BCL-2 inhibition has been shown to be effective in acute myeloid leukemia (AML) in combination with hypomethylating agents or low-dose cytarabine. However, resistance and relapse represent major clinical challenges. Thus, there is an unmet need to overcome resistance to current venetoclax-based strategies. We performed high-throughput drug screening to identify effective combination partners for venetoclax in AML. Overall, 64 anti-leukemic drugs were screened in 31 primary high-risk AML samples with or without venetoclax. Gilteritinib exhibited highest synergy with venetoclax in FLT3 wildtype AML. The combination of gilteritinib and venetoclax increased apoptosis, reduced viability, and was active in venetoclax-azacitidine resistant cell lines and primary patient samples. Proteomics revealed increased FLT3 wildtype signaling in specimens with low in-vitro response to the currently used venetoclax-azacitidine combination. Mechanistically, venetoclax with gilteritinib decreased phosphorylation of ERK and GSK3B via combined AXL and FLT3 inhibition with subsequent suppression of the antiapoptotic protein MCL-1. MCL-1 downregulation was associated with increased MCL-1 phosphorylation of serine 159, decreased phosphorylation of threonine 161 and proteasomal degradation. Gilteritinib and venetoclax were active in a FLT3 wildtype AML PDX model with TP53 mutation and reduced leukemic burden in four FLT3 wildtype AML patients receiving venetoclax-gilteritinib off-label after developing refractory disease under venetoclax-azacitidine. In summary, our results suggest that combined inhibition of FLT3/AXL potentiates venetoclax response in FLT3-wildtype AML by inducing MCL-1 degradation. Thus, the venetoclax-gilteritinib combination merits testing as potentially active regimen in high-risk AML patients with FLT3 wildtype.
Inter-patient variability and the similarity of healthy and leukemic stem cells have impeded the characterization of leukemic stem cells (LSCs) in acute myeloid leukemia (AML), and their differentiation landscape. Here, we introduce CloneTracer, a novel method that adds clonal resolution to single-cell RNA-seq datasets. Applied to samples from 19 AML patients, CloneTracer revealed routes of leukemic differentiation. While residual healthy cells dominated the dormant stem cell compartment, active leukemic stem cells resembled their healthy counterpart and retained erythroid capacity. By contrast, downstream myeloid progenitors were highly aberrant and constituted the disease-defining compartment: Their gene expression and differentiation state determined both chemotherapy response and the leukemia's ability to differentiate to transcriptomically normal monocytes. Finally, we demonstrated the potential of CloneTracer to identify surface markers mis-regulated specifically in leukemic cells by intra-patient comparisons. Taken together, CloneTracer revealed a differentiation landscape that mimics its healthy counterpart and determines biology and therapy response in AML.
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