Resistance to Selective FLT3 Inhibitors, Driven by FLT3 Ligand and FLT3 Point Mutations, Can Be Overcome with the Dual FLT3-Aurora Kinase Inhibitor CCT241736,
Authors: Moore Andrew S. , Faisal Amir , Bavetsias Vassilios , de Castro David Gonzalez , Sun Chongbo , Atrash Butrus , Valenti Melanie , de Haven Brandon Alexis , Avery Sian , Pearson Andrew DJ , Workman Paul , Blagg Julian , Raynaud Florence I , Eccles Suzanne A , Linardopoulos Spiros 
Published: October 2019

Abstract
Abstract 3492
Mutations of the FLT3 gene, arising from internal tandem duplications (−ITD) or point mutations of the tyrosine kinase domain (−TKD), cause constitutive activation of the FLT3 receptor tyrosine kinase resulting in stimulation of leukemic proliferation. FLT3-ITD mutations confer a poor prognosis in acute myeloid leukemia (AML), whilst the prognostic impact of FLT3-TKD mutations remains unclear. Small molecule inhibition of FLT3 kinase has been a focus of AML drug development for the past decade, but sustained clinical benefits have been limited by pharmacokinetic failures and resistance. Important mechanisms of resistance to small molecule inhibition of FLT3 include the development of secondary mutations in the FLT3 gene, elevated levels of FLT3 ligand (FL) and over-expression of the anti-apoptotic protein, Survivin. We have previously reported that high FL levels induce resistance to the selective FLT3 inhibitor MLN518 (tandutinib) in vitro. Furthermore, prolonged treatment of human FLT3-ITD positive AML cells (MOLM-13) with MLN518 caused resistance and the selection of cells doubly-mutated with FLT3-ITD and a FLT3-TKD (D835Y) point mutation. These resistant cells, termed MOLM-13-RES, also over-express Survivin. Here we demonstrate that such doubly-mutated AML cells are also relatively resistant to the FLT3 inhibitors Sorafenib and AC220 but not the dual FLT3-Aurora inhibitor CCT241736. CCT241736 is a novel, orally bioavailable, imidazo[4,5-b]pyridine derivative discovered at our Institute, highly selective for FLT3 and Aurora kinases with an S(10) selectivity score using KINOMEscan™ technology of 0.057 (fraction of 386 non-mutant kinases inhibited >90% when screened at 1 uM of CCT241736; San Diego, CA). In biochemical kinase studies, CCT241736 has IC50 values against FLT3, Aurora A and Aurora B of 0.035, 0.015 and 0.1 uM respectively. Furthermore, CCT241736 inhibits a wide range of FLT3 mutants, including FLT3-ITD, -D835Y, -D835H, -K663Q and –N841I. In cellular assays, CCT241736 inhibits viability of the human FLT3-ITD positive AML cell lines MOLM-13 and MV-4-11 with EC50 values of 0.1 and 0.27 uM respectively. Unlike the selective FLT3 inhibitors MLN518 and AC220, the in vitro cellular efficacy of CCT241736 is not affected by high levels of FL. In vivo, mouse tumor xenograft models of MOLM-13, MV-4-11, and the doubly-mutated cell line MOLM-13-RES are also sensitive to CCT241736 at well tolerated oral doses, with biomarker modulation consistent with dual inhibition of FLT3 and Aurora kinases. Based on allometric scaling from mouse and rat data, CCT241736 has favorable predicted human pharmacokinetics and phase I clinical trials are planned. In summary, dual inhibition of FLT3 and the critical mitotic kinase Aurora by CCT241736 may represent a novel treatment strategy for FLT3-mutated AML by overcoming the effects of high FL levels and limiting resistance caused by secondary mutations of the FLT3 gene.

Disclosures:
Moore: The Institute of Cancer Research: Employment. Faisal:The Institute of Cancer Research: Employment. Bavetsias:The Institute of Cancer Research: Employment. Gonzalez de Castro:The Institute of Cancer Research: Employment. Sun:The Institute of Cancer Research: Employment. Atrash:The Institute of Cancer Research: Employment. Valenti:The Institute of Cancer Research: Employment. de Haven Brandon:The Institute of Cancer Research: Employment. Avery:The Institute of Cancer Research: Employment. Pearson:The Institute of Cancer Research: Employment. Workman:The Institute of Cancer Research: Employment. Blagg:The Institute of Cancer Research: Employment. Raynaud:The Institute of Cancer Research: Employment. Eccles:The Institute of Cancer Research: Employment. Linardopoulos:The Institute of Cancer Research: Employment.