KU-60019

Ataxia telangiectasia mutated protein (ATM) is one of the key sensors of DNA damage and specific inhibitors of ATM are potent radiosensitizers. However, their clinical utility with radiation (RT) is limited because they lack tissue specificity and increase normal tissue injury. Pharmacologic (high dose) ascorbate (P-AscH^-) selectively increases oxidative stress in tumors while functioning as a donor antioxidant and reducing RT damage in normal tissues. We hypothesized that P-AscH^- could enhance the therapeutic index of ATM-inhibitor based chemoradiation (CRT) for colorectal cancer (CRC) by simultaneously enhancing efficacy and reducing RT bowel injury.

Human HCT116, SW480, and HT29 and murine CT26 and MC38 CRC models were used. Clonogenic survival was assessed following single-fraction RT (2-8 Gy) +/- P-AscH^- (5 pM/cell) +/- veliparib (PARP), VE821 (ATR), or KU60019 (ATM). Catalase expression was induced using HCT116 cells expressing a doxycycline inducible catalase transgene. DNA double strand breaks (DSBs) were quantified using neutral comet assays 0-24 hours post RT. Cell cycle phases were assessed using flow cytometry. ATM and pATM localization were assessed using IF. Jejunal toxicity was assessed using IHC in fixed tissues following single fraction (10 Gy) whole abdominal RT in c57bl/6 mice. Tumor growth delay was assessed following RT (5 Gy x 3) +/- drug treatment in unilateral flank tumors.

Veliparib, VE821, and KU60019 were potent radiosensitizers in HCT116, SW480, HT29, MC38, and CT26 CRC tumor models and P-AscH^- further reduced clonogenic survival with DRIs in all lines except for HT29. In contrast, P-AscH^- enhanced survival of cultured HUVEC and FHs-74 cells exposed to RT. Enhanced cell kill with P-AscH^- is H₂0₂ mediated as it is completely attenuated by inducible catalase expression. P-AscH^- significantly increased the number of DNA DSBs in tumors after RT in vitro. Despite the increase in DNA DSBs, P-AscH^-significantly decreased nuclear localization of activated pATM after RT and significantly decreased the fraction of cells in G2/M phases of the cell cycle. In vivo, RT + P-AscH^- + KU60019 induced more tumor growth delay/clearance than all other combinations in unilateral MC38 or HCT116 flank tumors. Finally, P-AscH^- significantly reduced loss of jejunal crypt cell density, epithelial architecture, and markers of lipid and protein oxidation following whole abdominal RT.

P-AscH^- selectively enhances the efficacy of ATM-based CRT in CRC tumor models while simultaneously decreasing RT-mediated small bowel toxicity. In tumors, P-AscH^- enhances DNA DSBs by stimulating an H₂0₂ flux and prevents activation of DNA repair pathways and cell cycle checkpoints by inhibiting RT-induced activation of ATM. Selective radioprotectors like P-AscH^- could facilitate the clinical translation ATM inhibitors as radiosensitizers.

Preterm infants are at greater risk for adverse drug effects due to hepatic immaturity. Multiple interventions during intensive care increases potential for drug interactions. In this setting, high-dose caffeine used for apnea in premature infants may increase acetaminophen toxicity by inhibiting ataxia telangiectasia mutated (ATM) gene activity during DNA damage response. To define caffeine and acetaminophen interaction, we modeled infantile prematurity in late-gestation fetal stage through human immortalized hepatocytes and liver organoids. The acute toxicity studies included assays for cell viability, mitochondrial dysfunction and ATM pathway-related DNA damage. Fetal cells expressed hepatobiliary properties, albeit with lower metabolic, synthetic and antioxidant functions than more mature hepatocytes. Acetaminophen in IC50 amount of 7.5 millimolar caused significant oxidative stress, mitochondrial membrane potential impairments, and DNA breaks requiring ATM-dependent repair. Caffeine markedly exacerbated acetaminophen toxicity by suppressing ATM activity in otherwise nontoxic 2.5 millimolar amount. Similarly, the specific ATM kinase antagonist, KU-60019, reproduced this deleterious interaction in 5 micromolar amount. Replicative stress from combined acetaminophen and caffeine toxicity depleted cells undergoing DNA synthesis in S phase and activated checkpoints for G0/G1 or G2/M restrictions. Synergistic caffeine and acetaminophen toxicity in liver organoids indicated these consequences should apply in vivo. The antioxidant, N-acetylcysteine, decreased oxidative damage, mitochondrial dysfunction and ATM pathway disruption to mitigate caffeine and acetaminophen toxicity. We concluded that hepatic DNA damage, mitochondrial impairment and growth-arrest after combined caffeine and acetaminophen toxicity will be harmful for premature infants. Whether caffeine and acetaminophen toxicity may alter outcomes in subsequently encountered hepatic disease needs consideration.