Abstract

17-AAG, an analogue of gelganamycin is an inhibitor of the molecular chaperone Hsp90, that results in apoptosis and inhibition of proliferation of myeloid or mixed lineage (MLL fusion gene induced) leukemia cells. The sensitivity to 17-AAG is highest in leukemia with FLT3 mutations, intermediate in wild type FLT3 and lowest in FLT3 negative leukemia cells. 17-AAG results in reduction of total levels of protein kinases FLT3, RAF, AKT and Chk1, a protein involved in DNA repair. Etoposide is a clinically effective agent in myeloid or mixed lineage leukemia therapy. Etoposide inhibits topoisomerase II and results in production of DNA double-strand breaks (DSBs). Normally these DSBs can be repaired by Rad51 via homologous recombination (HR). In DNA repair, Chk1 is required for HR through the interaction with Rad51. The effects of 17-AAG on Rad51 are unknown. Our current study evaluated the single and combined effects of 17-AAG and etoposide, and the mechanism of these effects in human leukemia cell lines with or without FLT3 mutations. Cell growth experiments using the MTT assay showed that the cell lines with MLL fusion genes and internal tandem duplication of FLT3 (ITD-FLT3) (Molm13 and MV4;11) were sensitive to both 17-AAG and etoposide. The IC50s for 17-AAG were 31 nM and 40 nM, respectively; the IC50s for etoposide were 37.7 nM and 45.6 nM, respectively. Wild-type FLT3 cells (HPB-Null and RS4;11) were less sensitive to both 17-AAG and etoposide; the IC50s for 17-AAG were 470 nM and 700 nM, respectively, and the IC50s for etoposide were 72.2 nM and 101.5 nM, respectively. The combination effects of 17-AAG and etoposide on cell proliferation were analyzed using Combination Index method (CalcuSyn software). Importantly, we observed synergistic inhibitory effects in FLT3-ITD cells (MV4;11 and Molm13) but only additive effects in wild type FLT3 cells (HPB-Null and RS4;11). Cell cycle analysis of MV4;11 and Molm13 cells showed that 17-AAG increased cells in G0/G1 phase after a 24 hrs treatment, while etoposide induced G2/M arrest only. Combined treatment with 17-AAG and etoposide results in Go/G1 arrest before the cells enter S phase, as with 17-AAG alone. Western-blotting showed that 17-AAG inhibited FLT3, Chk1 and in novel results Rad51 in ITD-FLT3 leukemia cells. To address the importance of FLT3 mutations on DNA repair proteins, Chk1 and Rad51, we used small interfering RNA (siRNA) targeted to FLT3. The results showed that Chk1 and Rad51 are dependent on constitutively activated FLT3 expression in ITD-FLT3 cells. In conclusion, the combination of 17-AAG with etoposide results in synergistic cellular inhibitory effects in ITD-FLT3 leukemia cells. The mechanism of the synergistic effects was found to be in part the result of inhibitory actions of 17-AAG on FLT3 dependent DNA repair proteins, Chk1 and in new findings Rad51 which are required for the repair of DNA damage induced by etoposide. 17-AAG, which is currently in clinical trials, combined with a topoisomerase II inhibitor, such as etoposide, has the potential to enhance therapeutic efficacy, particularly in high risk myeloid or mixed lineage leukemias with FLT3 mutation.

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