Abstract 1468: Chemotherapy-resistant bladder cancer: Involvement of homologous recombination repair

Abstract Introduction: Radical cystectomy after neoadjuvant cisplatin-based chemotherapy (NAC) remains the standard of care for localized and locally advanced muscle invasive bladder cancer (MIBC). However, around 65% of patients exhibit resistance to this regimen. HUS1, an important protein in the homologous recombination repair (HRR) pathway, has been associated with NAC resistance in different studies. While the HRR pathway, including proteins such as CHK1 and RAD51, is known to play a role in chemotherapy resistance, the specific mechanisms in MIBC remain underexplored. This study aims to investigate the role of HRR in cisplatin-based chemotherapy resistant bladder cancer. Materials & Methods: Cisplatin-resistant (CR) HT1197 and UMUC3 cell lines were developed via gradual cisplatin exposure for more than 3 months. To measure HRR in the cells, we developed a DRGFP assay. Using western blot and RNAseq, we compared differences in protein and RNA levels of DNA repair pathway members between the parental and CR cells. Using an Incucyte® imager, we analyzed cell proliferation after silencing or overexpressing HUS1 and RAD51 alone, or in combination with cisplatin treatment. Furthermore, we tested olaparib and radiation therapy in our models. Results: RAD51 and CHK1 protein levels are respectively 2.69 and 2.07 times higher expressed in our CR cells, underscoring enhanced HRR activity as a potential mechanism of resistance. These elevated RAD51 and CHK1 levels represent promising new biomarkers for predicting cisplatin response. Additionally, our DRGFP assay revealed a 2.5 times increased HRR activity in the CR cell lines compared to their parental lines. We observed a decreased proliferation in the parental (1.3x fold decrease), but not in the CR cells, when combining cisplatin treatment with silencing HUS1. In contrast, decreased proliferation was observed in both parental and CR cells when combining cisplatin with silencing RAD51 (fold decrease of respectively 4.5 and 2.25). Therefore, targeting RAD51 might be a novel approach to overcome chemotherapy resistance. Moreover, western blot data revealed a regulatory interaction between HUS1 and RAD51. Our data also indicated that CR cells exhibit increased resistance to other DNA-damaging agents, such as radiation therapy and the PARP inhibitor olaparib. Finally, RNAseq identified additional signatures potentially involved in chemotherapy resistance, including energy-related pathways and epithelial-mesenchymal transition markers. Conclusion: To the best of our knowledge, we are the first to show increased HRR in cisplatin-resistant bladder cancer cells and to give initial insights into the role of HRR in platinum-based chemotherapy resistance. Our findings propose promising therapeutic strategies to overcome chemotherapy resistance in bladder cancer. Citation Format: Nils C. van Creij, Francisco J. Roa, Florian Handle, Frédéric R. Santer, Per Sonne Holm, Zoran Culig, Renate Pichler. Chemotherapy-resistant bladder cancer: Involvement of homologous recombination repair [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 1468.