Targeting TET3 cytosine demethylase for immunogenic radiosensitizatioin - Abstract Colorectal cancer (CRC) is a leading cause of cancer mortality, and once metastatic, cure is rare. Mismatch repair-proficient (pMMR) CRC is particularly challenging because it fails to respond to immune checkpoint blockade. Radiotherapy (RT) can improve outcomes, yet durable control is limited by tumor-intrinsic resistance and adaptive immune escape. Beyond direct killing, RT generates cytosolic DNA, activates cGASSTING, and induces type I interferons that can drive anti-tumor immunity, but the same signaling induces PDL1 and other checkpoints that shield surviving cells from cytotoxic T-cell infiltration and promote resistance and recurrence. We have implicated the cytosine demethylase TET3, a gene often overexpressed in cancer and associated with poor outcomes, as a determinant of both DNA repair and adaptive resistance after RT. In CRC models, TET3 loss delays double-strand break (DSB) repair, increases cytosolic DNA, and heightens interferon signaling while failing to induce PD-L1 in response to STING activation or interferons. In vivo, TET3-deficient tumors are highly radiosensitive and display increased infiltration of cytotoxic T cells with elevated granzyme B. We hypothesize that TET3 supports tumor survival after RT by facilitating DSB repair and enabling interferon-mediated immune suppression. Temporarily disabling TET3 should potentiate RT by producing persistent, immunogenic DNA damage while preventing feedback inhibition of the anti-tumor response, and may also improve the efficacy of checkpoint blockade in pMMR CRC. Toward our aims, we will use CRISPR loss-of-function models in CRC cell lines and syngeneic tumors to quantify the effects of TET3 disruption on DSB repair kinetics, cytosolic DNA accumulation, cGAS-STING and interferon signaling, PD-L1 induction, and tumor control after RT. Vertebrate animal models are essential for this project because external-beam RT acts within vascularized tumors, where perfusion, oxygenation, stromal responses, and immune cell trafficking shape DNA damage, tumor cell survival, and therapeutic response. Syngeneic mouse tumor models further allow us to evaluate tumor-intrinsic TET3 function in the setting of an intact, dynamic immune system, including cytotoxic T-cell infiltration, checkpoint induction, antitumor immune control, and response to PD-1/PD-L1 blockade. In parallel, we will test small-molecule TET inhibitors with RT in vivo, measuring tumor growth, survival, and immune infiltration, and assess whether TET inhibition improves response to PD-1/PD-L1 blockade. This R21 will determine whether inhibiting TET3 creates a therapeutic opportunity to combine RT with immune activation in CRC. Positive results would support development of TET3-selective inhibitors or degraders as adjuvants to RT in CRC and other immunoresistant solid tumors.
