The Hsp90 Inhibitor 17-AAG and FLT3 Kinase Inhibitor GTP14564 Synergistically Inhibit MLL Fusion Gene Leukemias with FLT3 Mutations.

17-AAG, an inhibitor of the molecular chaperone Hsp90, results in apoptosis and inhibition of proliferation of certain leukemia cells. We have previously reported that responses to 17-AAG are highest in leukemia with FLT3 mutations, intermediate in leukemia with wild type FLT3 and lowest in FLT3 negative cells. 17-AAG operates primarily through reduction of total levels of FLT3 and other protein kinases, including RAF and AKT (Clin Cancer Res 2003, 4483). However, the inhibitory effects of 17-AAG on cells are partial. In this study we hypothesized that inhibition of FLT3 will enhance the effects of 17-AAG. GTP14564, an inhibitor of phosphorylation of FLT3 tyrosine kinase, was previously found to strongly inhibit proliferation of IL3-independent murine pro-B ITD-FLT3 Ba/F3 cells while proliferation of FLT3 ligand dependent Ba/F3 cells expressing wild type FLT3 was relatively resistant (JBC 2003, 32892). The current study evaluated the single and combined effects of 17-AAG and GTP14564, and the mechanism of the combined effects in human cell lines with MLL fusion genes with or without FLT3 mutations. We first determined the importance of FLT3 mutations using small interfering RNA (siRNA) targeted to FLT3. Cell growth experiments showed that a FLT3-ITD human MLL-AF4 fusion gene cell line (MV4;11) was very sensitive to FLT3 siRNA, while the FLT3-wild type amplified SEMK2-M1 cell line was intermediately sensitive and FLT3-wild type RS4;11 cell line was resistant. FLT3 siRNA sensitivities paralleled inhibitory responses to GTP14564 in cell proliferation assays. Importantly, we observed synergistic inhibitory effects on cell proliferation when 17-AAG and GTP14564 were combined in FLT3-ITD cells (MV4;11 and MOLM-13). Synergistic effects were also observed in cells with amplified wild type FLT3 (SEMK2-M1) but not in those with normal level of wild type FLT3 (RS4;11). Cell cycle analysis of MV4;11, SEMK2-M1 and RS4;11 showed that 17-AAG inhibited cells in G0/G1 phase with a reduced S phase fraction after a 24 hrs treatment. Apoptosis, represented by <2N fraction and cleaved PARP (by Western blotting), was induced by 17-AAG only in MV4;11 and SEMK2-M1 cells, but not in RS4;11 cells. GTP14564 induced G0/G1 arrest and apoptosis in MV4;11 and SEMK2-M1 cells, but in contrast to 17-AAG had no effect on RS4;11 cells. Combined treatment with 17-AAG and GTP14564 enhanced G0/G1 arrest and apoptosis in MV4;11 and SEMK2-M1, but not in RS4;11 cells. By Western blotting, 17-AAG reduced total cellular quantities of FLT3 and AKT in all cells tested while GTP14564 did not change these quantities. Significantly, both 17-AAG and GTP14564 alone and in combination reduced the levels of phosphorylated FLT3 and phosphorylated STAT5 in MV4;11 and SEMK2-M1 cells, but not in RS4;11 cells. In summary, GTP14564 inhibits cell proliferation and induces apoptosis of FLT3 mutated (but not FLT3 wild-type) MLL fusion gene leukemia cells and that the addition of 17-AAG results in synergistic inhibitory effects. The mechanism of the synergistic effects was found to be the result of complementary inhibitory actions of 17-AAG and GTP14564 both on FLT3 and on downstream phospho-proteins in the RAS/RAF/AKT and STAT signal transduction pathways. 17-AAG, which is currently in clinical trials, combined with FLT3 kinase inhibitors such as GTP14564 has the potential to enhance therapeutic efficacy, particularly in high risk MLL fusion gene leukemias with FLT3 mutation.