Abstract 639: Targeting TET3 potentiates an effective anti-tumor immune response after irradiation

Abstract The benefits of radiotherapy (RT) have long been appreciated as limited by formation and repair of DNA double-strand breaks (DSBs) in tumor cells, but recent understanding has identified a key role for RT and DNA damage in promoting inflammation that drives anti-tumor immune response. While chromosomal DSBs indeed directly limit cancer cell survival and proliferation, RT-induced damage leads to cytosolic DNA that activates cGAS/STING/IRF3 signaling and drives IFNα/β expression, recruiting cytotoxic T and NK cells to shrink tumors. In turn, along with DSB repair, another determinant of RT resistance is rebound immunosuppression. Here, IFNγ released by the infiltrating lymphocytes activates JAK/STAT to induce interferon-stimulated genes (ISGs) including PD-L1 that can reduce tumor inflammation and restore growth. Though combining PD-1/PD-L1 blockade with RT is sufficient to block this adaptive response in mice, the lack of benefit to patients suggests the need to more broadly target immunosuppressive ISG expression. Targeting IFNγ signaling by blocking JAK/STAT may limit immunosuppressive ISG expression but will impair T and NK cytotoxicity. Looking downstream, blocking JAK/STAT-dependent ISG promoter CpG demethylation by Ten Eleven Translocation (TET) dioxygenase enzymes may prevent PD-L1 expression without affecting cytotoxicity. In this study, we disrupted TET activity with small-molecule inhibitors and CRISPR RNP knockouts (KO) to evaluate effects on radiation response in vitro and in tumors. As predicted, TET inhibitors blocked PD-L1 induction in irradiated CT26 murine colon carcinoma cells. Assaying TET1, 2 and/or 3 KO CT26 cells for PD-L1 induction identified TET3 as the critical mediator. TET3 KO cells also exhibited slower DSB repair after irradiation, associated with markedly increased ssDNA resection and cytosolic DNA. The irradiated TET3 KO cells also displayed enhanced IFNα/β expression, suggesting intact cGAS/STING/IRF3 signaling. While TET3 KO CT26 cells formed tumors in syngeneic BALB/c mice much like CT26 controls, RT produced greater NK cell infiltrates by one week and increased tumor elimination by two weeks. TET3 KO 4T1 mammary carcinoma cells formed orthotopic tumors in BALB/c mice but produced fewer lung metastases compared to controls. In conclusion, TET3 may offer an attractive, druggable target for radiosensitization by slowing DSB repair, enhancing tumor inflammation, and blocking immunosuppressive ISG expression. Citation Format: Sera Averbek, Stephen J. Kron. Targeting TET3 potentiates an effective anti-tumor immune response after irradiation [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 639.