Radiosensitization by Kinase Inhibition Revealed by Phosphoproteomic Analysis of Pancreatic Cancer Cells

The proteomes and phosphoproteomes of radiosensitive and radioresistant PDAC cell lines were analyzed. Irrespective of the sensitivity of the cells, the phosphorylation-based radiation-responsive signaling network featured known DDR proteins and novel ATM substrates. Radioresistant cells displayed significant expression levels of apoptotic proteins, including NQO1, and elevated phosphorylation levels of proteins involved in actin dynamics and FAK activity. Sensitization of former resistant PDAC cells toward radiation was realized by pharmacological inhibition of FAK and CHEK by Defactinib and Rabusertib. [Display omitted] Highlights •Proteomes and phosphoproteomes of radiosensitive and radioresistant PDAC cell lines.•Common activation of DDR is proven by ATM activity on known and novel substrates.•Resistant cells bear raised NQO1 expression, actin dynamics including FAK activity.•Inhibitors of CHEK Rabusertib and FAK Defactinib radiosensitize PDAC cells. Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers and known for its extensive genetic heterogeneity, high therapeutic resistance, and strong variation in intrinsic radiosensitivity. To understand the molecular mechanisms underlying radioresistance, we screened the phenotypic response of 38 PDAC cell lines to ionizing radiation. Subsequent phosphoproteomic analysis of two representative sensitive and resistant lines led to the reproducible identification of 7,800 proteins and 13,000 phosphorylation sites (p-sites). Approximately 700 p-sites on 400 proteins showed abundance changes after radiation in all cell lines regardless of their phenotypic sensitivity. Apart from recapitulating known radiation response phosphorylation markers such as on proteins involved in DNA damage repair, the analysis uncovered many novel members of a radiation-responsive signaling network that was apparent only at the level of protein phosphorylation. These regulated p-sites were enriched in potential ATM substrates and in vitro kinase assays corroborated 10 of these. Comparing the proteomes and phosphoproteomes of radiosensitive and -resistant cells pointed to additional tractable radioresistance mechanisms involving apoptotic proteins. For instance, elevated NADPH quinine oxidoreductase 1 (NQO1) expression in radioresistant cells may aid in clearing harmful reactive oxygen species. Resistant cells also showed elevated phosphorylation levels of proteins involved in cytoskeleton organization including a