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Radiotherapy works by causing lethal DNA damage to cancer cells. However, in response to the DNA damage, cancer cells can activate DNA repair mechanisms, which may limit treatment efficacy. One promising strategy is thus to combine radiotherapy with inhibitors of DNA damage repair. Interestingly, recent studies suggest that inhibition of DNA repair can also affect anti-tumor immunity. Here, we have developed a large-scale flow cytometry screening method to identify compounds that inhibit DNA damage repair after radiation. Our aim is to explore how these DNA repair inhibitors affect tumor radiosensitivity and anti-tumor immune effects.

Cancer cells were harvested at 30 minutes and six hours after treatment with radiation and compound libraries, and DNA repair was assessed by levels of the DNA damage marker γH2AX. Barcoding with pacific blue staining was included to achieve highly accurate measurements of γH2AX levels. A pipetting robot and a flow cytometer equipped with a plate loader were used for screen automation. In follow-up studies of candidate hits, cell death was monitored by clonogenic survival, and DNA repair and immune signaling were investigated by immunoblotting and flow cytometry.

The screen was performed with more than 1900 compounds, from the Biomol kinase inhibitor, Enzo target and pathway, Cancer Selleck and Prestwick compound libraries, in A549 lung cancer and/or Reh leukemia cells. To verify the feasibility of our screening method for DNA repair, two repetitive screens were conducted in both cell lines with 357 of the compounds. The results were highly reproducible, and among the candidate hits were known regulators of DNA repair such as multiple HDAC inhibitors (e.g. SAHA). In addition, several previously unknown regulators of DNA repair were identified. Notably, largely similar results were obtained for the two cell lines. Results of screens performed in a single cell line will thus likely be widely applicable. A549 cells were further screened with the Cancer Selleck library, and Reh cells with the Prestwick library. Several screen results were validated by acquiring compounds from an independent source and testing the screen endpoints in independent experiments. Preliminary results suggest that some of the DNA repair inhibitors also promote anti-tumor immune effects after irradiation.

Altogether, our screens have identified more than 70 candidate hits. After further preclinical and clinical testing, the identified compounds may potentially be useful in combination with radiotherapy in future treatment approaches.