CRISPR-Cas9 Screen Identifies DYRK1A as a Target for Radiotherapy Sensitization in Pancreatic Cancer

Simple Summary Pancreatic cancer is the fourth leading cause of cancer-related death in Western countries. Although several therapeutic strategies have been developed for pancreatic cancer, radiation therapy has not yet yielded satisfactory results. Unraveling the mechanism of radioresistance in pancreatic cancer and developing new therapeutic targets has become a major challenge. Therefore, we applied kinome-wide CRISPR-Cas9 loss-of-function screening combined with the 3D cell culture method and identified DYRK1A as a sensitive target for radiotherapy. Additionally, we confirmed that DYRK1A-targeted inhibitors could enhance the efficacy of radiotherapy. Our results further support the use of CRISPR-Cas9 screening to identify novel therapeutic targets and develop new strategies to enhance radiotherapy efficacy in pancreatic cancer. Although radiation therapy has recently made great advances in cancer treatment, the majority of patients diagnosed with pancreatic cancer (PC) cannot achieve satisfactory outcomes due to intrinsic and acquired radioresistance. Identifying the molecular mechanisms that impair the efficacy of radiotherapy and targeting these pathways are essential to improve the radiation response of PC patients. Our goal is to identify sensitive targets for pancreatic cancer radiotherapy (RT) using the kinome-wide CRISPR-Cas9 loss-of-function screen and enhance the therapeutic effect through the development and application of targeted inhibitors combined with radiotherapy. We transduced pancreatic cancer cells with a protein kinase library; 2D and 3D library cells were irradiated daily with a single dose of up to 2 Gy for 4 weeks for a total of 40 Gy using an X-ray generator. Sufficient DNA was collected for next-generation deep sequencing to identify candidate genes. In this study, we identified several cell cycle checkpoint kinases and DNA damage related kinases in 2D- and 3D-cultivated cells, including DYRK1A, whose loss of function sensitizes cells to radiotherapy. Additionally, we demonstrated that the harmine-targeted suppression of DYRK1A used in conjunction with radiotherapy increases DNA double-strand breaks (DSBs) and impairs homologous repair (HR), resulting in more cancer cell death. Our results support the use of CRISPR-Cas9 screening to identify new therapeutic targets, develop radiosensitizers, and provide novel strategies for overcoming the tolerance of pancreatic cancer to radiotherapy.