Hahn, William CHagel, Kimberly Rose2022-06-0720222022-06-062022-05Hagel, Kimberly Rose. 2022. Systematic interrogation of tumor cell resistance to CAR T cell therapy in pancreatic cancer. Doctoral dissertation, Harvard University Graduate School of Arts and Sciences.29209089https://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37372137Chimeric antigen receptor (CAR) T cell therapy has been highly successful in the treatment of B cell malignancies, with response rates ranging from 50 to 90% and many patients achieving durable complete responses. This success engendered intense interest in the application of CAR T cell therapy for the treatment of solid tumors, which comprise 90% of cancers. Pancreatic ductal adenocarcinoma (PDAC) is a solid tumor with acute unmet need, causing the lowest relative 5-year survival rate of all cancers. As such, multiple CAR T cell therapies have been developed for PDAC, the majority of which target the glycosylphosphatidylinositol (GPI) linked surface protein mesothelin (MSLN). However, MSLN CAR T cell therapy has thus far failed to induce durable responses in solid tumors, with PDAC appearing particularly refractory. This lack of response is suggestive of basal resistance to MSLN CAR T cell therapy in PDAC and other solid tumors. To systematically explore tumor intrinsic modes of CAR T cell resistance and inform strategies to optimize CAR T cell therapy, we performed complimentary genome-scale loss-of-function screens. Specifically, we employed florescence activated cell sorting to identify genetic regulators of MSLN cell surface expression that represent putative mediators of antigen dependent MSLN CAR T cell evasion. Secondly, we performed co-culture screens with MSLN CAR T cells and MSLN-expressing PDAC cells to select for functional mediators of CAR T resistance. Together, these screens identified both antigen dependent and independent modes of MSLN CAR T cell evasion. For example, the GPI anchor pathway emerged as a prominent regulator of MSLN surface expression, with deficiencies in this pathway imparting resistance to MSLN CAR T cell cytotoxicity. We also identified the transcription factor TFAP4 as an antigen-independent effector of CAR T sensitivity, and downstream analyses highlighted NFκB signaling as a mediator of TFAP4-driven resistance. In total, our work explored the landscape of tumor-intrinsic resistance to MSLN CAR T cell therapy, identifying multiple genetic determinants of therapeutic efficacy.application/pdfenCAR T cellImmunotherapyMesothelinNfkBPancreatic cancerBiologyImmunologyThesis or Dissertation2022-06-070000-0002-2309-8720