Abstract 3199: Diverse non-FLT3 molecular mechanisms of crenolanib resistance
Authors: Haijiao Zhang, Anna M. Schultz, Samantha Savage, Daniel Bottomly, Beth Wilmot, Shannon K. McWeeney, Christopher Eide, Hoang Ho, Yee L. Lam, Richard Sweat, Jaime Faulkner, Evan Lind, Jeffrey W. Tyner
Journal: Cancer Research (2017)

Abstract FMS-like tyrosine kinase 3 (FLT3) activating mutations are primary molecular targets for the treatment of acute myeloid leukemia (AML) due to high prevalence and unfavorable prognosis. Several type II FLT3 inhibitors have shown clinical benefits but acquisition of secondary FLT3 mutations was reported as a common mechanism of resistance. Previous studies showed that crenolanib, a type I FLT3 inhibitor, had clinical activity without acquisition of secondary FLT3 mutations. To identify the molecular mechanisms associated with crenolanib sensitivity and resistance, we performed exome sequencing on crenolanib treated patients with FLT3-mutant multiply relapsed or refractory AML. Baseline mutational analysis revealed distinct mutational profiles in patients with prior FLT3 inhibitor exposure, especially in the following pathways: NRAS, IDH1, WT1 and RUNX1. Further analysis of patients who had poor response to crenolanib showed mutations in other cell signaling genes such as NRAS and PTPN11. Variant allele frequency (VAF) analysis showed that these mutations sometimes occurred in subclones independent of the FLT3 mutation-bearing clone or sometimes were acquired by the FLT3 mutation-bearing clone. To characterize the influence of these mutations on crenolanib sensitivity, we transduced genes of interest into cell lines harboring FLT3 activating mutations and treated these cells with crenolanib at various concentrations. Significantly increased crenolanib IC50 and IC90 were observed in NRAS G12V MOLM14 and PTPN11 A72D/FLT3 D835 Ba/F3 cells relative to the respective control NRAS WT MOLM14 and PTPN11 WT/FLT3 D835 Ba/F3 cells. Notably, addition of trametinib restored crenolanib sensitivity and demonstrated synergistic cytotoxic effects on cells with FLT3 and NRAS or PTPN11 mutations. In addition, increases in TET2 nonsense/frameshift mutations were observed in patients who did not respond to crenolanib. VAF analysis demonstrated that TET2 mutations co-occurred with FLT3 mutations. We also observed that bone marrow cells from FLT3-ITD knock-in/TET2 knock-out mice are resistant to crenolanib at low concentrations, but remain sensitive to azacytidine at the same level as FLT3-ITD knock-in/TET2 WT cells. The remaining patients exhibited a diverse spectrum of secondary mutations associated with chromatin modifiers, cohesion, spliceosomes and transcription factors which mostly expanded during treatment, suggesting an elaborate genetic/epigenetic mechanism of resistance to crenolanib. Our data suggest that comprehensive sequencing should be carried out on patient samples prior to treatment to identify and pre-emptively target problematic clones. In addition, even with high VAF FLT3 mutations, although FLT3 inhibitor monotherapy provide some clinical benefit, combining agents targeting cooperative lesions will be imperative to eradicate both the dominant clone and resistant subclones and improve patient responses. Citation Format: Haijiao Zhang, Anna M. Schultz, Samantha Savage, Daniel Bottomly, Beth Wilmot, Shannon K. McWeeney, Christopher Eide, Hoang Ho, Yee L. Lam, Richard Sweat, Jaime Faulkner, Evan Lind, Jeffrey W. Tyner. Diverse non-FLT3 molecular mechanisms of crenolanib resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3199. doi:10.1158/1538-7445.AM2017-3199