Synergistic Cytotoxicity of Gemcitabine, Clofarabine and Edelfosine (± DNA alkylating agent) in Lymphoma Cell Lines

Lymphomas are malignancies of the B or T lymphocytes. Due to the heterogeneity of this disease, finding an effective form of treatment is challenging. One treatment option is hematological stem cell transplantation (HSCT), whose success partly depends on the efficacy of the pretransplant regimen. Nucleoside analogs such as gemcitabine (Gem) and clofarabine (Clo) are being used as part of pretransplant conditioning therapy for lymphoma patients. To improve the cytotoxicity of these drugs, we studied their synergism with edelfosine (Ed), an alkyl phospholipid with different mechanisms of antitumor activity, using cell line models.  Exposure of the J45.01 and SUP-T1 (T-cell) and the OCI-LY10 (B-cell) lymphoma cell lines to IC₁₀ – IC₂₀ levels of the drugs resulted in strong synergistic cytotoxicity. While the individual drugs inhibited J45.01 cell proliferation by 3-19%, their combination inhibited proliferation by ~85%. 8-13% of J45.01 cells exposed to individual drugs underwent apoptosis, which increased to 71% for the triple-drug combination. Cell death correlated with increased phosphorylation of histone 2AX and KAP1, decreased mitochondrial transmembrane potential, increased production of reactive oxygen species, and release of pro-apoptotic factors.  Apoptosis was indicated by cleavage of PARP-1, caspases 3 and 8, MCL1 and ANP32B. Exposure of caspase 8-negative I9.2 cells to [Gem+Clo+Ed] resulted in 40% inhibition of proliferation (as compared with 85% inhibition in caspase 8-positive J45.01 cells), suggesting the relevance of caspases in the antitumor activity of the three-drug combination. These observations are consistent with a decreased level of phosphorylation of the prosurvival protein AKT in cells exposed to [Gem+Clo+Ed]. The three-drug combination also activated the SAPK/JNK stress signaling pathway, which resulted in the phosphorylation and heterodimerization of the transcription factors ATF2 and c-Jun, two proteins involved in the DNA-damage response and apoptosis.

        It is possible that [Gem+Clo+Ed] is stem cell sparing and should be investigated as salvage therapy in its own right; however, to further improve antitumor efficacy, we investigated the possible further synergism of the three drugs when combined with a DNA alkylating agent. Exposure of J45.01 to lower concentrations of the drugs (15 nM Gem, 25 nM Clo, 1.2 μg/ml Ed) in the presence of 6 μg/ml busulfan (Bu) or 0.18 µg/ml melphalan (Mel) resulted in stronger synergistic cytotoxicity.  While the lower concentrations of [Gem+Clo+Ed] caused a moderate DNA damage response and apoptosis, addition of either Bu or Mel greatly enhanced the drug-mediated effects. Overall, these results can be used as a mechanistic impetus for evaluating [Gem+Clo+Ed]±DNA alkylator as salvage therapy for refractory NHL either in conventional dosing or as part of preparative regimens for patients undergoing HSCT.