Abstract 1341: A novel ATR inhibitor combined with radiation sensitizes an immunologically cold tumor to immunotherapy

Abstract ATR is a major DNA damage response (DDR) protein and an attractive target to combine with radiotherapy to amplify radiosensitization and immune-sensitization. ATR has several roles in DDR, including DNA double-strand break (DSB) repair through homologous recombination (HR) and S/G2 cell-cycle checkpoint control. Cancer cells can survive radiation-induced complex DNA lesions by their capacity to repair DSB lesions. The disruption of the G2/M checkpoint by ATR inhibition in cells with elevated DNA damage caused by irradiation increases micronuclei numbers, which activate the cGAS-STING pathway, promoting immune signaling. We hypothesize that inhibition of ATR radiosensitizes cancer cells to photons and protons and amplifies immune activation. To test our hypothesis, we treated H1299, H460 and 4T1 cell lines with a novel ATR inhibitor in clinical development, ART0380, prior to irradiation with 6 MV x-rays (photons) or 3.9 keV/µm protons. We then analyzed clonogenic cell survival [sensitization enhancement ratio (SER) and relative biological effectiveness (RBE)], cell cycle populations at 6 and 24 after irradiation, DNA damage (γH2AX and 53BP1 foci) at 1 and 24 h after irradiation, inhibition of HR (Rad51 foci) at 2 and 6 h after irradiation, micronuclei and cGAS colocalized micronuclei at 24, 48 and 72 h after irradiation, and expression of proteins involved in the cGAS-STING pathway at 24 and 48 h after irradiation. For in-vivo studies, syngeneic leg allograft tumors in Balb/c mice with the 4T1 murine breast cancer cell line were irradiated with 6 MV x-rays or 3.9 keV/µm protons (3×8 Gy) in combination with 100 mg/kg ART0380 BID for 3 days on/4 days off for 2 weeks. Tumor volumes were monitored. Compared to radiation alone, radiation+ART0380 decreased the G2 population and increased the mitotic population. Cell lines were also radiosensitized by ART0380 treatment (SER>1) and RBE was modulated. ART0380 treatment increased persistence of the DNA damage caused by radiation. We observed significantly more micronuclei and cGAS colocalized micronuclei in the radiation+ART0380 groups compared to radiation alone, validating our hypothesis of increased immune activation. Interestingly, protons+ART0380 induced a higher effect compared to photons+ART0380. To assess if the increased immune activation induced by the combination of ART0380 with radiation is clinically actionable, we combined in vivo radiation+ART0380 to anti-CTLA4 antibody in a tumor model that is resistant to anti-CTLA4 alone. Remarkably, we detected sensitization to anti-CTLA4 when it was combined with both protons+ART0380 or photons+ART0380. Overall, radiation+ART0380 sensitized an immunologically cold tumor to anti-CTLA4, with protons inducing long-lasting tumor regression. Protons combined with ART0380 may be a novel strategy to sensitize radioresistant and immunologically cold tumors to immunotherapy. Citation Format: Mandira Manandhar, Scott J. Bright, Poliana C. Marinello, Jayesh B. Majithiya, Desirée Piscitello, Marco Ranzani, Helen Robinson, Graeme Smith, Gabriel O. Sawakuchi. A novel ATR inhibitor combined with radiation sensitizes an immunologically cold tumor to immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1341.