Targeting the DNA damage response in combination with radiation enhances type I interferon (T1IFN)-driven innate immune signaling. It is not understood, however, whether DNA-dependent protein kinase (DNA-PK), the kinase critical for repairing the majority of radiation-induced DNA double-strand breaks in cancer cells, is immunomodulatory. We show that combining radiation with DNA-PK inhibition increases cytosolic double-stranded DNA and tumoral T1IFN signaling in a cyclic GMP-AMP synthase (cGAS)- and stimulator of interferon genes (STING)-independent, but an RNA polymerase III (POL III), retinoic acid-inducible gene I (RIG-I), and antiviral-signaling protein (MAVS)-dependent manner. Although DNA-PK inhibition and radiation also promote programmed death-ligand 1 (PD-L1) expression, the use of anti–PD-L1 in combination with radiation and DNA-PK inhibitor potentiates antitumor immunity in pancreatic cancer models. Our findings demonstrate a novel mechanism for the antitumoral immune effects of DNA-PK inhibitor and radiation that leads to increased sensitivity to anti–PD-L1 in poorly immunogenic pancreatic cancers.

Our work nominates a novel therapeutic strategy as well as its cellular mechanisms pertinent for future clinical trials combining M3814, radiation, and anti-PD-L1 antibody in patients with pancreatic cancer.