P28
Genome-Scale CRISPR/Cas9 screening in tumor cells to identify mechanisms for overcoming cancer resistance in NK cell-based immunotherapy
M Schuldt(1,2) W Rackwitz(1,2) A Doyle(4) K Hölig(1,2) S R Künzel(1,2) T Tonn(1,2,3) J Eitler(1,2,3)
1:Experimental Transfusion Medicine, Faculty of Medicine Carl Gustav Carus, Dresden University of Technology, Dresden, Germany; 2:Institute for Transfusion Medicine Dresden, German Red Cross Blood Donation Service North-East, Dresden, Germany; 3:German Cancer Consortium (DKTK), Partner Site Dresden, Dresden, Germany; 4:Physics of Life, Dresden University of Technology, Dresden, Germany
Natural killer (NK) cells can rapidly identify and destroy malignant cells, making them a promising tool for cancer immunotherapy. Enhancing NK cell efficacy by expressing chimeric antigen receptors (CARs) or Fc receptors (CD16) in combination with monoclonal antibodies (ADCC) has shown significant potential. However, resistance to NK cell cytotoxicity, especially in solid tumors, remains a challenge.
To dissect the mechanisms of cancer cell resistance, we performed six genome-wide CRISPR-Cas9 knockout screens using leukemia, breast and pancreatic cancer cell lines in combination with the clinically relevant NK cell line NK-92 or its derivatives genetically engineered to express CAR specific for ErbB2 or high-affinity Fc receptors, in combination with therapeutic antibodies. Genomic DNA from surviving cancer cells was sequenced, and data was analyzed using Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK).
Molecular pathway analysis of the screening data revealed that cancer cells employ diverse and heterogeneous mechanisms to evade NK cell cytotoxicity. Each screen identified at least five genes with high-ranking scores for NK cell resistance or susceptibility. Validation of the top-ranked genes with high-throughput live-cell imaging cytotoxicity assays identified genes strongly responsible for the resistance to NK cell cytotoxicity. Among these, ICAM-1 was identified as a critical checkpoint required for natural and trastuzumab-mediated NK cell cytotoxicity, whereas anti-ErbB2-CAR targeting was able to overcome this resistance.
These findings highlight multiple clinically relevant resistance mechanisms to NK cell cytotoxicity, and provide valuable insights to enhance NK cell-based therapies for resistant cancers.