TP-0903, a Novel Axl Inhibitor with Activity in Drug Resistant FLT3-ITD+ AML through a Mechanism That Includes FLT3 Inhibition (2017)

Abnormal expression of receptor tyrosine kinase Axl has been associated with poor prognosis in different cancers including acute myeloid leukemia (AML) and has been linked to drug resistance against chemotherapy as well as targeted therapies including FLT3 inhibitors (Park et al. Leukemia 2015). With its role in tumorigenesis, Axl has been actively pursued as a potential therapeutic target with small molecule inhibitors. TP-0903, a type I inhibitor, was designed in silico on an Axl scaffold that does not clash with the Axl gatekeeper residue, Leu620 (Mollard et al. ACS Med Chem Lett 2011). Since Axl inhibitors are known to also interact with FLT3 (Myers et al. J Med Chem 2016), we evaluated the hypothesis that TP-0903 demonstrates preclinical activity against FLT3 -ITD+AML through a mechanism that potentially involves both Axl and FLT3 inhibition. TP-0903 was evaluated against Axl and FLT3 wildtype (WT), ITD and tyrosine kinase domain (TKD) mutations in binding and kinase assays. Inhibition of cell viability was evaluated in Ba/F3 cells transfected with GFP vector control (VC), FLT3 -ITD, or TKD mutations, FLT3 -ITD+ AML cell lines (MOLM13, MOLM13-Res harboring a ITD/D835Y mutation, and MV4-11), and primary diagnostic FLT3 -ITD+ blast samples. Inhibition of FLT3 signaling was determined by Western blot analysis. In vivo activity was evaluated in systemic MOLM13 and MOLM13-Res cell xenograft models in NOD scid gamma (NSG) mice; 1x106 luciferase expressing cells were injected intravenously to female NSG mice, and TP-0903 or vehicle was administered by oral gavage for 3 weeks, starting on day 7-10 post tail vein injection. TP-0903 40-60 mg/kg was administered once daily (qd), and 80 mg/kg was administered on Monday, Wednesday, and Friday (M/W/F). Progression of leukemia was monitored using bioluminescence imaging and Kaplan-Meir analysis was performed for animal survival. TP-0903 had potent binding affinities to Axl, FLT3 WT and all FLT3 mutants evaluated, expressed as Kd: Axl (8.2 nM), FLT3 WT (0.93 nM), ITD (5.6 nM), D835H (1.9 nM), D835V (2.1 nM), D835Y (1.4 nM), ITD/D835Y (0.79 nM), and ITD/F691L (1.9 nM). In a kinase assay, TP-0903 inhibited FLT3 ITD and D835Y with IC50 values of 3.9 nM and 0.12 nM, respectively. TP-0903 inhibited the viability of Ba/F3 transfected with VC (mean IC50 of 3 independent experiments, 47 nM), and activity was more potent against all cells expressing different FLT3 mutants: FLT3 -ITD (15 nM), D835H (16 nM), D835Y (22 nM), ITD/D835H (16 nM), ITD/D835Y (20 nM), and ITD/F691L (16 nM). Western blot analysis of lysates from TP-0903-treated Ba/F3 cells expressing ITD, D835Y, ITD/D835Y, or ITD/F691L mutants showed inhibition of phospho-FLT3 and phospho-STAT5 at 10-100 nM. In FLT3 -ITD+ AML cell lines, TP-0903 demonstrated potent activity with mean IC50 values of 21 nM, 16 nM, and 17 nM in MOLM13, MOLM13-Res, and MV4-11 cells, respectively; the IC50 in primary blast samples ranged from 38-80 nM. In vivo, TP-0903 delayed the outgrowth of MOLM13 cells and significantly prolonged survival in a dose/schedule dependent manner compared to vehicle-treated mice with the following median survival: vehicle (16 days), TP-0903 40 mg/kg qd (26 days, P=0.0039), TP-0903 60 mg/kg qd (32 days, P=0.002), and 80 mg/kg M/W/F (24 days, P=0.0015). In an ongoing MOLM13-Res xenograft model, TP-0903 60 mg/kg qd has suppressed the outgrowth of leukemic cells up to 11 days of treatment compared to vehicle-treated mice (P=0.0002). TP-0903, a novel Axl inhibitor with FLT3 inhibition activity, shows promising in vitro and in vivo activity in de novo and drug resistant FLT3 -ITD+ AML. Given that TP-0903 was initially designed to bind to the gatekeeper leucine residue in Axl, TP-0903 may overcome resistance to the clinically relevant FLT3 gatekeeper F691 substitution mutation to leucine, which most FLT3 inhibitors are vulnerable to. Further in vitro studies to evaluate mechanism of action and in vivo studies for dose optimization and efficacy evaluations in additional models of FLT3 -ITD+ AML are ongoing. Disclosures Whatcott: Tolero Pharmaceuticals, Inc.: Employment. Warner: Tolero Pharmaceuticals, Inc.: Employment. Blum: Boerhinger Ingelheim: Research Funding; Pfizer: Consultancy; Astellas: Consultancy.