Stathmin 1 inhibition amplifies ruxolitinib-induced apoptosis in JAK2V617F cells

The JAK/STAT pathway is constitutively activated in myeloproliferative neoplasms and can be inhibited by ruxolitinib, a selective JAK1/2 inhibitor. The JAK2^V617F mutation leads to constitutive STAT3 phosphorylation and potentially leads to inhibition of Stathmin 1 activity via STAT3. In support of this hypothesis, we found that, in HEL JAK2^V617F cells, ruxolitinib treatment decreased STAT3 and Stathmin 1 association, induced Stathmin 1 activation and microtubule instability. Silencing of Stathmin 1 significantly reduced cell proliferation and clonal growth, and increased apoptosis induced by ruxolitinib. Stathmin 1 silencing also prevented ruxolitinib-induced microtubule instability. To phenocopy the effect of Stathmin 1 inhibition, cells were treated with paclitaxel, a microtubule-stabilizing drug, in association or not with ruxolitinib; combined treatment significantly increased apoptosis, when compared to monotherapy. Notably, Stathmin 1 mRNA levels were highly expressed in CD34⁺ cells from primary myelofibrosis patients. We then proposed that an undesired effect of ruxolitinib treatment may constitute Stathmin 1 activation and microtubule instability in JAK2^V617F cells. Induction of microtubule stability, through Stathmin 1 silencing or paclitaxel treatment, combined with ruxolitinib could be an effective strategy for promoting apoptosis in JAK2^V617F cells.     

The PIM inhibitor AZD1208 synergizes with ruxolitinib to induce apoptosis of ruxolitinib sensitive and resistant JAK2-V617F-driven cells and inhibit colony formation of primary MPN cells

Classical myeloproliferative neoplasms (MPNs) are hematopoietic stem cell disorders that exhibit excess mature myeloid cells, bone marrow fibrosis, and risk of leukemic transformation. Aberrant JAK2 signaling plays an etiological role in MPN formation. Because neoplastic cells in patients are largely insensitive to current anti-JAK2 therapies, effective therapies remain needed. Members of the PIM family of serine/threonine kinases are induced by JAK/STAT signaling, regulate hematopoietic stem cell growth, protect hematopoietic cells from apoptosis, and exhibit hematopoietic cell transforming properties. We hypothesized that PIM kinases may offer a therapeutic target for MPNs. We treated JAK2-V617F-dependent MPN model cells as well as primary MPN patient cells with the PIM kinase inhibitors SGI-1776 and AZD1208 and the JAK2 inhibitor ruxolitinib. While MPN model cells were rather insensitive to PIM inhibitors, combination of PIM inhibitors with ruxolitinib led to a synergistic effect on MPN cell growth due to enhanced apoptosis. Importantly, PIM inhibitor mono-therapy inhibited, and AZD1208/ruxolitinib combination therapy synergistically suppressed, colony formation of primary MPN cells. Enhanced apoptosis by combination therapy was associated with activation of BAD, inhibition of downstream components of the mTOR pathway, including p70S6K and S6 protein, and activation of 4EBP1. Importantly, PIM inhibitors re-sensitized ruxolitinib-resistant MPN cells to ruxolitinib by inducing apoptosis. Finally, exogenous expression of PIM1 induced ruxolitinib resistance in MPN model cells. These data indicate that PIMs may play a role in MPNs and that combining PIM and JAK2 kinase inhibitors may offer a more efficacious therapeutic approach for MPNs over JAK2 inhibitor mono-therapy.     

Preclinical studies of Flonoltinib Maleate,a novel JAK2/FLT3 inhibitor,in treatment of JAK2V617F-induced myeloproliferative neoplasms

Janus kinase 2 (JAK2) hyperactivation by JAK2^V617F mutation leads to myeloproliferative neoplasms (MPNs) and targeting JAK2 could serve as a promising therapeutic strategy for MPNs. Here, we report that Flonoltinib Maleate (FM), a selective JAK2/FLT3 inhibitor, shows high selectivity for JAK2 over the JAK family. Surface plasmon resonance assays verified that FM had a stronger affinity for the pseudokinase domain JH2 than JH1 of JAK2 and had an inhibitory effect on JAK2 JH2V617F. The cocrystal structure confirmed that FM could stably bind to JAK2 JH2, and FM suppressed endogenous colony formation of primary erythroid progenitor cells from patients with MPNs. In several JAK2^V617F-induced MPN murine models, FM could dose-dependently reduce hepatosplenomegaly and prolong survival. Similar results were observed in JAK2^V617F bone marrow transplantation mice. FM exhibited strong inhibitory effects on fibrosis of the spleen and bone marrow. Long-term FM treatment showed good pharmacokinetic/pharmacodynamic characteristics with high drug exposure in tumor-bearing tissues and low toxicity. Currently, FM has been approved by the National Medical Products Administration of China (CXHL2000628), and this study will guide clinical trials for patients with MPNs.Subject terms: Myeloproliferative disease, Targeted therapies     

Burkitt leukemia with precursor B-cell features that developed after ruxolitinib treatment in a patient with hydroxyurea-refractory JAK2V617F-myeloproliferative neoplasm

A 62-year-old woman, who had a 16-year history of JAK2^V617F-mutated myeloproliferative neoplasm (MPN), developed Burkitt leukemia (BL) 16 months after treatment with ruxolitinib to control hydroxyurea-refractory conditions. BL cells were CD10⁺, CD19⁺, CD20⁻, CD34⁻, cytoplasmic CD79a⁺, and TdT⁺, and lacked surface immunoglobulins but expressed the cytoplasmic μ heavy chain. In the bone marrow, nuclear MYC⁺ BL cells displaced the MPN tissues. t(8;14)(q24;q32) occurred at a CG dinucleotide within MYC exon 1 and at the IGHJ3 segment, and an N-like segment was inserted at the junction. The V-D-J sequence of the non-translocated IGH allele had the unmutated configuration. DNA from peripheral blood at a time of the course of MPN exhibited homozygous JAK2^V617F mutation, while that at BL development included both JAK2^V617F and wild-type DNAs. Although the association between JAK1/2 inhibitor therapy for MPN and secondary development of aggressive B-cell neoplasm remains controversial, this report suggests that, in selected patients, close monitoring of clonal B-cells in the BM is required before and during treatment with JAK1/2 inhibitors.     

Imbalance of DNA‐dependent protein kinase subunits in polycythemia vera peripheral blood stem cells

Polycythemia vera (PV) is a clonal hematopoietic stem cell disease characterized by a trilinear accumulation of blood cells that has been recently associated with a JAK2^V617F point mutation. However, this molecular defect represents a rather late event in the disease progression, is not specific for this disease, and is not ascertained in all patients indicating that additional factors contribute to the specific phenotype of PV. Therefore, cDNA microarray analyses were performed on CD34⁺ peripheral blood stem cells (PBSC) with subsequent evaluation on mRNA and protein level of a larger cohort of PV patients. Microarray analyses revealed a significant dysregulation of 11 genes. KU86, a gene coding for a subunit of the DNA‐dependent protein kinase (DNA‐PK), displayed the strongest upregulation in all patients under study. This peculiarity was accompanied by downregulation of the catalytic DNA‐PK subunit DNA‐PKcs. Also Ku86 protein was upregulated and expressed in the vast majority of CD34⁺ PBSC nuclei while a weak nuclear expression was detected in only one blood donor. Differential expression of several genes, imbalance of the distinct subunits of DNA‐PK, and particularly the strong upregulation of Ku86 protein, are new findings in PV CD34⁺ PBSC. These factors may contribute to the accumulation of chromosomal aberrations, accumulation of hematopoietic cells (especially of erythropoiesis), and prolongation of CD34⁺ PBSC life span observed in PV. © 2008 Wiley‐Liss, Inc.     

Efficacy of NS-018, a potent and selective JAK2/Src inhibitor, in primary cells and mouse models of myeloproliferative neoplasms

Aberrant activation of Janus kinase 2 (JAK2) caused by somatic mutation of JAK2 (JAK2V617F) or the thrombopoietin receptor (MPLW515L) plays an essential role in the pathogenesis of myeloproliferative neoplasms (MPNs), suggesting that inhibition of aberrant JAK2 activation would have a therapeutic benefit. Our novel JAK2 inhibitor, NS-018, was highly active against JAK2 with a 50% inhibition (IC₅₀) of <1 n, and had 30–50-fold greater selectivity for JAK2 over other JAK-family kinases, such as JAK1, JAK3 and tyrosine kinase 2. In addition to JAK2, NS-018 inhibited Src-family kinases. NS-018 showed potent antiproliferative activity against cell lines expressing a constitutively activated JAK2 (the JAK2V617F or MPLW515L mutations or the TEL–JAK2 fusion gene; IC₅₀=11–120 n), but showed only minimal cytotoxicity against most other hematopoietic cell lines without a constitutively activated JAK2. Furthermore, NS-018 preferentially suppressed in vitro erythropoietin-independent endogenous colony formation from polycythemia vera patients. NS-018 also markedly reduced splenomegaly and prolonged the survival of mice inoculated with Ba/F3 cells harboring JAK2V617F. In addition, NS-018 significantly reduced leukocytosis, hepatosplenomegaly and extramedullary hematopoiesis, improved nutritional status, and prolonged survival in JAK2V617F transgenic mice. These results suggest that NS-018 will be a promising candidate for the treatment of MPNs.     

Characterization and Prognosis Significance of JAK2 (V617F), MPL,and CALR Mutations in Philadelphia-Negative Myeloproliferative Neoplasms

Background: The discovery of somatic acquired mutations of JAK2 (V617F) in Philadelphia-negative myeloproliferative neoplasms (Ph-negative MPNs) including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) has not only improved rational disease classification and prognostication but also brings new understanding insight into the pathogenesis of diseases. Dosage effects of the JAK2 (V617F) allelic burden in Ph-negative MPNs may partially influence clinical presentation, disease progression, and treatment outcome. Material and Methods: Pyrosequencing was performed to detect JAK2 (V617F) and MPL (W515K/L) and capillary electrophoresis to identify CALR exon 9.0 mutations in 100.0 samples of Ph-negative MPNs (38.0 PV, 55 ET, 4 PMF, and 3 MPN-U). Results: The results showed somatic mutations of JAK2 (V617F) in 94.7% of PV, 74.5% of ET, 25.0% of PMF, and all MPN-U. A high proportion of JAK2 (V617F) mutant allele burden (mutational load > 50.0%) was predominantly observed in PV when compared with ET. Although a high level of JAK2 (V617F) allele burden was strongly associated with high WBC counts in both PV and ET, several hematological parameters (hemoglobin, hematocrit, and platelet count) were independent of JAK2 (V617F) mutational load. MPL (W515K/L) mutations could not be detected whereas CALR exon 9.0 mutations were identified in 35.7% of patients with JAK2 negative ET and 33.3% with JAK2 negative PMF. Conclusions: The JAK2 (V617F) allele burden may be involved in progression of MPNs. Furthermore, a high level of JAK2 (V617F) mutant allele appears strongly associated with leukocytosis in both PV and ET.     

Spleen deflation and beyond: The pros and cons of Janus kinase 2 inhibitor therapy for patients with myeloproliferative neoplasms

The myeloproliferative neoplasms (MPNs) essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (MF) are malignancies that frequently harbor the recurrent somatic point mutation JAK2^V617F. The discovery of this mutation has fueled the development of Janus kinase 2 (JAK2) inhibitors. Available results have indicated that JAK2 inhibitors are particularly effective at reducing spleen size. However, the activity of these agents is multifaceted and also involves a marked improvement of systemic symptoms and, for those agents with dual JAK1 and JAK2 inhibitory activity, a marked reduction in the levels of circulating cytokines involved in the pathogenesis of the disease. Because JAK2 inhibitors are not specific for JAK2^V617F, responses have also been observed in JAK2^V617F‐negative MPNs because of the inhibition of wild‐type JAK2, which is also likely responsible for the induction of cytopenias in patients with MF and for the normalization of peripheral blood counts observed in patients with ET or PV. Given the distinct mortality and morbidity associated with ET, PV, and MF, the use of JAK2 inhibitors appears reasonable for patients with MF as well as for those with ET or PV who have become resistant or intolerant to hydroxyurea. Ongoing randomized, placebo‐controlled, phase 3 trials will further delineate the role of these agents in the management of patients with MPNs. The pros and cons of JAK2 kinase inhibitor therapy are herein discussed. Cancer 2012;. © 2011 American Cancer Society.     

Suppression of multiple anti‐apoptotic BCL2 family proteins recapitulates the effects of JAK2 inhibitors in JAK2V617F driven myeloproliferative neoplasms

Several lines of research suggest that Bcl‐xL‐mediated anti‐apoptotic effects may contribute to the pathogenesis of myeloproliferative neoplasms driven by JAK2V617F and serve as therapeutic target. Here, we used a knock‐in JAK2V617F mouse model and confirmed that Bcl‐xL was overexpressed in erythroid progenitors. The myeloproliferative neoplasm (MPN)‐induced phenotype in the peripheral blood by conditional knock‐in of JAK2V617F was abrogated by conditional knockout of Bcl2l1, which presented anemia and thrombocytopenia independently of JAK2 mutation status. Mx1‐Cre Jak2V617^W/VF/Bcl2l1^f/f mice presented persistent splenomegaly as a result of extramedullary hematopoiesis and pro‐apoptotic stimuli in terminally differentiated erythroid progenitors. The pan‐BH3 mimetic inhibitor obatoclax showed superior cytotoxicity in JAK2V617F cell models, and reduced clonogenic capacity in ex vivo assay using Vav‐Cre Jak2V617F bone marrow cells. Both ruxolitinib and obatoclax significantly reduced spleen weights in a murine Jak2V617F MPN model but did not show additive effect. The tumor burden reduction was observed with either ruxolitinib or obatoclax in terminal differentiation stage neoplastic cells but not in myeloid‐erythroid precursors. Therefore, disrupting the BCL2 balance is not sufficient to treat MPN at the stem cell level, but it is certainly an additional option for controlling the critical myeloid expansion of the disease.     

Identifying and addressing unmet clinical needs in Ph-neg classical myeloproliferative neoplasms: A consensus-based SIE,SIES, GITMO position paper

This article presents the results of group discussion among experts from SIE, SIES and GITMO societies aimed at highlighting unmet challenges in the management of Ph-neg myeloproliferative neoplasms (MPNs). The issues analyzed were: diagnosis of prefibrotic myelofibrosis; diagnosis of Ph-neg MPNs in the setting of splanchnic vein thrombosis (SVT); management of low-risk PV and low-risk ET patients with JAK2V617F mutation; molecular biomarkers in the prognostic evaluation of myelofibrosis (MF); ruxolitinib therapy in low-risk MF; therapy in patients with SVT-associated Ph-neg MPN; indications of splenectomy in MF. For each of these issues, proposals for advancement in clinical research were addressed.     

Selective JAK2/ABL dual inhibition therapy effectively eliminates TKI-insensitive CML stem/progenitor cells

Imatinib Mesylate (IM) and other tyrosine kinase inhibitor (TKI) therapies have had a major impact on the treatment of chronic myeloid leukemia (CML). However, TKI monotherapy is not curative, with relapse and persistence of leukemic stem cells (LSCs) remaining a challenge. We have recently identified an AHI-1-BCR-ABL-JAK2 protein complex that contributes to the transforming activity of BCR-ABL and IM-resistance in CML stem/progenitor cells. JAK2 thus emerges as an attractive target for improved therapies, but off-target effects of newly developed JAK2 inhibitors on normal hematopoietic cells remain a concern. We have examined the biological effects of a highly selective, orally bioavailable JAK2 inhibitor, BMS-911543, in combination with TKIs on CD34⁺ treatment-naïve IM-nonresponder cells. Combination therapy reduces JAK2/STAT5 and CRKL activities, induces apoptosis, inhibits proliferation and colony growth, and eliminates CML LSCs in vitro. Importantly, BMS-911543 selectively targets CML stem/progenitor cells while sparing healthy stem/progenitor cells. Oral BMS-911543 combined with the potent TKI dasatinib more effectively eliminates infiltrated leukemic cells in hematopoietic tissues than TKI monotherapy and enhances survival of leukemic mice. Dual targeting BCR-ABL and JAK2 activities in CML stem/progenitor cells may consequently lead to more effective disease eradication, especially in patients at high risk of TKI resistance and disease progression.     

Selective inhibition of JAK2-driven erythroid differentiation of polycythemia vera progenitors

Polycythemia Vera (PV) is a myeloproliferative disorder (MPD) that is commonly characterized by mutant JAK2 (JAK2V617F) signaling, erythrocyte overproduction, and a propensity for thrombosis, progression to myelofibrosis, or acute leukemia. In this study, JAK2V617F expression by human hematopoietic progenitors promoted erythroid colony formation and erythroid engraftment in a bioluminescent xenogeneic immunocompromised mouse transplantation model. A selective JAK2 inhibitor, TG101348 (300 nM), significantly inhibited JAK2V617F+ progenitor-derived colony formation as well as engraftment (120 mg/kg) in xenogeneic transplantation studies. TG101348 treatment decreased GATA-1 expression, which is associated with erythroid-skewing of JAK2V617F+ progenitor differentiation, and inhibited STAT5 as well as GATA S310 phosphorylation. Thus, TG101348 may be an effective inhibitor of JAK2V617F+ MPDs in clinical trials.     

Analysis of Common Driver Mutations in Philadelphia-Negative Myeloproliferative Neoplasms

BackgroundPhiladelphia-negative myeloproliferative neoplasms (MPNs) are a group of hematopoietic stem cell disorders that include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). This study examines the driver mutations among patients with MPNs in Kuwait.Patients and MethodsThis study was a retrospective review of 942 MPN cases with a driver mutation from July 2007 to June 2019 to examine their demographic, clinical, and laboratory attributes.ResultsThe annual incidence of MPNs is 1.6 per 100,000 persons, and ET is the most common subtype. The median age of our cohort was 55 years, and the patients were predominantly male. We found that the most frequent gene mutation of MPNs in our cohort was the JAK2V617F mutation, which was present in 90% of cases, followed by the CALR exon 9, MPLW515L/K, and JAK2 exon 12 mutations. In our cohort, thrombotic events were observed in 18.7% of cases.ConclusionAlthough Philadelphia-negative MPNs are rare hematologic malignancies, thrombosis is a relatively common initial presentation. The JAK2V617F mutation was the driver mutation in the majority of patients with MPN.     

Classical Philadelphia-negative myeloproliferative neoplasms: focus on mutations and JAK2 inhibitors

Classical Philadelphia- negative myeloproliferative neoplasms (MPNs) encompass three main myeloid malignancies: polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF). Phenotype-driver mutations in Janus kinase 2 (JAK2), calreticulin (CALR), and myeloproliferative leukemia virus oncogene (MPL) genes are mutually exclusive and occur with a variable frequency. Driver mutations influence disease phenotype and prognosis. PV patients with JAK2 exon 14 mutation do not differ in number of thrombotic events, risk of leukemic and fibrotic transformation, and overall survival to those with JAK2 exon 12 mutation. Type 2-like CALR-mutated ET patients have lower risk of thrombosis if compared with those carrying JAK2 or type 1-like CALR mutation. For ET, overall survival is comparable between patients with JAK2 and either type 1-like and type 2-like CALR mutations. For MF, better OS is demonstrated for patients harboring a type 1-like CALR mutation than those with type 2-like CALR or JAK2. The discovery of driver mutations in MPNs has prompted the development of molecularly targeted therapy. Among JAK2 inhibitors, ruxolitinib (RUX) has been approved for (1) treatment of intermediate-2 and high-risk MF and (2) PV patients who are resistant to or intolerant to hydroxyurea. RUX reduces spleen size and alleviates disease symptoms in a proportion of MF patients. RUX in MF leads to prolonged survival and reduces risk of death. RUX controls hematocrit, reduces spleen size and alleviates symptoms in PV. Adverse events of RUX are moderate, however, its long-term use may be associated with opportunistic infections. Trials with other JAK2 inhibitors are ongoing.     

JAK2V617F-mediated phosphorylation of PRMT5 downregulates its methyltransferase activity and promotes myeloproliferation

The JAK2V617F constitutively activated tyrosine kinase is found in most patients with myeloproliferative neoplasms. While examining the interaction between JAK2 and PRMT5, an arginine methyltransferase originally identified as JAK-binding protein 1, we found that JAK2V617F (and JAK2K539L) bound PRMT5 more strongly than did wild-type JAK2. These oncogenic kinases also acquired the ability to phosphorylate PRMT5, greatly impairing its ability to methylate its histone substrates, and representing a specific gain-of-function that allows them to regulate chromatin modifications. We readily detected PRMT5 phosphorylation in JAK2V617F-positive patient samples, and when we knocked down PRMT5 in human CD34+ cells using shRNA, we observed increased colony formation and erythroid differentiation. These results indicate that phosphorylation of PRMT5 contributes to the mutant JAK2-induced myeloproliferative phenotype.     

Myeloproliferative neoplasms: from JAK2 mutations discovery to JAK2 inhibitor therapies

Most BCR-ABL1-negative myeloproliferative neoplasms (MPN) carry an activating JAK2 mutation. Approximately 96% of patients with polycythemia vera (PV) harbors the V617F mutation in JAK2 exon 14, whereas the minority of JAK2 (V617F)-negative subjects shows several mutations in exon 12. Other mutation events as MPL, TET2, LNK, EZH2 have been described in chronic phase, while NF1, IDH1, IDH2, ASX1, CBL and Ikaros in blast phase of MPN. The specific pathogenic implication of these mutations is under investigation, but they may have a role in refinement of diagnostic criteria and in development of new prognostic models. Several trials with targeted therapy (JAK inhibitors) are ongoing mostly involving patients with PMF, post-PV MF and post-essential thrombocythemia (ET) MF. Treatment with ruxolitinib and TG101348 has shown clinically significant benefits, particularly in improvement of splenomegaly and constitutional symptoms in MF patients. On the other hand, JAK inhibitors have not thus far shown disease-modifying activity therefore any other deduction on these new drugs seems premature.     

Prevalence and clinicopathologic correlates of JAK2 exon 12 mutations in JAK2V617F-negative polycythemia vera.

After accounting for misdiagnosis and treatment effect, allele-specific (AS)-PCR detects the JAK2V617F mutation in >95% of polycythemia vera (PV) patients. Using database inquiry, we identified 6 of a total 220 cases with PV that were JAK2V617F-negative (prevalence=3%). Of these, five cases ( approximately 80%) were found to harbor one of the two JAK2 exon 12 mutations (F537-K539delinsL or N542-E543del) in bone marrow (BM) and/or peripheral blood cells. Similar screening of six additional cases - three each with idiopathic erythrocytosis (IE) or otherwise unexplained erythrocytosis (UE) - did not reveal either JAK2V617F or JAK2 exon 12 mutations. We found JAK2 exon 12 mutations in PV cases to be readily detected by both DNA sequencing and AS-PCR, regardless of whether BM or peripheral blood cells were used as the source for DNA. Although erythroid hyperplasia was the predominant histologic feature on BM examination, megakaryocyte abnormalities and reticulin fibrosis were noted in most PV patients harboring exon 12 mutations. However, similar BM morphologic changes can also be seen in some JAK2V617F-positive PV cases; therefore, distinct genotype-phenotype association cannot be established.