Activation Of JAK2 Tyrosine Kinase Research Articles

CXCR4- and BCR-triggered integrin activation in B-cell chronic lymphocytic leukemia cells depends on JAK2-activated Bruton's tyrosine kinase.

Bruton's tyrosine kinase (BTK) regulates the B-cell receptor (BCR) signaling pathway, which, in turn, plays a critical role in B-cell chronic lymphocytic leukemia (B-CLL) pathogenesis. The BTK-specific inhibitor Ibrutinib blocks BCR signaling and is now approved as effective B-CLL therapy. Chemokines, such as the homeostatic chemokine CXCL12, play a central role in B-CLL pathogenesis and progression, by regulating CLL cell interaction with the stromal microenvironment, leading to cells survival and proliferation. In this study, we investigated, in normal versus CLL B-lymphocytes, the role of BTK in signal transduction activated by the CXCL12-CXCR4 signaling axis and its involvement in rapid integrin activation. We show that BTK is rapidly activated by CXCL12 in healthy as well as CLL B-lymphocytes, with a kinetic of tyr-phosphorylation coherent with rapid adhesion triggering. BTK inhibition prevents CXCL12-induced triggering of lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) integrins. Furthermore, BTK inhibition blocks the activation of the small GTP-binding protein RhoA, controlling integrin affinity. Very importantly, we show that BTK tyr-phosphorylation and activation by CXCL12 depends on upstream activation of JAK2 tyrosine kinase. A comparative analysis of 36 B-CLL patients demonstrates that JAK2-dependent BTK regulatory role on integrin activation by CXCL12 is fully conserved in CLL cells. Finally, we show that the JAK2-BTK axis also regulates signaling to integrin activation by BCR. Thus, BTK and JAK protein tyrosine kinases (PTKs) manifest a hierarchical activity both in chemokine- as well as BCR-mediated integrin activation and dependent adhesion, potentially suggesting the possibility of combined therapeutic approaches to B-CLL treatment.

Preparation of Transmembrane Fragments Growth Hormone Receptor GHR in a Cell-Free Expression System for Structural Studies

Growth hormone somatotropin and its membrane receptor GHR, belonging to a superfamily of the type I receptors possessing tyrosine kinase activity, are involved in the intercellular signal transduction cascade and regulate a number of important physiological and pathological processes in humans. Binding with somatotropin triggers a transition of GHR between two alternative dimer states, resulting in an allosteric activation of JAK2 tyrosine kinase in the cell cytoplasm. Transmembrane domain of GHR directly involved in this complex conformational transition. It has presumably two dimerization interfaces corresponding to the "unliganded" and the active state of GHR. In order to study the molecular basis of biochemical signal transduction mechanism across the cell membrane, we have developed an efficient cell-free production system of a TM fragment of GHR, which contains its TM domain flanked by functionally important juxtamembrane regions (GHRtm residues 254-298). The developed system allows to obtain -1 mg per 1 ml of reaction mixture of 13C- and 15N-isotope-labeled protein for structural and dynamic studies of the GHR TM domain dimerization in the membrane-mimicking medium by high-resolution heteronuclear NMR spectroscopy.

Selective Anti Leukemic Activity Of Low Dose Decitabine In Combination With Ruxolitinib Against Stem/Progenitor Cells From Elderly AML Patients

Acute myeloid leukemia (AML) is a clonal hematopoietic disorder characterized by an accumulation of immature leukemic cells. The incidence of AML sharply increases with age. However outcomes for AML in the elderly are dismal with low response rates and high rates of relapse. Primitive AML leukemia stem cells (LSC) are resistant to conventional therapy, and potentially contribute to propensity to relapse. Therefore, new strategies to enhance targeting of AML LSCs and are required to improve outcomes. AML cells demonstrate several epigenetic alterations including enhanced promoter hypermethylation that may contribute to altered growth and self-renewal of LSC. The DNA methyltransferase (DNMT) inhibitor decitabine (DAC) has shown activity against AML LSC in preclinical studies. In clinical studies DAC is well tolerated as monotherapy and induces responses in approximately 20% of AML patients who are not candidates for conventional chemotherapy. Therefore there is considerable interest in developing combinations of DAC with other therapies for elderly patients with AML. We have previously shown that LSC from AML patients with poor risk cytogenetics demonstrate increased JAK2 tyrosine kinase activity and enhanced sensitivity to JAK2 inhibition. Since cross regulation between JAK/STAT and DNMT pathways has been reported, we evaluated the activity of the combination of DAC with the JAK2 tyrosine kinase inhibitor (TKI) Ruxolitinib (RUX) on CD34+ cells from elderly AML patients (>60 years). Exposure to low dose DAC (10 to 200nM) for 72 hours significantly inhibited the growth of primary AML CD34+ cells in a CellTitre Glo assay in a dose dependent manner (n=12, DAC 10nM 88±9% of control, 50nM 77±9%, 100nM 64±11% and 200nM 56±15%). DAC did not induce significant apoptosis in AML CD34+ cells (n=10, control, 22±5% of Annexin V positive cells vs 24±7% for the samples treated with 200nM of DAC) but dramatically and significantly reduced AML CD34+ cell growth in CFC assays (33±3%, 57±14%, 75±3% and 86±7% inhibition with 10, 50, 100 and 200nM of DAC respectively). The combination of RUX (100nM) and DAC (10 and 50nM) significantly enhanced inhibition of AML CD34+ cell growth compared to RUX or DAC alone (DAC 10nM 87±10% of control for, RUX 62±20%, DAC10+RUX 46±20%, DAC50nM 75±10%, and DAC50+RUX 43±20%, n=7, p<0.009). Combination of RUX (100nM) with 10 and 50nM DAC did not significantly enhance apoptosis in AML (n=10) or normal stem/progenitor cells (n=8, Ctrl 20.6±5%, DAC10nM 21.6±5%, RUX100nM 31.5±5%, DAC10-RUX 32.1±5%, DAC50nM 22.6±5% and DAC50-RUX 32.4±8%), but resulted in significantly enhanced inhibition of AML CD34+ CFC compared to RUX or DAC alone (n=4, CFC inhibition with DAC10 34±4%, RUX 46±15%, DAC10+RUX 68±17%, DAC50 56±15%, and DAC50+RUX 77±7%, p<0.004), but did not enhance inhibition of CB CFU-GM (n=4, CFC inhibition with DAC10 0±12%, RUX 24.3±16%, DAC10+RUX 4±9%, DAC50 9±16%, and DAC50+RUX 25±11%). These results indicate that combined targeting of both JAK/STAT and DNMT pathways can selectively inhibit the growth capacity of AML compared with normal stem/progenitor cells. We performed Western blotting on AML CD34+ cells (n=5) treated for 3 days with RUX 100nM, DAC 50nM or the combination of RUX and DAC to evaluate expression of DNMT1, DNMT3a, DNMT3b, phosphorylated STAT3 (Y705) and STAT5 (Y694) and SOCS3. Compared to the individual drugs, the combination resulted in greater reduction of p-STAT3 in 4 out 5 AML patient samples that was associated with increase in SOCS3 in 2 samples, suggesting a crosstalk between the JAK/STAT and DNMT pathways. Interestingly we did not observe enhanced inhibition of p-STAT5 with the combination. Two of five AML samples displayed decrease of DNMT1 expression and 3 of 5 samples decreased DNMT3a and DNMT3b expression upon treatment with the combination. In conclusion, our studies suggest that there may be a crosstalk between the JAK/STAT/SOCS3 signalling and DNA methylation mechanisms in CD34+ cells from elderly AML patients, and indicate that JAK2 TKI in combination with low doses of DAC selectively and significantly enhance inhibition of stem/progenitor cells from elderly AML patients compared to DNMT inhibitors alone. These results support further evaluation of this novel combination for the treatment of AML in the elderly. Disclosures:No relevant conflicts of interest to declare.

Effects of NVP-BSK805, a Novel JAK2 Inhibitor, on BCR-ABL and JAK2V617F Positive Cell Lines

Abstract 5002 Background:Janus kinases are critical components of cytokine signaling pathways that regulate hematopoiesis, growth, immunity, inflammation, and development. Oncogenic mutations of the non-receptor tyrosine kinase JAK2 are found in many Philadelphia chromosome negative myeloproliferative neoplasms. The V617F mutation in JAK2 occurs in 95% of patients with polycythemia vera, 50% of those with essential thrombocythemia and 50% of primary myelofibrosis patients. Preclinical results strongly support that JAK2 inhibitors could be effectively used in these three indications. Replacement of valine 617 with phenylalanine upregulates the tyrosine kinase activity of JAK2, causing constitutive activation of the JAK-STAT pathway and growth factor-independent cell proliferation. JAK2 has also been postulated to play an important role in BCR-ABL signal transduction. Therefore, inhibitors of the tyrosine kinase activity of JAK2 are under investigation as new therapy strategies for CMPNs. In this study the role of the novel JAK2 inhibitor, NVP-BSK805 (Novartis Pharmaceuticals), has been investigated in cells expressing either BCR-ABL or mutant JAK2. Possible synergistic effects between NVP-BSK805 and the already established tyrosine kinase inhibitors imatinib and nilotinib were assessed. Methods:The in vitro activity of NVP-BSK805 was analyzed in 12 hematopoietic cell lines, including 7 BCR-ABL positive (K562, KCL22, KU812, Lama87, BV173, EM3, SUP-B15), 4 JAK2 mutated (CHRF288, SET2, UKE1, HEL), the T-cell leukemia cell line Jurkat, and the neuroendocrine colonic tumour line LCC-18. Concentration kinetics from 0 up to 25 μM were established using XTT proliferation assays and flow cytometry for measuring apoptosis. Protein levels of JAK2, phospho-JAK2, STAT5, phospho-STAT5 and BCR-ABL were analyzed using Western blotting. NVP-BSK805 was also tested in combination with imatinib and nilotinib. JAK2 was sequenced in all cell lines in order to detect possible mutations in the gene. Results:Of the JAK2 mutated cell lines tested, 3 of 4 (CHRF288, SET2, UKE1) showed a significant reduction of proliferation, as well as viability, compared to the other cell lines. CHRF288 responded best to NVP-BSK805 with an IC50 value of 0.22 ± 0.04 μM. UKE1 and SET2 had similar values of 0.35 ± 0.03 μM and 0.37 ± 0.05 μM. Interestingly, HEL (V617F positive) cells showed only an IC50 value (1.8 ± 0.17 μM) for NVP-BSK805, comparable with that of the non-mutated BCR-ABL positive cell lines (1.5 to 2.7 μM). LCC-18 showed the weakest response of all cell lines tested, with an IC50 value of 9.93 ± 0.202 μM. Each cell line responded to concentrations higher than 5 μM with a strong reduction of proliferation due to inhibition of various kinases. Combination of the JAK2 inhibitor with imatinib and nilotinib showed no significant additive or synergistic effects, although all BCR-ABL positive cell lines responded well to both CML therapeutic agents. Western blotting of proteins of the JAK-STAT pathway confirmed the results of the proliferation and apoptosis tests showing a strong reduction of phoshorylated STAT5 in CHRF288 cells after a 30 min incubation even with NVP-BSK805 concentrations as low as 0.01 μM. UKE-1 and SET-2 showed reduction of pSTAT5 from 0.1 μM. Levels of total STAT5 were not affected. In all the other cell lines no changes were detected in any of the proteins tested. Conclusions:Here, we tested a novel JAK2 inhibitor in cells carrying the V617F mutation. Interestingly, not every cell line with the JAK2 V617F mutation showed a good response upon JAK2 inhibition, indicating that there are additional factors determining response. On the other hand, clinical trials with JAK inhibitors in myelofibrosis have shown responses in V617F-mutated and non-mutated patients, warranting further research to identify predictors of response. In BCR-ABL mutant cells not harbouring JAK2 mutations no significant inhibition of proliferation or apoptosis was detected following JAK2 inhibition, indicating that there are JAK2 independent signal transduction pathways of BCR-ABL to avoid apoptosis. Disclosures:le Coutre:Novartis Pharmaceuticals: Honoraria, Research Funding.

Abstract 5127: G6-mediated inhibition of Jak2 tyrosine kinase correlates with the cleavage and cellular reorganization of vimentin

Abstract Hyperkinetic Jak2 tyrosine kinase signaling has been implicated in several human diseases including various leukemias, lymphomas, myelomas and the myeloproliferative neoplasms. Using structure-based virtual screening, our lab previously identified a novel Jak2 inhibitor named G6. We showed that G6 has a specific inhibitory effect on Jak2 tyrosine kinase activity and potently suppresses Jak2-mediated cellular proliferation. To elucidate the biochemical and cellular mechanisms by which G6 inhibits Jak2-mediated cell proliferation, we treated Jak2-V617F expressing human erythroleukemia (HEL) cells for 12 hours with either vehicle control (DMSO) or 25 µM of the drug and then compared their protein expression profiles using two-dimensional gel electrophoresis. One protein that was found to be differentially expressed was the intermediate filament protein vimentin. Specifically, we found that full length vimentin was robustly expressed in untreated cells and absent in G6 treated cells. We hypothesized that inhibition of Jak2-V617F dependent pathogenic cell growth by G6 correlates with the cleavage and cellular reorganization of intermediate vimentin filaments. To this end, we found that the treatment of HEL cells with G6 induced a time- and dose-dependent cleavage of vimentin. This G6-induced cleavage of vimentin was both Jak2-dependent and calpain-mediated. Using indirect immunofluorescence, we found that G6 treatment resulted in a marked reorganization of vimentin filaments within cells. Finally, using a mouse model of Jak2-V617F mediated neoplasia, we found that G6 decreased the levels of vimentin protein within the bone marrow of G6 treated mice when compared to animals that received vehicle control. Overall, these results demonstrate that G6-mediated inhibition of Jak2-dependent pathogenic cell growth correlates with the proteolytic cleavage of full length vimentin and the reorganization of intermediate vimentin filaments within cells. As such, we may have identified a biochemical and cellular mechanism whereby G6 inhibits Jak2-V617F mediated cellular proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5127. doi:10.1158/1538-7445.AM2011-5127

STAT3-induced S1PR1 expression is crucial for persistent STAT3 activation in tumors

IL-6/Jak2 signaling is viewed critical for persistent Stat3 activation in cancer. However, IL-6-induced Stat3 activity is transient in normal physiology. Here we identify a mechanism important for persistent Stat3 activation in tumor cells and the tumor microenvironment. We show that sphingosine-1-phosphate receptor 1 (S1PR1), a G-protein-coupled receptor for lysophospholipid sphingosine-1-phosphate (S1P), is elevated in Stat3-positive tumors. Stat3 is a transcription factor for the S1pr1 gene. Enhanced S1pr1 expression activates Stat3 and upregulates Il6 gene expression, thereby accelerating tumor growth and metastasis. Conversely, silencing S1pr1 in tumor cells or immune cells inhibits tumor Stat3 activity, tumor growth and metastasis. S1P/S1PR1-induced Stat3 activation is persistent, in contrast to transient Stat3 activation by IL-6. S1PR1 activates Stat3 in part by upregulating Jak2 tyrosine kinase activity. We demonstrate that Stat3-induced S1pr1 expression, as well as S1P/S1PR1 pathway, is important for persistent Stat3 activation in cancer cells and the tumor microenvironment and for malignant progression.

A Component of a Heterodimeric Receptor Can Functionally Replace a Homodimeric Receptor in Supporting the Activation and Transforming Activity of JAK2-V617F

We have recently identified IL27Ra as a gene from an AML patient that transforms hematopoietic cells to a state of cytokine-independent growth. Importantly, we have found that the gene product of IL27Ra, intereukin-27 receptor (IL27R), is expressed on the cell surface of the leukemic cells of AML patients. IL27R, also know as TCCR and WSX-1, is a component if a heterodimeric type I cytokine receptor that functions as the ligand-binding subunit of the receptor for IL-27. Normally, IL27R functions with the gp130 co-receptor to induce signal transduction in response to IL-27. However, we have demonstrated that IL27R can induce the transformation of hematopoietic cells in a ligand- and gp130-independent manner. IL27R, which contains a JAK-binding Box 1 motif, requires JAK family kinase activity to transform cells. In a potential mechanism of IL27R-mediated transformation, IL27R may function as a homodimer to activate JAK family members. In an effort to determine if IL27R can function as a homodimer, we tested to see if IL27R could activate the V617F mutation of JAK2 commonly found in various myeloproliferative neoplasms (MPNs). It is believed that JAK2-V617F utilizes a homodimeric cytokine receptor complex as a scaffold in order to multimerize and become fully activated via transphosphorylation. Expression of IL27R activated the kinase activity of JAK2-V617F, suggesting that IL27R may form homodimers in cells. In addition, co-expression of IL27R and JAK2-V617F led to transformation of BaF3 cells to cytokine-independence, whereas IL27R expressed with wildtype JAK2 or JAK2-V617F expressed alone did not induce rapid cytokine-independent growth of these cells. This is similar data to that obtained with co-expression of JAK2-V617F with homodimeric receptors (e.g. EpoR, GCSFR), demonstrating that a component of a heterodimeric cytokine receptor can functionally replace a homodimeric receptor in mediating JAK2-V617F activation. Chemical crosslinking showed that IL27R could dimerize suggesting that IL27R may form a scaffold for JAK2-V617F-mediated transformation. In addition to providing a scaffold for binding and activation of JAK2-V617F, it has been proposed that homodimeric receptors also provide an important substrate of the JAK2 tyrosine kinase activity to induce cell signaling and transformation. Tyrosine phosphorylation of the scaffolding receptor presumably recruits signaling molecules (e.g. STATs) to the receptor where they can be activated by JAK2 phosphorylation. We have mutated the two tyrosine residues in the cytoplasmic domain of IL27R and used these receptor mutants to determine if tyrosine phosphorylation of IL27R is required to induce the transforming signal of JAK2-V617F. Upon initial cytokine removal, there is a slight lag in the growth of JAK2-V617F cells expressing an IL27R receptor that lacks both intracellular tyrosines, suggesting these tyrosines may play a role in signaling. However, this lag is transient and cytokine-independent growth becomes similar to cells expressing wildtype IL27R and JAK2-V617F. This suggests tyrosine phosphorylation of the receptor is not required to induce the transforming signal of JAK2-V617F. Our studies demonstrate for the first time that single chain components of heterodimeric receptors can functionally replace homodimeric receptors in mediating transformation of hematopoietic cells by JAK2-V617F. Our work also suggests that JAK2-V617F does not need to phosphorylate its receptor scaffold to mediate its transforming signal. The requirement for other endogenous cytokine receptors as substrates for activated JAK2-V617F receptors remains a possibility and is currently under investigation. Together, our studies demonstrate that IL27R, and perhaps other single chain components of heterodimeric receptors, may play unappreciated roles in myeloid diseases such as AML and MPNs.

Molecular Mechanisms of JAK2V617F Oncogenic Activation: The JAK2- V617F Mutant Utilizes the SH2 Domain for Constitutive Kinase Activity in the Presence of Unstimulated Heterodimeric Cytokine Receptor.

The JAK2-V617F mutation has been reported in the majority of MPDs including PV, ET, and IMF. This mutation leads to the constitutive activation of the JAK2 tyrosine kinase activity and overexpression of JAK2V617F renders hematopoietic cell lines growth factor-independent. However, the molecular mechanism leading to constitutive activation of JAK2V617F is largely unclear and the requirement of homodimeric or heterodimeric cytokine receptors needs to be determined. Here we show that oncogenic JAK2-V617F requires an intact SH2 domain for constitutive kinase activity. To this end we mutated the conserved arginine 426 within the SH2 domain to a lysine. Ba/F3 cells expressing JAK2V617F grew IL-3-independent and showed constitutive activation of JAK2, STAT5, and ERK1/2. In contrast, introduction of the SH2 mutation in JAK2V617F abrogated both transformation as well as constitutive activation of downstream signaling pathways. Accordingly, reconstitution of JAK2 mutants in a JAK2-negative cell line with IL-3R co-expression revealed reduced activation of JAK2 when the SH2 domain was mutated. It has been reported that JAK2 binding to homodimeric type I cytokine receptor may facilitate JAK2V617F-mediated transformation. Interestingly, co-expression of the homodomeric EpoR with SH2 mutated JAK2V617F rescues the phenotype indicating that the SH2 domain is required for JAK2 signaling in the presence of heterodimeric but not homodimeric cytokine receptors. Membrane localization studies showed equal membrane distribution of SH2-mutated and unmutated JAK2-V617F indicating that the SH2 domain mutation does not affect subcellular distribution of JAK2. However, co-IP experiments revealed a possible role for the SH2 domain in the dimerization and transphosphorylation of JAK2. Consequently, reduced transphosphorylation was seen in IL-3R- but not in EpoR-expressing cells. In a BM transplantation model we found that an intact SH2 domain in JAK2V617F was required for the induction of a MPD-like disease. Thus, our results points to an important role of the SH2 domain for the constitutive activation of JAK2V617F in cells expressing heterodimeric cytokine receptors.

A Novel Small-Molecule Approach To Inhibit Jak2 Tyrosine Kinase Signaling.

Jak kinases are non-receptor protein tyrosine kinases that play a pivotal role in cytokine/growth factor signaling through phosphorylation of specific proteins such as the Stat molecules. Activated Stats translocate to the nucleus where they mediate transcription of several target genes involved in cell cycle progression and survival (Bcl-xL, cyclin D1, c-myc, survivin. Many tumors have highly activated Stats that are associated with aberrant Jak2 regulation and recent studies have shown that activating mutations in Jak2 (V617F) play a key role in many myeloproliferative disorders such as polycythemia vera and essential thrombocythemia. Jak inhibitors may be useful in treating many diseases with aberrant Jak2/Stat signaling. The most commonly used inhibitor of Jak2 is the tyrphostin AG490, which inhibits Stat3 activation by preventing its tyrosine phosphorylation. However AG490 has limited in vivo efficacy and must be administered at high concentrations (>50 μM) for anti-tumor effects. We describe here a new class of compounds, termed degrasyns, that block Jak2 mediated activation of Stat3 in intact cells at high nM to low μM concentrations. Degrasyns (WP1130/CP2005) did not directly inhibit Jak2 tyrosine kinase activity but suppressed Stat3 activation by reducing the cytoplasmic levels of Jak2. Degrasyn-mediated Jak2 down-regulation was rapid (complete in 2 hrs) and not inhibited by proteasomal, lysosomal, or serine/threonine protease inhibitors. Biochemical studies and confocal microscopy show that degrasyn induces translocation of Jak2 from the plasma membrane/cytosolic fraction into the cytoskeletal fraction and this altered partitioning of Jak2 was associated with loss of cytokine-mediated Stat activation by degrasyn. Jak2 translocation was associated with tyrosine phosphorylation of specific proteins which complex with Jak2. Lyn kinase in the cytoskeletal fraction was highly activated by degrasyn in multiple hematopoetic tumors (multiple myeloma, mantle cell lymphoma, leukemias). Jak2 translocation and Stat inhibition by degrasyn is mechanistically distinct from “classical” Jak2 inhibitors and is not associated with a translocation of other kinases or cytokine signaling molecules in the Jak2 cascade (IL-6R, gp130, Lyn, Btk, Hck, Akt, PI-3K, Erk, Src, Jak1). Degrasyn induces cytoskeletal translocation of both wild-type and mutant (V617F) Jak2 and was associated with induction of apoptosis in HEL cells expressing the Jak2 V617F mutation. These results suggest that degrasyn suppresses Jak/Stat signaling through a unique mechanism involving translocation of Jak2 into a signal transduction incompetent compartment and may be used to investigate a novel form of Stat suppression. Degrasyn may also have anti-tumor effects on cells with aberrant activation of Jak/Stat signaling.

A potential role for HSP90 inhibitors in the treatment of JAK2 mutant-positive diseases as demonstrated using quantitative flow cytometry

The V617F mutation of the JAK2 tyrosine kinase is found in a majority of patients with myeloproliferative disorders. Flow cytometry assays for quantitation of phosphorylated and total protein for JAK2, STAT5, and heat shock proteins (HSPs) were developed to facilitate the study of the JAK/STAT pathway. A cell line homozygous for V617F (HEL) was treated with inhibitors of JAK2 tyrosine kinase activity and the HSP90 inhibitor 17-AAG. 17-AAG reduced HSP90 levels, but increased HSP70 levels. Phospho-STAT5, total STAT5, and total AKT levels were also reduced by17-AAG treatment. Further, phospho-JAK2, total JAK2, and cell viability were reduced to a greater extent by 17-AAG than by the pan-JAK kinase family inhibitor JKII or the JAK2-specific inhibitor AG490, and these inhibitors failed to synergize with 17-AAG. Flow-cytometry-based assays for JAK/STAT signaling pathway and HSPs are likely to have broad clinical utility for monitoring patients with abnormalities in the JAK2 pathway.

RPS19 and JAK2 Are Not Silenced by DNA Methylation in Diamond-Blackfan Anemia.

Diamond Blackfan Anemia (DBA) is a congenital disorder characterized by decreased red blood cell production accompanied by developmental abnormalities in 30% patients. Twenty-five percent of DBA patients display heterozygous mutations of ribosomal protein S19 (RPS19) on chromosome 19q13.2. No mutations were found in genes for other ribosomal proteins of the translation initiation complex. Although a second DBA locus has been proposed on in the region 8p23.3–8p22, the precise molecular defect is not known in 75% of DBA patients. The exact mechanism of how RPS19 mutations affect erythropoiesis remains unclear. Haploinsufficency of RPS19 may hamper translation machinery important for rapid erythroid differentiation. Reduced gene expression of a cluster of ribosomal proteins including RPS19 in DBA patients was recently reported. No causal therapy for DBA is available, with the exception of bone marrow transplantation. Some DBA patients benefit therapeutically from corticosteroids, cyclosporine A, or metoclopramide. Recently, a long-lasting remission was described in a DBA patient treated with valproic acid, a histone deacetylase inhibitor, suggesting epigenetic suppression of genes critical for erythropoiesis may be involved in the pathogenesis of DBA. DNA methylation of promoter-associated CpG islands is an epigenetic modification resulting in transcriptional silencing functionally equivalent to a loss-of-function mutation. Constitutive activation of JAK2 tyrosine kinase by a somatic V617F mutation leads to excessive erythropoiesis in polycythemia vera, an antonym of DBA. We hypothesized that silencing by DNA methylation of promoter-associated CpG island of the RPS19 or JAK2 genes may play a role in DBA. To test the hypothesis, we analyzed DNA methylation of RPS19 and JAK2 genes in 14 patients from the Czech DBA Registry. Genomic DNA isolated from blood cells of 3 DBA patients carrying heterozygous RPS19 mutations, 11 DBA patients without RPS19 mutation and 4 control samples was treated with bisulfite to convert all unmethylated cytosines to uracils while methylated cytosines were spared from the conversion. A region spanning 13 CpG sites positioned from 1–160 bases downstream from transcription start site (TSS) of RPS19 gene was PCR amplified and cloned in a sequencing vector. Individual bacterial clones were isolated and PCR inserts were sequenced in 8–12 clones per sample. Bisulfite cloning and sequencing revealed that more than 99% of CpG sites were converted to TpG and thus not methylated either in DBA samples (only 4/1466 CpG sites were methylated, methylation frequency was 0.3%) or control samples (2/555 CpG sites methylated, methylation frequency 0.4%). To explore a possibility of epigenetic suppression of erythropoietin signaling in DBA we analyzed DNA methylation of the CpG island of JAK2 tyrosine kinase gene in the same set of samples. Bisulfite-treated DNA was PCR amplified and T/C polymorphisms corresponding to unmethylated or methylated CpG sites were quantified by pyrosequencing. All DBA and control samples showed the absence of DNA methylation at four CpG sites located 12 to 25 bases downstream of TSS. We conclude that epigenetic silencing by DNA methylation is not involved in the expression of ribosomal structural protein RPS19; neither it affects the expression of a transducer of erythropoietin signaling JAK2 tyrosine kinase.

Homozygous JAK2 V617P Gain-of-Function Mutation Is Responsible for Constitutive JAK2/STAT5 Activation and Proliferation of HEL.92.1.7. Cell Line.

HEL (Human erythroleukemia) is a growth factor independent erythroleukemic cell line established from the bone marrow of a patient with relapsed Hodgkin disease after autologous bone marrow transplantation (Martin P & Papayannopoulou T: Science 1982; 216:1233–1235). HEL cells display a block in differentiation at the level of common erythroid-megakaryocytic progenitor and have been commonly used as a model to study erythroid and megakaryocytic differentiation. Liu RY et al. ( Blood 1999; 93:2369–2379) reported that constitutive activation of JAK2 tyrosine kinase in Dami/HEL cell line correlated with factor independent growth. Recently, several groups of investigators described a point mutation V617P in the autoinhibitory regulatory (JH2) domain of JAK2 tyrosine kinase in patients with myeloproliferative disorders (MPD). This acquired mutation results in constitutive activation of the JAK2/STAT5 pathway in hematopoietic cells. To date, the V617P mutation represents the most common causative genetic defect detected in patients with Philadelphia chromosome negative MPD. Interestingly, rare cases of patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) were found to have the same mutation. We tested several hematopoietic cell lines including HL-60, HEL, KG-1, K-562, TF-1, U937 and UT-7 for the presence of V617P mutation using RFLP and automatic DNA sequencing. We found the homozygous mutation V617P only in HEL cells. This cell line has a very complex karyotype suggesting that a defect of multiple oncogenes or antioncogenes could be responsible for sustained cell proliferation. We employed the JAK2 inhibitor, AG490 at concentrations ranging from 0 to 100 mM and measured proliferation of HEL and control K562 cell lines using MTT assay. We observed dose-dependent inhibition of HEL cell proliferation with low micromolar concentrations of AG490 in contrast to K562 control cell line, harboring bcr-abl fusion tyrosine kinase. IC50 was 13 mM for HEL cells and 65 mM for control K562 cells, respectively. Low concentrations of AG490 significantly decreased phosphorylation of JAK2 and STAT5 in HEL cells but not in control K562 cell line, where bcr-abl activates separately JAK2 and STAT5 pathway. Exposure of HEL cells to AG490 [0–50mM] induced apoptosis as measured by annexin V labeling in a concentration dependent fashion. No significant increase in apoptosis was detected in K562 cells using similar concentrations of the JAK2 inhibitor. Overall, these results suggest that the mechanism of proliferation of the HEL cell line is driven by constitutive activation of JAK2 tyrosine kinase secondary to V617P gain-of-function mutation. Our data provides further evidence that the JAK2/STAT5 intracellular signaling pathway is preserved in this cell line. Thus, HEL cells can serve as a model to test novel JAK2 specific inhibitors in preclinical studies.

Effect of obesity on insulin signaling through JAK2 in rat aorta

Pathway specific resistance to insulin signaling through PI 3-kinase/Akt/eNOS associated with a normal or hyperactivated MAP kinase signaling in vascular tissues has recently been proposed as a candidate link between cardiovascular disease and insulin resistance. Growth stimulatory pathways other than ERK/MAP kinase, such as JAK/STAT have not yet been investigated in vessels of animal models of insulin resistance. Here we have examined whether insulin is able to activate JAK2/STAT pathway in rat aorta and also the regulation of this pathway in an animal model of obesity/insulin resistance. Our results demonstrate that insulin activates JAK2 tyrosine kinase activity in rat aorta in parallel with the activation of STAT3 and STAT5a/b. Moreover, it is shown that, in obese animals, JAK2/STAT and MAP kinase pathways are hyperactivated in response to insulin, which occurs in association with a reduced activation of PI 3-kinase/Akt pathway in aorta. The results of the present study suggest that, besides ERK/MAP kinase pathway, another potentially pro-atherogenic pathway, JAK2/STAT is hyperactivated in vessels in a state of insulin resistance and this phenomenon, in association with the inhibition of the PI 3-kinase/Akt pathway, may play an important role in the pathogenesis of cardiovascular diseases.

Identification of 1,2,3,4,5,6-Hexabromocyclohexane as a Small Molecule Inhibitor of Jak2 Tyrosine Kinase Autophophorylation

The commercially available Jak2 inhibitor, alpha-cyano-3,4-dihydroxy-N-benzylcinnamide (AG490), has been used extensively to study Jak2 kinase function. While alpha-cyano-3,4-dihydroxy-N-benzylcinnamide is a potent Jak2 inhibitor, it can inhibit a number of other kinase signaling pathways as well. To circumvent this problem, we sought to identify novel small molecule inhibitors of Jak2 tyrosine kinase activity. For this, we constructed a homology model of the Jak2 kinase domain and identified solvent accessible pockets on the surface of the structure. Using the DOCK program, we tested 6451 compounds of known chemical structure in silico for their ability to interact with a pocket positioned adjacent to the activation loop. We attained the top seven scoring compounds from the National Cancer Institute and tested their ability to inhibit Jak2 autophosphorylation in vitro. Using Western blot analysis, we found that one of the compounds, 1,2,3,4,5,6-hexabromocyclohexane, was able to potently, and directly, inhibit Jak2 autophosphorylation. Characterization of this compound revealed that it inhibits Jak2 tyrosine autophosphorylation in both a time- and concentration-dependent manner. It greatly reduced growth hormone-mediated Jak2 autophosphorylation but did not block autophosphorylation of the epidermal growth factor receptor. Furthermore, doses as high as 100 muM were not toxic to cells as measured by their ability to exclude propidium iodide. As such, we believe that this compound could serve as a lead compound for a new generation of Jak2 inhibitors and, perhaps, be useful in elucidating the mechanisms of Jak2 kinase function.

Jak2 tyrosine kinase residues glutamic acid 1024 and arginine 1113 form a hydrogen bond interaction that is essential for Jak-STAT signal transduction.

Angiotensin II is a well-known vasoactive peptide, but it can also act as a potent growth factor, partially through activation of the tyrosine kinase Jak2. Activated Jak2 tyrosine phosphorylates and activates members of the Signal Transducers and Activators of Transcription (STAT) family of cytoplasmic transcription factors. Recently, we demonstrated that tryptophan 1020 and glutamic acid 1024 within the Jak2 activation loop are required for Jak2 tyrosine kinase activity. Here, we sought to elucidate the requirement of glutamic acid 1024 for Jak2 function. Using molecular modeling algorithms of the Jak2 kinase domain, we identified a putative interaction between glutamic acid 1024 and an arginine at position 1113. We generated a series of charge-based substitution mutations at position 1113 and found that conversion of arginine 1113 to glutamic acid, alanine, or lysine prevented Jak2 autophosphorylation. Furthermore, mutation of arginine 1113 prevented the following angiotensin II-dependent processes from occurring: (1) Jak2 tyrosine phosphorylation, (2) Jak2/AT1receptor co-association, (3) STAT1 recruitment to the Jak2/AT1receptor complex, (4) STAT1 tyrosine phosphorylation, and (5) STAT-mediated gene expression. We determined that the interaction between glutamic acid 1024 and arginine 1113 consists of two distinct hydrogen bonds. We conclude that these hydrogen bond interactions are critical for Jak2 kinase function and subsequent angiotensin II-dependent activation of the Jak/STAT signaling pathway.

Jak2 Tyrosine Kinase Mediates Oxidative Stress-induced Apoptosis in Vascular Smooth Muscle Cells

In vascular smooth muscle cells, Jak2 tyrosine kinase becomes activated in response to oxidative stress in the form of hydrogen peroxide. Although it has been postulated that hydrogen peroxide-induced Jak2 activation promotes cell survival, this has never been tested. We therefore examined the role that Jak2 plays in vascular smooth muscle cell apoptosis following hydrogen peroxide treatment. Here, we report that Jak2 tyrosine kinase activation by hydrogen peroxide is required for apoptosis of vascular smooth muscle cells. Upon treatment of primary rat aortic smooth muscle cells with hydrogen peroxide, we observed laddering of genomic DNA and nuclear condensation, both hallmarks of apoptotic cells. However, apoptosis was prevented by either the expression of a dominant negative Jak2 protein or by the Jak2 pharmacological inhibitor AG490. Moreover, expression of the proapoptotic Bax protein was induced following hydrogen peroxide treatment. Again, expression of a dominant negative Jak2 protein or treatment of cells with AG490 prevented this Bax induction. Following Bax induction by hydrogen peroxide, mitochondrial membrane integrity was compromised, and caspase-9 became activated. In contrast, in cells expressing a Jak2 dominant negative we observed that mitochondrial membrane integrity was preserved, and no caspase-9 activation occurred. These data demonstrate that the activation of Jak2 tyrosine kinase by hydrogen peroxide is essential for apoptosis of vascular smooth muscle cells. Furthermore, this report identifies Jak2 as a potential therapeutic target in vascular diseases in which vascular smooth muscle cell apoptosis contributes to pathological progression.

Modulation of Janus kinase 2 by cisplatin in cancer cells

Constitutive activation of Janus kinases (JAKs) is frequently detected in various human cancers. The activation of JAKs results in the phosphorylation and activation of signal transducers and activators of transcription (STATs). The constitutive activation of JAK/STAT pathway may play an important role in growth and survival of human cancer cells. In this study, we examined whether a chemotherapeutic agent cisplatin could inhibit the JAK/STAT pathway. In ovarian cancer and sarcoma cells that express constitutively active JAK2, cisplatin significantly inhibited tyrosine phosphorylation and kinase activity of JAK2 in a dose- and time-dependent manner. Meanwhile, cisplatin also inhibited Stat3 tyrosine phosphorylation and down-regulated BcL-XL anti-apoptotic protein in the cancer cells tested. In leukemia cells expressing high level of TEL-JAK2 fusion protein, cisplatin dramatically inhibited tyrosine phosphorylation of TEL-JAK2 as well. Furthermore, our results have shown that down-regulation of JAK2 by cisplatin might be through modulation of a tyrosine phosphatase SHP-1 but not SOCS family members. Taken together, our observations demonstrated that cisplatin down-regulated the JAK/STAT pathway through de-phosphorylation of JAK/STAT in cancer cells.

Jak2 is involved in c-Myc induction by Bcr-Abl.

We have previously shown that the Jak2 tyrosine kinase is activated in Bcr-Abl positive cell lines and blood cells from CML blast crisis patients by tyrosine phosphorylation. We are searching for downstream targets of Jak2 in Bcr-Abl positive cells. It is known that c-Myc expression is required for the oncogenic effects of Bcr-Abl, and that over-expression of c-Myc complements the transformation defect of the Bcr-Abl SH2 deletion mutant. Moreover, the Bcr-Abl SH2 deletion mutant and an Abl C-terminal deletion mutant are deficient in activating c-Myc expression. Since the Jak2 binds to the C-terminal domain of Bcr-Abl and optimal Jak2 activation requires the SH2 domain, we tested whether Jak2 was involved in c-Myc protein induction by Bcr-Abl. We treated the 32Dp210 Bcr-Abl cells with the Jak2 specific tyrosine kinase inhibitor, AG490, and found that this drug, like the Abl tyrosine kinase inhibitor STI-571, inhibited c-Myc protein induction by Bcr-Abl. Treatment of 32Dp210 Bcr-Abl cells with AG490 also inhibited c-MYC RNA expression. It is also known that c-Myc protein is a labile protein that is increased in amounts in response to various growth factors by a mechanism not involving new Myc protein formation. Treatment of 32Dp210 Bcr-Abl cells with both the proteasome inhibitor MG132 and AG490 blocked the reduction of the c-Myc protein observed by AG490 alone. An adaptor protein SH2-Bbeta is involved in the enhancement of the tyrosine kinase activity of Jak2 following ligand/receptor interaction. In this regard we showed that the Jak2/Bcr-Abl complex contains SH2-Bbeta. Expression of the SH2-Bbeta R555E mutant in 32Dp210 Bcr-Abl cells reduced c-Myc expression about 40% compared to a vector control. Interestingly, we found the reduction of the c-Myc protein in several clones of dominant-negative (DN) Jak2 expressing K562 cells correlated very well with the reduction of tumor growth of these cells in nude mice as compared to vector transfected K562 cells. Both STI-571 and AG490 also induced apoptosis in 32Dp210 cells. Of interest, IL-3 containing medium reversed the STI-571 induced apoptosis of 32Dp210 cells but did not reverse the induction of apoptosis by AG490, which strongly supports the specificity of the inhibitory effects of AG490 on the Jak2 tyrosine kinase. In summary, our findings indicate that Jak2 mediates the increase in c-Myc expression that is induced by Bcr-Abl. Our results indicate that activated Jak2 not only mediates an increase of c-MYC RNA expression but also interferes with proteasome-dependent degradation of c-Myc protein.

Redistribution of ERK/MAP kinase to uropod-like structures in interleukin-3-induced cell shape changes

Interleukin-3 (IL-3) is one of the cytokines of significance for the regulation of hematopoiesis and inflammation. Recently, we established IL-3-dependent Ba/F3 pro-B cells ectopically expressing RON tyrosine kinase, a receptor for macrophage-stimulating protein (MSP), and showed that MSP stimulation specifically promoted cell morphological changes through tyrosine phosphorylation of the IL-3 common β-chain receptor subunit (βc) by activated RON kinase without activation of JAK2 tyrosine kinase. Here we investigate the IL-3 signaling pathway leading to morphological changes through tyrosine phosphorylation of βc. Treatment of RON-expressing cells with PD98059 or U0126, inhibitors of mitogen-activated protein kinase kinase activity, blocked both IL-3- and MSP-induced morphological changes. Upon stimulation with IL-3 or MSP, extracellular-regulated kinase (ERK) and F-actin were redistributed in uropod-like structures. ERK and F-actin were colocalized within uropod-like structures, and a majority of F-actin were localized around the peripheries of accumulated ERK. Tyrosine phosphorylation of ERK was detected after stimulation with IL-3 or MSP, whereas treatment with U0126 specifically inhibited IL-3- or MSP-induced ERK phosphorylation but not tyrosine phosphorylation of βc. These results suggest that the activation and localization of ERK to uropod-like structures play a role in IL-3-induced morphological changes.

Mutation of glutamic acid residue 1046 abolishes Jak2 tyrosine kinase activity.

Jak2 is a member of the Janus family of tyrosine kinases and is known to be activated by a wide variety of ligands. Here, we sought to identify amino acid residues within Jak2 that are essential for its activation. We provide evidence that glutamic acid 1046 (E1046) is one such residue. Using molecular modeling algorithms of the Jak2 kinase domain, we identified a putative molecular interaction between E1046 and tryptophan 1020 (W1020). Conversion of E1046 to either arginine (E 1046R), alanine (E1046A), aspartic acid (E1046D) or glutamine (E1046Q) abolished Jak2 kinase activity as measured by autophosphorylation assays. Conversion of W1020 to glycine (W1020G) similarly abolished Jak2 kinase activity. Finally, we tested the ability of the E1046R mutant to activate the Jak/STAT signaling pathway in a ligand-dependent signaling system. The ability of angiotensin II to activate the Jak/STAT signaling pathway in cells expressing the E1046R mutant was severely compromised as measured by reduced (1) Jak2 autophosphorylation (2) Jak2 kinase activity (3) AT1/Jak2 co-association (4) Stat1 tyrosine phosphorylation and (5) angiotensin Il-mediated gene transcription. Thus, these studies demonstrate for the first time, the critical role of E1046 in mediating Jak2 activation and its subsequent downstream signaling events.