Gilteritinib is a potent and selective FLT3 kinase inhibitor with single-agent 
clinical efficacy in relapsed/refractory FLT3-mutated acute myeloid leukemia 
(AML). In this context, however, gilteritinib is not curative, and response 
duration is limited by the development of secondary resistance. To evaluate 
resistance mechanisms, we analyzed baseline and progression samples from 
patients treated on clinical trials of gilteritinib. Targeted next-generation 
sequencing at the time of AML progression on gilteritinib identified 
treatment-emergent mutations that activate RAS/MAPK pathway signaling, most 
commonly in NRAS or KRAS. Less frequently, secondary FLT3-F691L gatekeeper 
mutations or BCR-ABL1 fusions were identified at progression. Single-cell 
targeted DNA sequencing revealed diverse patterns of clonal selection and 
evolution in response to FLT3 inhibition, including the emergence of RAS 
mutations in FLT3-mutated subclones, the expansion of alternative wild-type FLT3 
subclones, or both patterns simultaneously. These data illustrate dynamic and 
complex changes in clonal architecture underlying response and resistance to 
mutation-selective tyrosine kinase inhibitor therapy in AML. SIGNIFICANCE: 
Comprehensive serial genotyping of AML specimens from patients treated with the 
selective FLT3 inhibitor gilteritinib demonstrates that complex, heterogeneous 
patterns of clonal selection and evolution mediate clinical resistance to 
tyrosine kinase inhibition in FLT3-mutated AML. Our data support the development 
of combinatorial targeted therapeutic approaches for advanced AML.See related 
commentary by Wei and Roberts, p. 998.This article is highlighted in the In This 
Issue feature, p. 983.