Bragg peak proton irradiation and ATM inhibition for rectal cancer.

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259 Background: Rectal cancer patients with a complete response (CR) after chemotherapy and radiotherapy may opt for non-operative management. Currently only 20-40% of patients reach a CR. Escalating the dose of radiation may increase the risk of toxicity. ATM inhibitors (ATMi) are potent radiosensitizers in colorectal cancer (CRC). Proton irradiation exhibits an entrance portion of the beam (ENT) where dose is deposited sparsely and a Bragg Peak (BP) with virtually no ‘exit dose’ thereafter. ATMi renders cells uniquely hypersensitive to proton BP, and BP+ATMi may increase the response of Mismatch Repair proficient (pMMR) rectal cancer to immunotherapy. Methods: Tumor efficacy and immunogenicity was assessed using clonogenic survival, γH2AX foci, micronuclei, and cytoplasmic cGas assays after indicated IR modalities +/-ATMi. Normal tissue toxicity was assessed via murine models of non-tumor bearing BALB/c after whole-abdominal irradiation (WAI) after indicated IR modalities +/- ATMi via acute weight loss, FITC/Dextran assay, and Radiation Injury Score (RIS, Scale 0-18) in jejunal and rectal samples at 4 days post-IR. Results: ATMi decreased clonogenic survival across all types of radiation, but the effect was most pronounced with BP protons. BP+ATMi also significantly increased unresolved γH2AX foci, micronuclei, and cytoplasmic cGas compared to ENT+ATMi. Normal tissue toxicity studies demonstrated that BP+ATMi did not significantly increase acute weight loss, RIS in jejunal or rectal tissue; but did increase FITC/Dextran intestinal permeability. Conclusions: Inhibition of ATM preferentially radiosensitized BP protons compared to x-rays or ENT protons in CRC but not in normal tissue based on weight loss and histopathological evaluation. Differential tumor efficacy and normal tissue toxicity after x-ray, entrance and Bragg Peak Protons +/- ATM inhibitor. A B C D E F G H I J DMSO 1 1 1 2.9 1.5 8764 96.4 10 0.3 0.2 ATMi 0.873 0.842 0.723 8.2 3.0 16300 98.5 5.8 0.7 0.8 x-ray 0.615 0.751 0.109 82.4 6.2 4.8 1.0 x-ray+ATMi 0.123 0.344 0.011 80.0 14.4 5.2 1.5 ENT protons 0.293 0.515 0.080 10.2 5.8 10429 78.5 6 2.9 1.5 ENT+ATMi 0.091 0.066 0.013 15.1 7.2 12274 73.0 23.8 5.2 3.8 BP protons 0.213 0.340 0.032 12.0 12.6 7192 75.6 24.6 4.1 3.7 BP+ATMi 0.074 0.038 0.004 22.7 16.2 43677 73.6 310.4 5.3 4.3 Clonogenic survival of pMMR colorectal cancer cell lines after IR (4 Gy) +/- 10nM AZD1390 (ATM inhibitor) ofA) HT29, B) CT26, and C) SW480 cell lines. D) γH2AX foci at 24 hours post-IR in HT29. E) Micronuclei, and F) cytoplasmic cGas integrated density per cell via immunofluorescence in SW480 72 hours post-IR. WAI (10Gy/1 fraction) was delivered to non-tumor bearing BALB/c mice which were followed for G) acute weight loss (% from baseline), and H) FITC/Dextran permeability assay. Tissue samples were graded for Radiation Injury Score in I) jejunal, and J) rectal samples at 4 days post-IR (n=5 per cohort, 3 samples per mouse). Figures are means of triplicate data.