CYT387, a novel JAK2 inhibitor, induces hematologic responses and normalizes inflammatory cytokines in murine myeloproliferative neoplasms.
ABSTRACT: Activating alleles of Janus kinase 2 (JAK2) such as JAK2(V617F) are central to the pathogenesis of myeloproliferative neoplasms (MPN), suggesting that small molecule inhibitors targeting JAK2 may be therapeutically useful. We have identified an aminopyrimidine derivative (CYT387), which inhibits JAK1, JAK2, and tyrosine kinase 2 (TYK2) at low nanomolar concentrations, with few additional targets. Between 0.5 and 1.5muM CYT387 caused growth suppression and apoptosis in JAK2-dependent hematopoietic cell lines, while nonhematopoietic cell lines were unaffected. In a murine MPN model, CYT387 normalized white cell counts, hematocrit, spleen size, and restored physiologic levels of inflammatory cytokines. Despite the hematologic responses and reduction of the JAK2(V617F) allele burden, JAK2(V617F) cells persisted and MPN recurred upon cessation of treatment, suggesting that JAK2 inhibitors may be unable to eliminate JAK2(V617F) cells, consistent with preliminary results from clinical trials of JAK2 inhibitors in myelofibrosis. While the clinical benefit of JAK2 inhibitors may be substantial, not the least due to reduction of inflammatory cytokines and symptomatic improvement, our data add to increasing evidence that kinase inhibitor monotherapy of malignant disease is not curative, suggesting a need for drug combinations to optimally target the malignant cells.
Project description:Current JAK2 inhibitors used for myeloproliferative neoplasms (MPN) treatment are not specific enough to selectively suppress aberrant JAK2 signalling and preserve physiological JAK2 signalling. We tested whether combining a JAK2 inhibitor with a series of serine threonine kinase inhibitors, targeting nine signalling pathways and already used in clinical trials, synergized in inhibiting growth of haematopoietic cells expressing mutant and wild-type forms of JAK2 (V617F) or thrombopoietin receptor (W515L). Out of 15 kinase inhibitors, the ZSTK474 phosphatydylinositol-3'-kinase (PI3K) inhibitor molecule showed strong synergic inhibition by Chou and Talalay analysis with JAK2 and JAK2/JAK1 inhibitors. Other pan-class I, but not gamma or delta specific PI3K inhibitors, also synergized with JAK2 inhibitors. Synergy was not observed in Bcr-Abl transformed cells. The best JAK2/JAK1 and PI3K inhibitor combination pair (ruxolitinib and GDC0941) reduces spleen weight in nude mice inoculated with Ba/F3 cells expressing TpoR and JAK2 V617F. It also exerted strong inhibitory effects on erythropoietin-independent erythroid colonies from MPN patients and JAK2 V617F knock-in mice, where at certain doses, a preferential inhibition of JAK2 V617F mutated progenitors was detected. Our data support the use of a combination of JAK2 and pan-class I PI3K inhibitors in the treatment of MPNs.
Project description:Proinflammatory cytokines such as TNF? are elevated in patients with myeloproliferative neoplasms (MPN), but their contribution to disease pathogenesis is unknown. Here we reveal a central role for TNF? in promoting clonal dominance of JAK2(V617F) expressing cells in MPN. We show that JAK2(V617F) kinase regulates TNF? expression in cell lines and primary MPN cells and TNF? expression is correlated with JAK2(V617F) allele burden. In clonogenic assays, normal controls show reduced colony formation in the presence of TNF? while colony formation by JAK2(V617F)-positive progenitor cells is resistant or stimulated by exposure to TNF?. Ectopic JAK2(V617F) expression confers TNF? resistance to normal murine progenitor cells and overcomes inherent TNF? hypersensitivity of Fanconi anemia complementation group C deficient progenitors. Lastly, absence of TNF? limits clonal expansion and attenuates disease in a murine model of JAK2(V617F)-positive MPN. Altogether our data are consistent with a model where JAK2(V617F) promotes clonal selection by conferring TNF? resistance to a preneoplastic TNF? sensitive cell, while simultaneously generating a TNF?-rich environment. Mutations that confer resistance to environmental stem cell stressors are a recognized mechanism of clonal selection and leukemogenesis in bone marrow failure syndromes and our data suggest that this mechanism is also critical to clonal selection in MPN.
Project description:Myeloproliferative neoplasms (MPNs) frequently have an activating mutation in the gene encoding Janus kinase 2 (JAK2). Thus, targeting the pathway mediated by JAK and its downstream substrate, signal transducer and activator of transcription (STAT), may yield clinical benefit for patients with MPNs containing the JAK2(V617F) mutation. Although JAK inhibitor therapy reduces splenomegaly and improves systemic symptoms in patients, this treatment does not appreciably reduce the number of neoplastic cells. To identify potential mechanisms underlying this inherent resistance phenomenon, we performed pathway-centric, gain-of-function screens in JAK2(V617F) hematopoietic cells and found that the activation of the guanosine triphosphatase (GTPase) RAS or its effector pathways [mediated by the kinases AKT and ERK (extracellular signal-regulated kinase)] renders cells insensitive to JAK inhibition. Resistant MPN cells became sensitized to JAK inhibitors when also exposed to inhibitors of the AKT or ERK pathways. Mechanistically, in JAK2(V617F) cells, a JAK2-mediated inactivating phosphorylation of the proapoptotic protein BAD [B cell lymphoma 2 (BCL-2)-associated death promoter] promoted cell survival. In sensitive cells, exposure to a JAK inhibitor resulted in dephosphorylation of BAD, enabling BAD to bind and sequester the prosurvival protein BCL-XL (BCL-2-like 1), thereby triggering apoptosis. In resistant cells, RAS effector pathways maintained BAD phosphorylation in the presence of JAK inhibitors, yielding a specific dependence on BCL-XL for survival. In patients with MPNs, activating mutations in RAS co-occur with the JAK2(V617F) mutation in the malignant cells, suggesting that RAS effector pathways likely play an important role in clinically observed resistance.
Project description:Myeloproliferative neoplasms (MPNs) often carry JAK2(V617F), MPL(W515L), or CALR(del52) mutations. Current treatment options for MPNs include cytoreduction by hydroxyurea and JAK1/2 inhibition by ruxolitinib, both of which are not curative. We show here that cell lines expressing JAK2(V617F), MPL(W515L), or CALR(del52) accumulated reactive oxygen species-induced DNA double-strand breaks (DSBs) and were modestly sensitive to poly-ADP-ribose polymerase (PARP) inhibitors olaparib and BMN673. At the same time, primary MPN cell samples from individual patients displayed a high degree of variability in sensitivity to these drugs. Ruxolitinib inhibited 2 major DSB repair mechanisms, BRCA-mediated homologous recombination and DNA-dependent protein kinase-mediated nonhomologous end-joining, and, when combined with olaparib, caused abundant accumulation of toxic DSBs resulting in enhanced elimination of MPN primary cells, including the disease-initiating cells from the majority of patients. Moreover, the combination of BMN673, ruxolitinib, and hydroxyurea was highly effective in vivo against JAK2(V617F)+ murine MPN-like disease and also against JAK2(V617F)+, CALR(del52)+, and MPL(W515L)+ primary MPN xenografts. In conclusion, we postulate that ruxolitinib-induced deficiencies in DSB repair pathways sensitized MPN cells to synthetic lethality triggered by PARP inhibitors.
Project description:The JAK2 mutation V617F is detectable in a majority of patients with Ph-negative myeloproliferative neoplasms (MPN). Enforced expression of JAK2 V617F in mice induces myeloproliferation and bone marrow (BM) fibrosis suggesting a causal role for the JAK2 mutant in the pathogenesis of MPN. However, little is known about mechanisms and effector molecules contributing to JAK2 V617F-induced myeloproliferation and fibrosis. Here we show that JAK2 V617F promotes expression of oncostatin M (OSM) in neoplastic myeloid cells. Correspondingly, OSM was found to be overexpressed in the BM and elevated in the serum of patients with JAK2 V617F+ MPN. In addition, OSM secreted by JAK2 V617F+ cells stimulated growth of fibroblasts and microvascular endothelial cells and induced the production of angiogenic and profibrogenic cytokines (HGF, VEGF, and SDF-1) in BM fibroblasts. All effects of MPN cell-derived OSM were blocked by a neutralizing anti-OSM antibody, whereas the production of OSM in MPN cells was effectively suppressed by a pharmacologic JAK2 inhibitor or RNAi-mediated knockdown of JAK2. In summary, JAK2 V617F-mediated upregulation of OSM may contribute to fibrosis, neoangiogenesis, and the cytokine storm observed in JAK2 V617F+ MPN, suggesting that OSM could serve as a novel therapeutic target molecule in these neoplasms. IMR90 cells were treated with a single dose of rh Oncostatin M (10ng/ml).
Project description:We determined the activity of hsp90 inhibitor, and/or Janus-activated kinase 2 (JAK2) tyrosine kinase inhibitor (TKI), against JAK2-V617F-expressing cultured mouse (Ba/F3-JAK2-V617F) and human (HEL92.1.7 and UKE-1) or primary human CD34(+) myeloproliferative neoplasm (MPN) cells.Following exposure to the hsp90 inhibitor AUY922 and/or JAK2-TKI TG101209, the levels of JAK2-V617F, its downstream signaling proteins, as well as apoptosis were determined.Treatment with AUY922 induced proteasomal degradation and depletion of JAK2-V617F as well as attenuated the signaling proteins downstream of JAK2-V617F, that is, phospho (p)-STAT5, p-AKT, and p-ERK1/2. AUY922 treatment also induced apoptosis of HEL92.1.7, UKE-1, and Ba/F3-hJAK2-V617F cells. Combined treatment with AUY922 and TG101209 caused greater depletion of the signaling proteins than either agent alone and synergistically induced apoptosis of HEL92.1.7 and UKE-1 cells. Cotreatment with AUY922 and TG101209 also induced significantly more apoptosis of human CD34(+) MPN than normal hematopoietic progenitor cells. As compared with the sensitive controls, JAK2-TKI-resistant HEL/TGR and UKE-1/TGR cells exhibited significantly higher IC(50) values for JAK2-TKI (P < 0.001), which was associated with higher expression of p-JAK2, p-STAT5, p-AKT, and Bcl-xL, but reduced levels of BIM. Unlike the sensitive controls, HEL/TGR and UKE/TGR cells were collaterally sensitive to the hsp90 inhibitors AUY922 and 17-AAG, accompanied by marked reduction in p-JAK2, p-STAT5, p-AKT, and Bcl-xL, with concomitant induction of BIM.Findings presented here show that cotreatment with hsp90 inhibitor and JAK2-TKI exerts synergistic activity against cultured and primary MPN cells. In addition, treatment with hsp90 inhibitor may overcome resistance to JAK2-TKI in human MPN cells.
Project description:Recent results have shown that myeloproliferative neoplasms (MPN) are strongly associated with constitutive activation of the Janus-activated kinase (JAK)2 tyrosine kinase. However, JAK2 inhibitors currently approved or under development for treating myeloproliferative neoplasms do not selectively deplete the malignant clone, and the inhibition of activity of the drug target (JAK2) has not been rigorously evaluated in the clinical studies. Therefore, in this study we developed an in vitro assay to gain insight into how effectively JAK2 activity is inhibited in the samples of patients.We treated primary cells from normal donors and patients with MPN with JAK2 inhibitors and measured phosphorylation of downstream targets STAT5 and STAT3 by flow cytometry. Obtained results were next correlated with JAK2 V617F allele burden and plasma cytokine level.We observed a dose-dependent decrease in pSTAT5 and pSTAT3 in ex vivo treated granulocytes. However, phosphorylation of STAT3 and STAT5 in cells from patients with myelofibrosis was significantly less inhibited when compared with cells from patients with polycythemia vera, essential thrombocythemia, and normal donors. Sensitivity to inhibition did not correlate with JAK2 V617F clonal burden. Mixing studies using plasma from patients with myelofibrosis did not transfer resistance to sensitive cells. Likewise, no single cytokine measured seemed to account for the observed pattern of resistance.Taken together, these observations suggest that there are cell intrinsic mechanisms that define a priori resistance to JAK2 inhibition in myelofibrosis, and the lesion is localized upstream of STAT3 and STAT5.
Project description:Patients with myeloproliferative neoplasms (MPN) have high levels of inflammatory cytokines, some of which drive many of the debilitating constitutional symptoms associated with the disease and may also promote expansion of the neoplastic clone. We report here that monocytes from patients with MPN have defective negative regulation of Toll-like receptor (TLR) signaling that leads to unrestrained production of the inflammatory cytokine tumor necrosis factor ? (TNF-?) after TLR activation. Specifically, monocytes of patients with MPN are insensitive to the anti-inflammatory cytokine interleukin 10 (IL-10) that negatively regulates TLR-induced TNF-? production. This inability to respond to IL-10 is a not a direct consequence of JAK2 V617F , as the phenotype of persistent TNF-? production is a feature of JAK2 V617F and wild-type monocytes alike from JAK2 V617F -positive patients. Moreover, persistent TNF-? production was also discovered in the unaffected identical twin of a patient with MPN, suggesting it could be an intrinsic feature of those predisposed to acquire MPN. This work implicates sustained TLR signaling as not only a contributor to the chronic inflammatory state of MPN patients but also a potential predisposition to acquire MPN.
Project description:Activation of Janus kinase 2 (JAK2), frequently as a result of the JAK2(V617F) mutation, is a characteristic feature of the classical myeloproliferative neoplasms (MPNs) polycythemia vera, essential thrombocythemia, and myelofibrosis, and it is thought to be responsible for the constitutional symptoms associated with these diseases. BMS-911543 is a JAK2-selective inhibitor that induces apoptosis in JAK2-dependent cell lines and inhibits the growth of CD34(+) progenitor cells from patients with JAK2(V617F)-positive MPN. To explore the clinical potential of this inhibitor, we tested BMS-911543 in a murine retroviral transduction-transplantation model of JAK2(V617F) MPN. Treatment was initiated at two dose levels (3 mg/kg and 10 mg/kg) when the hematocrit exceeded 70%. Following the first week, white blood cell counts were reduced to normal in the high-dose group and were maintained well below the levels in vehicle-treated mice throughout the study. However, BMS-911543 had no effect on red blood cell parameters. After 42 days of treatment, the proportion of JAK2(V617F)-positive cells in hematopoietic tissues was identical or slightly increased compared with controls. Plasma concentrations of interleukin 6, interleukin 15, and tumor necrosis factor ? were elevated in MPN mice and reduced in the high-dose treatment group, whereas other cytokines were unchanged. Inhibitor activity after dosing was confirmed in a cell culture assay using the plasma of dosed mice and phosphorylated signal transducer and activator of transcription 5 flow cytometry. Collectively, these results show that BMS-911543 has limited activity in this murine model of JAK2(V617F)-driven MPN and suggest that targeting JAK2 alone may be insufficient to achieve effective disease control.
Project description:Myeloproliferative neoplasm (MPN) patients frequently show co-occurrence of JAK2-V617F and mutations in epigenetic regulator genes, including EZH2 In this study, we show that JAK2-V617F and loss of Ezh2 in hematopoietic cells contribute synergistically to the development of MPN. The MPN phenotype induced by JAK2-V617F was accentuated in JAK2-V617F;Ezh2(-/-) mice, resulting in very high platelet and neutrophil counts, more advanced myelofibrosis, and reduced survival. These mice also displayed expansion of the stem cell and progenitor cell compartments and a shift of differentiation toward megakaryopoiesis at the expense of erythropoiesis. Single cell limiting dilution transplantation with bone marrow from JAK2-V617F;Ezh2(+/-) mice showed increased reconstitution and MPN disease initiation potential compared with JAK2-V617F alone. RNA sequencing in Ezh2-deficient hematopoietic stem cells (HSCs) and megakaryocytic erythroid progenitors identified highly up-regulated genes, including Lin28b and Hmga2, and chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) analysis of their promoters revealed decreased H3K27me3 deposition. Forced expression of Hmga2 resulted in increased chimerism and platelet counts in recipients of retrovirally transduced HSCs. JAK2-V617F-expressing mice treated with an Ezh2 inhibitor showed higher platelet counts than vehicle controls. Our data support the proposed tumor suppressor function of EZH2 in patients with MPN and call for caution when considering using Ezh2 inhibitors in MPN.