Bim and Mcl-1 exert key roles in regulating JAK2V617Fcell survival

Abstract

BackgroundThe JAK2V617F mutation plays a major role in the pathogenesis of myeloproliferative neoplasms and is found in the vast majority of patients suffering from polycythemia vera and in roughly every second patient suffering from essential thrombocythemia or from primary myelofibrosis. The V617F mutation is thought to provide hematopoietic stem cells and myeloid progenitors with a survival and proliferation advantage. It has previously been shown that activated JAK2 promotes cell survival by upregulating the anti-apoptotic STAT5 target gene Bcl-xL. In this study, we have investigated the role of additional apoptotic players, the pro-apoptotic protein Bim as well as the anti-apoptotic protein Mcl-1.MethodsPharmacological inhibition of JAK2/STAT5 signaling in JAK2V617F mutant SET-2 and MB-02 cells was used to study effects on signaling, cell proliferation and apoptosis by Western blot analysis, WST-1 proliferation assays and flow cytometry. Cells were transfected with siRNA oligos to deplete candidate pro- and anti-apoptotic proteins. Co-immunoprecipitation assays were performed to assess the impact of JAK2 inhibition on complexes of pro- and anti-apoptotic proteins.ResultsTreatment of JAK2V617F mutant cell lines with a JAK2 inhibitor was found to trigger Bim activation. Furthermore, Bim depletion by RNAi suppressed JAK2 inhibitor-induced cell death. Bim activation following JAK2 inhibition led to enhanced sequestration of Mcl-1, besides Bcl-xL. Importantly, Mcl-1 depletion by RNAi was sufficient to compromise JAK2V617F mutant cell viability and sensitized the cells to JAK2 inhibition.ConclusionsWe conclude that Bim and Mcl-1 have key opposing roles in regulating JAK2V617F cell survival and propose that inactivation of aberrant JAK2 signaling leads to changes in Bim complexes that trigger cell death. Thus, further preclinical evaluation of combinations of JAK2 inhibitors with Bcl-2 family antagonists that also tackle Mcl-1, besides Bcl-xL, is warranted to assess the therapeutic potential for the treatment of chronic myeloproliferative neoplasms.

Pro-Apoptotic Activity of Ruxolitinib Alone and in Combination with Hydroxyurea, Busulphan, and PI3K/mTOR Inhibitors in JAK2-Positive Human Cell Lines

“…In contrast, such effect was not observed in relation to cell lines, characterized by c-KIT mutations or transformed with the Bcr--Abl oncoprotein [6]. Although, up-regulation of the pro-apoptotic protein Bim and down-regulation of the anti-apoptotic Bcl-xL was observed, the exact mechanism of apoptosis related to JAK2 inhibitors remain to be elucidated [7][8][9].…”

“…Also, Rubert et al [8] suggest that Bim activation plays a key role in regulating the survival of SET-2 cells triggered by JAK2 inhibitor. They found that Bim activation following JAK2 inhibition led to enhanced sequestration of BclxL and Mcl-1 anti-apoptotic proteins.…”

“…Subsequently, the apoptotic index (AI) was assessed as a percentage of Ann-V positive cells. The mechanisms of apoptosis were judged by a drop of mitochondrial transmembrane potential (Δψ m ), activation of caspases (3,8,9) and expression of proteins involved in apoptosis regulation (Bax, Bad, Bid, Bcl-2, Mcl-1, XIAP, survivin, p53). The apoptotic, hypodiploid sub-G 1 fraction was evaluated on the basis of the DNA histogram.…”

Dysregulation of BCL-2 family proteins by leukemia fusion genes

“…BCL-2 and BCL-X L expression is increased in PV patients with the JAK2 V617F mutation, and higher JAK2 V617F expression in erythroid precursor cells from PV patients was associated with increased sensitivity to ABT-737 treatment (57). RNA interference-induced knockdown of BIM decreased sensitivity to JAK2 inhibition, implying that JAK2 signaling also represses BIM, whereas MCL-1 knockdown increased apoptosis in JAK2 V617F cells and increased sensitivity to JAK2 inhibition (58). Enforced expression of JAK2 V617F increased MCL-1 expression by STAT3-dependent transcription (59).…”

How does JAK2V617F contribute to the pathogenesis of myeloproliferative neoplasms?

“…Numerous STAT5 transcriptional targets have been reported in the literature, which, when dysregulated, can promulgate the malignant state in MPN. These include: (1) the PIM kinases PIM-1 and PIM-2, which stimulate cell proliferation and impair apoptosis 18 ; (2) c-MYC and JUNB, which provide an immediate early pro-proliferative response to growth stimuli 19,20 ; (3) CYCLIN D2, p27KIP, and CDC25A, which can disrupt the G 1 /S checkpoint and increase cell cycling 21,22 ; (4) the PU.1 and ID1 transcription factors, which function to increase myeloid differentiation 23,24 ; (5) BCL-XL and MCL-1, which inhibit apoptosis and promote cytokine-independent growth and erythropoietin-independent colony formation 25,26 ; and (6) RAD51, which increases rates of DNA repair and maintains genomic stability to facilitate rapid cell cycling. 27 Consistent with the central role of STAT5 in MPN pathophysiology, Jak2V617F expression is incapable of producing disease in a Stat5a/b-deficient mouse.…”