Abstract A065: Genome-scale CRISPR screens identify essential genes for tumor sensitivity to NK cells

Harnessing natural killer (NK) cells to attack tumors could improve immune-based cancer treatment strategies. However, mechanisms regulating sensitivity or resistance of cancer cells to the effector function of NK cells are incompletely understood. Here, we performed genome-scale CRISPR-Cas9 loss-of-function screens in human cancer cells to discover genes that influence susceptibility to primary human NK cells. To screen for genes essential for the interaction between NK cells 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 knockout clones 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 cancer types, 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-mediated lysis. This is consistent with missing-self recognition as a fundamental mechanism of NK cell activation. 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 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. Citation Format: Olli Dufva, Jay Klievink, Khalid Saeed, Matti Kankainen, Mette Ilander, Tiiina Hannunen, Sonja Lagstrom, Pekka Ellonen, Dean A Lee, Satu Mustjoki. Genome-scale CRISPR screens identify essential genes for tumor sensitivity to NK cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A065.