The Efficacy of Histone Deacetylase Inhibitor, Vorinostat against BCR-ABL Positive Leukemia Cells Include ABL Kinase Domain Mutation in Monotherapy and in Combination with Dasatinib

The development of imatinib are represented a major therapeutic advance in BCR-ABL positive diseases. However, a substantial number of patients are either primary refractory or acquire resistance to imatinib associated with ABL kinase domain mutation and this becomes a problematic in clinically. Histone acetylation and deacetylation is important for the accurate regulation of gene expression. Histone deacetylase (HDAC) inhibitors such as vorinostat (suberoylamide hydroxamic acid: SAHA) are the class of anticancer drugs currently in clinical trials for the treatment of hematological malignacies and solid tumors. Especially, vorinostat have shown efficacy in a wide range of cancers such as cutaneous T-cell lymphoma (CTCL). However, the molecular and functional consequences of vorinostat against BCR-ABL expressing cellswith Abl kinase mutation have not fully known. In this study, we investigated the vorinostat efficacy by using the murine Ba/F3 cell line which was transfected wild type (Wt) p210 BCR-ABL or imatinib resistant BCR-ABL mutants such as G250E, Q252H, Y253F, E255K, M294V, T315I, T315A, F317L, F317V, M351T and H396P. 48 hours treatment of vorinostat exhibits cell growth inhibition and proapoptotic activity murine Ba/F3 cells ectopically expressing wild type (Wt) p210 and imatinib resistant BCR-ABL mutants including T315I mutation in a dose dependent manner. IC50 of these cell lines are Wt(720nM), G250E(625nM), Q252H(220nM), Y253F(525nM), E255K(685nM), M294V(785nM), T315I(500nM), T315A(715nM), F317L(560nM), F317V(565nM), M351T(375nM) and H396P(485nM). Vorinostat is more effective in BCR-ABL mutant cells compared with Ba/F3 Wt BCR-ABL cells. We examined the intra cellular signaling by using these cell lines. We found that caspase 3, and poly (ADP-ribose) polymerase (PARP) were activated in a dose and a time dependent manner. Phosphorylation of Crk-L which is downstream target of BCRABL was reduced after vorinostat treatment. We also found the protein level of BCR-ABL was reduced after 48 hours vorinostat treatment in a dose dependent manner. BCR-ABL degradations in mutant cells were significantly enhanced compared with Ba/F3 Wt p210 BCR-ABL cells. Protein degradation of BCR-ABL was blocked by ubiquitin-proteasome inhibitor, Lactacystin suggested that this was involved ubiquitin-proteasome pathway. Dasatinib is a potent dual Src/BCR-ABL kinase inhibitor. Dasatinib is more than 100- fold more potent than imatinib against the ABL kinases and retains this low nanomolar inhibitory activity against the imatinib resistant mutations. Because dasatinib has shown efficacy against imatinib resistant BCR-ABL mutant, we investigated the efficacy between vorinostat and dasatinib by using these cell lines. We found that combination of vorinostat and dasatinib synergistically cell growth inhibition of Wt and BCR-ABL mutatants Ba/F3 cells in 48 hours treatment. Phosphorylation of Crk-L was reduced after vorinostat and dasatinib treatment in these cells. Caspase 3 and PARP activation were also synergistically increased after vorinostat and dasatinib treatment. Data from this study suggested that administration of the clinically available HDAC inhibitor vorinostat may be a powerful strategy against BCR-ABL mutant cells and enhance cytotoxic effects of dasatinib in those imatinib resistant BCR-ABL mutant cells and this approach warrants further examination in BCR-ABL related malignancies.