JAK2 Kinase Activity Research Articles

Phosphorylation of STAT2 on serine-734 negatively regulates the IFN-α-induced antiviral response.

Serine phosphorylation of STAT proteins is an important post-translational modification event that, in addition to tyrosine phosphorylation, is required for strong transcriptional activity. However, we recently showed that phosphorylation of STAT2 on S287 induced by type I interferons (IFN-α and IFN-β), evoked the opposite effect. S287-STAT2 phosphorylation inhibited the biological effects of IFN-α. We now report the identification and characterization of S734 on the C-terminal transactivation domain of STAT2 as a new phosphorylation site that can be induced by type I IFNs. IFN-α-induced S734-STAT2 phosphorylation displayed different kinetics to that of tyrosine phosphorylation. S734-STAT2 phosphorylation was dependent on STAT2 tyrosine phosphorylation and JAK1 kinase activity. Mutation of S734-STAT2 to alanine (S734A) enhanced IFN-α-driven antiviral responses compared to those driven by wild-type STAT2. Furthermore, DNA microarray analysis demonstrated that a small subset of type I IFN stimulated genes (ISGs) was induced more by IFNα in cells expressing S734A-STAT2 when compared to wild-type STAT2. Taken together, these studies identify phosphorylation of S734-STAT2 as a new regulatory mechanism that negatively controls the type I IFN-antiviral response by limiting the expression of a select subset of antiviral ISGs.

3D Modeling of Novel Transforming JAK Mutations in T-Cell Acute Lymphoblastic Leukemia Reveals Altered Pseudokinase-Kinase Domain Interactions That Result in Constitutive JAK Kinase Activity

Background:Many pediatric T-cell acute lymphoblastic leukemia patients harbor mutations in IL7Ra or downstream molecules encoded by JAK1, JAK3, N-RAS, K-RAS, NF1, AKT, and PTEN. These mutated signaling molecules can contribute to leukemia by disturbing a multitude of cellular processes such as the cell cycle, epigenetics, apoptosis, or affecting other important signal transduction pathways.Aims:We aimed to determine the overall incidence of JAK family mutations in a large cohort of T-ALL patients. We also aimed to generate a 3D JAK1 model including known and newly identified JAK mutations in order to better understand how these contribute to JAK kinase activity and the transformation of cells.Methods:We screened 146 pediatric T-ALL patient samples for mutations in the FERM, pseudokinase and kinase domains of the Janus kinase gene family (JAK1-3, TYK2). To establish a 3D JAK1 model, we superimposed individual pseudokinase and kinase crystallographic structures on the homologous TYK2 pseudokinase-kinase structure. We visualized JAK mutations and their effects on the 3D structure. We modified the IL3-dependent Ba/F3 cell line to express JAK mutant or wild type genes upon induction by doxycycline. We tested these Ba/F3 derivative lines for transforming ability, signaling, and resistance to various inhibitors in the absence of IL3.Results:JAK1 or JAK3 mutations were found in 10 patients; no mutations were found in JAK2 or TYK2. We found JAK1 and JAK3 mutations as previously reported, but also identified amino acid substitutions as a result of novel JAK1 mutations including V427M, L624YPILKV, E668Q, P815S, and T901G. Our novel 3D model of JAK1 places most mutations in one of two crucial pseudokinase-kinase interaction sites, which can weaken the interaction and facilitate constitutive kinase activity. One interaction is between the hinge region of the pseudokinase domain and the loop in the kinase domain, which is supported by four salt bridges. Mutations in T-ALL disrupting these salt bridges include E668Q, R724H and its JAK3 equivalent R657Q, and T901G. The second interaction with the kinase domain is formed by a helical domain in the pseudokinase domain, located just upstream of the conserved F575-F636-V658 triad. This F-F-V triad is predicted to act as a structural switch that controls the catalytic activity of JAK kinases. Various mutations occur in the direct vicinity and can affect the function of this switch. V658F in T-ALL and its JAK2 equivalent V617F in polycythemia vera patients are mutations in this triad. The frequent JAK3 mutation M511I in T-ALL flanks the F513 residue (equivalent of JAK1 F575) and also affects the F-F-V triad. The L624YPILKV insertion mutation is located in a loop near the helical domain, which may also subtly compromise the F-F-V triad structural switch leading to derepression of the kinase domain. Expression of mutant JAK genes-in contrast to the wild type genes-transforms Ba/F3 cells by supporting IL3-independent growth, and by activating downstream RAS-MEK-ERK and PI3K-AKT pathways. This pathway activation as a result of ligand-independent mutant JAK kinase activity was confirmed by measuring phospho-proteins including p-MEK, p-ERK, p-AKT, p-mTOR, and p-p70S6K, and can be blocked by JAK inhibitors. Notably, JAK3 mutants signal significantly weaker than JAK1 mutants, possibly due to different dependence on (endogenous) receptors that normally mediate wild type JAK signaling.Summary/Conclusion:In a 3D model, we show that JAK mutations are located in critical interface regions between the pseudokinase and kinase domains, maintaining the kinase in an open, active confirmation. The inducible Ba/F3 model system confirms the transforming capacity of JAK mutations, reveals constitutive active downstream signaling, and is also suitable to test the effect of various inhibitors. The visualization of various JAK mutations in a 3D model and how these contribute to kinase activity provides insight in how mutant JAK could be inhibited, helping guide the development of new small molecule inhibitors of mutant JAKs. DisclosuresBuijsman:Netherlands Translational Research Center B.V.: Equity Ownership, Other: founder and shareholder. Zaman:Netherlands Translational Research Center B.V.: Equity Ownership, Other: founder and shareholder.

Feed-Forward Reciprocal Activation of PAFR and STAT3 Regulates Epithelial-Mesenchymal Transition in Non-Small Cell Lung Cancer.

Platelet-activating factor receptor (PAFR), a G-protein-coupled receptor, has been implicated in tumorigenesis, but its contributions to metastatic progression have not been investigated. Here, we show that PAFR is overexpressed in non-small cell lung cancer (NSCLC) as well as in breast, colorectal, and gastric carcinomas. Expression of PAFR correlates closely with clinical stages, survival time, and distant metastasis. In human NSCLC cells, activation of the PAF/PAFR signaling axis accentuated malignant character, including by stimulating epithelial-mesenchymal transition (EMT). In contrast, silencing PAFR in aggressive NSCLC cells inhibited these effects. Mechanistic investigations showed that PAFR stimulated EMT by activating STAT3 via upregulation of G-protein-dependent SRC or JAK2 kinase activity. Notably, STAT3 transcriptionally elevated PAFR expression. Thus, activation of PAFR in NSCLC cells initiated a forward feedback loop responsible for mediating the aggressive malignant character of NSCLC cells in vitro and in vivo. Reinforcing this reciprocal activation loop, PAF/PAFR signaling also upregulated IL6 expression and thereby STAT3 activation. Overall, our results elucidated an important role for PAFR dysregulation in the pathogenicity of NSCLC and unraveled a forward feedback loop between PAFR and STAT3 that acts to drive the malignant progression of NSCLC.

Breaking resistance of pancreatic cancer cells to an attenuated vesicular stomatitis virus through a novel activity of IKK inhibitor TPCA-1

Vesicular stomatitis virus (VSV) is an effective oncolytic virus against most human pancreatic ductal adenocarcinoma (PDAC) cell lines. However, some PDAC cell lines are highly resistant to oncolytic VSV-ΔM51 infection. To better understand the mechanism of resistance, we tested a panel of 16 small molecule inhibitors of different cellular signaling pathways, and identified TPCA-1 (IKK-β inhibitor) and ruxolitinib (JAK1/2 inhibitor), as strong enhancers of VSV-ΔM51 replication and virus-mediated oncolysis in all VSV-resistant PDAC cell lines. Both TPCA-1 and ruxolitinib similarly inhibited STAT1 and STAT2 phosphorylation and decreased expression of antiviral genes MxA and OAS. Moreover, an in situ kinase assay provided biochemical evidence that TPCA-1 directly inhibits JAK1 kinase activity. Together, our data demonstrate that TPCA-1 is a unique dual inhibitor of IKK-β and JAK1 kinase, and provide a new evidence that upregulated type I interferon signaling plays a major role in resistance of pancreatic cancer cells to oncolytic viruses.

Targeting phosphatidylinositol-3-kinase pathway for the treatment of Philadelphia-negative myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPN) are a diverse group of chronic hematological disorders that involve unregulated clonal proliferation of white blood cells. Sevearl of them are associated with mutations in receptor tyrosine kinases or cytokine receptor associated tyrosine kinases rendering them independent of cytokine-mediated regulation. Classically they have been broadly divided into BCR-ABL1 fusion + ve (Ph + ve) or –ve (Ph-ve) MPNs. Identification of BCR-ABL1 tyrosine kinase as a driver of chronic myeloid leukemia (CML) and successful application of small molecule inhibitors of the tyrosine kinases in the clinic have triggered the search for kinase dependent pathways in other Ph-ve MPNs. In the past few years, identification of mutations in JAK2 associated with a majority of MPNs raised the hopes for similar success with specific targeting of JAK2. However, targeting JAK2 kinase activity has met with limited success. Subsequently, mutations in genes other than JAK2 have been identified. These mutations specifically associate with certain MPNs and can drive cytokine independent growth. Therefore, targeting alternate molecules and pathways may be more successful in management of MPNs. Among other pathways, phosphatidylinositol −3 kinase (PI3K) has emerged as a promising target as different cell surface receptor induced signaling pathways converge on the PI3K signaling axis to regulate cell metabolism, growth, proliferation, and survival. Herein, we will review the clinically relevant inhibitors of the PI3K pathway that have been evaluated or hold promise for the treatment of Ph-ve MPNs.

Src tyrosyl phosphorylates cortactin in response to prolactin

The hormone/cytokine prolactin (PRL) is implicated in breast cancer cell invasion and metastasis. PRL-induced pathways are mediated by two non-receptor tyrosine kinases, JAK2 and Src. We previously demonstrated that prolactin stimulates invasion of breast cancer cells TMX2-28 through JAK2 and its target serine/threonine kinase PAK1. We hypothesize herein that the actin-binding protein cortactin, a protein involved in invadopodia formation and cell invasion, is activated by PRL. We demonstrate that TMX2-28 cells are more invasive than T47D breast cancer cells in response to PRL. We determine that cortactin is tyrosyl phosphorylated in response to PRL in a time and dose-dependent manner in TMX2-28 cells, but not in T47D cells. Furthermore, we show that PRL mediates cortactin tyrosyl phosphorylation via Src, but not JAK2. Finally, we demonstrate that maximal PRL-mediated TMX2-28 cell invasion requires both Src and JAK2 kinase activity, while T47D cell invasion is JAK2- but not Src-dependent. Thus PRL may induce cell invasion via two pathways: through a JAK2/PAK1 mediated pathway that we have previously demonstrated, and Src-dependent activation and tyrosyl phosphorylation of cortactin.

JAK2 tyrosine kinase phosphorylates and is negatively regulated by centrosomal protein Ninein.

JAK2 is a cytoplasmic tyrosine kinase critical for cytokine signaling. In this study, we have identified a novel centrosome-associated complex containing ninein and JAK2. We have found that active JAK2 localizes around the mother centrioles, where it partly colocalizes with ninein, a protein involved in microtubule (MT) nucleation and anchoring. We demonstrated that JAK2 is an important regulator of centrosome function. Depletion of JAK2 or use of JAK2-null cells causes defects in MT anchoring and increased numbers of cells with mitotic defects; however, MT nucleation is unaffected. We showed that JAK2 directly phosphorylates the N terminus of ninein while the C terminus of ninein inhibits JAK2 kinase activity in vitro. Overexpressed wild-type (WT) or C-terminal (amino acids 1179 to 1931) ninein inhibits JAK2. This ninein-dependent inhibition of JAK2 significantly decreases prolactin- and interferon gamma (IFN-γ)-induced tyrosyl phosphorylation of STAT1 and STAT5. Downregulation of ninein enhances JAK2 activation. These results indicate that JAK2 is a novel member of centrosome-associated complex and that this localization regulates both centrosomal function and JAK2 kinase activity, thus controlling cytokine-activated molecular pathways.

Screening Active Compounds from Garcinia Species Native to China Reveals Novel Compounds Targeting the STAT/JAK Signaling Pathway.

Natural compounds from medicinal plants are important resources for drug development. In a panel of human tumor cells, we screened a library of the natural products from Garcinia species which have anticancer potential to identify new potential therapeutic leads and discovered that caged xanthones were highly effective at suppressing multiple cancer cell lines. Their anticancer activities mainly depended on apoptosis pathways. For compounds in sensitive cancer line, their mechanisms of mode of action were evaluated. 33-Hydroxyepigambogic acid and 35-hydroxyepigambogic acid exhibited about 1 μM IC50 values against JAK2/JAK3 kinases and less than 1 μM IC50 values against NCI-H1650 cell which autocrined IL-6. Thus these two compounds provided a new antitumor molecular scaffold. Our report describes 33-hydroxyepigambogic acid and 35-hydroxyepigambogic acid that inhibited NCI-H1650 cell growth by suppressing constitutive STAT3 activation via direct inhibition of JAK kinase activity.

Autocrine TNF-α Signaling in Hematopoietic Stem Cells Promotes Myeloproliferative Disease Progression through Activation of TNFR2

The JAK2V617F mutation is a unifying feature in the majority of patients with myeloproliferative neoplasms (MPNs). The presence of JAK2V617 in a hematopoietic stem (HSC) or progenitor cell confers a proliferative advantage over native JAK2 counterparts. Several inflammatory cytokines are elevated in MPN patients and in murine models of JAK2V617F-driven MPN. In particular, expression of tumor necrosis factor alpha (TNF-α) is increased by constitutive JAK2 kinase activity and imparts a competitive advantage on JAK2V617Fexpressing cells.To identify cell types responsible for increased levels of TNF-α we performed intracellular cytokine staining in leukocytes from myelofibrosis (MF) patients and normal controls. Using a panel of markers to define cellular subsets we found expression of TNF-α was uniformly low in unstimulated cells. However, when cells were treated with lipopolysaccharide (LPS), TNF-α expression was 16-fold higher in HSCs of MF patients compared to normal controls (p<0.005), while expression in mature populations was not different. In a murine model of JAK2V617F driven MPN, where JAK2V617F cells are identified by GFP, TNF-α expression was 3-fold higher in JAK2V617F-expressing HSCs (GFP+) compared to GFP-controls (p<0.005), irrespective of LPS stimulation.To evaluate anti-TNF therapy in MPNs, we treated JAK2V617F mice with etanercept, a soluble TNF receptor fusion protein that binds and inactivates TNF-α. Etanercept did not significantly restrain JAK2V617F-associated WBC or hematocrit increases over a 10-week period, despite suppression of TNF-α activity in plasma. As TNF-α may activate pro-apoptotic (predominantly through TNF receptor 1, TNFR1) and pro-survival pathways (predominantly through TNF receptor 2, TNFR2), we reasoned that global blockade of TNF-α may not shift the balance in favor of normal hematopoiesis. To investigate this we sorted primitive (Lin-, cKit+) cells from mice with JAK2V617F-induced MPN by TNF receptor expression. TNFR2+ cells showed significantly increased colony formation compared to TNFR1+ cells, demonstrating that TNFR2 expression is associated with increased clonogenic potential. Additionally we treated these primitive cells with specific antibodies blocking TNFR1 or TNFR2. Colony assays performed after 3 days in liquid culture with TNFR-blocking antibodies confirmed that interrupting TNFR2-mediated survival signaling resulted in decreased colony formation (61% reduction) while blocking TNFR1 increased colony formation (5% increase). In addition we tested CD34+ cells from MF patient samples with TNFR-blocking antibodies. As seen with the murine MPN cells, colony formation was decreased with the TNFR2 blocking antibody (30% reduction) and increased with the TNFR1 blocking antibody (48% increase) consistent with a differential activation of survival signals by TNF-α in MPN cells. These studies were performed without addition of exogenous TNF-α, which confirms the ability and requirement for these primitive JAK2V617Fcells to utilize TNF-α production to actively support survival.Our data suggest that TNF-α generated by primitive MPN cells promotes their survival through activation of TNFR2, while TNFR1 activation is suppressive. If selective inhibition of TNFR2 shifts the equilibrium toward normal hematopoietic cells, this would support the use of TNFR2 blockade to treat MF and other myeloproliferative neoplasms. Genotyping of human MF and murine MPN colonies cultured with TNF receptor blocking antibodies and experiments in normal human CD34+ cells are ongoing and will be presented. DisclosuresDeininger:BMS, Novartis, Celgene, Genzyme, Gilead: Research Funding; BMA, ARIAD, Novartis, Incyte, Pfizer: Advisory Board, Advisory Board Other; BMS, ARIAD, Novartis, Incyte, Pfizer: Consultancy.

JAK3 Mutants Transform Hematopoietic Cells through JAK1 Activation Causing T-Cell Acute Lymphoblastic Leukemia in a Bone Marrow Transplant Mouse Model

▪JAK3 is a cytosolic tyrosine kinase that associates with the common gamma chain in different cytokine receptors, in which the JAK1 kinase is another essential signaling protein. Large scale sequencing efforts recently identified mutations in the IL7R, JAK1 or JAK3 genes in about 25% of T-cell acute lymphoblastic leukemia (T-ALL) cases, with JAK3 being the most frequently (15% of T-ALL cases) mutated gene in this pathway. To determine if all mutations in JAK3 are true oncogenic mutations, we generated expression plasmids for 16 JAK3 mutants (M511I, A572T, A573V, R657Q, R657W, V674A, V678M, V678L, R775C, L857Q, Q865E, L875H, P906S, R925S, E958K, E1106G) and determined the in vitro and in vivo transforming properties, as well as their sensitivity to JAK kinase inhibitors.For 12 of 16 mutants expression in the IL3-dependent Ba/F3 cell line resulted in transformation to IL3 independent growth. Similarly, expression of the transforming mutants in IL7-receptor reconstituted 293T cells confirmed their ligand independent activation, while this was not observed for the non-transforming mutants. These 4 non-transforming mutants are likely to be passenger mutations, illustrating that results from sequencing always need to be confirmed by functional assays to distinguish driver mutations from passenger mutations. Most JAK3 mutants, except JAK3 L857Q and JAK3 L875H, were dependent on JAK1 kinase activity for their transforming capacities. In agreement with this, we observed that Ba/F3 cells transformed by the JAK1 dependent JAK3 mutants could be inhibited by ruxolitinib, a JAK1/JAK2 selective inhibitor, while the JAK3 L857Q and JAK3 L875H transformed cells were significantly less sensitive to ruxolitinib treatment. As expected, all JAK3 mutants were sensitive to the JAK3 selective inhibitor tofacitinib, except for JAK3 L875H, which showed resistance to all inhibitors tested.To determine the in vivo oncogenic properties of the JAK3 mutants, we expressed selected JAK3 mutants (M511I, A573V, L857Q, V674A and R657Q) in mouse hematopoietic cells through viral transduction. Mice transplanted with cells expressing JAK3 M511I, A573V or V674A showed a gradual increase of the WBC count and developed a T-ALL like disease within 120 to 200 days. In contrast, mice transplanted with cells expressing JAK3 L857Q or R657Q showed a lower increase in WBC count, and did present with severe splenomegaly and lymphadenopathy. Expression of JAK3 L857Q caused severe thymus hyperplasia, while the JAK3 R657Q mutant caused B-cell leukemia, illustrating that different JAK3 mutants seem to have variable oncogenic characteristics.Mice transplanted with cells expressing JAK3 M511I were treated with the JAK3 selective inhibitor tofacitinib and disease progression was followed by white blood cell count measurements. Treatment of the animals for 5 weeks with tofacitinib (oral gavage, 40 mg/kg/day) significantly decreased the disease progression compared to placebo treated mice. Moreover, we observed severe apoptosis of the leukemia cells in spleen and thymus in tofacitinib treated animals and not in placebo treated mice. However, tofacitnib treatment could not eradicate all leukemia cells, and the mice progressed when treatment was stopped.In conclusion, JAK3 is recurrently mutated in T-ALL patients, and we demonstrate that most JAK3 mutants are transforming proteins using In vitro and in vivo experiments. Our results show that JAK1 is an essential kinase for most JAK3 mutants, and that the majority of JAK3 mutants are sensitive to JAK3 and JAK1 specific inhibitors such as tofacitinib and ruxolitinib. However, some JAK3 mutants do show resistance to these inhibitors, which will need to be taken into account when trials are initiated for the treatment of ALL patients with JAK specific inhibitors. DisclosuresNo relevant conflicts of interest to declare.

JAK3 mutants transform hematopoietic cells through JAK1 activation, causing T-cell acute lymphoblastic leukemia in a mouse model

AbstractJAK3 is a tyrosine kinase that associates with the common γ chain of cytokine receptors and is recurrently mutated in T-cell acute lymphoblastic leukemia (T-ALL). We tested the transforming properties of JAK3 pseudokinase and kinase domain mutants using in vitro and in vivo assays. Most, but not all, JAK3 mutants transformed cytokine-dependent Ba/F3 or MOHITO cell lines to cytokine-independent proliferation. JAK3 pseudokinase mutants were dependent on Jak1 kinase activity for cellular transformation, whereas the JAK3 kinase domain mutant could transform cells in a Jak1 kinase–independent manner. Reconstitution of the IL7 receptor signaling complex in 293T cells showed that JAK3 mutants required receptor binding to mediate downstream STAT5 phosphorylation. Mice transplanted with bone marrow progenitor cells expressing JAK3 mutants developed a long-latency transplantable T-ALL–like disease, characterized by an accumulation of immature CD8+ T cells. In vivo treatment of leukemic mice with the JAK3 selective inhibitor tofacitinib reduced the white blood cell count and caused leukemic cell apoptosis. Our data show that JAK3 mutations are drivers of T-ALL and require the cytokine receptor complex for transformation. These results warrant further investigation of JAK1/JAK3 inhibitors for the treatment of T-ALL.

PAK1 tyrosine phosphorylation is required to induce epithelial-mesenchymal transition and radioresistance in lung cancer cells.

The p21-activated Ser/Thr kinase 1 (PAK1) kinase has an essential role in tumorigenesis and cell survival in many cancers, but its regulation is not fully understood. In this study, we showed that in response to irradiation of lung cancer cells, PAK1 was upregulated, tyrosine phosphorylated, and translocated to the nucleus. Tyrosine phosphorylation relied upon JAK2 kinase activity and was essential for PAK1 protein stability and binding to Snail. This radiation-induced JAK2-PAK1-Snail signaling pathway increased epithelial-mesenchymal transition (EMT) by regulating epithelial and mesenchymal cell markers. Notably, JAK2 inhibitors mediated radiosensitization and EMT blockade in a mouse xenograft model of lung cancer. Taken together, our findings offered evidence that JAK2 phosphorylates and stabilizes functions of PAK1 that promote EMT and radioresistance in lung cancer cells, with additional implications for the use of JAK2 inhibitors as radiosensitizers in lung cancer treatment.

BCR-ABL affects STAT5A and STAT5B differentially.

Signal transducers and activators of transcription (STATs) are latent cytoplasmic transcription factors linking extracellular signals to target gene transcription. Hematopoietic cells express two highly conserved STAT5-isoforms (STAT5A/STAT5B), and STAT5 is directly activated by JAK2 downstream of several cytokine receptors and the oncogenic BCR-ABL tyrosine kinase. Using an IL-3-dependent cell line with inducible BCR-ABL-expression we compared STAT5-activation by IL-3 and BCR-ABL in a STAT5-isoform specific manner. RNAi targeting of STAT5B strongly inhibits BCR-ABL-dependent cell proliferation, and STAT5B but not STAT5A is essential for BCL-XL-expression in the presence of BCR-ABL. Although BCR-ABL induces STAT5-tyrosine phosphorylation independent of JAK2-kinase activity, BCR-ABL is less efficient in inducing active STAT5A:STAT5B-heterodimerization than IL-3, leaving constitutive STAT5A and STAT5B-homodimerization unaffected. In comparison to IL-3, nuclear accumulation of a STAT5A-eGFP fusion protein is reduced by BCR-ABL, and BCR-ABL tyrosine kinase activity induces STAT5A-eGFP translocation to the cell membrane and co-localization with the IL-3 receptor. Furthermore, BCR-ABL-dependent phosphorylation of Y682 in STAT5A was detected by mass-spectrometry. Finally, RNAi targeting STAT5B but not STAT5A sensitizes human BCR-ABL-positive cell lines to imatinib-treatment. These data demonstrate differences between IL-3 and BCR-ABL-mediated STAT5-activation and isoform-specific effects, indicating therapeutic options for isoform-specific STAT5-inhibition in BCR-ABL-positive leukemia.

Combination of PIM and JAK2 inhibitors synergistically suppresses MPN cell proliferation and overcomes drug resistance.

Inhibitors of JAK2 kinase are emerging as an important treatment modality for myeloproliferative neoplasms (MPN). However, similar to other kinase inhibitors, resistance to JAK2 inhibitors may eventually emerge through a variety of mechanisms. Effective drug combination is one way to enhance therapeutic efficacy and combat resistance against JAK2 inhibitors. To identify potential combination partners for JAK2 compounds in MPN cell lines, we performed pooled shRNA screen targeting 5,000 genes in the presence or absence of JAK2 blockade. One of the top hits identified was MYC, an oncogenic transcription factor that is difficult to inhibit directly, but could be targeted by modulation of upstream regulatory elements such as kinases. We demonstrate herein that PIM kinase inhibitors efficiently suppress MYC protein levels in MPN cell lines. Importantly, overexpression of MYC restores the viability of PIM inhibitor-treated cells, revealing causal relationship between MYC down-regulation and cell growth inhibition by PIM compounds. Combination of various PIM inhibitors with a JAK2 inhibitor results in significant synergistic growth inhibition of multiple MPN cancer cell lines and induction of apoptosis. Mechanistic studies revealed strong downregulation of phosphorylated forms of S6 and 4EBP1 by JAK2/PIM inhibitor combination treatment. Finally, such combination was effective in eradicating in vitro JAK2 inhibitor-resistant MPN clones, where MYC is consistently up-regulated. These findings demonstrate that simultaneous suppression of JAK2 and PIM kinase activity by small molecule inhibitors is more effective than either agent alone in suppressing MPN cell growth. Our data suggest that JAK2 and PIM combination might warrant further investigation for the treatment of JAK2-driven hematologic malignancies.

The Role Of SOCS1 In BCR-ABL Mediated Transformation and Leukemogenesis

The BCR-ABL oncogene activates several signaling pathways, most notably by constitutive phosphorylation of the signal transducer and activator of transcription protein 5 (STAT5). After phosphorylation and nuclear translocation, STAT5 transcriptionally activates numerous genes responsible for proliferation, survival and differentiation of hematopoietic stem and progenitor cells. Among the STAT5 target genes are suppressor of cytokine signaling (SOCS) proteins. SOCS proteins inhibit JAK kinases by multiple mechanisms, thereby terminating cytokine signaling in a classical negative feedback loop. The SOCS family of proteins comprises eight members: cytokine-inducible SRC homology 2 (SH2) domain protein (CIS) and SOCS1–SOCS7. SOCS1 is frequently silenced by hypermethylation in multiple myeloma and inactivating mutations have been found in Hodgkin lymphoma with consecutive increase in JAK2 kinase activity. More recently, we identified SOCS1 as a “conditional oncogene” in the context of the FLT3-ITD oncogene (Reddy et al, Blood 2012): SOCS1 significantly enhanced FLT3-ITD-mediated myeloid transformation, both in vitro and in vivo. We hypothesized that this may be a more general mechanism of transformation and therefore analyzed the role of SOCS proteins in BCR-ABL mediated transformation and leukemogenesis.First, we investigated gene expression levels of SOCS proteins in BCR-ABL positive (versus BCR-ABL negative) cell lines and primary ALL long term-cultured cells. Upon treatment with the BCR-ABL inhibitor imatinib, mRNA expression levels of CIS and SOCS1-4 were reduced. SOCS5-7 did not exhibit any changes and were non-responsive to ABL-kinase inhibition. In lineage-depleted primary murine bone marrow retrovirally transduced with BCR-ABL, high induction of CIS and SOCS1-3 mRNA was detected, while SOCS4-7 showed only minor changes. When overexpressed in IL-3 dependent cell lines, SOCS1 led to a very rapid cell death within few days. Similar effects were demonstrated for CIS and SOCS2 overexpression, however, with a slower kinetics. In contrast, BCR-ABL transduced cells were insensitive to SOCS1 overexpression. In colony formation assays performed with primary hematopoietic cells, expression of SOCS1 led to a significant decrease of colony numbers. Interestingly, co-expression of SOCS1 and BCR-ABL (hereafter abbreviated as SOCS1/BCR-ABL) also lowered colony numbers compared to cells expressing BCR-ABL alone. However, when cells were subjected to interferon alpha or interferon gamma treatment, SOCS1/BCR-ABL positive cells displayed higher colony numbers and gained a growth advantage over BCR-ABL expressing cells, since anti-proliferative effects of the cytokines were inhibited by the presence of SOCS1. A careful analysis of the downstream signaling cascade of BCR-ABL and SOCS1/BCR-ABL expressing cells did not demonstrate any differences in the phosphorylation of AKT, ERK1/2 and STAT5. However, when BCR-ABL was inhibited by imatinib, STAT5 phosphorylation was significantly decreased in SOCS1/BCR-ABL transduced cells. Finally, the influence of SOCS1 in BCR-ABL mediated leukemia was investigated in a murine bone marrow transplantation model. BCR-ABL or SOCS1/BCR-ABL expressing cells led to disease formation with a chronic myeloid leukemia-like phenotype. Interestingly, the co-expression of SOCS1 and BCR-ABL prolonged disease latency, as opposed to the phenotype observed with FLT3-ITD (where SOCS1 co-expression shortened latency). In this setting SOCS1 acts as a tumor suppressor, protecting BCR-ABL transformed cells from rapid disease development, and a molecular analysis will be presented. Disclosures:No relevant conflicts of interest to declare.

Go6976, a Potent FLT3 Kinase Inhibitor, Exerts Antiproliferative Activity Against Acute Myeloid Leukemia Via Inhibition Of Survivin and Mcl-1 In The Presence Of Human Serum

The FMS-like tyrosine kinase 3 (FLT3) is a class III receptor tyrosine kinase involved in hematopoietic progenitor cell development. Mutations of FLT3 have been reported in about a third of patients with acute myeloid leukemia (AML). The presence of FLT3 mutations confers a poor prognosis, and thus recent studies are directed at developing and testing novel FLT3 inhibitors for the treatment of AML. Go6976 is an indolocarbazole with a simillar structural backbone to staurosporine. In the present study, we demonstrated that Go6976 displays a potent inhibitory activity against recombinant FLT3 using in vitro kinase assay. Its IC50 value is 0.7nM. We also tested the effect of Go6976 on several kinds of other kinases. Go6976 inhibited the kinase activity of Aurora-A, Aurora-B and JAK2 with IC50 values of 118.2 nM, 77.7 nM and 92.7 nM, respectively. Go6976 did not show the inhibitory activity against the FGFR3 even at 1 microM. These data indicated Go6976 preferentially and potently inhibit the FLT3. Go6976 significantly inhibited the proliferation of human leukemia cells having FLT3-ITD. The IC50 values of Go6976 against MV4-11 and MOLM-13 were 0.044 and 0.008 microM, respectively. In contrast, human leukemia HL-60 and U937 cells which lack FLT3-ITD showed strong resistance to Go6976 treatment. Furthermore, we observed that Go6976 shows minimal toxicity for purified human normal CD34(+) cells. Go6976 suppressed the phosphorylation of FLT3 in MV4-11 and MOLM13 cells. Consistent with FLT3 inhibition, Go6976 potently inhibited phosphorylation of constitutively activated STAT3/5, Erk1/2, and Akt. Western blotting analysis revealed that MV4-11 and MOLM13 cells possess abundant survivin and Mcl-1 protein. We hypothesized that the expression of survivin and Mcl-1 may be regulated by constitutive activation of FLT3. In order to test this hypothesis, the effect of siRNA for FLT3-ITD was examined. Indeed, we observed that siRNA-induced down-regulation of FLT3 decreased survivin and Mcl-1 expression in MOLM13 cells, suggesting that up-regulated survivin and Mcl-1 may be closely associated with FLT3 signaling. Interestingly, we found that both survivin mRNA and protein were rapidly downregulated by Go6976 treatment in MOLM13 and MV4-11 cells. It was also observed that Go6976 significantly suppressed Mcl-1 mRNA and protein. It has been reported that STAT-3 and STAT-5 signaling play a pivotal role in the regulation of survivin and Mcl-1, respectively. Thus, inhibitory effects of Go6976 on the expression Survivin and Mcl-1 may be a consequence of the suppression of phosphorylation of STAT-3 and STAT-5 by Go6976 in FLT3-ITD cells. This inhibition of anti-apoptotic proteins by Go6976 may be critical for its antiproliferative effect in FLT3-ITD cells. It has been known that previous FLT3 inhibitors such as PKC412 and CEP-701 bind to the human plasma protein, alpha1-acid glycoprotein, resulting in significantly diminished inhibitory activity against FLT3. Indeed, inhibitory effect of PKC412 on FLT3 was significantly decreased, when MOLM13 cells were treated with PKC412 in the presence of human serum. In contrast, we found that Go6976 potently inhibits phosphorylation of FLT3 and exerts cytotoxicity even in the presence of human serum or human alpha1-acid glycoprotein. In conclusion, our data indicate that Go6976 may have a unique therapeutic potential for FLT3-driven acute myeloid leukemia. Disclosures:No relevant conflicts of interest to declare.

Ruxolitinib Mediated Paradox JAK2 Hyperphosphorylation Is Due To The Protection Of Activation Loop Phosphotyrosines From Phosphatases

The JAK2 V617F mutation has been reported in the majority of bcr-abl negative myeloproliferative neoplasms (MPNs) including polycythemia vera (PV:90%), essential thrombocythemia (ET:50%) and idiopathic myelofibrosis (IMF:50%). Recently, ruxolitinib (Jakavi, INCB018424) a JAK2/JAK1 specific inhibitor has been approved by the FDA for the treatment of myelofibrosis. Unexpectedly, treatment of V617FJAK2 expressing cells with ruxolitinib causes paradoxical hyperphosphorylation of JAK2 at its two critical sites (tyr1007/tyr1008). The mechanism of ruxolitinib induced JAK2 hyperphosphorylation is not understood. It has been demonstrated that also the pseudokinase domain of JAK2 display kinase activity and plays a major role in regulating the kinase activity of JAK2. Deletion of the pseudokinase domain leads to hyperphosphorylation of JAK2. We thus hypothesized that ATP-competitive inhibitors might target the kinase activity of the pseudokinase domain. However, treatment of kinase dead pseudokinase domain JAK2V617F mutants with ruxolitinib also causes JAK2 hyperphosphorylation indicating that ruxolitinib mediated JAK2 hyperphosphorylation is not due to inhibition of the pseudokinase domain. We then reasoned that treatment with ruxolitinib might protect JAK2 from tyrosine phosphatases. Co-immunoprecipitation results however did not suggested that ruxolitinib treatment leads to the dissociation of phosphatases such as SHP-2. A Ruxolitinib resistant JAK2V617F mutant (V617F+L902QJAK2) and a JAK2V617F kinase dead mutant (V617F+K882RJAK2) both did not show paradox hyperphoshorylation after ruxolitinib treatment. This indicated that ruxolitinib mediated JAK2 hyperphosphorylation is mediated by a JAK2 intrinsic mechanism. Finally, native-immunocomplexes (non-denaturing IP) of JAK2V617F generated with a monoclonal antibody against ptyr1007/1008 could be blocked by the presence of ruxolitinib, although JAK2V617F was hyperphosphorylated at these sites. We therefore believe now that in the presence of ruxolitinib the JAK2 activation loop folds inside the kinase domain. This leads to the protection of ptyr1007/1008 sites from phosphatases. Most tyrosine kinases are structurally very plastic enzymes with large conformational differences observed between the inactive and active states of the kinase. These conformational changes are largely governed by disorder to order changes in N-lobe, activation loop and hydrophobic motif. When JAK2 is in an inhibited conformational restricted state, the phosphate group of tyr1007 and tyr1008 have an intermolecular interaction with Arg975 and Lys999. These interactions play a major role in the stabilization of the activation loop conformation inside the kinase domain. Mutation of Arg975 and Lys999 to Ala (V617F+R975A), (V617F+L999A) reduced the phosphorylation at both tyr1007/1008 residues. Importantly, ruxolitinib treatment did not lead to JAK2 hyperphosphorylation in this mutants. Taken together our results suggest a novel mode of kinase regulation by modulating kinase activity through conformational changes induced by ruxolutinib. Disclosures:No relevant conflicts of interest to declare.

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

Suppressor of Cytokine Signaling (SOCS)5 is thought to act as a tumour suppressor through negative regulation of JAK/STAT and epidermal growth factor (EGF) signaling. However, the mechanism/s by which SOCS5 acts on these two distinct pathways is unclear. We show for the first time that SOCS5 can interact directly with JAK via a unique, conserved region in its N-terminus, which we have termed the JAK interaction region (JIR). Co-expression of SOCS5 was able to specifically reduce JAK1 and JAK2 (but not JAK3 or TYK2) autophosphorylation and this function required both the conserved JIR and additional sequences within the long SOCS5 N-terminal region. We further demonstrate that SOCS5 can directly inhibit JAK1 kinase activity, although its mechanism of action appears distinct from that of SOCS1 and SOCS3. In addition, we identify phosphoTyr317 in Shc-1 as a high-affinity substrate for the SOCS5-SH2 domain and suggest that SOCS5 may negatively regulate EGF and growth factor-driven Shc-1 signaling by binding to this site. These findings suggest that different domains in SOCS5 contribute to two distinct mechanisms for regulation of cytokine and growth factor signaling.

Abstract 4308: A model of oncogene addiction: BCR-ABL hijacks signaling through a MEK-dependent process and inhibits growth factor-mediated signal transduction.

Abstract Background: Chronic myeloid leukemia (CML) is characterized by the presence of the Philadelphia (Ph) chromosome and the fusion gene BCR-ABL, which has been shown to activate key signaling pathways, including PI-3 kinase, RAS/ERK and STAT5. ABL tyrosine kinase inhibitors (TKIs) are highly effective clinical agents that inhibit downstream signaling, trigger CML cell apoptosis, and provide clinical evidence for the phenomenon of “oncogene addiction," whereby cell survival becomes critically dependent upon the activity of a particular oncogene product. TKI therapy has largely failed to achieve the same degree of clinical success in other activated kinase-driven malignancies, suggesting that BCR-ABL is relatively unique in its ability to establish a state of oncogene addiction. Results: To dissect the mechanism by which BCR-ABL coordinates STAT5 and RAS/ERK signaling to establish a state of oncogene addiction, JAK2 kinase activity and RAS-GTP levels were assessed in the CML patient-derived cell line K562. Surprisingly, basal levels of RAS-GTP were low and JAK2 kinase activity was not detectable. Treatment of K562 cells with erythropoietin (EPO) only very modestly activated JAK2. Similarly, EPO treatment after one hour of BCR-ABL inhibition also failed to rescue STAT5 or RAS/ERK activation. However, with more prolonged BCR-ABL kinase inhibition, we observed a time-dependent increase in the ability of EPO to rescue STAT5 and ERK phosphorylation. After 24hrs of BCR-ABL inhibition, EPO treatment led to a significant increase in both JAK2 activation and the GTP loading of RAS relative to vehicle treated cells. These data suggest that BCR-ABL kinase activity hampers the ability of JAK2 to activate STAT5 and RAS/MAPK . Further, 24hr treatment with the MEK inhibitor PD0325901 allowed for a comparable degree of EPO-mediated JAK2 activation, suggesting that the negative regulation of JAK2 is mediated at least in part by a MEK/ERK-dependent mechanism. These observations were subsequently validated in an isogenic system utilizing TF-1 cells. Similar to the EPO/JAK2 axis in K562 cells, TF1-BCR-ABL cells exhibit attenuated GM-CSF signaling relative to control TF1-puro cells, which was restored only after prolonged BCR-ABL inhibition. Significantly, we have observed that BCR/ABL-expressing cells irreversibly commit to apoptosis before growth factor signaling is fully restored. Conclusions: Our data suggest that BCR-ABL-mediated oncogene addiction is a consequence of a high level of physiologic negative feedback mechanisms that dampen growth factor receptor signaling. Our findings also help reconcile recent evidence that JAK2 inhibition restores the sensitivity of CML stem/progenitor cells to ABL TKI treatment in vitro with the observation that JAK2 is completely dispensable for BCR-ABL-induced myeloid disease in mice. Citation Format: Jennifer Gajan, Elisabeth Lasater, Cheryl Tajon, Charles Craik, Neil Shah. A model of oncogene addiction: BCR-ABL hijacks signaling through a MEK-dependent process and inhibits growth factor-mediated signal transduction. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4308. doi:10.1158/1538-7445.AM2013-4308

Abstract 931: Highly selective JAK1 kinase inhibitors suppress Y705-mediated STAT3 activation in a wide range of tumor types, with reduced hematological toxicity.

Abstract Constitutively activated pY705-STAT3 is oncogenic and prevalent in many tumor types, and has been shown to be driven in large part by constitutive autocrine and paracrine cytokine signaling. JAK kinases are critical mediators of cytokine signaling, and JAK1 has been shown to play an essential role downstream of IL-6 family cytokines, the upregulation of which has been documented in many tumor types. A JAK1 selective inhibitor should provide a therapeutic means of suppressing Y705 phosphorylation of STAT3 without incurring the hematological toxicity associated with inhibition of JAK2 and JAK3 activity, allowing for more intensive dosing and greater combinability with other agents, particularly those with hematological toxicities. We have developed highly selective inhibitors of JAK1 and used them to address the hypothesis that JAK1 is the primary driver of STAT3 mediated signaling in cancer cells. The compounds are highly selective in enzyme assays, with 1000-fold selectivity for JAK1 vs. other JAK family members, and minimal activity against other kinases at 1uM. In cell lines, they are over 500-fold selective for JAK1 vs. JAK2, measured using assays specific for JAK1-mediated STAT3 vs. JAK2-mediated STAT5 phosphorylation. In vivo, the compounds are also highly selective for JAK1 mediated signaling, measured by comparing inhibition of STAT3 phosphorylation in H1975 subcutaneous xenografts vs. inhibition of STAT5 phosphorylation in a disseminated TEL-JAK2 transformed BaF3 model. At doses where inhibition of STAT3 phosphorylation was greater than 90%, only minimal inhibition of STAT5 phosphorylation was observed. In a wide variety of human tumor lines (CRC, NSCLC, pancreatic, DLBCL) with a variety of genetic backgrounds (K-ras, b-raf, PI3K, EGFR mutant, PTEN null), complete knockdown of pSTAT3 (Y705) was observed with JAK1 selective compounds, at concentrations achievable in vivo. These effects are consistent with experiments where inhibition of Y705 phosphorylation of STAT3 was achieved with a JAK1-specific siRNA. At doses where in vivo activity was observed, the compounds had only minimal effect in an erythropoietin-driven mouse model of polycythemia and extramedullary erythropoiesis. These data support the hypotheses that in a variety of tumor types, pSTAT3-mediated signaling is dependent on JAK1 kinase activity, and that a selective JAK1 inhibitor can suppress STAT3 activation at doses that avoid the hematological toxicity associated with JAK2 and JAK3 inhibition. Citation Format: Richard Woessner, Geraldine Bebernitz, Kirsten Bell, Robert Caccese, Nakpangi Johnson, Jason Kettle, Caroline Rivard, Patricia Schroeder, Qibin Su, Haixia Wang, Minwei Ye, Weijia Zheng, Michael Zinda, Paul Lyne, Melissa Vasbinder, Dennis Huszar, Claudio Chuaqui. Highly selective JAK1 kinase inhibitors suppress Y705-mediated STAT3 activation in a wide range of tumor types, with reduced hematological toxicity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 931. doi:10.1158/1538-7445.AM2013-931