Abstract PR03: Use of CRISPR knockout screen to identify genes that regulate tumor cell sensitivity to NK cell-based immunotherapy

Natural killer (NK) cells play a key role in tumor immune surveillance by their ability to recognize and kill both hematologic and solid tumor cells. NK cell-based immunotherapy is emerging as a powerful treatment against certain malignancies. However, intrinsic and extrinsic mechanisms can lead tumor cells to develop resistance mechanisms to escape NK cell-mediated killing, including up- or downregulation of genes involved in the interaction between tumor and immune cells. Here, we use a “two cell type” (TCT) screening system to identify novel molecular targets regulating NK cell-mediated killing of glioblastoma stem cells (GSC), meningioma cells, as well as head and neck squamous cell carcinomas (HNSCC). By using a whole genome-scale CRISPR-Cas9 library, we induced mutations in tumor cells to mimic loss of function mutations involved in resistance or sensitivity to NK cells. After a round of selection with human peripheral blood NK cells, we profiled the genes that impaired the killing activity of NK cells or increased the sensitivity of tumor cells to NK cells. The most common genes lost in GSC and meningioma cells that lead to increased sensitivity to NK cells are involved in the ER-phagosome pathway, antigen presentation, and cellular localization. We then used GSC to validate our top genetic hits identified by the screening. One gene of interest, CHMP2A, is a component of ESCRT-III, a complex involved in multivesicular bodies and exosomes formation known to be involved in intercellular communications, mediating numerous physiologic processes among which are immunology and tumor biology. We confirmed that impaired expression of CHMP2A in GSC lines increases their sensitivity to NK cell-mediated killing. RNA sequencing demonstrates knockout (KO) of CHMP2A leads to increased expression of immune response genes, including certain chemokines. Indeed, NK cells demonstrated increased migration towards KO GSC corresponding to higher concentrations of CXCL10, CXCL12, and IL-8 in GSC-KO conditioned media. Since HNSCC are among the most highly immune-infiltrated cancer types, including a high degree of infiltrating NK cells, we next tested CHMP2A-KO-Cal27 cells as a model of HNSCC. Again, we observed an increased sensitivity to NK cells, higher levels of CXCL10, and increased migration of NK cells, demonstrating these mechanisms regulate NK cell-mediated killing of diverse tumor types. Additionally, in vivo studies using a Cal27 xenograft model and adoptive transfer of human NK cells in immunodeficient mice demonstrate increased NK cell-mediated killing of CHMP2A-KO-Cal27 cells compared to wild-type Cal27 cells. Together, these studies demonstrate the utility of a TCT screen to identify new mechanisms that mediate resistance of diverse tumor types to NK cell-mediated killing and allow us to now identify novel strategies to further improve this immunotherapeutic approach. This abstract is also being presented as Poster A25. Citation Format: Davide Bernareggi, Qi Xie, Briana Prager, Li Xiqing, Silvio Gutkind, Dan S. Kaufman. Use of CRISPR knockout screen to identify genes that regulate tumor cell sensitivity to NK cell-based immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr PR03.