Identification and Validation of Molecular Markers of Tumor Cell Resistance to Natural Killer Cells through CRISPR-Based Screens and Large-Scale Phenotypic Screens of Pooled Tumor Cell Lines

Natural killer (NK) cellsexhibit potent activity in pre-clinical models of diverse hematologic malignancies and solid tumors. The in vivo anti-tumor effects of NK cells are not confounded by graft vs. host disease, providing a key advantage over strategies exploiting T cell reactivity. Indeed, infusion of high numbers of NK cells, either autologous or allogeneic, after their ex vivo expansion and activation, has been feasible and safe in clinical studies. Because of these considerations, NK cells had been viewed as a promising therapeutic approach for a disease like multiple myeloma (MM), which remains incurable, even after initial responses to novel immunotherapies, including anti-CD38 antibodies; combinations of thalidomide derivatives with immune checkpoint blockade; and even CAR-T cells. However, our prior studies and more recent unpublished work indicate that tumor cells can exhibit decreased response to NK due to the protective effect of nonmalignant mesenchymal stromal cells and depending on the genetic background of the tumor cells. To our knowledge, there have been limited, if any, reports of systematic screens to quantify NK cell cytotoxicity against large panels of molecularly characterized tumor cell lines. Moreover, while there have been earlier RNAi-based studies, to the best of our knowledge there have not been any genome-wide CRISPR-based screens for candidate NK cell resistance genes. To address this void, we sought to identify and validate mechanisms regulating tumor cell response to NK cells, by performing orthogonal screens in 2 systems: (i) Using similar approaches as in other screens conducted by our lab in other models, we performed a genome-wide CRISPR gene editing screen (GECKO library) in the NK cell-sensitive colorectal Ca line HCT15 and identified candidate genes for which knockout confers resistance to the NK cell line KHYG1. (ii) We studied the cytotoxicity of NK cells (primary NK cells or KHYG1; at different time points and effector-to-tumor [E2T] ratios) against a pool of DNA-barcoded 568 lineage diverse solid tumor lines from the Cancer Cell Line Encyclopedia (CCLE), using the PRISM multiplexed cell viability screening technology. This PRISM screen expanded on a previous screen across 100 cancer cell lines and yielded consistent results supporting the reproducibility of the method. We correlated NK cell cytotoxicity with the CCLE genomic feature data set (mRNA expression, copy number variation, and gene mutational statues) for each of the 568 cell lines. The screens provided candidate "hits" which were concordant in both studies and included recognizable regulators of NK cell biology (e.g. loss-of-function of death receptors such as FAS or gain-of-function of HLA-E correlating with NK cell resistance); as well as other plausible, but previously under-appreciated candidates, which will be considered for further studies in this project. We did not observe significant tumor type-specific differences in NK cell responses in this screen, suggesting that candidate markers identified in these studies may be relevant across lineages, including MM and blood cancers (even though these lines were not included so far in our PRISM or CRISPR screens). Additional ongoing "multi-omic" analyses seek to further examine the links between the phenotypic responses to NK cells with the molecular features of these cell lines (e.g. integrating mutations, DNA methylation, proteomic [RPPA] etc.). Our study highlights the feasibility of combining CRISPR/Cas9-based functional genomic screens (on genome-wide and sub-genome scale) on individual cell lines with large scale phenotypic screens on pooled barcoded cell lines to identify candidate biomarkers of tumor cell sensitivity vs. resistance to NK cells. Notably our PRISM screen represents, to our knowledge, the largest effort to-date to correlate the molecular annotation of tumor cells with their response to uniform treatment, pre-clinical or clinical, to a cell-based immunotherapy.