Using in vivo (AAV)–SB-CRISPR screens and scRNAseq in four solid murine tumor models, Peng and Renauer et al. evaluated the impact of gene knockouts on tumor-infiltrating NK cells, identifying novel NK cells subpopulations with differentially expressed genes. Further, CALHM2 emerged as a potential checkpoint molecule. CALHM2 KO in human NK cells enhanced degranulation, cytokine production, and cytotoxicity in vitro, while CALHM2 KO in mouse NK or human CAR-NK cells improved tumor infiltration and cytotoxicity in vivo, even overcoming resistance in one CAR-NK model. CALHM2 knockout altered gene expression and related pathways both at baseline and upon stimulation.
Contributed by Lauren Hitchings
ABSTRACT: Natural killer (NK) cells have clinical potential against cancer; however, multiple limitations hinder the success of NK cell therapy. Here, we performed unbiased functional mapping of tumor-infiltrating NK (TINK) cells using in vivo adeno-associated virus (AAV)-SB (Sleeping Beauty)-CRISPR (clustered regularly interspaced short palindromic repeats) screens in four solid tumor mouse models. In parallel, we characterized single-cell transcriptomic landscapes of TINK cells, which identified previously unexplored subpopulations of NK cells and differentially expressed TINK genes. As a convergent hit, CALHM2-knockout (KO) NK cells showed enhanced cytotoxicity and tumor infiltration in mouse primary NK cells and human chimeric antigen receptor (CAR)-NK cells. CALHM2 mRNA reversed the CALHM2-KO phenotype. CALHM2 KO in human primary NK cells enhanced their cytotoxicity, degranulation and cytokine production. Transcriptomics profiling revealed CALHM2-KO-altered genes and pathways in both baseline and stimulated conditions. In a solid tumor model resistant to unmodified CAR-NK cells, CALHM2-KO CAR-NK cells showed potent in vivo antitumor efficacy. These data identify endogenous genetic checkpoints that naturally limit NK cell function and demonstrate the use of CALHM2 KO for engineering enhanced NK cell-based immunotherapies.
Author Info: (1) Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. System Biology Institute, Yale University, West Haven, CT, USA. Center for Cancer Systems Biolog
Author Info: (1) Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. System Biology Institute, Yale University, West Haven, CT, USA. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. (2) Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. System Biology Institute, Yale University, West Haven, CT, USA. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. Combined Program in the Biological and Biomedical Sciences, Yale University, New Haven, CT, USA. Molecular Cell Biology, Genetics, and Development Program, Yale University, New Haven, CT, USA. (3) Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. System Biology Institute, Yale University, West Haven, CT, USA. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. (4) Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. System Biology Institute, Yale University, West Haven, CT, USA. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. Combined Program in the Biological and Biomedical Sciences, Yale University, New Haven, CT, USA. Molecular Cell Biology, Genetics, and Development Program, Yale University, New Haven, CT, USA. (5) Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. System Biology Institute, Yale University, West Haven, CT, USA. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. (6) Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. System Biology Institute, Yale University, West Haven, CT, USA. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. (7) Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. System Biology Institute, Yale University, West Haven, CT, USA. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. Combined Program in the Biological and Biomedical Sciences, Yale University, New Haven, CT, USA. Molecular Cell Biology, Genetics, and Development Program, Yale University, New Haven, CT, USA. M.D.-Ph.D. Program, Yale University, West Haven, CT, USA. (8) Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. System Biology Institute, Yale University, West Haven, CT, USA. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. Combined Program in the Biological and Biomedical Sciences, Yale University, New Haven, CT, USA. Molecular Cell Biology, Genetics, and Development Program, Yale University, New Haven, CT, USA. M.D.-Ph.D. Program, Yale University, West Haven, CT, USA. (9) Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. System Biology Institute, Yale University, West Haven, CT, USA. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. (10) Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. System Biology Institute, Yale University, West Haven, CT, USA. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. (11) Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. System Biology Institute, Yale University, West Haven, CT, USA. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. (12) Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. System Biology Institute, Yale University, West Haven, CT, USA. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. Yale College, Yale University, New Haven, CT, USA. (13) Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. System Biology Institute, Yale University, West Haven, CT, USA. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. (14) Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. System Biology Institute, Yale University, West Haven, CT, USA. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. (15) Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. lupeng.ye@gmail.com. System Biology Institute, Yale University, West Haven, CT, USA. lupeng.ye@gmail.com. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. lupeng.ye@gmail.com. Nanjing University, Nanjing, China. lupeng.ye@gmail.com. (16) Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. sidi.chen@yale.edu. System Biology Institute, Yale University, West Haven, CT, USA. sidi.chen@yale.edu. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA. sidi.chen@yale.edu. Combined Program in the Biological and Biomedical Sciences, Yale University, New Haven, CT, USA. sidi.chen@yale.edu. Molecular Cell Biology, Genetics, and Development Program, Yale University, New Haven, CT, USA. sidi.chen@yale.edu. M.D.-Ph.D. Program, Yale University, West Haven, CT, USA. sidi.chen@yale.edu. Immunobiology Program, Yale University, New Haven, CT, USA. sidi.chen@yale.edu. Yale Comprehensive Cancer Center, Yale University School of Medicine, New Haven, CT, USA. sidi.chen@yale.edu. Department of Neurosurgery, Yale University School of Medicine, New Haven, CT, USA. sidi.chen@yale.edu. Yale Stem Cell Center, Yale University School of Medicine, New Haven, CT, USA. sidi.chen@yale.edu. Yale Liver Center, Yale University School of Medicine, New Haven, CT, USA. sidi.chen@yale.edu. Yale Center for Biomedical Data Science, Yale University School of Medicine, New Haven, CT, USA. sidi.chen@yale.edu. Yale Center for RNA Science and Medicine, Yale University School of Medicine, New Haven, CT, USA. sidi.chen@yale.edu.
