Genome Scale CRISPR Screens Identify Essential Genes for Sensitivity to Natural Killer Cells in Hematological Malignancies
Year: 2018

Immunotherapy is a promising approach to improve treatment responses in hematological malignancies. Accumulating evidence suggests a role for natural killer (NK) cells in controlling hematological malignancies. However, mechanisms regulating sensitivity or resistance of hematologic cancer cells to the effector function of NK cells are incompletely understood. Here, we performed genome scale CRISPR Cas9 loss of function screens to systematically map genes that regulate sensitivity of hematologic malignancies to NK cells. To screen for genes involved in the interaction between NK and cancer cells, we infected human cancer cells expressing Cas9 with a genome scale lentiviral guide RNA library  The resulting pool of knockout cells was exposed to NK cells expanded from peripheral blood of healthy donors. Enriched and depleted knockouts were detected by next generation sequencing of the integrated sgRNA cassettes, enabling identification of genes conferring resistance or susceptibility to NK cell mediated lysis. The screens were performed in cell lines from diverse hematological malignancies, including chronic myeloid leukemia (CML) B cell acute lymphoblastic leukemia, diffuse large B cell lymphoma (DLBCL) and multiple myeloma. We recovered several known mechanisms of NK cell/cancer cell interactions, demonstrating feasibility of the screening approach. Loss of genes encoding components of the MHC class I complex (B2M, HLA A, HLA C, HLA E) sensitized multiple cancer cell lines to NK cell lysis, consistent with missing self recognition. Furthermore, knockout of IFN JAK STAT signaling mediators led to increased tumor cell lysis, suggesting that MHC class I induction in response to NK cell derived IFN gamma enables NK cell evasion by tumor cells. We also identified genes essential for effective NK cell mediated lysis. NCR3LG1, encoding the B7 H6 ligand for the NKp30 activating NK cell receptor, was essential for NK cell lysis of CML cells. In contrast, knockout of apoptotic mediators and TRAIL pathway components conferred resistance to NK cell cytotoxicity in DLBCL cells, indicating heterogeneity in NK cell/cancer cell interactions between cancer types. Our data support a view that distinct mechanisms regulate sensitivity to NK cell cytotoxicity in different hematologic cancers. Importantly, our results indicate that loss of function mutations in the antigen presenting machinery and the IFN JAK STAT pathway sensitize tumors to NK cell effector function. As alterations in these genes are associated with resistance to T cell immunotherapies such as PD 1 blockade, NK cell based therapies could be employed to overcome resistance in these patients. In summary, we suggest that systematic identification of mechanisms governing tumor immune susceptibility has the potential to uncover novel immunotherapy targets. Merck, Sharp, and Dohme: Consultancy;  Equity Ownership, Membership on an entity9s Board of Directors or advisory committees; CytoSen Therapeutics: Equity Ownership, Membership on an entity9s Board of Directors or advisory committees, Pfizer: Honoraria, Research Funding; Novartis: Honoraria,  Honoraria;  Research Funding; Bristol Myers Squibb: Honoraria,