Validation of ITD mutations in FLT3 as a therapeutic target in human acute myeloid leukaemia

Abstract

Effective targeted cancer therapeutic development depends upon distinguishing disease-associated 'driver' mutations, which have causative roles in malignancy pathogenesis, from 'passenger' mutations, which are dispensable for cancer initiation and maintenance. Translational studies of clinically active targeted therapeutics can definitively discriminate driver from passenger lesions and provide valuable insights into human cancer biology. Activating internal tandem duplication (ITD) mutations in FLT3 (FLT3-ITD) are detected in approximately 20% of acute myeloid leukaemia (AML) patients and are associated with a poor prognosis. Abundant scientific and clinical evidence, including the lack of convincing clinical activity of early FLT3 inhibitors, suggests that FLT3-ITD probably represents a passenger lesion. Here we report point mutations at three residues within the kinase domain of FLT3-ITD that confer substantial in vitro resistance to AC220 (quizartinib), an active investigational inhibitor of FLT3, KIT, PDGFRA, PDGFRB and RET; evolution of AC220-resistant substitutions at two of these amino acid positions was observed in eight of eight FLT3-ITD-positive AML patients with acquired resistance to AC220. Our findings demonstrate that FLT3-ITD can represent a driver lesion and valid therapeutic target in human AML. AC220-resistant FLT3 kinase domain mutants represent high-value targets for future FLT3 inhibitor development efforts.

Figure 1. Mutation screen of FLT3-ITD reveals secondary kinase domain mutations that cause varying degrees of resistance to AC220

a, Numbers of independent AC220-resistant Ba/F3 FLT3-ITD subpopulations with amino acid substitution at the indicated residue obtained from a saturation mutagenesis assay (n = 97 clones). b, Normalized cell viability of Ba/F3 populations stably expressing FLT3-ITD mutant isoforms after 48 h in various concentrations of AC220 (error bars represent standard deviations of triplicates from the same experiment). c, Western blot analysis using anti-phospho-FLT3 (pFLT3) or anti-FLT3 antibody performed on lysates from IL-3-independent Ba/F3 populations expressing the FLT3-ITD mutant isoforms indicated. Cells were exposed to AC220 at the indicated concentrations for 90 min.

Figure 2. Modelling of FLT3–AC220 interactions

a, Docking model of the AC220-bound FLT3 kinase domain: AC220 (blue); activation loop (orange) and DFG motif (green); amino acid residues that confer AC220 resistance when mutated (F691, D835 and Y842) and that interact with AC220 (F691, C694 and F830) in sticks. b, Surface and stick presentation of AC220 and AC220-interacting residues on FLT3: the carbonyl oxygen of C694 interacts with one of the AC220 amide groups; F691 forms a π–π stacking interaction with AC220; F830 interacts with AC220 through a perpendicular aromatic–aromatic interaction. c, Structure of the activation loop: residues D835,Y842 and interacting residues on FLT3 depicted in sticks.