Abstract 7159: Characterizing the role of a <i>KDM3C</i> germline polymorphism in chemoradiotherapy outcomes for rectal and head and neck cancers

Abstract Background: Predictive biomarkers for chemoradiotherapy (CRT) are crucial for personalized cancer treatment. We have identified a germline missense polymorphism in lysine modifier, KDM3C that significantly associates with CRT outcomes in locally advanced rectal cancer (LARC) and head and neck squamous cell carcinoma (HNSCC). KDM3C regulates cellular processes including double-strand break (DSB) repair and is the first lysine modifier to directly influence BRCA1-mediated DSB repair. We hypothesized that the identified polymorphism, a serine to threonine substitution, modulates CRT responses by altering protein interactions necessary for BRCA1-recruitment to DSB sites. Methods: Isogenic rectal (RCM-1) and head and neck (Cal-27, SCC9) cancer lines were generated via CRISPR/Cas9-editing; wild-type (WT) and SNP pairs. Immunofluorescence (IF) staining of DSB repair proteins, bulk RNA-sequencing, and colony survival assays were performed. Structure-function studies were conducted using AlphaFold-Multimer (v2.3) and AlphaFold3. Results: SNP-bearing cells exhibited significantly reduced survival compared to WT cells following treatment with various DNA-damaging agents, including irradiation, platinum-based and other chemotherapeutics, and PARP inhibitors (p&amp;lt;0.001, one-way ANOVA). RNA-sequencing revealed downregulation in DNA repair and upregulation in immune response genes in SNP-bearing cells compared to the WT (q&amp;lt;0.05). To elucidate the mechanisms behind these results, we explored the regulation of the RNF8-RAP80-BRCA1 pathway in DSB repair, directly influenced by KDM3C. AlphaFold modeling of the FHA domain of RNF8 in complexes with KDM3C peptides (Ser/Thr at SNP position, with/without phosphorylation) showed significantly more stable complexes with KDM3C phospho-peptides, with an order of stability pT&amp;gt;pS&amp;gt;&amp;gt;T&amp;gt;S; aligning with prior studies on FHA peptide binding showing a preference for pThr over pSer. In fact, multiple proteins in BRCA1-mediated DSB repair involve FHA-phospho-peptide interactions, and KDM3C SNP region is implicated to play an important role in modulating this process. Supporting AlphaFold predictions, significantly reduced colocalization of RAP80-BRCA1 was observed in SNP-cells versus WT upon irradiation, indicating deficiency in DSB repair (p&amp;lt;0.01, Mann-Whitney test). Further, SNP-bearing cells exhibited increased colocalization of cGAS with micronuclei, a marker of genomic instability (p&amp;lt;0.05, one-way ANOVA), alongside an upregulated expression of genes associated with innate immune response (p&amp;lt;0.05, unpaired t-test). Conclusion: Results highlight the role of the KDM3C SNP in DSB repair, influencing the response to DNA-damaging therapy and triggering broader genomic and immune responses. These findings position this polymorphism as a potential stratification marker to improve CRT efficacy in LARC and HNSCC. Citation Format: Adria Hasan, Pragya Priyadarshini, Elena V. Demidova, Philip Czyzewicz, Takahiko Murayama, Shreya Shah, Mark Andrake, Yan Zhou, Roland Dunbrack, Israel Cañadas, Thomas J. Galloway, Margret B. Einarson, Barbara Burtness, Erica A. Golemis, Joshua E. Meyer, Sanjeevani Arora. Characterizing the role of a KDM3C germline polymorphism in chemoradiotherapy outcomes for rectal and head and neck cancers [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 7159.