Characterizing and Overriding the Structural Mechanism of the Quizartinib-Resistant FLT3 "Gatekeeper" F691L Mutation with PLX3397

Abstract

Tyrosine kinase domain mutations are a common cause of acquired clinical resistance to tyrosine kinase inhibitors (TKI) used to treat cancer, including the FLT3 inhibitor quizartinib. Mutation of kinase "gatekeeper" residues, which control access to an allosteric pocket adjacent to the ATP-binding site, has been frequently implicated in TKI resistance. The molecular underpinnings of gatekeeper mutation-mediated resistance are incompletely understood. We report the first cocrystal structure of FLT3 with the TKI quizartinib, which demonstrates that quizartinib binding relies on essential edge-to-face aromatic interactions with the gatekeeper F691 residue, and F830 within the highly conserved Asp-Phe-Gly motif in the activation loop. This reliance makes quizartinib critically vulnerable to gatekeeper and activation loop substitutions while minimizing the impact of mutations elsewhere. Moreover, we identify PLX3397, a novel FLT3 inhibitor that retains activity against the F691L mutant due to a binding mode that depends less vitally on specific interactions with the gatekeeper position.

Significance: We report the first cocrystal structure of FLT3 with a kinase inhibitor, elucidating the structural mechanism of resistance due to the gatekeeper F691L mutation. PLX3397 is a novel FLT3 inhibitor with in vitro activity against this mutation but is vulnerable to kinase domain mutations in the FLT3 activation loop.

Figure 1. Co-Crystal structure of quizartinib bound to FLT3 and inferred binding poses of other FLT3 Inhibitors

A. Overview of FLT3 kinase domain in complex with quizartinib. B. A close-up view of quizartinib inside FLT3 (colors correspond to the structural features indicated in a) highlighting key active site residues, water-mediated hinge interactions, anchoring of the urea group, and the intramolecular hydrogen bond within the morpholinoethoxy solubilizing group. C. Inferred binding pose of sorafenib using sorafenib-KDR co-structure (PDB:4ASD) as a template. D. A view of quizartinib bound to native FLT3. E. F691L creates steric clashes with quizartinib (middle ring and urea linker); F. F691L is incompatible with the urea linker of sorafenib. G. Inferred binding pose of ponatinib using ponatinib-KIT co-structure as a template (PDB:4U0I). H. The steric effect of F691L is better tolerated by ponatinib.

Figure 2. PLX3397 is an inhibitor with activity against the FLT3-ITD/F691L mutation

A. The chemical structure of PLX3397. B. PLX3397 inhibition of FLT3-ITD-driven human AML MV4;11 xenografts in nude mice. Mice were injected with MV4;11 cell suspension in 100 μL PBS + 100 μL Matrigel (5×10⁶ cells per mouse) in the lower left abdominal flank. Treatments (vehicle and PLX3397 at two dose levels, all given daily via oral gavage) started when the average tumor size reached 125 mm³. PLX3397 at 10mg/kg dose resulted in tumor stasis whereas PLX3397 at 30mg/kg dose caused tumor regression. Mean tumor sizes of each treatment group (+standard errors) are plotted over the course of 21-day dosing. C. Binding pose of PLX3397 in FLT3 inferred from its co-structure with CSF1R (PDB:4R7H). D. PLX3397 binding is minimally affected by F691L. E. Proliferation of Molm14, Molm14/F691L, MV4;11 and K562 cells after 48 hours in various concentrations of PLX3397 (error bars represent s.d. of triplicates from the same experiment). F. Western blot analysis for phosphotyrosine and total FLT3 performed after immunoprecipitation using anti-FLT3 antibody from lysates prepared from Molm14 and Molm14/F691L cells. Western Blot analysis using anti-phospho-S6, anti-phospho-STAT5, anti-S6 and anti-STAT5 antibody performed on whole cell lysates prepared from Molm14 and Molm14/F691L cells. Cells were exposed for 120 minutes to healthy control plasma with the indicated concentrations of PLX3397. Data shown represents a single experiment.

Figure 3. Mutation Screen of FLT3-ITD and FLT3-ITD/F691L Reveals Kinase Domain Mutations that Cause Resistance to PLX3397

A. Numbers of independently-derived PLX3397-resistant Ba/F3/FLT3-ITD subpopulations with amino acid substitution at the indicated residue obtained from a saturation mutagenesis assay for FLT3-ITD and B. FLT3-ITD/F691L. C. IC₅₀s for proliferation of Ba/F3 cells expressing PLX3397-resistant mutants in FLT3-ITD and FLT3-ITD/F691L backgrounds. Bars with slanted lines indicate mutations observed only in clones with the ITD+F691L background. Error bars represent s.d. of 3 or more independent experiments. D. Western blot analysis using anti-phospho-FLT3, anti-phospho-STAT5, anti-FLT3 and anti-STAT5 antibody performed on lysates from IL-3-independent Ba/F3 populations expressing the FLT3-ITD mutant isoforms indicated. Cells were exposed to PLX3397 at the indicated concentrations for 90 minutes.

Figure 4. Structural mapping of the PLX3397-resistant mutations in FLT3

A. The residues for which mutations have been identified from the in vitro saturation mutagenesis screen are represented as spheres (colors correspond to the structural features indicated in Figure 1a). Details of the mutations can be found in Figure 3 and Supplemental Table 4. No mutation makes a direct contact with the inhibitor. B. A majority of the mutations were found in the activation loop (yellow, depicted in the PLX3397-bound, inactive state). The red ribbon traces the extended conformation of activation loop during active state (modeled using the coordinates of active KIT structure, PDB:1PKG). The proximity of M664 suggests a role in biasing the activation loop conformation. C. Close-up view of the hydrogen bond network formed by N676, H671, E692 and K826 at the back of the ATP-binding pocket. N676S disengages this network, thus releasing a molecular brake that keeps the enzyme in an inactive state(30). D. Close-up view of D698 and its role in nucleating the structure between the end of hinge and the beginning of the αD helix. The long-range structural effect of D698N on state of the enzyme remains unknown.

Figure 5. Plasma Inhibitory Assay Shows PLX3397 is Active Against FLT3-ITD/F691L at Clinically Achievable Plasma Concentrations

Western blot analysis for phosphotyrosine and total FLT3 performed after immunoprecipitation using anti-FLT3 antibody on lysates prepared from parental Molm14 cells and Molm14 cells expressing the FLT3-ITD/F691L mutation. Cells were exposed for 120 minutes to pre-treatment (PRE) and steady-state plasma from cycle 2 day 1 (C2D1) obtained from 2 patients (7.08 and 7.11) treated with PLX3397 at the phase 2 dose of 3000mg daily. Data shown represents a single experiment.