Abstract 3763: Genome-wide CRISPR screens identify critical targets to enhance CAR-NK cell antitumor potency

Abstract Adoptive cell therapy using engineered natural killer (NK) cells has emerged as a promising new treatment, with gene editing offering opportunities to further increase antitumor potency. However, strategies to address both intrinsic and extrinsic therapeutic barriers remain largely unexplored. To address this, we developed a pooled CRISPR discovery platform to identify genetic perturbations that can be exploited for CAR-NK cellular engineering strategies. Using primary human NK cells, we performed multiple genome-wide CRISPR screens under conditions of repeated tumor challenge, relevant to the tumor microenvironment (TME). These screens uncovered novel NK cell checkpoints that regulate resistance to tumor-induced and TME-driven immunosuppression. High-content screening in NK cells with enhanced degranulation capacity validated these findings, identifying phenotypic correlates of how reprogrammed NK cells acquire enhanced potency. Cross-validation of top screen hits in a large patient cohort across 17 diverse cancer types demonstrated transcriptional upregulation of top hits compared to healthy donors, underscoring their clinical relevance. Key pathways enriched among the hits included components of the Cul5-RING ubiquitin ligase, the Mediator and the SAGA complexes, as well as genes involved in chromatin remodeling and negative regulation of NFκB. These findings highlight transcriptional reprogramming and post- translational modification (PTM) components as means to optimize NK cell effector functions for enhanced anticancer immunity. Targeted ablation of top hits MED12, ARIH2 and CCNC markedly improved the antitumor potency of both non-transduced and CAR/IL-15-engineered NK cells against multiple hard-to-treat cancers using two different IL-15-armored CAR receptors targeting CD70 and TROP2. ARIH2 and CCNC deficient TROP2/IL-15-CAR-NK cells displayed enhanced metabolic fitness and a marked increase in activated/cytotoxic clusters (DNAM-1high, Granzyme Bhigh, Perforinhigh, CD69high), with a concomitant decrease in inhibitory subsets (NKG2Ahigh, Granzyme Blow, CD69low), as shown by mass cytometry. Multiplexed ARIH2/CCNC editing of TROP2/IL-15-CAR-NK cells significantly increased NK cell expansion, tumor bed infiltration, release of cytotoxic granules and antitumor efficacy in an orthotopic in vivo mouse model of pancreatic cancer. This was accompanied by the induction of cytotoxic (CD16high, Granzyme Bhigh, Perforinhigh) and activated (DNAM-1high, CD69high, NKG2Dhigh) NK cell subsets. In conclusion, by applying high-content genome-wide CRISPR screening in primary human NK cells, this study revealed novel regulators of NK cell function, including MED12, ARIH2 and CCNC. Targeting these regulators enhances CAR-NK cell metabolic fitness, cytotoxicity and in vivo antitumor efficacy, offering new opportunities for optimizing CAR-NK therapies. Citation Format: Alexander Biederstädt, Rafet Basar, Jeong-Min Park, Merve Dede, John G. Watts, Nadima Uprety, Rejeena Shrestha, Francia Reyes-Silva, May Daher, Bin Liu, Hind Rafei, Pinaki Prosad Banerjee, Ping Li, Sunil Acharya, Sanjida T. Islam, Huihui Fan, Donghai Xiong, Vernikka C. Woods, Paul Lin, Silvia Tiberti, Ana K. Nunez Cortes, Xin Ru Jiang, Inci Biederstädt, Patrick C. Zhang, Ye Li, Seema Rawal, Enli Liu, Luis H. Muniz-Feliciano, Gary M. Deyter, Elizabeth J. Shpall, Natalie W. Fowlkes, Ken Chen, Katayoun Rezvani. Genome-wide CRISPR screens identify critical targets to enhance CAR-NK cell antitumor potency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3763.