Manipulation of Redox Metabolism Using Pharmacologic Ascorbate Opens a Therapeutic Window for Radio-Sensitization by ATM Inhibitors in Colorectal Cancer

Abstract

Purpose: Ataxia telangiectasia mutated kinase (ATM) inhibitors are potent radiosensitizers that regulate DNA damage responses and redox metabolism, but they have not been translated clinically because of the potential for excess normal tissue toxicity. Pharmacologic ascorbate (P-AscH^-; intravenous administration achieving mM plasma concentrations) selectively enhances H₂O₂-induced oxidative stress and radiosensitization in tumors while acting as an antioxidant and mitigating radiation damage in normal tissues including the bowel. We hypothesized that P-AscH^- could enhance the therapeutic index of ATM inhibitor-based chemoradiation by simultaneously enhancing the intended effects of ATM inhibitors in tumors and mitigating off-target effects in adjacent normal tissues.

Methods and materials: Clonogenic survival was assessed in human (human colon tumor [HCT]116, SW480, HT29) and murine (CT26, MC38) colorectal tumor lines and normal cells (human umbilical vein endothelial cell, FHs74) after radiation ± DNA repair inhibitors ± P-AscH^-. Tumor growth delay was assessed in mice with HCT116 or MC38 tumors after fractionated radiation (5 Gy × 3) ± the ATM inhibitor KU60019 ± P-AscH^-. Intestinal injury, oxidative damage, and transforming growth factor β immunoreactivity were quantified using immunohistochemistry after whole abdominal radiation (10 Gy) ± KU60019 ± P-AscH^-. Cell cycle distribution and ATM subcellular localization were assessed using flow cytometry and immunohistochemistry. The role of intracellular H₂O₂ fluxes was assessed using a stably expressed doxycycline-inducible catalase transgene.

Results: KU60019 with P-AscH^- enhanced radiosensitization in colorectal cancer models in vitro and in vivo by H₂O₂-dependent oxidative damage to proteins and enhanced DNA damage, abrogation of the postradiation G2 cell cycle checkpoint, and inhibition of ATM nuclear localization. In contrast, concurrent P-AscH^- markedly reduced intestinal toxicity and oxidative damage with KU60019.

Conclusions: We provide evidence that redox modulating drugs, such as P-AscH^-, may facilitate the clinical translation of ATM inhibitors by enhancing tumor radiosensitization while simultaneously protecting normal tissues.