Novel potent and selective inhibitors targeting FLT3 for AML therapy.

Abstract

6542 Background: Acute Myeloid Leukemia (AML) is a malignancy frequently driven by mutations in the FMS-like tyrosine kinase 3 (FLT3) gene. The FLT3 internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations, particularly D835 and F691, appear in approximately 30% of AML patients, often leading to poor prognosis and resistance to existing therapies. Gilteritinib and Quizartinib are two FDA-approved FLT3 inhibitors, with the former approved only for relapsed/refractory AML and the latter approved only for newly diagnosed AML. Quizartinib does not target TKD resistance mutations, whereas Gilteritinib’s efficacy on FLT3-ITD-D835Y is limited and it is not effective against FLT3-ITD-F691L. Consequently, there is a critical need for next-generation FLT3 inhibitors that can address all of these mutations. Methods: We have characterized efficacy of two novel FLT3 inhibitors, CCM-405 and CCM-445. In vitro enzymatic binding affinities were determined by the KdELECT assay, and cellular IC 50 s were determined by the Cell-Titer Glo assay. In vivo antitumor activity of CCM-405 / 445 was evaluated in mutant cell line-derived xenograft (CDX) models of AML. Tumor growth inhibition (TGI) was measured in the FLT3-ITD luciferase-expressing MV4-11 (MV4-11-luc) systemic xenograft model as well as subcutaneous xenograft models of FLT3-ITD F691L and D835Y mutants. Efficacy of novel inhibitors was compared with Gilteritinib. In vitro efficacy was compared with experimental FLT3 TKD mutant inhibitor Luxeptinib. Results: Enzymatically, CCM-405 / 445 inhibit FLT3-ITD, FLT3-ITD-D835V and FLT3-ITD-F691L with K d s of 12 nM / 4.1 nM, 1.9 nM / 0.39 nM and 1.6 nM / 0.4 nM, respectively (Luxeptinib K d s: ITD-D835V: 550 nM; ITD-F691L: 97 nM). CCM-405 / 445 inhibit the proliferation of Ba/F3 FLT3-ITD and FLT3-ITD D835Y cell lines with potency comparable to Gilteritinib, and FLT3-ITD F691L with potency superior to Gilteritinib, and are significantly less toxic to Ba/F3 FLT3 WT than to the mutants (Luxeptinib: negligible cellular mutant/WT selectivity). CCM-405 / 445 are also potent against human AML cell lines MV4-11 and MOLM-13. In vivo , in the systemic FLT3-ITD model, CCM-405 induced ~90% tumor regression (> 100% TGI) and was significantly more effective than Gilteritinib (p < 0.001), which did not regress the tumor, when these agents were administered orally at doses corresponding to equal fractions of their maximum tolerated doses (MTDs). In both FLT3-ITD-F691L and FLT3-ITD-D835Y CDX models, CCM-405 induced almost complete tumor regression (>100% TGI). Both novel inhibitors were significantly more efficacious than Gilteritinib (45% and 4% TGI, respectively). Conclusions: Novel FLT3 inhibitors have been developed that can both target FLT3-ITD and potentially overcome mutational resistance to FDA-approved FLT3 inhibitors. These agents are significantly more effective than Gilteritinib and have potential clinical applications.