Abstract 2555: The mechanism of action of bendamustine alone or in combination with nucleoside analogs in chronic lymphocytic leukemia
Authors: Kost Sara E.F. , Bouchard Eric D.J. , Liang William S. , Banerji Versha , Gibson Spencer B. , Katyal Sachin , Johnston James B. 
Published: August 2015

Abstract
Bendamustine (BEN) is a promising new treatment for chronic lymphocytic leukemia (CLL) and is believed to 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, likely due to the inhibition of repair of BEN DNA cross-linking by FLU. However, both BEN and FLU are marrow-toxic, limiting the use of this combination in the clinic. The nucleoside analog, pentostatin (PEN), is an adenosine deaminase inhibitor that causes the accumulation of deoxyadenosine (dADO) and is less marrow-suppressive than FLU. However, the cytotoxic effectiveness of dADO/PEN in combination with BEN is not known.
Flow cytometry analysis using annexin V/7-ADD and the MTT assay were performed to determine the effectiveness of BEN treatment in primary CLL cells as compared to or in combination with CLB, FLU, or dADO/PEN at varying drug concentrations and time points. Using the MTT assay, there was a linear relationship between cytotoxicity and drug concentration, similar to that seen with CLB. In vitro treatment of CLL cells with BEN, CLB, FLU, or dADO/PEN induced apoptosis, the degree being time- and concentration-dependent. However, the sensitivity of CLL cells to BEN or CLB varied, suggesting different mechanisms of action. Enhanced cell kill was seen with the combination of BEN or CLB with FLU or dADO/PEN, with the extent of increased cytotoxicity being similar for FLU or dADO/PEN. Cell death mechanisms were analyzed via staining for surface expression of death receptors (DR4 and DR5) and mitochondrial stress (DICO6 and DHE). γ-H2AX staining was used to measure DNA double-strand breaks (DSBs) and the alkaline comet assay was used to measure both DNA DSBs and single-strand breaks (SSBs). An increase in DR4 and DR5 surface expression, loss of mitochondrial membrane potential, and increased reactive oxygen species production was observed with all agents. There was an increase in DNA SSB and DSB following FLU, as compared to BEN and CLB, while a combination of BEN and FLU further increased the number of breaks (especially SSBs) compared to FLU alone.
Thus, BEN has the properties of an alkylating agent, rather than a nucleoside analog, but has a different spectrum of antitumor activity to CLB. BEN induces apoptosis through both the death receptor and mitochondrial pathways. Increased cell kill is seen on combining BEN with dADO/PEN suggesting that this might be a useful combination in the clinic.
Citation Format: Sara E.F. Kost, Eric D.J. Bouchard, William S. Liang, Versha Banerji, Spencer B. Gibson, Sachin Katyal, James B. Johnston. The mechanism of action of bendamustine alone or in combination with nucleoside analogs in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2555. doi:10.1158/1538-7445.AM2015-2555