Abstract 2086: Dual inhibitors of Flt3 and c-Fest tyrosine kinases potently inhibit proliferation of AML cells expressing an active Flt3 mutant. (2013)

Abstract Acute myelogenous leukemia (AML) is a hematologic cancer associated with advancing age and relatively poor prognosis. Although diverse genetic changes are associated with AML, about one-third of cases involve activating mutations in the receptor tyrosine kinase, Flt3. Clinical trials of most Flt3 tyrosine kinase inhibitors have been disappointing, indicating that additional signaling pathways contribute to the transformed phenotype. Previous studies have linked the c-Fes protein tyrosine kinase to Flt3 in AML, and RNAi-knockdown studies suggest that c-Fes may be required for Flt3 function. Recently, our laboratory reported several classes of inhibitors with favorable selectivity profiles for c-Fes both in vitro and in cell-based assays. In this study, we tested c-Fes inhibitors from four different chemical classes for their growth-suppressive activity against myeloid leukemia cells lines with an activating mutation in Flt3 (MV4-11 cells) vs. wild-type Flt3 (THP-1). All c-Fes inhibitors tested potently inhibited the growth of MV4-11 AML cells, but were far less active against THP-1 cells. The IC50 values for inhibition of MV4-11 cell proliferation by c-Fes inhibitors of the diaminopyrimidine, phenoxyquinazoline and pyrrolopyrimidine classes ranged from 25-140 nM, rivaling the potency of the clinical Flt3 inhibitors lestaurtinib and tandutinib. In contrast, two pyrazolopyrimidine c-Fes inhibitors were less potent in MV4-11 cells, with IC50 values for growth arrest in the low micromolar range. KINOMEscan analysis, an indirect binding assay which provides a relative measure of inhibitor selectivity across the kinome, suggested that the more potent compounds may have dual specificity for both Flt3 and c-Fes while the less potent compounds may be more selective for c-Fes alone. To test this idea, we performed in vitro kinase assays with recombinant purified c-Fes and Flt3 proteins. We observed that the most potent inhibitors of MV4-11 AML cell proliferation inhibited both c-Fes and Flt3 kinase activity, with IC50 values in the 0.2-1.5 μM range for c-Fes and in the 0.2-4.0 μM range for Flt3. For example, the phenoxyquinazoline analog TL02-59 showed similar potency against both c-Fes (IC50 of 1.0-1.5 μM) and Flt3 (IC50 of 0.6-4.0 μM) in vitro, and potently blocked MV4-11 cell proliferation (IC50 of 26.5 nM) and induced apoptosis. In contrast, the pyrazolopyrimidine inhibitor WZ4-49-8, which exhibited 20-fold greater selectivity for c-Fes over wild-type Flt3 in vitro, was less active against MV4-11 cell proliferation (IC50 of 1.86 μM) and produced a lower apoptotic response than TL02-59. Interestingly, the established Flt3 inhibitor lestaurtinib was also a relatively potent inhibitor of c-Fes, yielding IC50 values in the 0.4-0.8 μM range. These results suggest that dual inhibition of both Flt3 and c-Fes may provide a therapeutic advantage for the treatment of AML. Citation Format: Mark Weir, Sabine Hellwig, Nathanael S. Gray, Thomas E. Smithgall. Dual inhibitors of Flt3 and c-Fest tyrosine kinases potently inhibit proliferation of AML cells expressing an active Flt3 mutant. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2086. doi:10.1158/1538-7445.AM2013-2086