and cross-phosphorylation of JAK2V617F at the cell membrane. Forced overexpression of cytokine receptors could rescue the JAK2V617F SH2 mutant supporting a critical role of JAK2V617F abundance for constitutive activation. However, under physiologic cytokine receptor expression the SH2 domain is absolutely necessary for oncogenic JAK2V617F activation. This is demonstrated in a bone marrow transplantation model, in which an intact SH2 domain in JAK2V617F is required for the induction of an MPN-like disease. Thus, our results points to an indispensable role of the SH2 domain in JAK2V617F-induced MPNs.

BACKGROUND: Activating mutations in JAK1 and JAK2 have been described in patients with various hematologic malignancies including acute lymphoblastic leukemia and myeloproliferative neoplasms, leading to clinical trials with JAK inhibitors. While there has been a tremendous effort towards the development of specific JAK inhibitors, mutations conferring resistance to such drugs have not yet been observed. DESIGN AND METHODS: Taking advantage of a model of spontaneous cellular transformation, we sequenced JAK1 in selected tumorigenic BaF3 clones and identified 25 de novo JAK1 activating mutations, including 5 mutations already described in human leukemias. We further used this library of JAK1 mutation-positive cell lines to assess their sensitivity to ATP-competitive inhibitors. RESULTS: While most JAK1 mutants were sensitive to ATP-competitive JAK inhibitors, mutations targeting Phe958 and Pro960 in the hinge region of the kinase domain rendered JAK1 constitutively active but also resistant to ALL tested JAK inhibitors. Furthermore, mutation of the homologous Tyr931 in JAK2 wild-type or JAK2 V617F mutant found in patients with myeloproliferative neoplasms also conferred resistance to JAK inhibitors, such as INCB018424, which is currently in clinical use. CONCLUSIONS: Our data indicate that some activating mutations not only promote autonomous cell proliferation but also confer resistance to ATP-competitive inhibitors. In vivo, such a mutation can potentially occur as primary JAK-activating mutations but also as secondary mutations combining oncogenicity with drug resistance.

It has been well established that disruption of JAK2 signaling regulation is involved in various hematopoietic disorders; however, the detailed mechanism by which abnormal activation of JAK2 exhibits transforming activity remains to be elucidated. The somatic JAK2 mutation (V617F) was identified in most patients with polycythemia vera (PV). Here, we show that JAK2 V617F mutant was constitutively active and exhibited tumorigenesis activity as a potent oncogene when erythropoietin receptor (EpoR) was co-expressed. To clarify the signaling pathway of JAK2 V617F mutant, we investigated the functional role of downstream transcription factor STAT5 in its induced cellular transformation and tumorigenesis in nude mice. Interestingly, JAK2 V617F mutant failed to exhibit transforming activity when STAT5 activation was inhibited utilizing EpoR mutant (HM). Furthermore, the expression of constitutively active STAT5 mutant (1*6) exhibited transforming activity. Taking these observations together, it is concluded STAT5 plays an essential role in EpoR-JAK2 V617F mutant-induced hematopoietic disorder and would be a good target for the treatment of PV.

The JAK2(V617F) mutation is present in the majority of patients with polycythemia vera and one-half of those with essential thrombocythemia and primary myelofibrosis. JAK2(V617F) is a gain-of-function mutation resulting in constitutive JAK2 signaling involved in the pathogenesis of these diseases. JAK2(V617F) has been shown to promote S-phase entry. Here, we demonstrate that the CDC25A phosphatase, a key regulator of the G1/S cell-cycle transition, is constitutively overexpressed in JAK2(V617F)-positive cell lines, JAK2-mutated patient CD36(+) progenitors, and in vitro-differentiated proerythroblasts. Accordingly, CDC25A is overexpressed in BM and spleen of Jak2(V617F) knock-in mice compared with wild-type littermates. By using murine FDC-P1-EPOR and human HEL and SET-2 cell lines, we found that JAK2(V617F)-induced CDC25A up-regulation was caused neither by increased CDC25A transcription or stability nor by the involvement of its upstream regulators Akt and MAPK. Instead, our results suggest that CDC25A is regulated at the translational level through STAT5 and the translational initiation factor eIF2alpha. CDC25A inhibition reduces the clonogenic and proliferative potential of JAK2(V617F)-expressing cell lines and erythroid progenitors while moderately affecting normal erythroid differentiation. These results suggest that CDC25A deregulation may be involved in hematopoietic cells expansion in JAK2(V617F) patients, making this protein an attracting potential therapeutic target.

Constitutive activation of STAT3 is a common feature in many solid tumors including non-small cell lung carcinoma (NSCLC). While activation of STAT3 is commonly achieved by somatic mutations to JAK2 in hematologic malignancies, similar mutations are not often found in solid tumors. Previous work has instead suggested that STAT3 activation in solid tumors is more commonly induced by hyperactive growth factor receptors or autocrine cytokine signaling. The interplay between STAT3 activation and other well-characterized oncogenic "driver" mutations in NSCLC has not been fully characterized, though constitutive STAT3 activation has been proposed to play an important role in resistance to various small-molecule therapies that target these oncogenes. In this study we demonstrate that STAT3 is constitutively activated in human NSCLC samples and in a variety of NSCLC lines independent of activating KRAS or tyrosine kinase mutations. We further show that genetic or pharmacologic inhibition of the gp130/JAK2 signaling pathway disrupts activation of STAT3. Interestingly, treatment of NSCLC cells with the JAK1/2 inhibitor ruxolitinib has no effect on cell proliferation and viability in two-dimensional culture, but inhibits growth in soft agar and xenograft assays. These data demonstrate that JAK2/STAT3 signaling operates independent of known driver mutations in NSCLC and plays critical roles in tumor cell behavior that may not be effectively inhibited by drugs that selectively target these driver mutations.

Approximately 10% of B-cell acute lymphoblastic leukemias (B-ALLs) overexpress the cytokine receptor subunit CRLF2, which may confer a poor prognosis. CRLF2 binds its ligand thymic stromal lymphopoietin (TSLP) as a heterodimer with IL7R. Subsets of CRLF2-overexpressing B-ALLs also have a gain-of-function CRLF2 F232C mutation or activating mutations in JAK2. Whether these mutant alleles confer differences in signaling has not been addressed. Through a domain mutation analysis, we demonstrate a distinct dependence on the CRLF2 intracellular tyrosine Y368 in signaling by CRLF2 F232C, but not signaling induced by TSLP or through CRLF2/mutant JAK2. In contrast, CRLF2 signaling in each context is strictly dependent on both the CRLF2 box1 domain and the intracellular tryptophan W286. Using a global quantitative analysis of tyrosine phosphorylation induced by TSLP, we previously identified TSLP-induced phosphorylation of multiple kinases implicated in B-cell receptor signaling, including Lyn, Btk, Hck, Syk, MAPK8, MAPK9, and MAPK10. We now demonstrate that cells dependent on CRLF2/mutant JAK2 have reduced phosphorylation at these targets, suggesting that the kinases promote TSLP-mediated proliferation but serve as negative regulators of CRLF2/mutant JAK2 signaling. Thus, targetable nodes downstream of CRLF2 differ based on the presence or absence of additional mutations in CRLF2 signaling components.

EZH2 (enhancer of zeste homolog 2) is a critical enzymatic subunit of the polycomb repressive complex 2 (PRC2), which trimethylates histone H3 (H3K27) to mediate gene repression. Somatic mutations, overexpression and hyperactivation of EZH2 have been implicated in the pathogenesis of several forms of cancer. In particular, recurrent gain-of-function mutations targeting EZH2 Y641 occur most frequently in follicular lymphoma and aggressive diffuse large B-cell lymphoma and are associated with H3K27me3 hyperactivation, which contributes to lymphoma pathogenesis. However, the post-translational mechanisms of EZH2 regulation are not completely understood. Here we show that EZH2 is a novel interactor and substrate of the SCF E3 ubiquitin ligase beta-TrCP (FBXW1). beta-TrCP ubiquitinates EZH2 and Jak2-mediated phosphorylation on Y641 directs beta-TrCP-mediated EZH2 degradation. RNA interference-mediated silencing of beta-TrCP or inhibition of Jak2 results in EZH2 stabilization with attendant increase in H3K27 trimethylation activity. Importantly, the EZH2(Y641) mutants recurrently implicated in lymphoma pathogenesis are unable to bind beta-TrCP. Further, endogenous EZH2(Y641) mutants in lymphoma cells exhibit increased EZH2 stability and H3K27me3 hyperactivity. Our studies demonstrate that beta-TrCP has an important role in controlling H3K27 trimethylation activity and lymphoma pathogenesis by targeting EZH2 for degradation.

During the past 10 years, major progress has been accomplished with the discovery of activating mutations that are associated with the vast majority of BCR-ABL negative human myeloproliferative neoplasms (MPNs). The identification in 2005 of JAK2 V617F triggered great interest in the JAK2-STAT5/STAT3 pathway. Discovery in 2006 of mutants of thrombopoietin receptor (TPO-R/MPL) and later on of mutants in negative regulators of JAK-STAT pathway led to the notion that persistent JAK2 activation is a hallmark of MPNs. In 2013, mutations in the gene coding for the chaperone calreticulin were reported in 20-30 % of essential thrombocythemia and primary myelofibrosis patients. Here, we will address the question: what do we know about calreticulin that could help us understand its role in MPNs? In addition to oncogenic driver mutations, certain MPNs also exhibit epigenetic mutations. Targeting of both oncogenic drivers and epigenetic defects could be required for effective therapy.

Stem cell factor (SCF) is a hematopoietic growth factor that interacts with the receptor tyrosine kinase, c-kit. We have found that SCF-stimulates rapid and transient tyrosine phosphorylation of JAK2 in human and murine cell lines, as well as in normal human progenitor cells. JAK2 and c-kit were associated in unstimulated cells with further recruitment of JAK2 to the c-kit receptor complex after SCF stimulation. Treatment of cells with JAK2 antisense oligonucleotides resulted in a 46% decrease in SCF-induced proliferation. These data demonstrate that SCF induces tyrosine phosphorylation of JAK2 and suggest that JAK2 is a component of the SCF signal transduction pathway.

The vav proto-oncogene product (Vav), which is specifically expressed in hematopoietic cells, contains multiple structural motifs commonly used by intracellular signaling molecules. Although a variety of stimuli including erythropoietin (Epo) have been shown to tyrosine phosphorylate Vav, little is known about the Vav signal transduction pathway. Here, we have investigated the role of Vav in the Epo signaling pathway by characterizing its interaction with other proteins, using the human Epo-responsive cell line, F-36P. Immunoprecipitation and immunoblot analyses have demonstrated that Vav was associated with the Epo receptor (EpoR) in an Epo-independent manner and was tyrosine-phosphorylated after Epo stimulation. Furthermore, two phosphotyrosine proteins (pp70 and pp100) co-immunoprecipitated with the regulatory subunit of phosphatidylinositol 3-kinase (PI3-kinase) (p85) were identified as EpoR and Vav, respectively. The interaction between Vav and p85 was shown to be mediated through the SH2 domains of p85 by an in vitro binding assay and confirmed by the presence of in vitro PI3-kinase activity associated with Vav. Treatment of the cells with antisense-vav and -p85 abrogated Epo-induced cell proliferation and PI3-kinase activity. Finally, we found that JAK2 was associated with Vav in vivo and that Vav could be tyrosine-phosphorylated by activated JAK2 in vitro. These results suggest the possible role of JAK2 for tyrosine phosphorylation of Vav and involvement of Vav and PI3-kinase in Epo-induced proliferative signals.