Multi-Omic Single-Cell Sequencing Reveals Genetic and Immunophenotypic Clonal Selection in Patients With FLT3-Mutated AML Treated With Gilteritinib/Venetoclax

Background: The combination of the FLT3 inhibitor gilteritinib with the BCL-2 inhibitor venetoclax (VenGilt) is highly efficacious in relapsed/refractory (R/R) FLT3-mutated acute myeloid leukemia (AML). In a recent phase 1b trial, VenGilt demonstrated a modified complete response rate of 75% (Daver et al, J Clin Oncol. 2022; published online). However, many patients (pts) relapsed after VenGilt. Proposed resistance mechanisms include RAS pathway mutations (Ven and Gilt) and monocytic immunophenotype (Ven). Using single cell (SC) multi-omic profiling, we sought to identify drivers of resistance to this combination. Methods: We analyzed 35 serial samples from 12 pts with R/R AML treated with VenGilt on the phase 1b trial (NCT03625505). We performed high-throughput SC DNA sequencing (Mission Bio Tapestri) on all samples and concurrent multi-omic SC cell-surface immunophenotyping on 18 samples. Results: We analyzed 107,528 cells (median 8960 cells/pt) from 12 pts with 2-5 serial timepoints. Eight pts had previously received tyrosine kinase inhibitors (TKI). Nine pts had clinical responses to VenGilt with a median relapse-free time of 8.3 mo. Of these 9 pts, 3 proceeded to transplant. Each pt had ≥1 genetically distinct clone identified (median 4.5, range 2-6). Fourteen FLT3mut clones were detected, with 2 pts having 2 separate FLT3mut clones. In all 12 pts, the FLT3mut clones were reduced with VenGilt. In 7 pts with a total of 9 clones, the FLT3mut population decreased to an undetectable level. Three pts had FLT3-IDH1/2 co-mutated clones, and the double mutant clones were reduced to undetectable levels in all 3. One pt developed a dominant IDH2mut clone at relapse without a FLT3 co-mutation. Nine pts demonstrated an initial decrease in non-FLT3mut clones, including clones with DNMT3A, NPM1, IDH1/2, TET2, EZH2, WT1, and NRAS mutations, suggesting some activity of VenGilt against non-FLT3mut populations. In 3 pts, all clones aside from those associated with clonal hematopoiesis of indeterminant potential (CHIP) were undetectable at follow-up. Five pts had ≥1 pre-existing NRAS- or KRAS-mutated clone. In 4/5 pts, the RASmutclone expanded on therapy and was dominant at relapse in 3/4 pts, with the fourth pt having a dominant IDH2mut clone. All 4 pts with expanding RASmutclones had prior TKI, while the pt whose RASmutclone decreased did not. Pre-existing RAS populations were small (0.001-18.1% of total cells) and were undetectable by bulk sequencing in 2/4 cases. RAS was not co-mutated with FLT3 in the same single cell in any cases. In 2 pts, FLT3mut clones present before VenGilt initially decreased but subsequently expanded on therapy. No de novo mutations within resistant/expanding FLT3mut clones were detected. We demonstrated immunophenotypic evolution for individual genotypes, including both FLT3mut and FLT3WT clones. For 5 pts with serial multi-omic SC samples, increased CD11b and CD13 expression was observed over time on a single cell basis in ≥1 genotype within individuals, suggesting increased monocytic-like features. These features were observed among 4 RASmut, 2 FLT3mut, and 1 TET2mut clones. At baseline, these 5 pts had heterogenous immunophenotypic profiles, with genetic clones occupying monocytic, immature, and mixed phenotypes; however, all 5 pts demonstrated ≥1 clone with increased monocytic immunophenotypic features after VenGilt treatment. Conclusion: Multi-omic SC sequencing enabled direct measurement of genetic and immunophenotypic clonal architecture and evolution with VenGilt treatment. VenGilt was highly effective against FLT3-mutated clones, even in the presence of co-mutations. Genetic resistance was associated with outgrowth of pre-existing subclones, primarily RAS mutations, rather than acquistion of de novo mutations. This differs from prior studies of Gilt monotherapy, in which both outgrowth of parallel clones and secondary mutations within the FLT3-mutated clone were associated with resistance. These data suggest the addition of Ven may suppress secondary resistance mutations, possibly due to enhanced apoptosis and cytotoxicity against FLT3-mutated cells. Multiple clones, predominatly those with a RAS mutation, developed monocytic features under VenGilt pressure. It is unknown whether monocytic immunophenotype is an independent resistance mechanism or a bystander to other mechanisms, such as increased MCL1 or RAS pathway gene expression. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal