Vorinostat Synergistically Potentiates MK-0457 Lethality in Chronic Myelogenous Leukemia (CML) Cells Sensitive and Resistant to Imatinib Mesylate.

Abstract

MK-0457/VX-680 was developed as a pan-aurora kinase inhibitor that induces apoptosis in tumor cells and suppresses tumor growth in vivo. Recently, it was found that MK-0457 is also a potent inhibitor of both wild type and mutated Bcr/Abl kinases, including the T315I mutant which confers resistance to first- (i.e., imatinib mesylate/IM) and second-generation kinase inhibitors (e.g., dasatinib and nilotinib). Moreover, MK-0457 exhibits significant activity in patients with T315I phenotype-refractory CML or Ph-positive ALL. Here we examined interactions between MK-0457 and the histone deacetylase inhibitor (HDACI) vorinostat/SAHA in Bcr/Abl+ leukemia cells, including those resistant to IM through various mechanisms, particularly the T315I mutation. Co-administration of non- or subtoxic concentrations of vorinostat (0.5–2 μM) increased the lethality of MK-0457 (5–100 nM) in a highly synergistic manner in K562 and LAMA84 cells. Similar interactions were observed in primary CD34+ CML cells, whereas the combination largely spared normal bone marrow mononuclear cells. Moreover, the MK-0457/vorinostat regimen markedly induced cell death in IM-resistant K562 cells exhibiting a Bcr/Abl-independent, Lyn-dependent form of resistance, a phenomenon accompanied by Lyn inactivation. Notably, the regimen was highly active against BaF/3 cells bearing various Bcr/Abl point mutations (i.e., T315I, E255K, and M351T) conferring resistance to IM. These events were associated with inactivation and downregulation of wild type and mutated Bcr/Abl (particularly T315I), reduced expression of phospho-CrkL, and vorinostat-mediated interruption of interactions between Bcr/Abl and Hsp90. Moreover, exposure of cells to marginally toxic doses of MK-0457 resulted in accumulation of cells with greater than 4N DNA content, resulting from induction of endoreduplication. Co-administration of vorinostat strikingly enhanced aurora kinase inhibition by MK-0457, reflected by markedly diminished expression of phospho-histone H3 (Ser 10), accompanied by preferential killing of polyploid cells. Interestingly, vorinostat also interacted with a selective inhibitor of aurora kinase A and B, at concentrations that inhibited histone H3 phosphorylation, to potentiate apoptosis in CML cells without modifying expression of phospho-Bcr/Abl or -CrkL. These results suggest that aurora kinase inhibition plays a functional role in synergistic interactions between HDACI and aurora kinase inhibitors. Finally, vorinostat markedly induced expression of the pro-apoptotic BH3-only protein Bim in various CML cell lines, including BaF/3 cells expressing the T315I Bcr/Abl mutation, as well as in primary CML cells. Knockdown of Bim by siRNA significantly protected K562 cells from apoptosis induced by vorinostat but not MK-0457. Furthermore, siRNA-mediated Bim downregulation blocked induction of Bim expression by vorinostat and dramatically diminished the capacity of this agent to potentiate MK-0457 lethality. In addition, blockade of apoptosis by Bim siRNA did not modify downregulation of Bcr/Abl or phospho-histone H3 in cells exposed to the vorinostat/MK-0457 regimen, arguing against the possibility that the latter phenomena are secondary to activation of apoptotic cascade. Together, these findings indicate that vorinostat dramatically increases MK-0457 lethality in IM-sensitive and -resistant CML cells, and suggest that inactivation of Bcr/Abl kinase and aurora kinases, as well as Bim induction by vorinostat, contribute to this phenomenon.