In vivo perturbomics identifies CALHM2 as a checkpoint of NK cell therapy [bulkRNAseq]

Natural killer (NK) cells are an innate immune cell type that serves at the first level of defense against pathogens and cancer. NK cells have clinical potential; However, multiple current limitations exist that naturally hinder the successful implementation of NK cell therapy against cancer, including their effector function, persistence, and tumor infiltration. To unbiasedly identify genes underlying critical NK cell characteristics against cancer, we perform functional mapping of tumor infiltrating NK (TINK) cells by both in vivo AAV-CRISPR screens and single-cell sequencing. We establish a strategy with AAV-SleepingBeauty(SB)-CRISPR screening leveraging a focused high-density sgRNA library, and perform four independent in vivo tumor infiltration screens of primary NK cells in mouse models of melanoma, breast cancer, pancreatic cancer, and glioblastoma. In parallel, we characterize single-cell transcriptomic landscapes of tumor-infiltrating NK cells, which identifies previously unexplored sub-populations of NK cells with distinct expression profiles, a shift from immature to mature NK (mNK) cells in the tumor microenvironment (TME), and decreased expression of mature marker genes in mNK cells. CALHM2, a calcium homeostasis modulator previously not linked to NK function, emerges as a convergent hit from both CRISPR screen and single-cell data. CALHM2 knockout NK cells show enhancement of in vitro cytotoxicity and in vivo tumor infiltration in both mouse primary NK and human chimeric antigen receptor (CAR)-NK cells. Re-introduction of CALHM2 mRNA reverses CALHM2 knockout phenotype of NK cytotoxicity and degranulation. Importantly, in a solid tumor model that is completely resistant to adoptive cell therapy of unmodified CAR-NK cells, CALHM2 knockout CAR-NK cells show potent in vivo anti-tumor efficacy. Human primary NK cell study with multiple donors reveals that CALHM2 knockout enhances cytotoxicity, degranulation and cytokine production of NK cells. Transcriptomics profiling of human primary NK cells reveal multiple enriched pathways of downstream genes upon CALHM2 knockout in both baseline and stimulated conditions. These data identify endogenous cellular genetic checkpoints that naturally limit NK cell function, and pinpoint CALHM2 as a key factor in which genetic modification can be engineered to enhance NK cell-based immunotherapies.