Mechanism of Action of MK-0457 Efficacy in Monotherapy and in Combination with Vorinostat in Wild Type and T315I Mutant BCR/ABL Positive Leukemia Cells.

Abstract

Mutation in the ABL kinase domain is the principal mechanism of imatinib resistance in patients with BCR/ABL-positive leukemia. MK-0457 is a small molecule inhibitor of the Aurora kinase family with potent cross-reactivity towards wild type and T315I BCR/ABL and FLT-3, and the compound has shown activity in preclinical models of solid tumors and FLT-3 driven leukemia. MK-0457 is presently being evaluated in a clinical trial in chronic myelogenous leukemia (CML) patients with imatinib resistance such as T315I mutation, but the mechanism of MK-0457 is not fully evaluated. In this study, the gene expression profiles of CML cell line, K562, exposed to imatinib or MK-0457 were analyzed and compared. When their gene expression profiles were compared, 937 genes in imatinib and 895 genes in MK-0457 were increased and 625 genes were overlapped. In contrast, 597 genes in imatinib and 537 genes in MK-0457 were decreased and 396 genes were overlapped. These down regulated genes included heat shock proteins (HSPs) suggesting these results may relate to the BCR/ABL stability. Next, we examined the intracellular signaling of MK-0457 in BCR/ABL positive cells with T315I mutation by using BAF-3 p185 BCR/ABL (p185) and BAF-3 BCR/ABL T315I (T315I) cell lines. MK-0457 potently induced apoptosis of p185 cells and T315I cells in 72 hours treatment. IC50 of MK-0457 was 100 nM (p185) and 250 nM (T315I). We found that caspase 3, and poly (ADP-ribose) polymerase (PARP) were activated and BCR/ABL phosphorylation was reduced after MK-0457 treatment in a dose dependent manner. It has been reported that histone deacetylase inhibitor (e.g., vorinostat) also depleted BCR/ABL, as well as induced apoptosis and sensitized BCR/ABL-expressing leukemia cells to apoptosis induced by imatinib. These data suggest that MK-0457 may be synergistic with histone deacetylase inhibitors in light of recent publications that have shown that vorinostat induces acetylation of the heat shock protein (HSP), depletes the HPS90 client proteins, including BCR/ABL, and enhances the imatinib-induced apoptosis in BCR/ABL-expressing cells. We found that combination of MK-0457 and vorinostat synergistically increased the apoptosis of p185 cells and T315I cells in 72 hours treatment. Caspase 3 and PARP activation were also synergistically increased after vorinostat and MK-0457 treatment. We examined the intracellular signaling by using these cell lines. Phosphorylation of BCR/ABL, signal-transducing activators of transcription 5 (STAT5), one of the src kinase, Lyn, Crk-L were reduced after treatment with MK-0457 + vorinostat. We also found that cyclin D1 was decreased after MK-0457 or vorinostat and MK-0457 treatment. Histone H4 is also acetylated after vorinostat treatment, but phosphorylation of BCR/ABL was not reduced directly. We evaluated the activity of MK-0457 and vorinostat in primary Ph positive acute lymphoblastic leukemia cells with the T315I mutation. We found that MK-0457 potently induced cell growth inhibition of primary T315I cells in 72 hours treatment. Moreover, combination of vorinostat and MK-0457 synergistically increased the extent of apoptosis in primary T315I cells. This study provides further insight into the mechanism of action of MK-0457 in wild type and T315I mutant BCR-ABL cells and provides mechanistic rational for the synergistic induction of apoptosis observed for the combination of MK-0457 + vorinostat.