Muscle-invasive bladder cancer (MIBC) treatment depends on histological subtypes. While urothelial carcinoma (UC) benefits from novel therapies, options beyond radical cystectomy for rare subtypes such as squamous cell carcinoma (SCC) remain limited. Since we previously demonstrated ATR inhibitor (ATRi) enhanced radiation sensitivity in vitro, we aimed to further decipher the therapeutic impact of ATRi and compensatory pathways bypassing ATRi-resistance in patient-derived ex vivo cultures (PDCs).

PDCs (p-SCC, p-UC, n = 6) were established and characterized by immunohistochemistry, qPCR, and whole-exome sequencing. Independent ATRi-resistant cell models (p-SCC^ATRi) were generated through long-term ATRi treatment (Ceralasertib) and characterized by multi-dimensional profiling. Drug responses were analyzed via cell viability (IC50) and clonogenic survival assays ± ionizing radiation (IR). DNA repair capacity was measured via γH2AX immunofluorescence, comet assays, qPCR, and immunoblotting. ATR siRNA knockdown and ATRi short-term studies validated the ATR-RAD51 axis. RAD51 inhibitor (RAD51i) B02 was tested in p-SCC^ATRi by cell cycle analysis and in ovo tumor growth of chorioallantoic membrane (CAM) xenografts, complemented by apoptosis staining.

ATRi treatment sensitized cells to IR, reducing IC50 values up to 2.5-fold (at 8 Gy: 0.52 µM in SCC, 0.82 µM in UC). Clonogenic assays and γH2AX staining confirmed impaired DNA repair (γH2AX foci at 8 Gy: 11-fold in SCC, 15-fold in UC). In resistant p-SCC^ATRi models, ATRi-adaptation triggered various compensatory and potentially epigenetic regulated DNA repair pathways, particularly homologous recombination (HR) repair involving genes like BRCA1 and RAD51. Downstream consequences of functional ATR loss also affected non-DNA repair processes such as cell cycle, chromatin reorganization, and immunomodulation. As a therapeutic strategy, RAD51i overcame resistance by lowering IC50 by 40–80%, increasing DNA damage (2.2-fold γH2AX foci), and inducing G2/M arrest (2.4-fold). Finally, in ovo, RAD51i significantly induced apoptosis impairing tumor growth in p-SCC^ATRi xenografts by up to 37%.

Our results propose ATR as a promising target in bladder cancer by (1) enhancing radio-sensitivity through classical ATR inhibition and (2) exploiting resistant ATRi-adaptation as a vulnerability by targeting compensatory HR repair reliance through RAD51 inhibition. These findings on the ATR–HR axis suggest novel strategies to improve bladder cancer treatment and addressing therapy resistance.