Single-Cell Functional Genomics of Natural Killer Cell Interaction with Blood Cancers

Natural killer (NK) cells are emerging as a promising therapeutic option in hematological malignancies. To better understand how NK cells interact with blood cancer cells, we studied co-cultures of NK and blood cancer cells using single-cell and genome-scale functional genomics screens.

We performed multiplexed single-cell RNA-seq (scRNA-seq) on co-cultures of primary expanded and non-expanded NK cells and 26 cell lines representing diverse hematological malignancies (AML, CML, MM, B- and T-ALL, BCL) to study how both cell types respond transcriptomically to their interaction. We correlated the scRNA-seq based cell state changes with sensitivity of the cell lines to NK cell killing quantified using screens of pooled DNA-barcoded cell lines relying on the PRISM system to understand the relations of the phenotypic changes NK cell cytotoxicity. We further integrated the results with genetic, epigenetic, and transcriptomic data of the cell lines to uncover molecular regulators of the scRNA-seq-based responses and cancer cell susceptibility to NK cells. We performed 12 genome-scale CRISPR loss-of-function and gain-of-function screens of cancer-cell intrinsic NK cell resistance mechanisms across 7 blood cancer cell lines and investigated the mechanisms-of-action of 65 genome-scale screen hits using CRISPR screens with scRNA-seq readout (CROP-seq) in both tumor and NK cells to functionally dissect the gene regulatory networks driving the observed cell state changes in interacting NK and cancer cells.

At single-cell resolution, exposure of NK to cancer cells induced a spectrum of distinct activation states. Depending on the cancer cell lineage and molecular phenotype, NK cells transitioned either into an activated state characterised by expression of genes encoding receptors such as TNFRSF9 (4-1BB), TNFRSF18 (GITR), and the inhibitory checkpoints HAVCR2 (TIM-3) and TIGIT, or an state marked by type I interferon (IFN) signature including OAS1, OAS2, and MX1, or remained relatively unaltered. Transition to the activated or type I IFN states correlated with improved tumor cell killing across a panel of cell lines, ranging from more sensitive myeloid to more resistant B-lymphoid cancers, indicating that these molecular states drive tumor elimination. Tumor cells responded to NK cell attack by activating interferon gamma (IFNy) signaling and resulting in MHC class I induction.

Molecular correlates of increased sensitivity included higher expression of activating receptor ligands NCR3LG1, PVR, and ULBP1 in myeloid cancers, whereas resistance correlated with higher expression of the antigen-presenting machinery in B-lymphoblastic leukemia cells as well as mutations in the NF-kB regulator TRAF3 in multiple myeloma. Genome-scale CRISPR screens confirmed the functional roles of these correlates, uncovering cancer cell-intrinsic genes including activating receptor ligands and mediators of antigen presentation and interferon gamma signaling as key regulators of susceptibility to NK cells. CRISPR screens with scRNA-seq readout identified MHC-I, IFNy, and NF-κB regulation as underlying mechanisms. Knocking out mediators of interferon gamma signaling such as JAK1 prevented cancer cells from responding to NK cell attack by inducing MHC class I, resulting in increased transition of NK cells to the activated state as measured by scRNA-seq. Cancer cell knockout of positive regulators of NK cell response (CD58,NCR3LG1) induced an inactive NK cell state, providing experimental evidence how cancer cell-intrinsic genetic alterations can shape the molecular profile of attacking immune cells to promote immune evasion.

Collectively, our findings shed light at single-cell resolution on the dynamic transcriptomic changes in interacting immune effector and cancer cells and provide insights on the cancer cell-intrinsic mechanisms regulating these processes. The adaptive responses in both cell types highlight potential therapy targets for enhancing NK cell-based treatments. In addition, the integration of these studies with multi-omics data provides potential genomic and phenotypic biomarkers for responses to NK cell-based treatments.

Mitsiades:H3 Biomedicine: Research Funding; Novartis: Research Funding; Springworks: Research Funding; Janssen/Johnson & Johnson: Research Funding; Oncopeptides: Consultancy; Secure Bio: Consultancy; FIMECS: Consultancy; Fate Therapeutics: Consultancy; Ionis Pharmaceuticals: Consultancy; BMS: Research Funding; Sanofi: Research Funding; Nurix: Research Funding; Karyopharm: Research Funding; Arch Oncology: Research Funding; Abbvie: Research Funding; EMD Serono: Research Funding; TEVA: Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding.