Abstract A033: Targeting SETD2 in Combination with Radiotherapy in Rectal Cancer

Abstract Background: Histone modifications are central to regulating gene expression, chromatin organization, and DNA repair. Tri-methylation of histone H3 on lysine 36 (H3K36me3), catalyzed exclusively by the methyltransferase SETD2, is critical for the proper execution of homologous recombination and DNA damage repair processes. In KRAS-mutant colorectal cancers, radioresistance represents a significant therapeutic hurdle. Our preliminary studies indicate that oncogenic KRAS upregulates SETD2 expression, suggesting that the KRAS-SETD2-H3K36me3 axis might contribute to the DNA repair proficiency underlying radiation resistance. Hypothesis: We propose that inhibition of SETD2 will disrupt H3K36me3-dependent recruitment of DNA repair complexes, thereby sensitizing KRAS-mutant colorectal cancer cells to ionizing radiation. Methods: To test our hypothesis, we performed comprehensive in vitro studies using three established human KRAS-mutant colorectal cancer cell lines. Genetic inhibition was achieved via RNA interference (RNAi) targeting SETD2, and pharmacologic inhibition was conducted using EZM0414, a selective, orally bioavailable SETD2 inhibitor in preclinical development. Following treatment with either RNAi or EZM0414, cells were exposed to ionizing radiation. Radiosensitivity was quantified using clonogenic assays, while the extent of DNA damage was assessed through immunofluorescence detection of γH2AX foci. Additionally, western blot analyses were performed to evaluate the expression profiles of key DNA damage response proteins. Results: Both genetic and pharmacologic abrogation of SETD2 resulted in a marked enhancement of radiation-induced DNA damage compared to radiation treatment alone. A significant increase in γH2AX foci was observed in cells treated with EZM0414 in combination with ionizing radiation, indicating an elevated accumulation of double-strand breaks. Furthermore, clonogenic assays revealed that inhibition of SETD2 substantially impaired cell survival following radiation exposure. These data collectively support the contention that SETD2-mediated H3K36me3 serves a protective role in the DNA damage response, thereby promoting radioresistance in KRAS-mutant colorectal cancer cells. Conclusion and Translational Impact: Our findings establish a functional link between oncogenic KRAS signaling, SETD2 activity, and DNA repair competence in colorectal cancer, highlighting the SETD2-H3K36me3 axis as a key mediator of radiation resistance. By targeting SETD2, our study provides compelling preclinical evidence for a novel radiosensitization strategy. These results have immediate translational relevance and underscore the potential for integrating SETD2 inhibitors into therapeutic regimens to overcome radioresistance, ultimately improving outcomes in patients with treatment-refractory KRAS-mutant colorectal cancer. Citation Format: Lokesh Akana, Saaimatul Huq, Henrique Rodrigues, Adam R. Wolfe. Targeting SETD2 in Combination with Radiotherapy in Rectal Cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr A033.