A flow cytometry-based screening platform for identifying candidate radiosensitizers targeting DNA repair

Radiotherapy induces cytotoxic DNA damage, but activation of DNA repair pathways and cell-cycle checkpoints can limit therapeutic efficacy. Here, we developed a high-throughput, flow cytometry-based screening platform to identify compounds that inhibit radiation-induced DNA repair and checkpoint activation. Reh leukemia and A549 lung cancer cells were irradiated and screened against up to 700 bioactive compounds, with DNA damage persistence quantified by {gamma}H2AX levels across independent screens. Cell barcoding using Pacific Blue staining was incorporated to enable highly accurate quantification of {gamma}H2AX across treatment conditions. The platform yielded robust and reproducible results and supported multiparametric analysis, including assessment of G2 checkpoint activation by phospho-histone H3. Largely overlapping candidate radiosensitizers were identified in both cell lines, including the multi-kinase inhibitor 5-iodotubercidin and the PI3K/mTOR inhibitor omipalisib. Validation studies in lung cancer and glioblastoma models confirmed screen performance. Mechanistically, omipalisib reduced phosphorylation of the non-homologous end-joining protein DNA-PK, consistent with impaired double-strand break repair. Both compounds enhanced radiosensitivity in clonogenic survival assays. Notably, 5-iodotubercidin increased radiosensitivity in glioblastoma cells despite previous reports of radioprotective effects in normal brain tissue. Together, these findings establish a robust barcoded screening approach for identifying radiosensitizers that target DNA damage repair and checkpoint responses.