Triad-SL: A new synthetic lethality paradigm combining two distinct DNA damage response pathway inhibitors and particle radiotherapy

Current synthetic lethality (SL) strategies in cancer therapy utilize tumor-specific deficiencies in the DNA damage response (DDR)-such as homologous recombination deficiency (HRD) from BRCA1/2 mutations-to selectively eradicate malignant cells by inhibiting backup repair pathways, most notably using PARP inhibitors. However, these vulnerabilities occur in only a small subset of patients, as many tumors exhibit complex or mosaic mutational profiles that only partially resemble classical BRCAness. To overcome this limitation, we propose that engineered synthetic lethality can be achieved by inducing spatially and temporally controlled DNA damage with high-precision particle radiotherapy (RT), combined with dual targeting of distinct DDR pathways. To this end, we systematically evaluated the effect of RT with a combinatorial matrix of potent small-molecule inhibitors targeting ATM, ATR, DNA-PK, and PARP, establishing the Triad-SL paradigm as a framework for next-generation cancer treatment. Single and dual drug concentrations causing minimal or no toxicity when used alone were established by titration experiments to ensure effective cell killing occurs only in the presence of RT and synergistic dual DDR inhibition. Interestingly, we found that conventional viability assays underestimated DDR-dependent effects, especially when combined with RT. When assessed using clonogenic survival assays, the same Triad-SL combinations exhibited up to 1000-fold greater sensitivity. Within their respective pathway classes, DDR inhibitors demonstrated comparable efficacy and synergized effectively with photon- or carbon ion irradiation, respectively. Of note, combined treatment with PARP- and ATM inhibitors produced strong, cell line-specific cytotoxicity, substantially reducing survival fractions even in the absence of irradiation, indicating combined cytotoxic potential. Among all combinations tested, ATM, DNA-PK, and PARP inhibitors yielded the most promising outcomes, producing strong radiosensitizing effects across multiple DDR inhibitor pairings. By leveraging DDR inhibitors with differing biodistribution, pharmacokinetics, and target selectivity, potent synergistic effects may be achieved at reduced doses-particularly when combined with focused irradiation. In conclusion, the multimodal Triad-SL strategy, integrating selective DDR inhibition with tailored radiation quality, represents a promising approach to enhance therapeutic efficacy and broaden the applicability of synthetic lethality in cancer treatment.