Abstract 5379: Mechanism of fludarabine and oxaliplatin combined activity depends on the activity of XPF endonuclease

Abstract

Abstract We set out to determine the mechanism by which fludarabine and oxaliplatin exert more than additive cell killing in chronic lymphocytic leukemia (CLL). We postulate that enhanced activity of oxaliplatin and fludarabine combination is based on termination by fludarabine of DNA re-synthesis associated with nucleotide excision repair (NER) following removal of oxaliplatin adducts. The blocked repair would result in single-strand and double-strand DNA breaks which would activate cell DNA damage signaling and apoptosis. More than additive apoptotic killing was observed in 49% (±22) of CLL cells after exposure to oxaliplatin and fludarabine in vitro, although single agents showed minimal increase in cell death (3% ±9 for oxaliplatin, 13% ±14 for fludarabine), as demonstrated by Annexin V and PI staining at 24 hr (expected vs. observed p=0.0003; n=10). Oxaliplatin induced a 2-fold increase in DNA synthesis in CLL cells, which was inhibited by fludarabine, as determined by thymidine incorporation (n=5). Knockdown of XPF, the NER 5′ endonuclease, in CLL patient samples (n=3) eliminated oxaliplatin induced DNA synthesis (3-fold increase in thymidine incorporation with scramble siRNA compared to 1.4-fold increase with XPF siRNA). Wild type CHO (AA8) cells showed more than additive killing after combination treatment similar to the observation with CLL patient samples (predicted survival 50%; observed 21%). In contrast, only additive killing was observed in CHO cells lacking XPF (UV41) with equitoxic doses of oxaliplatin and fludarabine (predicted survival 56%; observed 54%) suggesting that XPF function is essential for the cooperative interaction of the combination. Alkaline assays demonstrated that inhibition of repair by fludarabine was accompanied by DNA strand break formation within 12 hr (n=4) in CLL patients samples. The increase in Olive tail moment ranged from 2- to 5-fold, confirming induction of single-strand breaks. The double-strand break repair proteins ATM, SMC1, H2AX and DNA-PKcs were phosphorylated as early as 2 hr post-treatment with the combination and persisted up to 24 hr (3-fold, 24-fold, 10-fold and 10-fold increase, respectively; n=4). This response suggests that DNA damage generated by fludarabine and oxaliplatin exposure was processed to double-strand DNA breaks in CLL cells. Our work provides evidence that the combination of oxaliplatin and fludarabine results in the formation of toxic DNA damage and that their combined activity depends on a functional NER pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5379.