Clonal Selection with RAS Pathway Activation Mediates Secondary Clinical Resistance to Selective FLT3 Inhibition in Acute Myeloid Leukemia

Abstract

Gilteritinib is a potent and selective FLT3 kinase inhibitor with single-agent clinical effi cacy 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 identifi ed 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 identifi ed 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.

Gilteritinib is a clinically active FLT3 inhibitor with broad activity against FLT3 kinase domain mutations

“…In a recent report from our group, treatment-emergent Ras/MAPK pathway mutations were acquired in 15 of 41 (36.6%) patients treated with gilteritinib, including activating mutations in NRAS (13/41 patients; 31.7%), KRAS (3/41 patients; 7.3%), PTPN11 (3/41 patients; 7.3%), CBL (2/41 patients; 4.9%), and BRAF (1/41 patients; 2.4%). 37 We also identified 2 patients with new BCR-ABL1 fusions, consistent with prior reports. 37,38 These results suggest that acquisition of new or parallel oncogenic driver mutations will be common, as has been reported with crenolanib.…”

“…37 We also identified 2 patients with new BCR-ABL1 fusions, consistent with prior reports. 37,38 These results suggest that acquisition of new or parallel oncogenic driver mutations will be common, as has been reported with crenolanib. 32,37 Ultimately, although gilteritinib has activity against a wide variety of FLT3 mutations and appears less clinically vulnerable to on-target resistance, rational combination therapies will be required to suppress outgrowth of heterogeneous resistant leukemic clones.…”

“…32 This finding is congruent with results of our study and reinforces that type I inhibitors may be overall less susceptible to on-target resistance, as is suggested by prior mutagenesis studies which often identify less resistance mutations associated with type I inhibitors 17,33 than type II inhibitors 12,15,25,26 (supplemental Table 2) as well as by our recent report of non-FLT3 dependent gilteritinib resistance mechanisms. 37 The increased vulnerability of type II inhibitors to TKD mutations is likely attributable to the fact that type II inhibitors bind to allosteric sites in the inactive kinase conformation. Type II inhibitors are therefore susceptible both to mutations which change kinase conformation as well as mutations at drug contact sites.…”

Single-cell mutational profiling enhances the clinical evaluation of AML MRD

“…Cell capture rates were between 5% and 10%; however, the microdroplet cell capture process is not selective or size dependent, and should not introduce biases in resolving the underlying clonal architecture. 17 The limit of detection of the platform is 0.1%, which has been reported with cell line spike-in experiments prepared at different ratios. 13 Baseline patient characteristics are provided in Table 1, and additional clinical information is provided in supplemental Table 2.…”

“…9 The lower limit of detection by SCS was similar to published results using the Tapestri platform. 17 We have previously reported on the heterogeneity of relapsed AML, showing multiple distinct patterns of clonal evolution, including relapse with the predominant clone from diagnosis, with a minor subclone from diagnosis, or with further clonal evolution. 18 Individual cases from the current cohort also display diverse patterns of clonal evolution with similar patterns as our initial findings.…”