Dynamic genetic and nongenetic RAS pathway activation drives resistance to FLT3 and BCL2 inhibitor therapy

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