Individualized Mitochondrial Functional Approach to Combination of BCL-2 and MCL-1 Antagonism in Acute Myeloid Leukemia

BCL-2 antagonist venetoclax combined with hypomethylating agents or low-dose cytarabine is a new standard of care for treatment-naive elderly or unfit AML patients. Despite the striking overall response of 70%, only 30% achieved MRD negative status with a short remission duration of 11.3 months. This highlights the need for new agents that can overcome venetoclax resistance. We exploited mitochondrial functional assay called BH3 profiling to identify predictors of clinical responses and to discover combination partners of venetoclax. BH3 profiling measures apoptotic priming of a cell (proximity to the apoptotic threshold) by measuring mitochondrial response to a standardized panel of pro-apoptotic BH3 oligopeptides. By using pretreatment myeloblasts of patients (N=16) from clinical trial of venetoclax and azacitidine, we found that response to HRK + MS1 peptides (infers BCL-XL and MCL-1 dependency, respectively) inversely correlates with the achievement of remission. The serial sampling on treatment revealed the emergence of differential patterns of anti-apoptotic dependency that correlated with patient response. Of note, mitochondrial sensitivity to the MS-1 peptide, an indication of MCL-1 dependence, increased from 0% at diagnosis to 40% at relapse, suggesting an opportunity for MCL-1 antagonism in the relapsed setting.

First, we exposed 6 AML cell lines with a BCL-2 (venetoclax) and MCL-1 antagonist (S63845). As previously reported, combination treatment resulted in synergistic cell line killing. Venetoclax treatment (within an hour) led to a dynamic increase in mitochondrial sensitivity to the MCL-1 selective MS-1 peptide, while S63845 caused increased mitochondrial susceptibility to the BCL-2 and BCL-XL selective BAD peptide. There was a correlation between % of priming caused by MS-1 and BAD peptide with Loewe synergy score (Spearman r=0.79, p<0.05). This suggests that BH3 profiling can provide a rapid pharmacodynamic marker of the activity of BH3 mimetics.

We next investigated combination MCL-1 and BCL-2 antagonism in vivo in venetoclax resistant AML PDXs. We transplanted NSG mice with human AML cells and started them on venetoclax (100mg/kg, PO) until leukemia relapsed (N=6 models). We subjected resistant myeloblasts to BH3 profiling and identified dependency on BCL-2 and MCL-1 to guide therapeutic choices. As a single agent, S63845 showed limited activity in venetoclax resistance models (p<0.01 vs. vehicle; N=2/4 models). We next tested combinations at 3 different dosing schedules where BH3 mimetics were given either in a simultaneous week, alternative weeks or switched based on leukemia burden. Mice receiving venetoclax and S63845 in the same week showed the greatest anti-leukemia effects (p<0.01 to p<0.0005; N=4/4 models). We found superior benefit of BCL-2 + MCL-1 combination antagonism over MCL-1 alone in venetoclax resistance settings, implies that resistant myeloblasts escape apoptosis by switching their anti-apoptotic dependencies.

Next, we applied the dynamic BH3 profiling technique that measures drug-induced changes in mitochondrial priming to discover additional agents that may overcome venetoclax resistance. We compared mitochondrial priming signals of 40 targeted agents in isolated parental and resistant cells. Although venetoclax resistant cells gained cross resistance to most of the agents, inhibitors of the FLT-3 pathway, including quizartinib, crenolanib, and gilteritinib retained similar priming responses as in parental cells. To validate this, we re-transplanted venetoclax resistant cells and found that quizartinib treatment maintained anti-leukemia efficacy compared to venetoclax or vehicle-treated mice (p<0.0001). In an unbiased whole transcriptome analysis, venetoclax resistant PDXs showed enrichment for JAK/STAT, RAS/MAPK, and PI3K/AKT/mTOR pathways, and corresponding upregulation in protein levels of p-FLT3, p-STAT5, p-MAPK, and p-AKT. In addition we identified that FLT3-ITD mutation measured via capillary electrophoresis was maintained in resistant clones while point mutation in BCL2 (BCL2G101V) was undetectable.

In conclusion, our study illustrates the power of mitochondrial measurements as a predictive biomarker for BH3 mimetic based therapy. We provide an evidence for the persistent activity of FLT-3 inhibitors in venetoclax resistance settings, a discovery made possible by dynamic BH3 profiling.