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Inhibition of the ATR kinase abrogates the G2 checkpoint and suppresses DNA repair after irradiation, resulting in micronucleus formation and increased cell death. Recent studies suggest that ATR inhibition may also increase antitumor immune responses, but the underlying mechanisms remain poorly understood. Inhibitors of apoptotic caspases could potentially also be used to increase antitumor immune effects, but their roles in radiation-induced immunogenicity are not well understood. We have examined whether ATR inhibition can potentiate radiation-induced immunogenic cell death and interferon signaling in human cancer cells, and whether caspase activation contributes to regulate these responses.

Human lung cancer and osteosarcoma cell lines (SW900, H1975, A549, H460, U2OS) were treated with radiation and ATR inhibitors (VE822, AZD6738) in the absence and presence of a pan-caspase inhibitor (Q-VD-OPh). Caspase activation and IFN signaling were measured by immunoblotting of cell extracts at 1-6 days after treatment. IFN-β secretion was measured by ELISA. Transfection with siRNAs (CGAS, STING1) was used to explore the hypothesis that cytosolic DNA from ruptured micronuclei activates the cGAS-STING-IFN pathway. Hallmark factors of immunogenic cell death were assessed by immunoblotting of cell growth medium (HMGB1 release), the CellTiter-Glo assay (ATP secretion) and flow cytometry (surface-presentation of calreticulin).

Co-treatment with radiation (5 Gy) and ATR inhibitors abrogated the G2 checkpoint in all cell lines, accompanied by micronucleus formation. In absence of the caspase inhibitor, the radiation-induced IFN response was moderately increased by ATR inhibition in four out of five cell lines. Consistent with activation of the cGAS-STING-IFN pathway, depletion of cGAS or STING abolished the induction of IFN response. Addition of the caspase inhibitor to cells treated with ATR inhibitor and radiation resulted in markedly increased IFN response in all cell lines. The response was dependent on the co-treatment, as no or only a small increase in IFN was found when caspase inhibition was combined with either ATR inhibition or irradiation alone. The ATR inhibitors also increased the release of HMGB1 after irradiation, but this effect was suppressed upon caspase inhibition. Secretion of ATP was increased by ATR inhibition to a variable extent in the different cell lines, and slightly further increased by caspase inhibition. Surface-presentation of calreticulin was increased after irradiation, but not further increased by the ATR or caspase inhibitors.

ATR inhibition can increase IFN signaling and two hallmark factors of immunogenic cell death in irradiated human cancer cells, but the responses vary between cell lines. Treatment-induced caspase activity counteracts the IFN response, but promotes HMGB1 release. Caspase activation thus appears to play both suppressive and stimulative roles in radiation-induced immunogenicity.