A Mixed Type 1 and Type 2 Kinase Inhibitor That Overrides FLT3 F691 and D835 Resistance Mutations

Introduction: Mutations in FLT3, a class III receptor tyrosine kinase predominantly expressed on hematopoietic progenitor cells, represent the most common genetic alteration in patients with acute myeloid leukemia (AML). Approximately 25% of patients with AML harbor internal tandem duplications (ITD) within the juxtamembrane domain of FLT3. Quizartinib, the first selective FLT3 tyrosine kinase inhibitor (TKI) to show clinical activity, frequently achieves a composite complete remissions (CRc) rate of approximately 50% in relapsed and refractory FLT3-ITD⁺AML patients. However, patient samples obtained at the time of relapse with quizartinib revealed resistance-conferring FLT3 mutations at two kinase domain residues (F691L and D835V/Y/F). The crystal structure of the FLT3-quizartinib complex indicates that quizartinib binding relies on essential edge-to-face aromatic interactions with the gatekeeper F691 residue, and with F830 within the DFG motif in the activation loop (A-loop). This reliance makes quizartinib responses critically vulnerable to gatekeeper (F691) and A-loop (D835) substitutions. This has led to the identification of TKIs that have activity against quizartinib-resistant FLT3 mutants, including type 1 inhibitors (e.g. crenolanib, ASP2215) targeting specifically D835 mutants, and type 2 inhibitors (e.g. pexidartinib, ponatinib) active against F691L. Until now, no FLT3 inhibitor has demonstrated potent inhibition of both classes of resistance mutations.

Methods: To identify FLT3 inhibitors with activity against both gatekeeper and A-loop mutants, we used a modular approach to systematically alter the constituents of quizartinib to optimize interactions with the mutant proteins guided by X-ray structures and modeling. The compounds were profiled through a panel of in vitro growth assays and in vivo studies using Ba/F3 cells engineered to express FLT3-ITD/F691L and FLT3-ITD/D835Y, and MOLM14 quizartinib-resistant clones (MOLM14/F691L and MOLM14/D835Y) that have acquired the same mutations after chronic exposure to quizartinib. When injected into the tail vein of nude mice, the engineered Ba/F3 cells home to the spleen and proliferate, causing marked splenomegaly, whereas MOLM14 parental cells and MOLM14 resistant clones significantly reduce the overall survival of mice. Ba/F3-induced splenomegaly is directly dependent on the activity of the expressed mutant FLT3 and inhibition of splenomegaly measures target engagement in vivo. Analysis of efficacy in the MOLM14 survival models was done by Kaplan-Meier estimates and the logrank statistic.

Results: Structure-guided lead optimization generated a series of FLT3 inhibitors with a mixed type 1 and type 2 binding mode and equal potency against the two classes of resistant mutants. Further optimization to improve pharmaceutical properties and maximize in vivo efficacy led to development candidate PLX3623. PLX3623 potently inhibited the growth of two Ba/F3 cell lines expressing F691L and D835Y mutants, with IC₅₀ values of 0.18 (±0.04) and 0.15 (±0.03) nM, respectively. PLX3623 had a comparable antiproliferative effect on the MOLM14 resistant clones, with IC₅₀values of 0.52 (±0.14) and 0.26 (±0.08) nM for the F691L and D835Y clones, respectively. In vivo PLX3623 treatment resulted in dose-dependent reduction in Ba/F3-induced splenomegaly, achieving >90% inhibition at 3 mg/kg dose. PLX3623 at 3mg/kg increased the length of survival of MOLM14 parental cell recipients by 120%, compared to the 80% increase afforded by 3 mg/kg quizartinib. At 3mg/kg dose, PLX3623 extended the survival of mice that received the two MOLM14 resistant clones by 80%; quizartinib at the same dose provided no survival benefit.

Conclusion: Clonal evolution and tumor heterogeneity present a major challenge to targeted therapy and personalized cancer medicine. While acquired mutations individually may be actionable targets, often multiple inhibitors are required to combat the clonal diversity in a refractory setting. PLX3623, a FLT3 inhibitor with a mixed type 1 and type 2 binding mode, is the first TKI that is active against mutations conferring resistance through independent structural mechanisms. The in vitro and in vivo potency and favorable nonclinical safety profile of PLX3623 support its clinical development as a potentially best-in-class therapy to overcome clinical resistance to first generation FLT3 inhibitors.