Identification of Novel Regulators of Natural Killer Cell Activity

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Project Summary/Abstract: Natural killer (NK) cells are a key part of the innate immune system with the ability to kill both hematological malignancies and solid tumors. NK cell-based therapies are rapidly gaining clinical interest. However, mechanisms that NK cells use to mediate anti-tumor activity remain unclear. Here, we specifically investigate a novel CRISPR/Cas9-mediated screening method to identify new immunologic (NK cell) targets in head and neck squamous cells carcinoma (HNSCC) cells. We chose HNSCC for these studies as NK cells are known to highly infiltrate HNSCC and a high degree of NK cells infiltration positively correlates with HNSCC patient survival. Additionally, the anti-EGFR antibody cetuximab and checkpoint inhibitor antibodies are immune therapies approved for HNSCC. Despite these therapeutic advances, this malignancy often becomes refractory to these immune-based therapies as selective pressures push tumor cells to develop resistance mechanisms to escape NK cell-mediated killing. For this project we will determine how somatic mutations in tumor cells can change their response to NK cell-based immunotherapy. We hypothesize that this CRISPR/Cas9-based genetic screening system will identify mechanisms of resistance that lead to better targeting by NK cells. We will use a genome wide CRISPR/Cas9 KO library to mimic loss of function mutations in HNSCC. After a round of selection with NK cells, those genes that increase resistance or sensitivity of tumor cells to NK cells will be profiled. Afterwards the top targets will be validated in vitro and RNA sequencing will be performed to define the molecular pathways involved. These studies will also use top hits obtained from studies of CRISPR/Cas9 screening on glioblastoma stem cells that identified novel regulators of NK cell-mediated killing. This combined analysis of HNSCC and glioblastoma increases the power to identify key genes that regulate NK cell activity against diverse tumors. We also aim to identify and specifically prioritize novel targets where pharmacological agents impact molecular pathways that enhance NK cell-mediated killing. The most promising genetic hits will be further studied in vivo using both a xenograft mouse model of engineered human tumors treated with human NK cells and a syngeneic system that allows us to validate the ability of proposed genes to regulate NK cell-mediated activity against HNSCC in an immunocompetent mouse model. These complementary in vitro, in silico and in vivo approaches will allow us to test pharmacological and genetic strategies to up or down-regulate tumor antigens and NK cell receptors that can be translated into clinical trials to improve anti-HNSCC and likely other anti-tumor activity.