Ye et al. developed catalytically dead-guide RNA based genome-wide gain-of function (GOF) CRISPR activation screen, and identified PRODH2 as a lead target to boost primary CD8+ T cell effector functions. Genomic knock-in or lentiviral overexpression of PRODH2 in CAR T cells reprogrammed T cell metabolism, activated the proline metabolic pathway, enhanced mitochondria biogenesis and oxidative metabolism, increased T cell effector function, and enhanced CAR T cell therapy in vitro and in vivo. PRODH2 GOF CAR T cells showed a memory-like phenotype after long-term antigen stimulation.
Contributed by Shishir Pant
ABSTRACT: Chimeric antigen receptor (CAR)-T cell-based immunotherapy for cancer and immunological diseases has made great strides, but it still faces multiple hurdles. Finding the right molecular targets to engineer T cells toward a desired function has broad implications for the armamentarium of T cell-centered therapies. Here, we developed a dead-guide RNA (dgRNA)-based CRISPR activation screen in primary CD8(+) T cells and identified gain-of-function (GOF) targets for CAR-T engineering. Targeted knockin or overexpression of a lead target, PRODH2, enhanced CAR-T-based killing and in vivo efficacy in multiple cancer models. Transcriptomics and metabolomics in CAR-T cells revealed that augmenting PRODH2 expression reshaped broad and distinct gene expression and metabolic programs. Mitochondrial, metabolic, and immunological analyses showed that PRODH2 engineering enhances the metabolic and immune functions of CAR-T cells against cancer. Together, these findings provide a system for identification of GOF immune boosters and demonstrate PRODH2 as a target to enhance CAR-T efficacy.
Author Info: (1) System Biology Institute, Integrated Science & Technology Center, West Haven, CT 06516, USA; Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, US
Author Info: (1) System Biology Institute, Integrated Science & Technology Center, West Haven, CT 06516, USA; Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA; Center for Cancer Systems Biology, Integrated Science & Technology Center, West Haven, CT 06516, USA. (2) System Biology Institute, Integrated Science & Technology Center, West Haven, CT 06516, USA; Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA; Center for Cancer Systems Biology, Integrated Science & Technology Center, West Haven, CT 06516, USA; Yale M.D.-Ph.D. Program, 367 Cedar Street, New Haven, CT 06510, USA; Combined Program in the Biological and Biomedical Sciences, Yale University, New Haven, CT 06510, USA; MCGD Program, Yale University, New Haven, CT 06510, USA. (3) System Biology Institute, Integrated Science & Technology Center, West Haven, CT 06516, USA; Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA; Center for Cancer Systems Biology, Integrated Science & Technology Center, West Haven, CT 06516, USA. (4) System Biology Institute, Integrated Science & Technology Center, West Haven, CT 06516, USA; Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA; Center for Cancer Systems Biology, Integrated Science & Technology Center, West Haven, CT 06516, USA. (5) System Biology Institute, Integrated Science & Technology Center, West Haven, CT 06516, USA; Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA; Center for Cancer Systems Biology, Integrated Science & Technology Center, West Haven, CT 06516, USA; Yale M.D.-Ph.D. Program, 367 Cedar Street, New Haven, CT 06510, USA; Combined Program in the Biological and Biomedical Sciences, Yale University, New Haven, CT 06510, USA; MCGD Program, Yale University, New Haven, CT 06510, USA. (6) System Biology Institute, Integrated Science & Technology Center, West Haven, CT 06516, USA; Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA; Center for Cancer Systems Biology, Integrated Science & Technology Center, West Haven, CT 06516, USA; Yale M.D.-Ph.D. Program, 367 Cedar Street, New Haven, CT 06510, USA; Combined Program in the Biological and Biomedical Sciences, Yale University, New Haven, CT 06510, USA; MCGD Program, Yale University, New Haven, CT 06510, USA; Immunobiology Program, Yale University, New Haven, CT 06520, USA; Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06520, USA. (7) System Biology Institute, Integrated Science & Technology Center, West Haven, CT 06516, USA; Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA; Center for Cancer Systems Biology, Integrated Science & Technology Center, West Haven, CT 06516, USA; The College, Yale University, New Haven, CT 06520, USA. (8) System Biology Institute, Integrated Science & Technology Center, West Haven, CT 06516, USA; Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA; Center for Cancer Systems Biology, Integrated Science & Technology Center, West Haven, CT 06516, USA; Yale M.D.-Ph.D. Program, 367 Cedar Street, New Haven, CT 06510, USA; Combined Program in the Biological and Biomedical Sciences, Yale University, New Haven, CT 06510, USA; MCGD Program, Yale University, New Haven, CT 06510, USA. (9) System Biology Institute, Integrated Science & Technology Center, West Haven, CT 06516, USA; Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA; Center for Cancer Systems Biology, Integrated Science & Technology Center, West Haven, CT 06516, USA. (10) System Biology Institute, Integrated Science & Technology Center, West Haven, CT 06516, USA; Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA; Center for Cancer Systems Biology, Integrated Science & Technology Center, West Haven, CT 06516, USA. (11) System Biology Institute, Integrated Science & Technology Center, West Haven, CT 06516, USA; Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA; Center for Cancer Systems Biology, Integrated Science & Technology Center, West Haven, CT 06516, USA. (12) System Biology Institute, Integrated Science & Technology Center, West Haven, CT 06516, USA; Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA; Center for Cancer Systems Biology, Integrated Science & Technology Center, West Haven, CT 06516, USA. (13) System Biology Institute, Integrated Science & Technology Center, West Haven, CT 06516, USA; Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA; Center for Cancer Systems Biology, Integrated Science & Technology Center, West Haven, CT 06516, USA. (14) System Biology Institute, Integrated Science & Technology Center, West Haven, CT 06516, USA; Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA; Center for Cancer Systems Biology, Integrated Science & Technology Center, West Haven, CT 06516, USA. (15) System Biology Institute, Integrated Science & Technology Center, West Haven, CT 06516, USA; Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA; Center for Cancer Systems Biology, Integrated Science & Technology Center, West Haven, CT 06516, USA. (16) System Biology Institute, Integrated Science & Technology Center, West Haven, CT 06516, USA; Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA; Center for Cancer Systems Biology, Integrated Science & Technology Center, West Haven, CT 06516, USA. (17) System Biology Institute, Integrated Science & Technology Center, West Haven, CT 06516, USA; Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA; Center for Cancer Systems Biology, Integrated Science & Technology Center, West Haven, CT 06516, USA. (18) Advanced Cell Therapy Laboratory, Yale University School of Medicine, New Haven, CT 06520, USA; Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT 06520, USA. (19) Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT 06520, USA; Department of Epidemiology of Microbial Diseases, Yale University School of Public Health, New Haven, CT 06520, USA; Department of Pathology, Yale University School of Medicine, New Haven, CT 06520, USA; Department of Rheumatology, Yale University School of Medicine, New Haven, CT 06520, USA; Center for Biomedical Data Science, Yale University School of Medicine, New Haven, CT, USA; Comprehensive Cancer Center, Yale University, New Haven, CT 06510, USA. (20) System Biology Institute, Integrated Science & Technology Center, West Haven, CT 06516, USA; Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA; Center for Cancer Systems Biology, Integrated Science & Technology Center, West Haven, CT 06516, USA; Combined Program in the Biological and Biomedical Sciences, Yale University, New Haven, CT 06510, USA; MCGD Program, Yale University, New Haven, CT 06510, USA; Immunobiology Program, Yale University, New Haven, CT 06520, USA; Center for Biomedical Data Science, Yale University School of Medicine, New Haven, CT, USA; Comprehensive Cancer Center, Yale University, New Haven, CT 06510, USA; Department of Neurosurgery, Yale University School of Medicine, New Haven, CT, USA; Yale Stem Cell Center, Yale University School of Medicine, New Haven, CT, USA; Yale Liver Center, Yale University School of Medicine, New Haven, CT, USA; Center for RNA Science and Medicine, Yale University, New Haven, CT 06510, USA. Electronic address: sidi.chen@yale.edu.
