Abstract 1338: System biological approach identifies heterochromatin modification as an important mechanism of acquired cancer radioresistance

Abstract Radiotherapy (RT) is an integral modality for the treatment of cancer, with >70% patients requiring RT. However, tumor responses to RT are heterogeneous and influenced by several intrinsic and extrinsic factors. Clonal dynamics have identified that in some cancers, radioresistance is dominated by a process of clonal selection and adaptation, thereby suggesting that tumor cells harbor an adaptive pro-survival mechanism to RT. To understand the mechanisms underpinning this process, we generated four radioresistant (RR) cancer models - prostate (22Rv1, DU145) and head and neck (FaDu, HK1) cancer cell lines by exposing them to 90 Gy over 45 fractions delivered over 12 weeks, which mimicked the fractionated radiotherapy schema employed in the clinic. First, WES revealed an enrichment of mismatch repair mutational signatures (SBS6, 14, 15, 20) across our RR models and identified several non-synonymous single nucleotide variants in RAD51AP1, SMC1A, and APC2 genes that are associated with pro-survival pathways. At the transcriptome level, we observed several up- and down-regulated genes across the 4 models, but were able to identify five common overexpressed genes - ATP8A1, BAHD1, CTSD, JAK1, and MYLIP. Over-representation pathway analyses highlighted the association of the BAHD1 gene with an activated heterochromatin formation pathway (rich factor=0.01, adjusted P-value=0.02). This corroborated WB and IF analyses showing an increased expression of heterochromatin markers (H3K9me3 and H3K27me3) in the RR cells post-irradiation, but not in their respective wild-type (WT) parentals. Knockdown of BAHD1 by siBAHD1 treatment consequently reduced the expression of H3K9me3 and H3K27me3 and reversed the RR phenotypes (Clonogeic SF2Gy siBAHD1:control=1.72 [FaDu-RR], SF4Gy siBAHD1:control=1.63 [22Rv1-RR]). Collectively, these findings point to the model that BAHD1-dependent heterochromatin formation is an acquired phenomenon integral to radioresistance in our models. Simultaneously, parallel high-throughput drug screening of our RR and WT cancer cells identified susceptibility to Panobinostat (Pano) - a pan-HDAC inhibitor in vitro and validated in vivo. Combinatorial Pano and AZD7762 - a Chk1 inhibitor exhibited stronger anti-tumor activity against the RR than WT cells (IC50: 0.45 nm [RR] vs 0.60 nm [WT]). Downstream analyses of Pano-treated RR compared with WT tumors further indicated higher apoptosis and reduced proliferation in the former. Finally, knockdown of class I and II HDACs pointed to reversal of HDAC6 expression as the primary target of Pano. Herein, our systems approach of investigations of our RR cancer models uncovered the importance of the heterochromatin in acquired cancer radioresistance, and presented a potential new molecular vulnerability of RR cancer cells to targeting by epigenetic drugs. Citation Format: Chaw Yee Beh, Sharon Pei Yi Chan, Celestia Pei Xuan Yeo, Boon Hao Hong, Evelyn Mui Cheng Tan, Dewi Susanti, Pei Ling Tai, Eugenia Li Ling Yeo, Khee Chee Soo, Edward Kai-Hua Chow, Melvin Lee Kiang Chua. System biological approach identifies heterochromatin modification as an important mechanism of acquired cancer radioresistance [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 1338.