Targeting ATR offers multifaceted treatment strategies involving RAD51-mediated compensatory DNA repair in bladder cancer

Background: Muscle-invasive bladder cancer treatment depends on histological and molecular subtypes. While urothelial carcinoma (UC) benefits from diverse therapies, options beyond radical cystectomy for rare subtypes such as squamous cell carcinoma (SCC) remain limited. We previously demonstrated that ATR inhibitor (ATRi) Ceralasertib, enhanced UC and SCC treatment efficacy in vitro. Thus, we investigated the therapeutic impact of ATRi, its downstream effects and compensatory pathways bypassing ATR dysfunction in patient-derived ex vivo models. Results: Patient-derived ex vivo ATRi-adapted models (p-SCCATRi) were generated through long-term ATRi treatment (Ceralasertib) and characterized via RNA-seq profiling. In p-SCCATRi, ATRi adaptation led to decreased sensitivity (up to 3.3-fold IC50 increase), with compensatory upregulation of DNA repair, particularly homologous recombination (HR) genes like BRCA1 and RAD51, plus chromatin reorganization and immune downregulation. HR upregulation was targetd with application of RAD51 inhibitor (B02). Conclusion: Our results propose ATR as a promising target in bladder cancer by (1) enhancing radiosensitivity through classical ATR inhibition, and (2) exploiting ATRi-adaptation as a vulnerability by targeting compensatory HR activation through RAD51 inhibition. These findings offer novel strategies for improving bladder cancer treatment. Overall design: For patient-derived ex vivo models, cells were isolated from fresh SCC cystectomy tissue and cultured in supplemented MEBM medium with 2% matrigel. Dysfunctional ATR models in p-SCCs (p-SCCATRi) were generated by long-term treatment (>6 months) with the ATR inhibitor Ceralasertib (AZD6738), with the concentration gradually increased from 0.05 µM to 0.60 µM over time. Parallel control cultures were treated with DMSO. Two ATRi-adapted models (p-SCC1ATRi and p-SCC2ATRi) and their respective DMSO-treated controls (p-SCC1DMSO and p-SCC2DMSO) were profiled for transcriptomic changes using RNA-seq.