The Histone Deacetylase Inhibitor SAHA Sensitizes Lymphoma Cells to Combination of Cladribine and Gemcitabine with DNA Alkylating Agent Busulfan

Hematopoietic stem cell transplantation (HSCT) is an effective treatment for patients with refractory lymphomas. Nucleoside analogues (NA) and DNA alkylating agents are efficacious in treating hematologic malignancies. To design an efficient, safe and more economical pre-transplant conditioning regimen for lymphoma patients, we investigated the cytotoxicity of cladribine (Clad), gemcitabine (Gem), busulfan (Bu) and the histone deacetylase inhibitor suberoylanilide hydroxamic acid, SAHA, as single agents, or in combination, in the lymphoma cell lines J45.01 and U937. [Clad+Gem+Bu] combinations synergistically inhibited the proliferation of both cell lines to 40-60% relative to the control. Addition of SAHA to this combination increased the inhibition to 55-70%. Investigation of the underlying mechanisms of their synergism indicated that exposure of lymphoma cells to [Clad+Gem+Bu] combination resulted in DNA damage, activation of the ATM-CHK2 pathway, histone modifications, decrease in the level of anti-apoptotic proteins, and decrease in mitochondrial membrane potential which caused leakage of apoptosis-inducing factors such as cytochrome c and Smac/Diablo into the cytosol. These cytocidal effects were greatly enhanced by SAHA. Pre-treatment of J45.01 cells with a pan-caspase inhibitor, Z-VAD-FMK, prior to exposure to the [Clad+Gem+Bu+SAHA] combination blocked the activation of caspases, which alleviated DNA damage and apoptosis, as shown by decreased phosphorylation of histone 2AX and cleavage of PARP-1. Exposure of J45.01 cells to SAHA 4 hours prior to the addition of the [Clad+Gem+Bu] combination resulted in 58% inhibition of proliferation relative to control, while continuous exposure to [Clad+Gem+Bu] combination alone or 4-hour pre-exposure to SAHA followed by washing then addition of [Clad+Gem+Bu] resulted in 40%, and 46% inhibition, respectively (P = 0.005). These results underscore the importance of optimizing the sequence of drug exposure. Our results provide a basis for using [Clad+Gem+Bu] as a pre-transplant conditioning regimen for lymphoma patients undergoing HSCT, and further addition of an epigenetic modifier, such as SAHA, may play a role as a sensitizer to optimize the efficacy of the chemotherapy regimen.