Clofarabine acts synergistically with ionizing radiation in vitro and in vivo by inhibiting the repair of DNA damage

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Clofarabine, a second generation nucleoside analogue, was the first new drug approved by the FDA for the treatment of pediatric leukemia in more than a decade. Clofarabine combines the favorable characteristics of fludarabine and cladribine, but is more hydrolytically stable and resistant to phosphorylase cleavage. The active form of clofarabine, clofarabine triphosphate, is a potent inhibitor of DNA polymerase α and ribonucleotide reductase. It can block DNA synthesis and possibly inhibit DNA repair. Therefore we hypothesized that clofarabine could work synergistically with radiotherapy to increase the therapeutic index of cancer. We found that, low doses of clofarabine (< 10nM) alone did not induce significant γ-H2AX foci formation, an early event of the ionizing radiation (IR)-induced DNA damage response, but prolonged their presence up to 24 hrs after 6Gy IR. Low doses of clofarabine also increased IR-induced γ-H2AX foci formation. In the meantime, larger doses (100-1000nM) of clofarabine alone were able to induce significant DNA double strand breaks. These data suggest that clofarabine could inhibit DNA repair following IR, thereby sensitizing cells to radiation. To investigate clofarabine-induced radiosensitization, Hela cells were treated with increasing doses of clofarabine (10-1000nM) and IR (0-6Gy). A significant decrease in clonogenic survival was observed in irradiated cells treated with clofarabine, demonstrating a strong synergistic effect between clofarabine and IR. Finally, clofarabine-induced radiosensitization was established in vivo using a colorectal cancer model, DLD-1, in athymic nude mice. When used alone with a moderate dose (30mg/kg, X12, i.p. injection) in the mouse model, Clofarabine had limited effects on tumor growth. However, when combined with fractionated radiotherapy, it led to a significant increase in tumor growth inhibition. These results suggest that clofarabine has the ability to act as a potent radiosensitizer both in vitro and in vivo by perpetuating DNA damage culminating in decreases in tumor survival and can be readily translated into clinical studies.