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bioRxiv · 10.1101/2025.08.14.666406

JNK knockdown enhances CAR-T cell cytotoxicity

Kuhlmann, C. J.·Jepson, C.·Blucas, M.·Suleiman, F.·Manda, A.·Nagaoka-Kamata, Y.·Kamata, M.
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Abstract

BackgroundBoosting 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 that the strategic regulation of NFAT activity by targeting c-Jun N-terminal Kinases (JNK) can bolster the tumor-eradicating potential of CAR-T cells. MethodsWe 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. ResultsJNK 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 led to elevated levels of granzyme B expression which could be recapitulated through overexpression of NFATc1, suggesting an NFATc1 dependent mechanism of action. ConclusionsJNK signaling is a significant regulator of CAR-T cell cytotoxicity, offering a potential strategy to directly enhance CAR-T effectiveness in human cancer therapies.

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Kuhlmann, C. J., Jepson, C., Blucas, M., Suleiman, F., Manda, A., Nagaoka-Kamata, Y., Kamata, M.. 2025-08-15. JNK knockdown enhances CAR-T cell cytotoxicity. https://doi.org/10.1101/2025.08.14.666406

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