DNA repair initiated in chronic lymphocytic leukemia lymphocytes by 4-hydroperoxycyclophosphamide is inhibited by fludarabine and clofarabine

Purpose: Chronic lymphocytic leukemia (CLL) lymphocytes respond to DNA alkylation by excision repair, with the extent of repair increasing as the cells acquire resistance to alkylating agents. Because incorporation of nucleotide analogues into the repair patches elicits death signals in quiescent cells, the increased capacity for excision repair in alkylator-resistant cells could facilitate incorporation of nucleotide analogues. We hypothesized that the mechanism-based interaction of nucleoside analogues with alkylating agents could elicit greater than additive killing of CLL cells. Experimental Design: Lymphocytes from 50 patients with CLL that were not refractory to alkylators were treated in vitro with 4-hydroperoxycyclophosphamide (4-HC) with or without prior incubation with fludarabine nucleoside (F-ara-A) or with clofarabine (Cl-F-ara-A). DNA damage repair kinetics were determined by the single-cell gel electrophoresis (comet) assay. Cytotoxicity was assessed by staining with annexin V. Results: CLL lymphocytes promptly initiated and completed excision repair in response to 4-HC. A 2-h preincubation with 10 μM F-ara-A or 10 μM Cl-F-ara-A inhibited the repair initiated by 4-HC, with inhibition peaking at the intracellular concentrations of 50 μM F-ara-ATP or 5 μM Cl-F-ara-ATP. Combining 4-HC with either F-ara-A or Cl-F-ara-A produced more than additive apoptotic cell death than the sum of each alone. The increase in cytotoxicity was proportional to the initial magnitude of the DNA incision and to the extent of repair inhibition by the nucleoside analogues, suggesting close correlation between the repair inhibition and induction of cell death. Conclusions: DNA repair, which is active in CLL lymphocytes, may be a biological target for facilitating the incorporation of nucleoside analogues and increasing their cytotoxicity. Thus, the increased repair capacity associated with resistant disease may be manipulated to therapeutic advantage.