The Histone Deacetylase Inhibitor SAHA Sensitizes AML Cells to a Combination of Nucleoside Analogs and the DNA-Alkylating Agent Busulfan

DNA alkylating agent busulfan (Bu) and nucleoside analogs clofarabine (Clo) and fludarabine (Flu) are commonly used as part of the pre-transplant conditioning regimen in allogeneic hematopoietic stem cell transplantation (allo-HSCT). We previously reported that a combination of [Bu+Clo+Flu] has a synergistic cytotoxicity in AML cells. We hypothesized that combination of [Bu+Clo+Flu] with histone deacetylase inhibitor SAHA will further enhance the cytotoxicity. We exposed the AML cell lines KBM3/Bu250⁶ (KBu) to Bu (10 μg/ml), Clo (10 nM), Flu (0.2 μM) and SAHA (0.7 μM),  and OCI-AML3 to Bu (1.5 μg/ml), Clo (10 nM), Flu (0.5 μM) and SAHA (0.7 μM) alone or in various combinations. Exposure of these cells to Bu, Clo, Flu or SAHA alone, or to  two-drug combinations [Bu+Clo], [Bu+Flu] and [Clo+Flu], did not significantly affect cell proliferation relative to the control. The combination of [Bu+Clo+Flu] resulted in 20-30% inhibition of cell proliferation. Addition of SAHA to the [Bu+Clo+Flu] mixture further enhanced the inhibition of proliferation of KBu and OCI-AML3  by 60% and 80%, respectively, suggesting synergistic cytotoxicity. Biochemical analyses suggest that this cytotoxicity may be attributed to (1) activated DNA-damage response and cell cycle checkpoint through ATM-CHK2-P53 pathway, (2) histone 3 and histone 4 modifications, and (3) activation of both the intrinsic and extrinsic pathways of apoptosis. The pro-apoptotic proteins BAX and cleaved BID increased in the mitochondria when cells were exposed to [Bu+Clo+Flu+SAHA], and further the pro-survival protein MCL-1 was cleaved and yielded a pro-apoptotic 28-kDa protein, while the level of the pro-survival protein BCL-xL decreased. Finally, phosphorylated and pan P53 increased in the mitochondria when OCI-AML3 cells that harbor wild type P53 were exposed to [Bu+Clo+Flu] or [Bu+Clo+Flu+SAHA]. These changes in the level of proteins involved in mitochondrial control of apoptosis may consequently cause mitochondrial outer membrane permeabilization (MOMP). The mitochondrial membrane potential, as a marker for MOMP, decreased by 40% or 80% when cells were exposed to [Bu+Clo+Flu] or [Bu+Clo+Flu+SAHA], respectively, and this change caused leakage of cytochrome cand SMAC/DIABLO into the cytosol leading to caspase activation, and release of apoptosis-inducing factor (AIF) into the nucleus, inducing nuclear fragmentation and cell death. These results provide a mechanistic basis for introducing SAHA as a complement to our double nucleoside-busulfan combination in pre-transplant conditioning therapy for AML patients undergoing allo-HSCT. We hypothesize that SAHA-associated enhancement of apoptosis will result in increased antitumor efficacy with retained safety in a clinical transplant program.