Accumulating evidence suggests that exogenous cellular stress induces PD-L1 upregulation in cancer. A DNA double-strand break (DSB) is the most critical type of genotoxic stress, but the involvement of DSB repair in PD-L1 expression has not been investigated. Here we show that PD-L1 expression in cancer cells is upregulated in response to DSBs. This upregulation requires ATM/ATR/Chk1 kinases. Using an siRNA library targeting DSB repair genes, we discover that BRCA2 depletion enhances Chk1-dependent PD-L1 upregulation after X-rays or PARP inhibition. In addition, we show that Ku70/80 depletion substantially enhances PD-L1 upregulation after X-rays. The upregulation by Ku80 depletion requires Chk1 activation following DNA end-resection by Exonuclease 1. DSBs activate STAT1 and STAT3 signalling, and IRF1 is required for DSB-dependent PD-L1 upregulation. Thus, our findings reveal the involvement of DSB repair in PD-L1 expression and provide mechanistic insight into how PD-L1 expression is regulated after DSBs.

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Clonogenic assays are the gold standard for determining radiosensitivity, which governs tumor response to radiation therapy. Although multiple studies of clonogenic assays on cancer cell lines have been published, the robustness of this technique has not been examined by comparative analysis of data from different studies. To address this issue, we investigated the inter-assay precision of clonogenic assays by analyzing in-house and published data on A549, a cell line frequently studied in this context. The coefficients of variation for SF2, the surviving fraction after 2 Gy irradiation, and D10, the radiation dose that reduces survival to 10%, were below 30% for both in-house data obtained from 20 independent experiments performed under consistent experimental settings (i.e., radiation type, dose rate, and timing of cell seeding) and data collected from 192 publications using diverse experimental settings. Multivariate analyses of the published data revealed that timing of cell seeding significantly affected SF2. These data indicate that SF2 and D10 of clonogenic assay have acceptable inter-assay precision, and that timing of cell seeding influences the inter-assay precision of SF2. These results provide a rationale for combined analysis of published clonogenic assay data, which may help to discover robust biological properties associated with tumor radiosensitivity.