Identification of Novel Regulators of Natural Killer Cell Activity

项目概要/摘要： 自然杀伤（NK）细胞是先天免疫系统的关键部分，具有杀死血液和淋巴细胞的能力。 恶性肿瘤和实体瘤。基于NK细胞的疗法正在迅速获得临床关注。然而，在这方面， NK细胞用于介导抗肿瘤活性的机制仍不清楚。在这里，我们专门调查了一个 新的CRISPR/Cas9介导的筛选方法，以识别头颈部新的免疫（NK细胞）靶点 鳞状细胞癌（HNSCC）细胞。我们选择HNSCC进行这些研究，因为已知NK细胞具有高度的 浸润HNSCC和高度NK细胞浸润与HNSCC患者存活正相关。 此外，抗EGFR抗体西妥昔单抗和检查点抑制剂抗体是免疫疗法 批准用于HNSCC。尽管这些治疗进展，这种恶性肿瘤往往成为难治性的，这些 基于免疫的治疗作为选择性压力推动肿瘤细胞发展抵抗机制以逃避 NK细胞介导的杀伤。在这个项目中，我们将确定肿瘤细胞中的体细胞突变如何改变它们的细胞周期。 对基于NK细胞的免疫疗法的反应。我们假设这种基于CRISPR/Cas9的遗传筛查 系统将识别导致NK细胞更好靶向的抗性机制。我们将使用一个基因组 广泛的CRISPR/Cas9 KO文库以模拟HNSCC中的功能丧失突变。经过与NK的一轮选拔后 在NK细胞中，将分析增加肿瘤细胞对NK细胞的抗性或敏感性的那些基因。后来 将在体外验证最佳靶点，并进行RNA测序以确定分子途径 涉案这些研究还将使用从胶质母细胞瘤CRISPR/Cas9筛选研究中获得的最高命中率 干细胞鉴定了NK细胞介导的杀伤的新调节因子。HNSCC的联合分析和 胶质母细胞瘤增加了识别调节NK细胞活性对抗各种肿瘤的关键基因的能力。我们 还旨在确定和特别优先考虑药理学药物影响分子的新靶点， 增强NK细胞介导的杀伤的途径。最有希望的遗传命中将在体内进一步研究 使用用人NK细胞处理的工程化人肿瘤的异种移植小鼠模型和同基因的 系统，使我们能够验证拟议的基因调节NK细胞介导的活性的能力， 免疫活性小鼠模型中的HNSCC。这些互补的体外、计算机模拟和体内方法 将使我们能够测试药理学和遗传学策略，以上调或下调肿瘤抗原和NK细胞 受体，可以转化为临床试验，以提高抗HNSCC和可能的其他抗肿瘤活性。

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.