Functional Genomic Landscape of Natural Killer Cell Evasion in Multiple Myeloma

Natural killer (NK) cell-based therapies are considered promising future approaches for multiple myeloma (MM) treatment, but immune evasion mechanisms are poorly understood. To determine the mechanisms regulating MM cell response to NK cells, we performed genome-wide (GW) and targeted CRISPR screens in MM cell lines. To further investigate the transcriptional impact of genes identified as regulators of sensitivity to NK cells, we performed additional pooled CRISPR screens with a single-cell (sc) transcriptome readout using the CROP-seq platform and integrated these findings with data from phenotypic assessment of NK cell response of pooled “DNA-barcoded” cell line including 15 MM cell lines (PRISM platform) and molecular profiling of MM patient samples. Our loss-of-function (LOF) and gain-of-function (GOF) GW-CRISPR screens were performed in the MM1.S, LP1, KMS11 MM lines treated with ex vivo expanded NK cells vs untreated control. The top LOF hits were validated using a focused library of ~600 genes. As expected, LOF of class I HLA /antigen presentation machinery genes, transcriptional regulators of HLA and IFNg pathway genes sensitized MM cells to NK cells, confirming that these pathways represent prominent suppressors of NK cell killing. Moreover, LOF of death receptors or downstream effectors was associated with NK resistance; while LOF of the negative regulators of death receptor signaling (e.g. CFLAR, and XIAP) sensitized to NK cell killing. To mechanistically dissect the transcriptional impact of genes identified as regulators of sensitivity to NK cells, we performed scRNAseq using the CROP-seq platform. Pools of MM1.S and LP1 expressing single-guide (sg)RNAs targeting 31 select hits were co-cultured with NK cells for 24 h or left untreated, followed by scRNA-seq and sgRNA detection, differential gene-expression analysis and patient data correlation. The single cell transcriptional profiling of each CRISPR-based LOF for genes of interest documented that disruption of TRAF2, NFKBIA or NFKBIB enhanced NF-kB signaling and was associated with increased expression of the death receptor FAS, a key mediator of NK cell sensitivity (Figure), and changes in expression of BIRC3, CD70, and CXCL10(the latter may further increase immune reactivity through recruitment of T and NK cells). Interestingly, in our PRISM data, high transcriptional NF-kB signatures and/or presence of TRAF3 mutations (an activator of NF-kB signaling) correlated with decreased NK cell resp