Bim and Mcl-1 exert key roles in regulating JAK2V617F cell survival.
BACKGROUND
The 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.


METHODS
Pharmacological 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.


RESULTS
Treatment 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.


CONCLUSIONS
We 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.
Disease Area Oncology, Novartis Institutes for BioMedical Research, Basel, Switzerland.
Joëlle
J
Rubert
Zhiyan
Z
Qian
Rita
R
Andraos
Daniel A
DA
Guthy
Thomas
T
Radimerski
D019943:Amino Acid Substitution; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000072224:Bcl-2-Like Protein 11; D015153:Blotting, Western; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics / Q000502:physiology; D005434:Flow Cytometry; D006801:Humans; D053614:Janus Kinase 2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D009154:Mutation; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D010766:Phosphorylation / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011810:Quinoxalines / Q000494:pharmacology; D034622:RNA Interference; D015398:Signal Transduction / Q000187:drug effects
D051017:Apoptosis Regulatory Proteins; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; D061945:Caspase Inhibitors; D008565:Membrane Proteins; D064549:Myeloid Cell Leukemia Sequence 1 Protein; C553194:NVP-BSK805; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011810:Quinoxalines; C507924:JAK2 protein, human; D053614:Janus Kinase 2; D020169:Caspases
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