OAB-019: CRISPR screens with single-cell transcriptome readout reveal potential mechanisms of response to natural killer cell treatment in multiple myeloma

Background Natural killer (NK) cell-based therapy is a promising approach to improve treatment responses in multiple myeloma (MM), which remains incurable. However, genetic determinants and mechanisms of sensitivity or resistance to NK cells in MM are incompletely understood. Here, we investigated the transcriptional impact of genes identified as regulators of sensitivity to NK cells in genome-wide CRISPR screens and integrated the findings with patient-derived data. Methods To systematically dissect the mechanisms by which select genes identified in our previous genome-wide studies influence MM cell response to NK cells, we performed pooled CRISPR screens with a single-cell (sc) transcriptome readout using the CROP-seq platform. Based on genome-wide screen ranking and biological relevance, 31 genes were selected. Pools of MM1.S and LP1 expressing sgRNAs targeting select hits were co-cultured with NK cells for 24 h or left untreated, followed by scRNA-seq and sgRNA detection and assignment to cells, followed by differential gene-expression analysis and patient data correlation. Results The analysis of the transcriptomic responses in MM cells upon NK cell exposure revealed that engagement by NK cells resulted in activation of IFNy-JAK-STAT signaling and upregulation of HLA class I genes, including the inhibitory NKG2A ligand HLA-E, as well as HLA class II. We then evaluated the impact of each sgRNA perturbation on gene expression in each condition, compared to the untreated control. Silencing of IFNy signaling mediators (JAK1, JAK2, STAT1, IFNGR2) prevented the induction of the IFNy response upon NK cell exposure, providing a mechanism for the observed sensitization to NK cells via reduced expression of HLA I genes. Disruption of TRAF2, NFKBIA, or NFKBIB, which also sensitized MM1.S to NK cells, induced NF-kB signaling. The induced NF-kB signature included increased expression of the death receptor FAS, pivotal for susceptibility to NK cells in the MM1.S CRISPR screen. We then correlated the findings with patient data from the CoMMpass dataset. Mutations in the NF-kB negative regulators TRAF2 and NFKBIA were associated with increased expression of NF-kB target genes also identified experimentally by CROP-seq. Also, NFKBIA mutations were linked to reduced HLA-E expression in both MM patients and CROP-seq, suggesting a potential explanation for the NK-sensitizing effect of NFKBIA disruption. Interestingly, patients with NLRC5 mutations had lower HLA-E expression consistent with CROP-seq data, indicating that although rare, NLRC5 mutations may predispose MM cells to higher NK-sensitivity. Conclusions Our data shed light on mechanisms of response to NK cells in MM and identify potential therapeutic targets for combination treatment. Furthermore, patient data correlation highlights subgroups that might have an increased susceptibility to NK cell treatment, highlighting the potential of such studies in the identification of predictive biomarkers.