Abstract A029: Mechanistic insight into a germline <i>KDM3C</i> polymorphism associated with chemoradiotherapy outcomes in cancer

Abstract Background: Chemoradiotherapy (CRT) is a standard-of-care treatment for several solid tumors, including locally advanced rectal cancer (LARC) and head and neck squamous cell carcinoma (HNSCC), yet the absence of predictive biomarkers impedes patient stratification and therapeutic optimization. CRT induces DNA double-strand breaks (DSBs), and repair of these lesions is influenced by chromatin architecture, where histone lysine methylation plays a critical role. We identified a germline missense single nucleotide polymorphism (SNP) in KDM3C, a lysine demethylase, that significantly correlates with CRT outcomes. We hypothesize that this serine-to-threonine SNP modulates CRT response by disrupting protein interactions required for BRCA1 recruitment to DNA damage sites. Methods: Isogenic SNP and wild type (WT) rectal (RCM-1) and head and neck (Cal-27, SCC-9) cancer cell lines were generated via CRISPR/Cas9. Functional analyses included immunofluorescence staining of DSB repair proteins, bulk RNA sequencing, and colony survival assays. Structural modeling was conducted using AlphaFold-Multimer (v2.3) and AlphaFold3. Results: SNP-bearing cells exhibited significantly reduced survival after treatment with irradiation, platinum-based and other chemotherapeutics, and PARP inhibitors (p&amp;lt;0.001, unpaired T-test). Bulk RNA-sequencing revealed downregulation of DNA repair and upregulation in immune response genes in SNP-bearing cells (q&amp;lt;0.05). Upon irradiation, SNP cells showed impaired colocalization of RAP80-BRCA1 foci (p&amp;lt;0.001, Mann-Whitney test), indicating defective homologous recombination. TCGA rectal tumors stratified by KDM3C genotype (A/A, A/T, T/T) were analyzed for COSMIC single-base substitution (SBS) mutational signatures. SBS3, a hallmark of homologous recombination deficiency, was absent in A/A (WT) tumors, modestly present in A/T heterozygotes, and most enriched in T/T homozygotes. AlphaFold2 modeling revealed that KDM3C comprises three structured domains: a N-terminal SH3-like β-barrel, a central zinc-binding module (residues 1694-1938) with a KDM3-specific zinc-binding domain and ZZ domain, and a C-terminal JmjC catalytic domain (residues 2150-2450). The rest of KDM3C including the SNP site, is predicted to be intrinsically disordered. Modeling of RNF8’s FHA domain in complex with KDM3C peptides (Ser/Thr at the SNP site, ±phosphorylation) demonstrated higher predicted binding stability for phosphorylated Thr over Ser (order of stability pT &amp;gt; pS &amp;gt;&amp;gt; T &amp;gt; S), consistent with known FHA domain preferences. Additionally, RNF8 FHA domain is predicted to interact with SH3-like domain of KDM3C, further implicating this polymorphism in modulating DSB repair via BRCA1 recruitment. Conclusion. Our findings highlight the functional role of KDM3C SNP in regulating DSB repair and CRT response. This SNP could serve as a stratification marker to enhance CRT efficacy in LARC, HNSCC and potentially other tumors. Citation Format: Adria Hasan, Pragya Priyadarshini, Elena V. Demidova, Philip Czyzewicz, Leonny Gathuka, Takahiko Murayama, Shreya Shah, Gavin Hearne, Mark Andrake, Yan Zhou, Margret B. Einarson, Thomas J. Galloway, Roland Dunbrack, Israel Cañadas, Gail L. Rosen, Barbara Burtness, Erica A. Golemis, Johnathan R. Whetstine, Joshua E. Meyer, Sanjeevani Arora. Mechanistic insight into a germline KDM3C polymorphism associated with chemoradiotherapy outcomes in 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 A029.