ATR inhibition sensitizes pancreatic cancer cells to cytotoxic and immunogenic effects of X-ray and carbon ion irradiation.

Abstract

Pancreatic ductal adenocarcinoma (PDAC) remains a clinical challenge characterized by an alarmingly low survival rate. Despite surgical advances and new chemotherapy combinations, currently available treatment fails to improve the overall survival of PDAC patients largely due to an immunosuppressive tumor microenvironment. Radiation can induce cell death and reprogramme the tumor microenvironment by promoting anti-tumor immune response. The cGAS-STING and RIG-I-MAVS pathways are central components of the innate immune system that detect cytosolic nucleic acids and initiate type I interferon production. This study aims to leverage radiation-induced DNA damage together with inhibition of DNA repair and cell cycle checkpoints to potentiate type I interferon response and thereby enhance anti-tumor immunogenicity in PDAC. Two different PDAC cell lines (KRAS wild-type BxPC-3 and KRAS-mutated PANC-1) were used to test two different radiation modalities (X-rays and carbon ions) and regimens (single and hypofractionated dose) in combination with ATR and CHK1 inhibitors as well as the STING agonist diABZI. Cell survival, immunogenic cell death, accumulation of cytosolic dsDNA and micronuclei, gene expression profiles, and STING- and NF-κB-dependent immune signaling were assessed. Immune activation was evaluated by incubating immune cells with supernatants from PDAC cells, followed by analysis of activation markers using spectral flow cytometry. Here we demonstrate that ATR inhibition sensitizes BxPC-3 cells to the cytotoxic effects of radiation and potentiates the immunogenic effects of carbon ions and hypofractionated X-rays (3x8 Gy). Increased accumulation of cytosolic dsDNA and micronuclei was coupled with STING-dependent IFNB1 secretion and genome-wide induction of inflammatory gene expression programs, ultimately resulting in the activation of monocytes. This was not the case for PANC-1 cells, where radiation alone exerted immunosuppressive effects on monocytes. Our results support further evaluation of ATR inhibition in combination with radiotherapy in KRAS wild-type pancreatic cancer.