Treatment with Hsp90 Inhibitor, 17-AAG Overcomes Resistance to Small Molecule FLT3-Inhibitors in FLT3/ITD-Positive Leukemia Cells Harboring N676K-Mutation.

The receptor tyrosine kinase FLT3 is mutated in ~30% of acute myeloid leukemia (AML) and small molecules that selectively inhibit FLT3 kinase activity induce apoptosis in blasts from AML patients with FLT3 mutations and prolong survival in animal models of FLT3-induced myeloproliferative disease. Therefore, targeting mutated FLT3 is an attractive therapeutic strategy, and early clinical trials testing FLT3-inihibitors have shown measurable clinical responses. However, most of these responses were transient and in some cases an additive single amino acid substitution (such as N676K) within FLT3 kinase domain was reported to be a possible cause for the temporary response. We previously showed a novel FLT3 inhibitor, FI-700, selectivity suppresses the growth of leukemia cells with FLT3 mutations (Kiyoi, et al., Clin Cancer Res 07). In order to examine how we can overcome drug resistance due to additional kinase mutations, we cultured MOLM-13 (FLT3/ITD-positive leukemia cell line) cells with increasing doses of FI-700 to generate several FI-700-resistant sublines. In 6 of 6 clones with resistance to more than 1 μM FI-700, FLT3N676K mutation was detected in one allele, and these were also resistant to small molecule FLT3-inhibitors such as AG1295 and AG1296. In a drug-screening for this kind of subline (MOLM-13/FLT3N676K), sub-molar treatment with molecular chaperon Hsp90 inhibitor, 17-AAG, inhibited growth as revealed by MTT-assays, and a combination of 17-AAG and FI-700 synergistically induced cell death as indicated by DNA-histograms and PI/Annexin-V assays. Expression of FLT3 by FACS and phosphorylation of FLT3 and its substrate molecules (such as STAT5), were significantly inhibited by the combined treatment of FI-700 and 17-AAG. To reveal the detailed mechanisms underlying these synergistic effects, we are currently investigating the binding of FLT3 and Hsp90, the degradation and the localization of FLT3 in these cells. We are also investigating the in vivo-effects of combined treatment in NOD/SCID mice inoculated with MOLM-13/FLT3N676K. These results imply that treatment of FLT3/ITD-positive leukemia cells with 17-AAG is a promising strategy for overcoming the drug resistance of cells with mutations in the FLT3 kinase domain.