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Bim and Mcl-1 exert key roles in regulating JAK2V617Fcell survival
© Rubert et al; licensee BioMed Central Ltd. 2011
Received: 5 August 2010
Accepted: 19 January 2011
Published: 19 January 2011
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.
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.
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.
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.
The somatic activating JAK2V617F mutation is found in nearly every patient with the chronic myeloproliferative neoplasm (cMPN) polycythemia vera (PV) and roughly half of those patients affected by essential thrombocythemia (ET) and primary myelofibrosis (PMF) . At the molecular level, it is thought that the V617F mutation in the JAK2 pseudokinase alleviates some of the negative regulation that this domain normally elicits on the kinase domain , allowing for increased kinase autoactivation . Clinical trials with JAK inhibitors in primary myelofibrosis patients are underway and have shown rapid suppression of splenomegaly and improvement of constitutional symptoms . However, up to now effects on mutant allele burden have been modest and bone marrow fibrosis appears to persist , warranting continued pre-clinical and clinical research in order to improve therapeutic outcome of JAK inhibitors in cMPNs. Mutant JAK2V617F, which arises at the level of the hematopoietic stem cell , likely provides progenitor cells with both a proliferation and a survival advantage . Hence, a potential avenue for enhanced JAK2V617F cell killing by JAK2 inhibitors may lie in simultaneous perturbation of survival mechanisms. Importantly, several studies have found that the anti-apoptotic Bcl-2 family member Bcl-xL plays a role in PV erythroblast survival [8, 9]. Along these lines, Bcl-xL depletion induced apoptosis in JAK2V617F mutant cells and the BH3 (Bcl-2-homology domain 3)-mimetic ABT-737 was shown to preferentially kill JAK2V617F mutant PV erythroid precursors as compared to healthy subject erythroblasts [9, 10]. The BH3-only pro-apoptotic protein Bad has been implicated in regulating JAK2V617F mutant cell survival  and engages anti-apoptotic Bcl-2, Bcl-xL and Bcl-w, but not Mcl-1 . Mcl-1 protein is normally short-lived due to rapid proteasome-mediated destruction but contributes to resistance to cell-death stimuli if its levels are elevated [12, 13].
In this study we focused on elucidating potential roles of pro-apoptotic Bim and anti-apoptotic Mcl-1 in regulating JAK2V617F mutant cell survival. In contrast to Bad, Bim can engage all Bcl-2 pro-survival family members, including Mcl-1 . Both Bim and Mcl-1 were readily detectable in JAK2V617F mutant cell lines and co-immunoprecipitated. JAK2 inhibition led to changes in Bim-EL Ser69 phosphorylation, along with a drop in total Mcl-1 levels and concomitant induction of programmed cell death. In support of a key role in regulating JAK2V617F cell survival, Mcl-1 depletion by RNAi was found to severely compromise cell viability and sensitized cells to JAK2 inhibition. Taken together, we show that Mcl-1 appears to be critical for JAK2V617F mutant cell survival, and corroborate that cell death induced by JAK2 inhibition requires Bim activation. Our findings suggest that combinations of JAK2 inhibitors with Bcl-2 family antagonists that tackle both Bcl-xL and Mcl-1 merit further preclinical evaluation of the therapeutic potential for the treatment of cMPNs.
Compounds and formulations
NVP-BSK805 (free base) was synthesized internally , 10 mM stock solutions were prepared in dimethyl sulfoxide (DMSO) and aliquots were stored at -20°C until use. The ethyl-ester of the pan-caspase inhibitor Z-VAD-FMK was synthesized internally. UO126 (# 1144, Tocris Bioscience, Ellisville, MO, USA) was prepared as a 10 mM stock solution in DMSO and stored at -20°C until use. Obatoclax mesylate (# S1057, Selleck Chemicals, Houston, TX, USA) was prepared as a 10 mM stock solution in DMSO and stored at -20°C until use.
SET-2 cells (generously provided by Prof. Hans Drexler, DSMZ, Braunschweig, Germany) were cultured in standard RPMI medium supplemented with 10% of fetal calf serum (FCS), 2 mM L-glutamine and 1% (v/v) penicillin/streptomycin. MB-02 cells (generously provided by Prof. Doris Morgan, Drexel University, Philadelphia, PA, USA) were grown in RPMI medium as described above, supplemented with 10 ng/ml recombinant human GM-CSF (granulocyte-macrophage colony-stimulating factor), 10 ng/ml recombinant human SCF (stem cell factor) and 10 mM sodium pyruvate. TF-1 cells (American Type Culture Collection) were cultured in RPMI medium, supplemented with 20% of fetal bovine serum, 1 mM L-glutamine, 5 g/l sodium bicarbonate, 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/l D-glucose, 1% (v/v) penicillin/streptomycin and 2 ng/ml GM-CSF.
The following stealth™ RNAi oligonucleotides (Invitrogen, Carlsbad, CA, USA) were used; BAD: duplex 1 5'-GCUCCGGAGGAUGAGUGACGAGUUU-3', duplex 2 5'-GGACUCCUUUAAGAAGGGACUUCCU-3' and duplex 3 5'-UCUUCCAGUCCUGGUGGGAUCGGAA-3'; Bim: duplex 1 5'-UGAGUGUGACCGAGAAGG UAGACAA-3', duplex 2 5'-CAUGAGUUGUGACAAAUCAACACAA-3' and duplex 3 5'-CACGAAUGGUUAUCUUACGACUGUU-3'; Mcl-1: duplex 1 5'-GAAAGUAUCACAGACGUUCUCGUAA-3', duplex 2 5'-CGGGACUGGCUAGUUAAACAAAGAG-3' and duplex 3 5'-GGUUUGUGGAGUUCUUCCAUGUAGA-3', and a non-targeting control stealth™ RNAi oligo 5'-GAUGAAGGGAGGGUGUACCAACUUA-3'. Cells were transfected with RNAi oligonucleotides using Nucleofactor™ Solution V (Amaxa GmbH, Cologne, Germany) and the Amaxa system according to the instructions of the manufacturer.
Real-Time Quantitative PCR
Mcl-1 mRNA levels were determined by real-time quantitative PCR using the Applied Biosystems Taqman Gene Expression kit (# Hs03043899_m1, Applied Biosystems, Carlsbad, CA, USA). Total RNA from cells was isolated with the RNeasy Mini Kit (# 74104, Qiagen, Hilden, Germany), accompanied by an on-column DNase (# 79254, Qiagen) digestion. Expression levels of the housekeeping gene GAPDH (# 4310884E, Applied Biosystems) were also measured as an endogenous normalization control. Mcl-1 and GAPDH signals were measured with FAM (6-carboxy-fluorescein) and VIC fluorescent reporter dye labeling, respectively. The volume of each reaction was 10 μl per well (384-well plate), which consisted of 5 μl 2 × reaction buffer and 0.05 μl 200 × Euroscript RT (reverse transcriptase) enzyme and RNase inhibitor mix from the one-step RT-qPCR MasterMix Plus (# RT-QPRT-032X, Eurogentec, Seraing, Belgium), 0.5 μl 20 × Taqman Gene Expression mix together with 2 μl of 50 ng RNA as amplification template. The ROX reference dye was present in the RT-qPCR reaction buffer. RT-qPCR was carried out on the ABI 7900HT Fast Real-Time PCR system (Applied Biosystems, SDS2.3 software). The reaction mixtures were incubated at 48°C for 30 minutes, during which the reverse transcription took place, 95°C for 10 minutes to activate HotGoldStar DNA polymerase (Eurogentec), followed by 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. Samples were measured in triplicate. Cycle threshold (Ct) values were used to determine the relative amounts of Mcl-1 and GAPDH mRNA levels in the samples. 2-Ct Mcl-1 values were computed and normalized to mean 2-Ct GAPDH values. Mcl-1 mRNA levels were depicted as fold change compared to DMSO vehicle control by dividing normalized 2-Ct values of compound treated samples by those of vehicle treated samples.
Cells were extracted in lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 25 mM β-glycerophosphate, 25 mM NaF, 5 mM EGTA, 1 mM EDTA, 15 mM pyrophosphate PPI, supplemented freshly with 1% Nonidet P-40, 1 x protease inhibitor cocktail (Complete Mini, Roche), 1 mM DTT, 0.2 mM sodium-vanadate and 1 mM PMSF) by passing through a 1 ml syringe connected to a 23-gauge needle. Cell debris were pelleted by centrifugation. Typically, 20 μg of protein lysates were resolved by NuPAGE Novex 4-12% Bis-Tris Midi Gels (Invitrogen, Carlsbad, CA, USA) and transferred to PVDF membranes by semi-dry blotting. The following antibodies were used to probe blots: Anti-cleaved caspase 3 (# 9664), 7 (# 9491), 8 (# 9496), 9 (# 9501), Bad (#9292), Bak (# 3814), Bax (# 2772), Bcl-xL (# 2762), Bim (# 2933), phospho-Bim (Ser55 (Homo sapiens: Ser59) (# 4550), phospho-Bim (Ser69) (# 4581), ERK1/2 (# 9102), phospho-ERK1/2 (Thr201/Tyr204) (9101), Mcl-1 (# 4572), PARP (# 9542), phospho-STAT5 (# 9359) and phospho-tyrosine (# 9411) were from Cell Signaling Technology (Beverly, MA, USA). Anti-Bim (# 202000) from Calbiochem (San Diego, CA, USA) was also used. The STAT5 antibody (# sc-835) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The β-tubulin (# T4026) and Mcl-1 (# AAP-240) antibodies were from Sigma (St. Louis, MO, USA) and Assay Designs (Ann Arbor, MI, USA), respectively. Antibodies were typically incubated overnight at 4°C followed by washes and incubation with the corresponding HRP-conjugated secondary antibodies. Immunoreactive bands were revealed with enhanced chemiluminescence reagents.
Immunoprecipitation and co-immunoprecipitation assays
Cells were extracted either in CHAPS lysis buffer  or in Triton/glycerol ("TG") lysis buffer  (the latter for Bcl-xL/Bax co-immunoprecipitation studies), lysates were kept on ice and protein content was determined by Bradford assay. Immediately thereafter, typically 500 μg total protein input were subject to immunoprecipitation using the following antibodies: Anti-Bim (# 2933), anti-Bcl-xL (# 2762) and anti-Bax (# 2772) were from Cell Signaling Technology (Beverly, MA, USA), anti-Mcl-1 (# 559027) from BD Biosciences (Franklin Lakes, NJ, USA). Co-immunoprecipitation assays were carried out using 1.5 ml Eppendorf protein LoBind Tubes (# 0030 108.116, Eppendorf, Hamburg, Germany). Bound fractions were released by heating at 70°C for 10 minutes in 20 μl NuPAGE LDS sample buffer. The supernatant containing the bound fraction was resolved by gradient gel electrophoresis and transferred to PVDF membranes for Western blot analysis as described above.
Anti-proliferative activity of the JAK2 inhibitor NVP-BSK805 was determined by incubating SET-2 cells or MB-02 cells with an 8 point concentration range of compound and cell proliferation relative to DMSO treated cells was measured (typically after 72 hours for SET-2 cells and after 96 hours for MB-02 cells, unless specified otherwise) using the colorimetric WST-1 (Roche Diagnostics GmbH, Penzberg, Germany) cell viability readout. Of each triplicate treatment the mean was calculated and these data were plotted in XLfit 4 (XLfit 4, ID Business Solutions Ltd, Guildford, Surrey, UK) to determine the respective half-maximal growth-inhibitory concentration (GI50) values.
Cultured cells were collected after treatments, washed once with PBS and resuspended in propidium iodide buffer (1 mM sodium citrate (pH 4.0), 1.5 mM NaCl, 5 mM EDTA, 5 mM EGTA, 0.1% NP40, 4 μg of propidium iodide/ml and 80 μg/ml of RNaseA in PBS). After 30 minutes of incubation in the dark on ice, cellular DNA content was measured with a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). For detection of activated Bak, cells were washed in PBS and then fixed at RT for 5 minutes in 0.25% paraformaldehyde (diluted in PBS). After washing twice with PBS the cell pellet was resuspended in 200 μl PBS containing 0.1% digitonin (# D141, Sigma) and then 10 μl mouse anti-Bak antibody (# AM03 (Ab-1), Calbiochem) were added followed by incubation on ice for 30 minutes. After washing twice with PBS the cell pellet was resuspended in 100 μl PBS and incubated at room temperature in the dark for 40 minutes with 5 μl fluorescein isothiocyanate (FITC)-conjugated anti-mouse antibody (# sc-2010, Santa Cruz) followed by two washes with PBS and flow cytometry analysis. The shifts in fluorescent channel 1-height (FL1-H) fluorescence intensity compared to DMSO vehicle controls were quantified and represented as fold change over DMSO control.
The t-test was conducted to determine statistical significance between two groups. The significance level was set at p < 0.05. Statistical analysis was performed using SigmaPlot v11.0 (Systat Software Inc, Chicago, IL, USA).
NVP-BSK805 JAK2 inhibitor triggered cell death requires activation of caspase cascades and is overcome by caspase inhibition
Key role of Bim in JAK2 inhibitor induced apoptosis in JAK2V617Fcells
JAK2 inhibition in JAK2V617Fcells modulates the post-translational modification of Bim and levels of Mcl-1
As alluded to above, Bim-EL levels were readily detectable in SET-2 and MB-02 cell lines at baseline and did not increase appreciably upon JAK2 inhibitor treatment (Figure 2A and B). This was reminiscent of the modest changes in Bim-EL levels reported in IL-3 dependent mouse pro-B FL5.12 cells following IL-3 deprivation . Thus, we investigated if the association of Bim with Mcl-1 and/or Bcl-xL [15, 16] would be impacted by JAK2 inhibition. Using SET-2 JAK2V617F mutant cell extracts, we found that Mcl-1 co-immunoprecipitated with Bim and vice versa (Figure 5B). Importantly, despite a drop in total and immunoprecipitatable Mcl-1 levels in JAK2V617F mutant cells treated with NVP-BSK805, the relative ratio of Bim immunoprecipitated with Mcl-1 appeared constant or even increased compared to control cell extracts, indicating enhanced association of Bim and Mcl-1 upon JAK2 inhibition (Figure 5B). Interestingly, the amounts of Mcl-1 that could be immunoprecipitated from cells treated with NVP-BSK805 were already strongly reduced at the 4 hours time point (Figure 5B), at which total levels in whole cell extracts were not yet substantially lower compared to control cells (Figure 5C). The importance of Bcl-xL in regulating survival of JAK2V617F cells has already been recognized [10, 27, 28], hence, we also assessed its interaction with Bim . Similar to the results obtained with Mcl-1, the relative amounts of Bcl-xL co-immunoprecipitated with Bim were comparable between extracts prepared from control and JAK2 inhibitor treated cells (Figure 5D), despite reduced overall levels of Bcl-xL after 24 hours of drug treatment (Figure 5C). Using an antibody that recognizes an amino-terminal epitope of human Bax, there was a pronounced increase in the amounts of detergent-soluble Bax that could be immunoprecipitated after treatment of SET-2 cells with NVP-BSK805 (Figure 5D), while the total levels of Bax were unchanged (Figure 5C). Levels of detergent-soluble Bax that could be immunoprecipitated reached a plateau by 48 hours following JAK2 inhibition (Additional file 2). These findings imply either a change of Bax conformation, or a change of multi-protein complexes containing Bax, or both upon JAK2 inhibition. In support of changes in Bim/Bcl-xL/Bax complexes following JAK2 inhibition, lower amounts of Bax co-immunoprecipitated with Bcl-xL from cells treated with NVP-BSK805 (Figure 5D). Mcl-1 was not found to co-immunoprecipitate Bax (data not shown). Importantly, besides Bax also Bak needs to be activated to trigger mitochondrial cell death  and Mcl-1 has been described to antagonize Bak at the mitochondrial membrane . Since both Bax and Bak are expressed in SET-2 cells (Figure 5C) we investigated Bak activation following JAK2 inhibition. To this end, we carried out co-immunoprecipitation experiments to study the interaction of Bak with either Mcl-1 or Bcl-xL. Unfortunately, these analyses were confounded by unspecific binding of Bak to the beads. Thus, we assessed Bak activation by flow cytometry using a conformation-specific Bak antibody . These analyses revealed significant Bak activation in SET-2 cells starting at 24 hours following JAK2 inhibition (Figure 5E).
Mcl-1 is required for survival of JAK2V617Fcells
In malignant and normal cells the balance between pro-apoptotic and anti-apoptotic signals determines cell survival. The JAK2V617F mutation was identified with high frequencies in the MPNs PV, ET as well as PMF, and is thought to provide mutant progenitor cells with a proliferation and survival advantage . In the present study, we have focused on assessing the roles of the pro-apoptotic protein Bim and the anti-apoptotic protein Mcl-1 in JAK2V617F mutant cells. We report that Bim depletion by RNAi suppresses JAK2 inhibitor-induced apoptosis, while Mcl-1 depletion profoundly affects JAK2V617F mutant cell viability and sensitizes cells to JAK2 inhibition. The BH3-only protein Bim plays an important role in hematopoietic homeostasis  and has been shown to be regulated by factors that activate JAK2 signaling [26, 35]. Two cooperating pathways downstream of JAK2 activation have been reported to keep Bim activity in check; On one hand, PI3K/AKT signaling regulates the expression of the Bim gene via the forkhead transcription factor FOXO3A [36, 37], whereas on the other hand, MEK/ERK signaling promotes Bim phosphorylation on Ser69 and triggers its degradation by the proteasome . Furthermore, it was recently found that Bim expression in erythroblasts is suppressed by the LRF transcription factor (itself being a direct target of GATA1) in the process of erythroid maturation . Mcl-1 is a member of five anti-apoptotic proteins (Bcl-2, Bcl-xL, Bcl-W, Mcl-1 and A1) that antagonize the pro-apoptotic proteins Bak and Bax . Mcl-1 has a chief role in regulating the survival of hematopoietic stem cells and early hematopoietic progenitors . Bcl-xL has an important role in protecting hematopoietic cells and maturing erythroid cells from cell death [41, 42] and is a target gene of EpoR/JAK2 signaling . Mcl-1 and Bcl-xL sequester Bak and Bax until their displacement is promoted by the action of activated BH3-only proteins to trigger subsequent mitochondrial cell death .
Here we show that JAK2 inhibition in JAK2V617F mutant cells led to post-translational changes in Bim that affected its interaction with other Bcl-2 family members. We detected enhanced association of Bim-EL with Mcl-1 upon JAK2 inhibition, seemingly consistent with earlier findings of apoptosis induction by serum withdrawal . Furthermore, there was a sharp increase in the levels of immunoprecipitable Bax following JAK2 inhibition. In various settings, Bim-EL activation also involves loss of MEK/ERK pathway-mediated Ser69 phosphorylation, whereby Bim evades proteasomal degradation . Loss of Bim-EL Ser69 phosphorylation following JAK2 inhibition in the JAK2V617F mutant cell lines analyzed in this study likely plays a role in Bim activation, in agreement with a recent study by Will et al. . However, Will et al. reported that Bim protein levels were up-regulated in JAK2V617F mutant cells following JAK2 inhibition , which we did not see in our analyses. These differences might be attributable to different experimental settings. In fact, using factor-independent Ba/F3 pro-B cells stably expressing EpoR and JAK2V617F we also detected low basal levels of Bim-EL and a marked up-regulation upon JAK2 inhibition (data not shown), as found by Will et al. However, Ba/F3 cells do not represent the hematopoietic lineage in which the JAK2V617F mutation arises and regulation of Bim activity may be cell lineage-specific . Taken together, our findings imply that Bim is in a latent complex with the Bcl-2 family pro-survival proteins Mcl-1 and Bcl-xL in viable JAK2V617F mutant cells. Both Mcl-1 and Bcl-xL govern survival of JAK2V617F mutant cells by keeping Bax and Bak in check. In turn, JAK2 inhibition is postulated to affect Bim complexes such that Mcl-1 and Bcl-xL are neutralized. This is proposed to drop anti-apoptotic activity in JAK2V617F mutant cells below a critical threshold, unleashing Bak and Bax to drive mitochondrial cell death. Upon inhibition of JAK2/STAT signaling the expression of Bcl-xL  and Mcl-1 [25, 45] is suppressed, along with subsequent reduction of Bcl-xL and Mcl-1 protein levels, thereby contributing to the loss of pro-survival activity. Hence, as in CML [46–48] and FLT-3 mutant  AML cells, Bim is also emerging as a central cell death driver in JAK2V617F mutant cells (, and this report).
Polycythemia vera patients with high JAK2V617F mutant allele burden were described to have increased levels of Bcl-2 as well as Bcl-xL, and the Bcl-2/Bcl-W/Bcl-xL inhibitor ABT-737 was shown to preferentially inhibit proliferation and induce mitochondrial depolarization in JAK2V617F mutant erythroblasts as compared to those from healthy subjects . However, at the level of the individual MPN patient, Zeuner et al. did not detect a strict correlation between Bcl-2 or Bcl-xL expression and drug resistance, indicating that response to therapy may be determined by additional underlying anti-apoptosis mechanisms. Our findings suggest that combinations of JAK2 inhibitors with Bcl-2 family antagonists that also tackle Mcl-1, besides Bcl-xL [21, 50], merit further preclinical evaluation of the therapeutic potential for the treatment of cMPNs. Importantly, partial inhibition of Mcl-1 may be sufficient to sensitize cells to JAK2 inhibition. This could be important in order to minimize the impact on normal cells, such as e.g. on B and T lymphocytes, in which Mcl-1 plays a key role, as revealed by conditional knock-out studies . Furthermore, it will be of particular interest to explore if combinations of JAK2 inhibitors with Bcl-2 family antagonists result in enhanced killing of the MPN mutant clone. Thus, follow-up experiments in suitable preclinical MPN animal models [52–54] would be important for proof of concept in vivo and to support the translation of potentially promising therapeutic modalities into the clinical setting. Encouragingly, clinical assessment of JAK inhibitors in MPN patients is underway , as well as intense drug discovery and development efforts to identify Mcl-1 antagonists [32, 56].
Bim and Mcl-1 were found to have opposing roles in regulating JAK2V617F cell survival. JAK2 inhibition in JAK2V617F mutant cells led to loss of Bim-EL Ser69 phosphorylation, with concomitant enhanced sequestration of the Bcl-2 family proteins Mcl-1 and Bcl-xL. Consistent with a key role of Bim in regulating apoptosis in JAK2V617F mutant cells, depletion of the BH3-only protein by RNAi markedly suppressed JAK2 inhibitor-induced cell death. Vice versa, RNAi-mediated Mcl-1 depletion sensitized JAK2V617F mutant cells to JAK2 inhibition. Thus, further preclinical assessment of combinations of JAK2 inhibitors with Bcl-2 family antagonists in models of cMPNs is warranted and antagonizing Mcl-1, besides Bcl-xL, should be an integral part of such strategies.
The authors wish to thank Prof. Hans Drexler and Prof. Doris Morgan for the generous gift of SET-2 and MB-02 cell lines, respectively. The experimental advice from Dr. Débora Bonenfant and Dr. Johannes Voshol is greatly appreciated. Finally, we would like to thank Dr. Elisabeth Buchdunger and Dr. Francesco Hofmann for critical reading of the manuscript.
- Nelson ME, Steensma DP: JAK2 V617F in myeloid disorders: What do we know now, and where are we headed?. Leuk Lymphoma. 2006, 47: 177-194. 10.1080/10428190500301348.View ArticlePubMedGoogle Scholar
- Saharinen P, Vihinen M, Silvennoinen O: Autoinhibition of Jak2 Tyrosine Kinase Is Dependent on Specific Regions in Its Pseudokinase Domain. Mol Biol Cell. 2003, 14: 1448-1459. 10.1091/mbc.E02-06-0342.View ArticlePubMedPubMed CentralGoogle Scholar
- Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJP, Boggon TJ, Wlodarska I, Clark JJ, Moore S, et al: Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell. 2005, 7: 387-397. 10.1016/j.ccr.2005.03.023.View ArticlePubMedGoogle Scholar
- Vannucchi AM: How do JAK2-inhibitors work in myelofibrosis: An alternative hypothesis. Leukemia Res. 2009, 33: 1581-1583. 10.1016/j.leukres.2009.06.001.View ArticleGoogle Scholar
- Mesa R, Gale RP: Hypothesis: How do JAK2-inhibitors work in myelofibrosis. Leukemia Res. 2009, 33: 1156-1157. 10.1016/j.leukres.2009.04.011.View ArticleGoogle Scholar
- Jamieson CH, Gotlib J, Durocher JA, Chao MP, Mariappan MR, Lay M, Jones C, Zehnder JL, Lilleberg SL, Weissman IL: The JAK2 V617F mutation occurs in hematopoietic stem cells in polycythemia vera and predisposes toward erythroid differentiation. Proc Natl Acad Sci USA. 2006, 103: 6224-6229. 10.1073/pnas.0601462103.View ArticlePubMedPubMed CentralGoogle Scholar
- Laubach JP, Fu P, Jiang X, Salter KH, Potti A, Arcasoy MO: Polycythemia vera erythroid precursors exhibit increased proliferation and apoptosis resistance associated with abnormal RAS and PI3K pathway activation. Exp Hematol. 2009, 37: 1411-1422. 10.1016/j.exphem.2009.09.009.View ArticlePubMedPubMed CentralGoogle Scholar
- Silva M, Richard C, Benito A, Sanz C, Olalla I, Fernandez-Luna JL: Expression of Bcl-x in Erythroid Precursors from Patients with Polycythemia Vera. N Engl J Med. 1998, 338: 564-571. 10.1056/NEJM199802263380902.View ArticlePubMedGoogle Scholar
- Zeuner A, Pedini F, Francescangeli F, Signore M, Girelli G, Tafuri A, De Maria R: Activity of the BH3 mimetic ABT-737 on polycythemia vera erythroid precursor cells. Blood. 2009, 113: 1522-1525. 10.1182/blood-2008-03-143321.View ArticlePubMedGoogle Scholar
- Gozgit JM, Bebernitz G, Patil P, Ye M, Parmentier J, Wu J, Su N, Wang T, Ioannidis S, Davies A, et al: Effects of the JAK2 Inhibitor, AZ960, on Pim/BAD/BCL-xL Survival Signaling in the Human JAK2 V617F Cell Line SET-2. J Biol Chem. 2008, 283: 32334-32343. 10.1074/jbc.M803813200.View ArticlePubMedGoogle Scholar
- Adams JM, Cory S: The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene. 2007, 26: 1324-1337. 10.1038/sj.onc.1210220.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhang B, Gojo I, Fenton RG: Myeloid cell factor-1 is a critical survival factor for multiple myeloma. Blood. 2002, 99: 1885-1893. 10.1182/blood.V99.6.1885.View ArticlePubMedGoogle Scholar
- Kaufmann SH, Karp JE, Svingen PA, Krajewski S, Burke PJ, Gore SD, Reed JC: Elevated Expression of the Apoptotic Regulator Mcl-1 at the Time of Leukemic Relapse. Blood. 1998, 91: 991-1000.PubMedGoogle Scholar
- Pissot-Soldermann C, Gerspacher M, Furet P, Gaul C, Holzer P, McCarthy C, Radimerski T, Regnier CH, Baffert F, Drueckes P, et al: Discovery and SAR of potent, orally available 2,8-diaryl-quinoxalines as a new class of JAK2 inhibitors. Bioorg Med Chem Lett. 2010, 20: 2609-2613. 10.1016/j.bmcl.2010.02.056.View ArticlePubMedGoogle Scholar
- Gomez-Bougie P, Bataille R, Amiot M: The imbalance between Bim and Mcl-1 expression controls the survival of human myeloma cells. Eur J Immunol. 2004, 34: 3156-3164. 10.1002/eji.200424981.View ArticlePubMedGoogle Scholar
- Ewings KE, Hadfield-Moorhouse K, Wiggins CM, Wickenden JA, Balmanno K, Gilley R, Degenhardt K, White E, Cook SJ: ERK1/2-dependent phosphorylation of BimEL promotes its rapid dissociation from Mcl-1 and Bcl-xL. EMBO J. 2007, 26: 2856-2867. 10.1038/sj.emboj.7601723.View ArticlePubMedPubMed CentralGoogle Scholar
- Baffert F, Régnier CH, De Pover A, Pissot-Soldermann C, Tavares GA, Blasco F, Brueggen J, Chène P, Drueckes P, Erdmann D, et al: Potent and Selective Inhibition of Polycythemia by the Quinoxaline JAK2 Inhibitor NVP-BSK805. Mol Cancer Ther. 2010, 9: 1945-1955. 10.1158/1535-7163.MCT-10-0053.View ArticlePubMedGoogle Scholar
- Uozumi K, Otsuka M, Ohno N, Moriyama T, Suzuki S, Shimotakahara S, Matsumura I, Hanada S, Arima T: Establishment and characterization of a new human megakaryoblastic cell line (SET-2) that spontaneously matures to megakaryocytes and produces platelet-like particles. Leukemia. 2000, 14: 142-152. 10.1038/sj.leu.2401608.View ArticlePubMedGoogle Scholar
- Morgan DA, Gumucio DL, Brodsky I: Granulocyte-macrophage colony-stimulating factor-dependent growth and erythropoietin-induced differentiation of a human cell line MB-02. Blood. 1991, 78: 2860-2871.PubMedGoogle Scholar
- Adams JM: Ways of dying: multiple pathways to apoptosis. Genes Dev. 2003, 17: 2481-2495. 10.1101/gad.1126903.View ArticlePubMedGoogle Scholar
- Will B, Siddiqi T, Jorda MA, Shimamura T, Luptakova K, Staber PB, Costa DB, Steidl U, Tenen DG, Kobayashi S: Apoptosis induced by JAK2 inhibition is mediated by Bim and enhanced by the BH3 mimetic ABT-737 in JAK2 mutant human erythroid cells. Blood. 2010, 115: 2901-2909. 10.1182/blood-2009-03-209544.View ArticlePubMedPubMed CentralGoogle Scholar
- Chao J-R, Wang J-M, Lee S-F, Peng H-W, Lin Y-H, Chou C-H, Li J-C, Huang H-M, Chou C-K, Kuo M-L, et al: mcl-1 Is an Immediate-Early Gene Activated by the Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) Signaling Pathway and Is One Component of the GM-CSF Viability Response. Mol Cell Biol. 1998, 18: 4883-4898.View ArticlePubMedPubMed CentralGoogle Scholar
- Maurer U, Charvet C, Wagman AS, Dejardin E, Green DR: Glycogen Synthase Kinase-3 Regulates Mitochondrial Outer Membrane Permeabilization and Apoptosis by Destabilization of MCL-1. Mol Cell. 2006, 21: 749-760. 10.1016/j.molcel.2006.02.009.View ArticlePubMedGoogle Scholar
- Zhong Q, Gao W, Du F, Wang X: Mule/ARF-BP1, a BH3-Only E3 Ubiquitin Ligase, Catalyzes the Polyubiquitination of Mcl-1 and Regulates Apoptosis. Cell. 2005, 121: 1085-1095. 10.1016/j.cell.2005.06.009.View ArticlePubMedGoogle Scholar
- Moulding DA, Quayle JA, Hart CA, Edwards SW: Mcl-1 Expression in Human Neutrophils: Regulation by Cytokines and Correlation With Cell Survival. Blood. 1998, 92: 2495-2502.PubMedGoogle Scholar
- Harada H, Quearry B, Ruiz-Vela A, Korsmeyer SJ: Survival factor-induced extracellular signal-regulated kinase phosphorylates BIM, inhibiting its association with BAX and proapoptotic activity. Proc Natl Acad Sci USA. 2004, 101: 15313-15317. 10.1073/pnas.0406837101.View ArticlePubMedPubMed CentralGoogle Scholar
- Garcon L, Rivat C, James C, Lacout C, Camara-Clayette V, Ugo V, Lecluse Y, Bennaceur-Griscelli A, Vainchenker W: Constitutive activation of STAT5 and Bcl-xL overexpression can induce endogenous erythroid colony formation in human primary cells. Blood. 2006, 108: 1551-1554. 10.1182/blood-2005-10-009514.View ArticlePubMedGoogle Scholar
- Zhao R, Follows GA, Beer PA, Scott LM, Huntly BJP, Green AR, Alexander DR: Inhibition of the Bcl-xL Deamidation Pathway in Myeloproliferative Disorders. N Engl J Med. 2008, 359: 2778-2789. 10.1056/NEJMoa0804953.View ArticlePubMedGoogle Scholar
- Willis SN, Chen L, Dewson G, Wei A, Naik E, Fletcher JI, Adams JM, Huang DCS: Proapoptotic Bak is sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3-only proteins. Genes Dev. 2005, 19: 1294-1305. 10.1101/gad.1304105.View ArticlePubMedPubMed CentralGoogle Scholar
- Luciano F, Jacquel A, Colosetti P, Herrant M, Cagnol S, Pages G, Auberger P: Phosphorylation of Bim-EL by Erk1//2 on serine 69 promotes its degradation via the proteasome pathway and regulates its proapoptotic function. Oncogene. 2003, 22: 6785-6793. 10.1038/sj.onc.1206792.View ArticlePubMedGoogle Scholar
- Wiggins CM, Band H, Cook SJ: c-Cbl is not required for ERK1/2-dependent degradation of BimEL. Cell Signal. 2007, 19: 2605-2611. 10.1016/j.cellsig.2007.08.008.View ArticlePubMedPubMed CentralGoogle Scholar
- Nguyen M, Marcellus RC, Roulston A, Watson M, Serfass L, Murthy Madiraju SR, Goulet D, Viallet J, Bélec L, Billot X, et al: Small molecule obatoclax (GX15-070) antagonizes MCL-1 and overcomes MCL-1-mediated resistance to apoptosis. Proc Natl Acad Sci USA. 2007, 104: 19512-19517. 10.1073/pnas.0709443104.View ArticlePubMedPubMed CentralGoogle Scholar
- Tse C, Shoemaker AR, Adickes J, Anderson MG, Chen J, Jin S, Johnson EF, Marsh KC, Mitten MJ, Nimmer P, et al: ABT-263: A Potent and Orally Bioavailable Bcl-2 Family Inhibitor. Cancer Res. 2008, 68: 3421-3428. 10.1158/0008-5472.CAN-07-5836.View ArticlePubMedGoogle Scholar
- Bouillet P, Metcalf D, Huang DC, Tarlinton DM, Kay TW, Köntgen F, Adams JM, Strasser A: Proapoptotic Bcl-2 Relative Bim Required for Certain Apoptotic Responses, Leukocyte Homeostasis, and to Preclude Autoimmunity. Science. 1999, 286: 1735-1738. 10.1126/science.286.5445.1735.View ArticlePubMedGoogle Scholar
- Abutin RM, Chen J, Lung TK, Lloyd JA, Sawyer ST, Harada H: Erythropoietin-induced phosphorylation/degradation of BIM contributes to survival of erythroid cells. Exp Hematol. 2009, 37: 151-158. 10.1016/j.exphem.2008.10.008.View ArticlePubMedGoogle Scholar
- Dijkers PF, Medema RH, Lammers J-W, Koenderman L, Coffer PJ: Expression of the pro-apoptotic Bcl-2 family member Bim is regulated by the forkhead transcription factor FKHR-L1. Curr Biol. 2000, 10: 1201-1204. 10.1016/S0960-9822(00)00728-4.View ArticlePubMedGoogle Scholar
- Essafi A, Fernandez de Mattos S, Hassen YAM, Soeiro I, Mufti GJ, Thomas NSB, Medema RH, Lam EWF: Direct transcriptional regulation of Bim by FoxO3a mediates STI571-induced apoptosis in Bcr-Abl-expressing cells. Oncogene. 2005, 24: 2317-2329. 10.1038/sj.onc.1208421.View ArticlePubMedGoogle Scholar
- Maeda T, Ito K, Merghoub T, Poliseno L, Hobbs RM, Wang G, Dong L, Maeda M, Dore LC, Zelent A, et al: LRF Is an Essential Downstream Target of GATA1 in Erythroid Development and Regulates BIM-Dependent Apoptosis. Dev Cell. 2009, 17: 527-540. 10.1016/j.devcel.2009.09.005.View ArticlePubMedPubMed CentralGoogle Scholar
- Gélinas C, White E: BH3-only proteins in control: specificity regulates MCL-1 and BAK-mediated apoptosis. Genes Dev. 2005, 19: 1263-1268.View ArticlePubMedGoogle Scholar
- Opferman JT, Iwasaki H, Ong CC, Suh H, Mizuno S, Akashi K, Korsmeyer SJ: Obligate Role of Anti-Apoptotic MCL-1 in the Survival of Hematopoietic Stem Cells. Science. 2005, 307: 1101-1104. 10.1126/science.1106114.View ArticlePubMedGoogle Scholar
- Motoyama N, Wang F, Roth KA, Sawa H, Nakayama K, Nakayama K, Negishi I, Senju S, Zhang Q, Fujii S, Loh DY: Massive cell death of immature hematopoietic cells and neurons in Bcl-x-deficient mice. Science. 1995, 267: 1506-1510. 10.1126/science.7878471.View ArticlePubMedGoogle Scholar
- Wagner KU, Claudio E, Rucker EB, Riedlinger G, Broussard C, Schwartzberg PL, Siebenlist U, Hennighausen L: Conditional deletion of the Bcl-x gene from erythroid cells results in hemolytic anemia and profound splenomegaly. Development. 2000, 127: 4949-4958.PubMedGoogle Scholar
- Richmond TD, Chohan M, Barber DL: Turning cells red: signal transduction mediated by erythropoietin. Trends Cell Biol. 2005, 15: 146-155. 10.1016/j.tcb.2005.01.007.View ArticlePubMedGoogle Scholar
- Quelle FW, Wang J, Feng J, Wang D, Cleveland JL, Ihle JN, Zambetti GP: Cytokine rescue of p53-dependent apoptosis and cell cycle arrest is mediated by distinct Jak kinase signaling pathways. Genes Dev. 1998, 12: 1099-1107. 10.1101/gad.12.8.1099.View ArticlePubMedPubMed CentralGoogle Scholar
- Puthier D, Bataille R, Amiot M: IL-6 up-regulates Mcl-1 in human myeloma cells through JAK/STAT rather than Ras/MAP kinase pathway. Eur J Immunol. 1999, 29: 3945-3950. 10.1002/(SICI)1521-4141(199912)29:12<3945::AID-IMMU3945>3.0.CO;2-O.View ArticlePubMedGoogle Scholar
- Kuribara R, Honda H, Matsui H, Shinjyo T, Inukai T, Sugita K, Nakazawa S, Hirai H, Ozawa K, Inaba T: Roles of Bim in Apoptosis of Normal and Bcr-Abl-Expressing Hematopoietic Progenitors. Mol Cell Biol. 2004, 24: 6172-6183. 10.1128/MCB.24.14.6172-6183.2004.View ArticlePubMedPubMed CentralGoogle Scholar
- Aichberger KJ, Mayerhofer M, Krauth M-T, Vales A, Kondo R, Derdak S, Pickl WF, Selzer E, Deininger M, Druker BJ, et al: Low-Level Expression of Proapoptotic Bcl-2-Interacting Mediator in Leukemic Cells in Patients with Chronic Myeloid Leukemia: Role of BCR/ABL, Characterization of Underlying Signaling Pathways, and Reexpression by Novel Pharmacologic Compounds. Cancer Res. 2005, 65: 9436-9444. 10.1158/0008-5472.CAN-05-0972.View ArticlePubMedGoogle Scholar
- Kuroda J, Puthalakath H, Cragg MS, Kelly PN, Bouillet P, Huang DCS, Kimura S, Ottmann OG, Druker BJ, Villunger A, et al: Bim and Bad mediate imatinib-induced killing of Bcr/Abl+ leukemic cells, and resistance due to their loss is overcome by a BH3 mimetic. Proc Natl Acad Sci USA. 2006, 103: 14907-14912. 10.1073/pnas.0606176103.View ArticlePubMedPubMed CentralGoogle Scholar
- Nordigarden A, Kraft M, Eliasson P, Labi V, Lam EWF, Villunger A, Jonsson J-I: BH3-only protein Bim more critical than Puma in tyrosine kinase inhibitor-induced apoptosis of human leukemic cells and transduced hematopoietic progenitors carrying oncogenic FLT3. Blood. 2009, 113: 2302-2311. 10.1182/blood-2008-07-167023.View ArticlePubMedGoogle Scholar
- Dai Y, Grant S: Targeting Multiple Arms of the Apoptotic Regulatory Machinery. Cancer Res. 2007, 67: 2908-2911. 10.1158/0008-5472.CAN-07-0082.View ArticlePubMedGoogle Scholar
- Opferman JT, Letai A, Beard C, Sorcinelli MD, Ong CC, Korsmeyer SJ: Development and maintenance of B and T lymphocytes requires antiapoptotic MCL-1. Nature. 2003, 426: 671-676. 10.1038/nature02067.View ArticlePubMedGoogle Scholar
- James C, Ugo V, Le Couedic J-P, Staerk J, Delhommeau F, Lacout C, Garcon L, Raslova H, Berger R, Bennaceur-Griscelli A, et al: A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature. 2005, 434: 1144-1148. 10.1038/nature03546.View ArticlePubMedGoogle Scholar
- Wernig G, Kharas MG, Okabe R, Moore SA, Leeman DS, Cullen DE, Gozo M, McDowell EP, Levine RL, Doukas J, et al: Efficacy of TG101348, a Selective JAK2 Inhibitor, in Treatment of a Murine Model of JAK2V617F-Induced Polycythemia Vera. Cancer Cell. 2008, 13: 311-320. 10.1016/j.ccr.2008.02.009.View ArticlePubMedGoogle Scholar
- Pikman Y, Lee BH, Mercher T, McDowell E, Ebert BL, Gozo M, Cuker A, Wernig G, Moore S, Galinsky I, et al: MPLW515L Is a Novel Somatic Activating Mutation in Myelofibrosis with Myeloid Metaplasia. PLoS Med. 2006, 3: e270-10.1371/journal.pmed.0030270.View ArticlePubMedPubMed CentralGoogle Scholar
- Verstovsek S: Therapeutic Potential of Janus-activated Kinase-2 Inhibitors for the Management of Myelofibrosis. Clin Cancer Res. 2010, 16: 1988-1996. 10.1158/1078-0432.CCR-09-2836.View ArticlePubMedGoogle Scholar
- Lessene G, Czabotar PE, Colman PM: BCL-2 family antagonists for cancer therapy. Nat Rev Drug Discov. 2008, 7: 989-1000. 10.1038/nrd2658.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/24/prepub
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