Page 192 - Haematologica March 2020

L. Han et al.
of AML progenitors, while normal progenitors were only minimally affected.
CyTOF has proven to be a powerful approach for iden- tifying functional proteins in diverse cell populations at single-cell levels.39 Several groups, including ours, have studied the feasibility of CyTOF in AML.26,40,41 In this study, we utilized CyTOF combined with SPADE soft- ware42 to investigate the efficacy of cobimetinib and vene- toclax in two primary patients’ samples: one responder and one non-responder. BCL2 was highly expressed in CD34+ stem/progenitor cells compared to the CD34– cells, underlying the critical need for BCL2 inhibition to elimi- nate LSC. The venetoclax-sensitive sample displayed a higher level of BCL2 protein than the resistant sample. Cobimetinib inhibited G-CSF-induced pERK irrespective of response status. In line with several studies reporting that suppression of mTORC1 and its downstream path- ways (specifically S6) predicted sensitivity to MEK inhibi- tion,21,22 our data also demonstrated that the pS6 signaling pathway was suppressed in the cobimetinib-responding sample, suggesting that S6 phosphorylation may be a more predictive pharmacodynamic marker for MEK inhi- bition. However, the latter requires validation in a larger cohort of samples from patients.
The distinct response patterns in AML cell lines and patients’ samples led us to search for additional pharmaco- dynamic markers correlating with drug responses using proteomic and transcriptomic profiling. In line with the findings of an extensive study of MEK inhibition,20 we observed bypass induction of pMEK signaling upon MEK inhibition, which was more pronounced in cobimetinib- resistant cell lines. Several signaling pathways were highly activated in cobimetinib-sensitive cell lines, including pS6, pERK, p38MAPK, and pPTEN. Lauchle and colleagues demonstrated that leukemia clones with pre-existing resistance to MEK inhibition displayed reduced p38 kinase activity and increased RasGRP1 levels.13 It was also previ- ously reported that the RSK signaling pathway, which is downstream of MAPK, regulates an mTOR-independent pathway to induce S6 phosphorylation.43 Western blotting analysis performed to validate the RPPA data showed that S6 phosphorylation at both Ser235/236 and Ser240/244 sites was markedly suppressed in cobimetinib-sensitive OCI-AML3 and MV4-11 cells. In OCI-AML3 cells, the combination treatment resulted in significant cell death characterized by elevated levels of cleaved PARP, which could be attributed to disruption of BCL2:BIM complexes, releasing BIM to trigger apoptosis. We also observed BIM induction in MV4-11 cells, underscoring its critical role in the efficacy of the combination of BCL2 and MEK inhibitors.37,38,44 Although RPPA data showed no modula- tion of MCL1 after cobimetinib treatment, both western blot and Meso Scale Discovery assays showed downregu- lation of MCL1 in OCL-AML3 cells and upregulation of MCL1 after venetoclax treatment. These data suggest that
increased MCL1 levels induced by venetoclax favor the formation of MCL1:BIM complexes were disrupted, free- ing BIM to initiate apoptosis. Consistent with these find- ings, we recently showed that MCL-1 degradation associ- ated with MDM2 inhibition occurs through MEK/ERK suppression and GSK3 activation.45 The downregulation of MYC levels by cobimetinib also suggests a MEK/ERK- GSK3β link, as ubiquitination and degradation of MYC requires phosphorylation at T58 by GSK3β.46 Furthermore, RNA sequencing analyses revealed enhanced expression of MYC and E2F target genes in cells demonstrating a syner- gistic response to the cobimetinib-venetoclax combination. This finding is consistent with a previous report that MEK inhibition sensitized cells to ABT-263-induced apoptosis by promoting a G1 cell cycle arrest.37 Glycolysis and oxida- tive phosphorylation (OXPHOS) are known to be regulat- ed by ERK signaling through RNK126-mediated ubiquiti- nation of pyruvate dehydrogenase kinase.30 Potent anti- tumor efficacy has been demonstrated in melanoma cells through combined inhibition of BCL2, OXPHOS and MAPK signaling.47 Alterations in p53 and UPR pathways identified by transcriptome analysis may also account for synergy between MEK and BCL2 inhibition.48 These pro- posed mechanisms of actions are summarized in Online Supplementary Figure S10. However these models require further validation in controlled mechanistic studies.
The potency of the cobimetinib and venetoclax combi- nation was further demonstrated in vivo using models established with OCI-AML3 (resistant to venetoclax) and MOLM13 (resistant to cobimetinib) leukemia cells. Although we observed strong synergistic effects in both cell lines in vitro, the combination did not confer significant survival benefits in the in vivo models. This may be due to protection against cell death provided by the microenvi- ronment, as we have observed in patients’ samples cul- tured in cytokine-rich medium. Similar to our in vitro obser- vations, the OCI-AML3 xenograft model is hypersensitive to cobimetinib, and we found no significant survival differ- ences between animals that received single-agent cobime- tinib and those that received the combination. In the very aggressive MOLM13 model, in which untreated mice die 3 weeks after cell injection, the combination reduced but did not eliminate leukemia burden markedly on day 17.
In summary, combinatorial blockade of the MAPK and BCL2 pathways promotes cell death and suppresses pro- liferation in the majority of primary AML cells. This anti- leukemia efficacy is associated with the simultaneous inhibition of BCL2 by venetoclax and the downregulation of MCL1 mediated by cobimetinib, which together enable the release of the pro-death protein BIM. These preclinical data provided a strong mechanistic rationale for evaluat- ing the combination of cobimetinib with venetoclax in a phase I trial now enrolling elderly patients with relapsed/refractory AML (NCT02670044), and initial data have included objective clinical responses.49
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