Bulk sequencing of relapsed tumors reveals mutations associated with resistance to cancer therapy but is insufficient to fully assess all causes of relapse. Due to inherent tumor heterogeneity, on-treatment tumor evolution may select for genetically distinct clones or shifts in malignant transcriptional states not resolvable by bulk sequencing. We performed multiomic single cell (SC) DNA/protein and RNA/protein profiling of a clinical trial cohort of acute myeloid leukemia (AML) patients treated on the Phase 1b clinical trial of the BCL2 inhibitor venetoclax and the FLT3 inhibitor gilteritinib (Ven/Gilt) to characterize immunophenotypic, transcriptional, and genetic clonal evolution driving resistance. We found that while Ven/Gilt effectively eliminated FLT3 mutant clones, resistance was associated with RAS activation via multiple mechanisms including selection for RAS mutant clones, non-mutational upregulation of RAS transcriptional programs and a shift to RAS-associated monocytic AML differentiation. In an in vitro model of monocytic differentiation associated with non-mutational RAS transcriptional activation, we demonstrated that RAS pathway inhibition re-sensitized to Ven/Gilt. These data illustrate that convergent resistance pathways in patients can be activated via diverse genetic and non-genetic mechanisms. These results underscore that RAS signaling is central to FLT3 and BCL2 inhibitor resistance, is tightly coupled to AML monocytic differentiation and highlight RAS pathway inhibition as a viable clinical strategy to combat resistance. CT# NCT03625505
Kennedy et al. (Mon,) studied this question.