Genome-Scale CRISPR-Cas9 Screen Identifies Genes Essential for T Cell-Based Cancer Therapies

Identification: Restifo, N.P.


Genome-Scale CRISPR-Cas9 Screen Identifies Genes Essential for T Cell-Based Cancer Therapies

S.J. Patel1, N. Sanjana2, A. Eidizadeh1, S. Vodnala1, M. Cam1, A.J. Leonardi1, R. Kishton1, T. Yamamoto1, R.L. Eil1, M. Sukumar1, F. Zhang2 and N.P. Restifo1

1NCI, National Institutes of Health, Bethesda, MD, USA; 2Massachusetts Institute of Technology and Broad Institute, Cambridge, MA USA

Somatic gene mutations and dysregulations enable T cell recognition of tumor but can also alter the vulnerability of a cancer cell to T cell-based immune rejection. To systematically study genes that regulate the sensitivity of cancer cells to killing by cytolytic T cells, we developed a two cell-type functional mutagenesis platform by combining human T cell engineering and CRISPR-Cas9 genome editing. We targeted all known human coding genes and miRNA’s using CRISPR libraries containing 123,411 distinct single guide RNAs (sgRNA). Using ‘second best guide’ and RNAi Gene Enrichment Ranking (RIGER) analysis we catalogued genes and pathways whose loss in tumor cells impaired killing by tumor-specific T cells. Using transcriptome data from 11,409 patients from The Cancer Genome Atlas (TCGA) database, we correlated the gene set found using the CRISPR screens with a set of genes having high Peason correlation coefficients with cytolytic effector function (EFT) in 36 cancer histologies. We validated 9 uncharacterized candidate genes with individual gene knock-outs, using sgRNA’s, human target cell lines and human gene-engineered T cells that were not in the screening libraries. We assessed the in vivo relevance of one of these validated candidates, APLNR, using an in vivo adoptive cell T-cell transfer in a mouse model. We found that Aplnr loss in B16 tumors reduced the efficacy of T cell-mediated tumor clearance in immunocompetent hosts in vivo. Finally, we observed mutations of the APLNR G protein–coupled receptor in patients with cancer. Collectively, this study provides a genome-scale functional profile of essential genes in tumors for the cytolytic effector function whose deficiency likely acts as an immune evasion strategy. Further, these data may serve as a guide for patient stratification in cancer immunotherapies.


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