LAUSR

Abstract Radiotherapy is the most widely used cancer treatment and improvements in its efficacy and safety are highly sought-after. Peposertib (also known as M3814), a potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor, effectively suppresses the repair of radiation-induced DNA double-strand breaks (DSB) and regresses human xenograft tumors in preclinical models. Irradiated cancer cells devoid of p53 activity are especially sensitive to the DNA-PK inhibitor, as they lose a key cell-cycle checkpoint circuit and enter mitosis with unrepaired DSBs, leading to catastrophic consequences. Here, we show that inhibiting the repair of DSBs induced by ionizing radiation with peposertib offers a powerful new way for improving radiotherapy by simultaneously enhancing cancer cell killing and response to a bifunctional TGFβ “trap”/anti-PD-L1 cancer immunotherapy. By promoting chromosome misalignment and missegregation in p53-deficient cancer cells with unrepaired DSBs, DNA-PK inhibitor accelerated micronuclei formation, a key generator of cytosolic DNA and activator of cGAS/STING-dependent inflammatory signaling as it elevated PD-L1 expression in irradiated cancer cells. Triple combination of radiation, peposertib, and bintrafusp alfa, a fusion protein simultaneously inhibiting the profibrotic TGFβ and immunosuppressive PD-L1 pathways was superior to dual combinations and suggested a novel approach to more efficacious radioimmunotherapy of cancer. Implications: Selective inhibition of DNA-PK in irradiated cancer cells enhances inflammatory signaling and activity of dual TGFβ/PD-L1 targeted therapy and may offer a more efficacious combination option for the treatment of locally advanced solid tumors.