Abstract 281: Bendamustine has the biochemical properties of an alkylating agent that synergizes with nucleoside analogues in chronic lymphocytic leukemia
Authors: Kost Sara EF , Bouchard Eric DJ , LaBossière Élise , Ye Xibiao , Queau Michelle L. , Liang William S. , Banerji Versha , Gibson Spencer B. , Katyal Sachin , Johnston James B. 
Published: July 2016

Abstract
Bendamustine (BEN) is a promising new treatment for chronic lymphocytic leukemia (CLL) and may function as an alkylating agent (eg, chlorambucil, CLB) while sharing structural similarities to a nucleoside analogue (eg, fludarabine, FLU). Enhanced cell death has been observed with the combination of BEN and FLU in primary CLL cells in vitro, but both BEN and FLU are marrow-toxic, potentially limiting the clinical value of this combination. While the nucleoside analogue pentostatin (PEN) is active in CLL and less marrow-toxic than FLU, it is unknown whether it produces synergy when combined with BEN. PEN acts by inhibiting adenosine deaminase, resulting in the accumulation of deoxyadenosine (dADO), which is toxic to CLL cells. In the present study, we evaluated the activity of BEN against CLL cells in vitro, as compared to CLB, FLU or dADO/PEN, alone and in combination. Cell death was assessed using annexin V/7-ADD staining and the MTT assay. We observed that BEN, CLB, FLU, or dADO/PEN induced apoptosis, the extent of which was time- and concentration-dependent. In general, cross-resistance was observed to all agents, with previously treated patients or those with a del 17p being most resistant. Enhanced cell killing was seen when combining BEN or CLB with FLU or dADO/PEN, with the extent of synergy being similar for FLU or dADO/PEN. To correlate mechanism of cell death as a result of treatment with these agents, we analyzed the expression of death receptors (DR4 and DR5) and mitochondrial stress (DICO6 and DHE). An increase in DR5 (but not DR4) surface expression, loss of mitochondrial membrane potential, and increased reactive oxygen species production was observed following treatment indicating that all agents induced death through the death receptor and mitochondrial apoptotic pathways. However, using γH2AX and the alkaline comet assay, FLU produced a greater number of DNA double-strand breaks (DSB) than BEN or CLB. Thus, all these agents induce apoptosis through the mitochondrial and death receptor pathways with cross-resistance being observed in most cases. BEN is more similar to CLB in producing fewer DNA DSBs than FLU and demonstrating synergy when combined with FLU or dADO/PEN. The latter observation suggests that combining BEN with PEN might be useful clinically.
Citation Format: Sara EF Kost, Eric DJ Bouchard, Élise LaBossière, Xibiao Ye, Michelle L. Queau, William S. Liang, Versha Banerji, Spencer B. Gibson, Sachin Katyal, James B. Johnston. Bendamustine has the biochemical properties of an alkylating agent that synergizes with nucleoside analogues in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 281.