Evaluation of bendamustine in combination with fludarabine in primary chronic lymphocytic leukemia cells

Abstract

Key Points• The fludarabine and bendamustine combination is cytotoxic to CLL cells even in the presence of a protective microenvironment.• H2AX activation was maximum with the combination, and unscheduled DNA synthesis induced by bendamustine was blocked by fludarabine.The fludarabine and cyclophosphamide couplet has become the backbone of the chronic lymphocytic leukemia (CLL) standard of care. Although this is an effective treatment, it results in untoward toxicity. Bendamustine is a newly approved and better-tolerated alkylating agent. We hypothesized that similar to cyclophosphamide, bendamustineinduced DNA damage will be inhibited by fludarabine, resulting in increased cytotoxicity.To test this hypothesis and the role of the stromal microenvironment in this process, we treated CLL lymphocytes in vitro with each drug alone and in combination. Simultaneous or prior addition of fludarabine to bendamustine resulted in maximum cytotoxicity assayed by 3,39-dihexyloxacarbocyanine iodine negativity, annexin positivity, and poly (adenosine 59-diphosphate-ribose) polymerase cleavage. Cytotoxicity elicited by combination of both agents was similar in these malignant B cells cultured either in suspension or on marrow stroma cells. Cell death was associated with DNA damage response, which was determined by phosphorylation of H2AX and unscheduled DNA synthesis. H2AX activation was maximum with the drug combination, and unscheduled DNA synthesis induced by bendamustine was blocked by fludarabine. In parallel, ATM, Chk2, and p53 were phosphorylated and PUMA was induced. Cell death was caspase independent; however, caspases did decrease levels of Mcl-1 survival protein. These data provide a rationale for combining fludarabine with bendamustine for patients with CLL. (Blood. 2014;123(24):3780-3789) IntroductionThe most efficacious therapies in chronic lymphocytic leukemia (CLL) include alkylating agents and the combination of these DNA-damaging drugs with purine nucleoside analogs. In fact, the combination of cyclophosphamide and fludarabine or pentostatin has long been the standard of care for CLL.Bendamustine is a newly approved alkylating agent. Chemically, bendamustine is 4-{5-[bis(2-chloroethyl)amino]-1-methyl-2-bezimidazolyl} butyric acid hydrochloride.1 Structurally, it is an alkylating agent with a benzimidazole ring and a butyric acid side chain, which improves water solubility.2 The nitrogen mustard group of bendamustine resembles a similar group on chlorambucil and cyclophosphamide, the 2 most commonly used alkylating agents for CLL.When chlorambucil, which is among the oldest drugs used for the treatment of CLL, 3 was compared with bendamustine for efficacy and toxicity profiles, the overall response rate to bendamustine was 68%, which was more than double the observed rate with chlorambucil. 4 Based on these results, bendamustine was approved by the US Food and Drug Administration for the treatment of CLL. 5 In another randomized clinical study of untreated CLL, fludarabine resulted in higher response rates and ...

Idelalisib and bendamustine combination is synergistic and increases DNA damage response in chronic lymphocytic leukemia cells

“…The primary mechanism of cell death is damage of DNA, genotoxic stress, and apoptosis [14, 20]. While idelalisib-induced apoptosis was minor (Figure 1A), the couplet of bendamustine and idelalisib resulted in synergistic combination at many different concentrations (Figure 1D).…”

“…Phosphorylation of p53, Chk2, and H2AX are mediated through active ATM. [14, 23, 24] It is worthy to mention that none of the patient samples studied had 11q or 17p deletion, the sites for ATM and p53, respectively.…”

“…Both drugs were used in micromolar concentrations that were chosen on the basis of reported plasma concentrations of the free drug in patients. [1, 2, 5, 12, 14, 20] For bendamustine we used generally 20 μM exogenous drug. Bendamustine peak levels are 28 μM [53] or 20 - 24 μM at 90 and 120 mg/m 2 [54, 55].…”

“…Previously, we have demonstrated utility of bendamustine in preclinical setting for primary CLL cells and shown synergistic or additive interactions with fludarabine [14]. Importantly, both p53 positive and p53 mutated CLL responded similarly to bendamustine [15, 16].…”

“…Because PI3K inhibitors, including idelalisib also result in DNA damage, we postulated that combination of idelalisib to bendamustine may result in additive or synergistic cytotoxicity due to enhanced DNA damage. When used alone, bendamustine was more active in suspension cultures of CLL but stromal microenvironment protected CLL cells from bendamustine-mediated cytotoxicity [14]. One of the primary pathways of microenvironment-induced CLL cell survival is BCR axis that includes the PI3K/Akt cassette.…”