Genome-Wide CRISPR Screen Reveals Cancer Cell Resistance to NK Cells Induced by NK-Derived IFN-γ

Abstract

The anti-leukemia activity of NK cells helps prevent relapse during hematopoietic stem cell transplantation (HSCT) in leukemia patients. However, the factors that determine the sensitivity or resistance of leukemia cells in the context of NK-mediated cytotoxicity are not well-established. Here, we performed a genome-wide CRISPR screen in the human chronic-myelogenous-leukemia (CML) cell line K562 to identify genes that regulate the vulnerability of leukemia cells to killing by primary human NK cells. The distribution of guide RNAs (gRNAs) in K562 cells that survived co-incubation with NK cells showed that loss of NCR3LG1, which encodes the ligand of the natural cytotoxicity receptor NKp30, protected K562 cells from killing. In contrast, loss of genes that regulate the antigen-presentation and interferon-γ-signaling pathways increased the vulnerability of K562 cells. The addition of IFN-γ neutralizing antibody increased the susceptibility of K562 cells to NK-mediated killing. Upregulation of MHC class I on K562 cells after co-incubation with NK cells was dependent on IFNGR2. Analysis of RNA-seq data from The Cancer Genome Atlas (TCGA) showed that low IFNGR2 expression in cancer tissues was associated with improved overall survival in acute myeloid leukemia (AML) and Kidney Renal Clear Cell Carcinoma (KIRC) patients. Our results, showing that the upregulation of MHC class I by NK-derived IFN-γ leads to resistance to NK cytotoxicity, suggest that targeting IFN-γ responses might be a promising approach to enhance NK cell anti-cancer efficacy.

Type I and II Interferons in the Anti-Tumor Immune Response

“…In contrast, in an HBV-associated hepatocellular carcinoma (HCC) model, IFNγ produced by NK cells was found to induce STAT1-dependent expression of epithelial cell adhesion molecules, promoting the epithelial-to-mesenchymal transition of HBV surface antigen-positive hepatocytes and increasing HCC incidence in vivo [ 169 ]. In another study, NK-derived IFNγ-induced the expression of MHC class I molecules in leukemia cells and decreased their susceptibility to NK cytotoxicity [ 170 ], while in melanoma cells loss of IFNγ signaling components increased tumor cell sensitivity to NKs [ 171 ]. Using several mouse tumor models, IFNγ was shown to be essential for CTL-driven development of immune-resistant cancer cell clones through increased tumor cell genetic instability [ 172 ].…”

Interrogating immune cells and cancer with CRISPR-Cas9

“…Altogether, these studies have confirmed key roles for antigen processing and presentation, IFNγ signaling, and the TNF response in antitumor immunity and identified novel members of these pathways that are mutated in patient tumors and may represent new therapeutic avenues for activating the immune response to cancer (Box 3 and Figure 2). These screens have also been extended to additional tumor-immune cell interactions, including with natural killer (NK) cells [54][55][56] and chimeric antigen receptor (CAR) T cells [57]. A significant caveat is that many of these screens have been performed in vitro using two-cell interaction systems, or in immunodeficient in vivo models where exogenous tumor-specific T cells are provided (due to the inherent immunogenicity of CRISPR components in immune-competent mice), which does not account for the complex multicellular interactions that occur during an antitumor immune response.…”

CRISPR/Cas9 in Cancer Immunotherapy: Animal Models and Human Clinical Trials

“…Another genome-wide CRISPR/Cas9 screen allowed to reveal genes responsible for resistance of human chronic-myelogenous-leukemia cells to NK cells induced by IFN-γ. This process was discovered to be governed by loss of gene encoding a ligand responsible for cytotoxicity, as well as by a gain of function mutation affecting antigen presentation that protects wild type cells [ 55 ]. Nonetheless, IFN-γ-mediated responses are of great concern, limiting ongoing research, due to IFN-γ having both tumorigenic and antitumorigenic characteristics [ 56 ].…”