Synergy between 17 AAG and Arsenic Trioxide in the Induction of Apoptosis in Myeloma Cell Lines.

Arsenic trioxide (ATO) is an effective chemotherapeutic agent for the treatment of acute promyelocytic leukemia and is being tested in phase II studies in various types of hematological malignancies and solid tumors. We have previously shown that ATO is a potent inducer of apoptosis in multiple myeloma cells, engaging primarily the intrinsic apoptotic pathway in cells expressing w.t. p53. In contrast, in cells expressing mutant p53, both the intrinsic and extrinsic apoptotic pathways are engaged. These findings were further supported by a recent study using a p53 temperature sensitive (p53Ts) mutant cell line, BRK, expressing w.t. p53 at 32C and mutant p53 phenotype at 37C (Akay et al. Akay et al., AACR; Abstract #5344, 2005). Furthermore, myeloma cells expressing w.t. p53 transfected with SiRNA for p53 or p21 behaved like cells with mutant p53 (Kircelli et al. ASH presentation, 2005). Employing the Affymetrix Microarray technology to compare global gene expression in myeloma cell lines we identified a number of new genes affected by ATO (Gazitt et al. ASH presentation, 2005). One of these genes was heat shock protein 90 (HSP90). We therefore hypothesized that treatment of myeloma cells with blockers of HSP90 such as geldanamycin or its newly discovered potent derivative; 17-allylamino-17-demethoxygeldanamycin (17-AAG) in combination with ATO will result with synergy in the induction of apoptosis in these cells. Indeed, treatment of IM9 myeloma cells (w.t. p53) and U266 myeloma cells (mutant p53) with 17 AAG (0 to 3uM) resulted with a time/dose induced apoptosis to a maximum of 25% apoptosis by annexin V. Treatment with ATO alone at 2.5 uM resulted with 22% apoptosis following 24 hours of treatment. However, ATO synergized with 17 AAD to induce 2–3 fold higher apoptosis compared to the sum of the individual drugs in each dose tested. In contrast, only additive effect was observed between 17 AAG and ATO in the induction of mitochondrial membrane (MM) depolarization as measured by TMRE fluorescence and in the depletion of glutathione measured by MCB fluorescence. Interestingly, 17 AAG did not have any effect on generation of reactive oxygen species as measured by DHR fluorescence. Finally, 17 AAG induced mild arrest of cells at G2/M with marked increase in G2/M arrest when combined with ATO, which by itself did not increase the percentage of cells at G2/M. Similar trend was observed in U266 cells, in which apoptosis, MM depolarization, depletion of glutathione and G2/M arrest were much higher with 17 AAG alone or with ATO alone, and hence only additive effect was observed between the 2 drugs, at the same dosing used for the IM9 cells. Western immunoblot analysis of the levels of HSP 90 -alpha (90kd) and beta (81kd) subunits revealed slight inhibition with 17 AAG alone and ATO alone with marked decreased in HSP90 inhibition observed in cells treated with both drugs. Furthermore, analysis of the proteins involved in the intrinsic and extrinsic apoptosic pathways revealed a shift from activation of the intrinsic to activation of the extrinsic apoptotic pathway in IM9 cells treated with the combination of 17 AAG and ATO, similar to the pattern observed in U266 treated with ATO alone. These results strongly suggest that 17 AAG could potentiate the effect of ATO, in myeloma patients treated with the combination of the 2 drugs.