Molecular Mechanism Of Pd-L1 Upregulation In Cancer Cells After X-Ray Irradiation

Treatment using immune checkpoint inhibitors have recently demonstrated marked clinical efficacy in cancer treatment. One such strategy is the use of anti-programmed death 1 (PD-1) antibody, which blocks interaction between the PD-1 receptor and its ligand, programmed death ligand-1 (PD-L1). Previous reports reveal that PD-L1 expression is upregulated in cancer cells after X-irradiations. When combined with anti-PD-1 therapy, radiotherapy synergistically reduces tumor growth in vivo, suggesting that the immune suppression caused by PD-L1 upregulation in cancer cells is critically involved in tumor growth. However, the molecular mechanism underlying PD-L1 upregulation after X-ray irradiations is not fully elucidated. In this study, we hypothesized that DNA damage signaling, particularly DNA double strand break (DSB) repair, is involved in PD-L1 upregulation following X-ray irradiations. U2OS cells were irradiated to induce DSBs, and PD-L1 expression was examined by immunoblotting, real-time PCR, and immunofluorescent analysis. Specific inhibitors against Ataxia telangiectasia mutated (ATM), ataxia telangiectasia, and Rad3 related protein (ATR), or Chk1 were examined for their involvement in PD-L1 upregulation after DSBs. To identify the genes inducing PD-L1 upregulation after X-ray irradiations, siRNA screen targeting DSB repair was conducted by immunoblotting. Furthermore, PD-L1 levels in neoplastic samples were examined by TCGA data set analysis to investigate potential correlations between PD-L1 expression and mutations in DSB repair genes. PD-L1 upregulation was observed 24–48 hours after 10 Gy X-ray irradiation and inhibition of the ATM/ATR/Chk1 pathway significantly suppressed this upregulation. The siRNA screen revealed that depletion of either BRCA2 or Ku80 enhanced PD-L1 upregulation after X-ray irradiation. This upregulation required ATM/Chk1 kinase activity in BRCA2 depleted cells. We also found that the upregulation in Ku80-depleted cells requires EXO1-nuclease dependent DNA end resection followed by Chk1 activation In addition, TCGA analysis revealed that neoplastic samples having mutations in BRCA2 or Ku70/80 genes exhibited higher levels of PD-L1 than in those without mutations. Our study reveals that DNA repair pathways are involved in the upregulation of PD-L1 in response to X-rays. PD-L1 expression in cancer cells may be predictive of responses to anti-PD-1 therapy, particularly following radiotherapy. In addition, these data suggest that the efficacy of anti-PD-1 therapy is further enhanced in DSB repair defective cancer cells in combination with radiotherapy.