Homologous Recombination and Alternative End-Joining Repair Pathways are Important Determinants of Radiosensitivity to Proton Radiation Therapy.

Proton beam radiation therapy (PBT) offers superior accuracy of dose deposition, reducing the risk of adverse effects to surrounding healthy tissues. However, despite high medical need, molecular and cellular determinants of radiosensitivity to PBT remain underexplored, and prognostic biomarkers and therapeutic targets informing precision medicine strategies for PBT are mostly missing. This study aimed to investigate the role of DNA double-strand break (DSB) repair pathways in shaping tumor response to proton versus photon radiation. The study employed genetic and pharmacologic methods to impair DSB repair, including CRISPR-Cas9 gene editing to generate DSB repair deficient (ATM, PARP1, and BRCA2 knockout) A549 and HCT116 cell lines, and pharmacologic inhibitors of ATM and PARP using KU55933 or AZD1390 and olaparib, respectively. Cellular responses to photon (x-rays) and proton irradiation were evaluated through clonogenic survival assays, crystal violet proliferation, and annexin V/7AAD apoptosis assays. To investigate DNA repair mechanisms, U2OS reporter systems were employed, complemented by chromosomal aberration analysis, and pulsed-field gel electrophoresis. Finally, the translational relevance of the findings was validated using the chorioallantoic membrane assay closer representing an in vivo situation. PBT triggered a stronger activation of resection-dependent DNA repair pathways, primarily homologous recombination and alternative end-joining (alt-EJ), compared with photon irradiation. This increased activation was further supported by classical cytogenetics results. Tumor cells deficient in BRCA2, ATM, or PARP1 showed significantly increased sensitivity to PBT, highlighting enhanced relative biological effectiveness in both, in vitro and in the chorioallantoic membrane model. Importantly, combining PBT with olaparib, AZD1390 or KU55933 potentiated tumor cell killing, even in repair-proficient models, showing synergy not observed with photons. The observed genotype-specific or drug-induced increase in radiosensitivity toward PBT highlights the promise of genetic profiling of DSB repair defects for biology-driven patient stratification and the use of PARP inhibitors in guiding personalized proton radiation therapy strategies.