AbstractChimeric antigen receptor (CAR) T-cell therapy can lead to dramatic clinical responses in B-cell malignancies. However, early clinical trials with CAR T-cell therapy in non–B-cell malignancies have been disappointing to date, suggesting that tumor-intrinsic features contribute to resistance. To investigate tumor-intrinsic modes of resistance, we performed genome scale CRISPR-Cas9 screens in mesothelin (MSLN)-expressing pancreatic cancer cells. Co-culture with MSLN-targeting CAR T cells identified both antigen-dependent and antigen-independent modes of resistance. In particular, loss of the majority of the genes involved in the pathway responsible for GPI-anchor biosynthesis and attachment abrogated the ability of CAR T cells to target pancreatic cancer cells, suggesting that disruption of this pathway may permit MSLN CAR T-cell evasion in the clinic. Antigen-independent mediators of CAR T-cell response included members of the death receptor pathway as well as genes that regulate tumor transcriptional responses, including TFAP4 and INTS12. TFAP4-mediated CAR T resistance depended on the NFκB transcription factor p65, indicating that tumor resistance to CAR T-cell therapy likely involves alterations in tumor-intrinsic states. Overall, this study uncovers multiple antigen-dependent and -independent mechanisms of CAR T-cell evasion by pancreatic cancer, paving the way for overcoming resistance in this disease that is notoriously refractory to immunotherapy.Significance:The identification and validation of key determinants of CAR T-cell response in pancreatic cancer provide insights into the landscape of tumor cell intrinsic resistance mechanisms and into approaches to improve therapeutic efficacy.