JNK knockdown enhances CAR-T cell cytotoxicity through NFATc1 in preclinical models of ovarian cancer

Background: Boosting the performance of chimeric antigen receptor T (CAR-T) cell therapy in solid tumors may provide a substantial advantage for cancer patients. Recognizing the vital role of the nuclear factor of activated T cells (NFAT) in T cell function, we hypothesized the strategic regulation of NFAT activity by targeting c-Jun N-terminal Kinases (JNK) can bolster the tumor-eradicating potential of CAR-T cells. Methods: We developed a lentivirally encoded short-hairpin RNA (shRNA) for stable knockdown of JNK in CAR-T cells. CAR-T cells targeting human epidermal growth factor receptor 2 (HER2) were produced from human peripheral blood. Functionality was tested in vitro and in two xenograft models of human ovarian cancer. Results: JNK knockdown in CAR-T cells suppressed antigen-induced stimulation and helper T cell cytokine production, while enhancing anti-tumor cytotoxicity in vitro and in ovarian cancer xenograft experiments. Mechanistically, JNK knockdown altered NFAT signaling to facilitate NFATc1-dependent transcription, leading to elevated levels of granzyme B expression. Conclusions: JNK signaling is a significant regulator of CAR-T cell cytotoxicity, offering a potential strategy to directly enhance CAR-T effectiveness in human cancer therapies.