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L. Han et al.
data in four cell lines representing different response pat- terns (Table 1, Figure 3C and Online Supplementary Figure S5C). Suppression of pERK by cobimetinib was observed in both sensitive (OCI-AML3 and MV4;11) and resistant (MOLM13 and U937) cells at 0.01 mM, irrespective of response patterns. pS6 (Ser235/236 or Ser240/244) was inhibited by a low dose (0.1 mM) of cobimetinib in sensi- tive OCI-AML3 cells but not in resistant U937 cells (Figure 3C), consistent with our RPPA findings noted above. These data also indicate direct suppression of mTORC1 signaling by cobimetinib as the Ser240/244 site is regulat- ed exclusively by mTORC1. MYC was downregulated by cobimetinib alone or in combination with venetoclax in OCI-AML3, but not in U937 cells. Cell death character- ized by elevated levels of cleaved PARP was observed in the combination group in OCI-AML3 cells, consistent with pro-apoptotic synergy (Figure 3C). To capture the dynamic interactions of pro- and anti-apoptotic BCL2 family members, a Meso Scale Discovery assay was per- formed in OCI-AML3 cells (Figure 3D). High basal levels of BCL2 and BCL2:BIM complexes were identified. After venetoclax treatment, BCL2:BIM complexes were disrupt- ed and MCL1 protein levels were upregulated, resulting in increased MCL1:BIM complexes. The combination of cobimetinib with venetoclax suppressed both BCL2:BIM and MCL1:BIM complexes, enabling release of free BIM to induce cell death (Figure 3D). In addition, cobimetinib treatment induced total BIM protein levels in MV4;11 cells, thereby priming the cells for death (Online Supplementary Figure S7).
To refine our search for potential biomarkers correlating with response to the venetoclax-cobimetinib combina- tion, we performed RNA sequencing and evaluated differ- ential gene expression after exposure to the drugs. The aim was to identify hallmark cancer pathways significant- ly altered specifically in cells sensitive to the drug combi- nation (Figure 3E and Online Supplementary Table S2). Our analysis demonstrated that several downstream path- ways, including MYC, E2F and their target genes, were significantly altered after treatment in cells that responded synergistically to the combination. Consistent with west- ern blot data, mTORC1 signaling was also altered in cells showing synergistic responses. Hypoxia and unfolded protein response (UPR) pathways were also significantly enriched, possibly downstream of mTOR/4EBP1/eIF4E signaling, which directs protein synthesis of HIF-1α,28 and can trigger the UPR.29 Glycolysis, another enriched path- way, is regulated by the ERK signaling pathway through RNK126-mediated ubiquitination of pyruvate dehydroge- nase kinase, which may account for resistance to apopto- sis.30
The combination of cobimetinib and venetoclax reduces leukemia burden in acute myeloid leukemia models in vivo
To test the efficacy of cobimetinib and venetoclax in vivo, we induced leukemia in NSGS mice by injecting the animals with genetically engineered OCI-AML3/Luc/GFP cells. Leukemia engraftment was confirmed 1 week after injection using BLI. Mice were randomly distributed into four arms and dosed orally with vehicle, cobimetinib (10 mg/kg), venetoclax (100 mg/kg), or cobimetnib plus vene- toclax daily for 28 days. BLI demonstrated that the leukemia burden was significantly reduced in treated groups compared to controls over time (Figure 4A). At
week 5, the tumor reduction was significantly greater in the groups that received single-agent cobimetinib (P<0.001) or cobimetnib plus venetoclax (P<0.001) than in the control group (Figure 4B). The tumor reduction was greater following combination treatment than following venetoclax (P<0.05) or cobimetinib (P<0.05) alone. All drug treatments, including combinations, were tolerated in vivo based on minimal changes in body weights (data not shown).
As a second AML cell line-derived xenograft model, we introduced genetically engineered MOLM3/Luc/GFP cells into NSGS mice and initiated treatment as for the OCI- AML3/Luc/GFP model. Again, BLI demonstrated signifi- cantly reduced leukemia burden in the treated groups compared to controls and the reduction was more pro- nounced in the groups treated with single-agent veneto- clax or cobimetinib plus venetoclax (Figure 4C, D). Additionally, human CD45 engraftment and cell counts in both bone marrow and spleen demonstrated a trend toward decreased tumor burden in mice treated with the drug combination compared to that in mice treated with either agent alone (Online Supplementary Figure S8A, B). As in the OCI-AML3/Luc xenograft model, all drug treat- ments were tolerated based on minimal changes in body weights. We performed additional PDX studies in NSG mice using an AML PDX generated from primary sample AML11 (Table 2). The mice were treated with the same doses of drugs as those used in the cell line models. As shown in Online Supplementary Figure S9, the combination therapy extended survival in the AML11 PDX model. These data demonstrate that the combination of cobime- tinib plus venetoclax potently suppresses leukemia burden in tumor-bearing mice in vivo at tolerable doses.
Discussion
Although gain-of-function mutations often represent secondary events in the pathogenesis of AML,31,32 they are required for AML maintenance and are therefore attrac- tive therapeutic targets.33 While MEK inhibitors have demonstrated limited activity in AML as single agents,15,34 preclinical studies with first generation MEK and BCL2 inhibitors demonstrated synergistic induction of apoptosis by suppression of MCL1 following MEK inhibition.11,35
In this study, five of the 11 AML cell lines tested were sensitive to cobimetinib, including two that harbored a FLT3-ITD mutation (MOLM14 and MV4;11) and one with an NRAS mutation (OCI-AML3). Consistent with previ- ous reports, the baseline levels of ERK phosphorylation did not correlate with response to cobimetinib.21,36 Venetoclax as a single agent had activity in five of the cell lines tested while the combination with cobimetinib was synergistic in seven of the cell lines, including those that were resistant to each agent alone.
To extend our preliminary findings in cell lines, we stud- ied a selection of genetically diverse primary AML patients’ samples. Venetoclax induced pronounced apop- tosis (>50%) in only three of the samples (16.7%), a rate similar to that of clinical responses to venetoclax monotherapy (19%),10 possibly reflecting protective prop- erties of the tumor microenvironment, as our culture con- ditions were cytokine-rich. Cobimetinib induced very lim- ited cell death in all AML samples, consistent with previ- ous reports that MEK inhibitors preferentially suppress
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haematologica | 2020; 105(3)