CRISPR Screening of CAR T Cells and Cancer Stem Cells Reveals Critical Dependencies for Cell-Based Therapies [GSC]

Glioblastoma (GBM) is the most prevalent primary malignant brain tumor, containing self-renewing stem-like GBM stem cells (GSCs) that have been a focus of immunotherapies. Chimeric antigen receptor (CAR) T cell therapy has shown evidence of clinical activity, but overall limited responses in patients with GBMs. Here, we interrogated molecular determinants of CAR T cell-mediated GBM killing through whole-genome CRISPR screens in both CAR T cells and patient-derived GSCs. CRISPR screening of CAR T cells identified dependencies for their effector functions, including TLE4 and IKZF2. Targeted knockout of these genes in CAR T cells robustly enhanced antitumor efficacy against GBM patient-derived xenografts (PDXs). Bulk and single cell-RNA sequencing of edited CAR T cells revealed transcriptional profiles of superior effector function and inhibited exhaustion responses. Reciprocal screening of GSCs identified genes essential for their susceptibility to CAR-mediated killing, including RELA and NPLOC4, the knockout of which altered the tumor-immune signaling axis and increased responsiveness of CAR therapy. Overall, CRISPR screening of CAR T cells and GSCs are promising strategies to discover avenues and inform potential combinatorial approaches for enhancing CAR T cell therapeutic efficacy against GBM, and can be extended to reveal key mediators of immunotherapy responses across solid tumors.胶质母细胞瘤（Glioblastoma, GBM）是最常见的原发性恶性脑肿瘤，其携带有自我更新能力的干细胞样胶质母细胞瘤干细胞（GBM stem cells, GSCs），此类细胞长期以来都是免疫治疗的核心研究靶点。嵌合抗原受体（Chimeric antigen receptor, CAR）T细胞疗法已被证实具备临床活性，但在胶质母细胞瘤患者中整体应答效果有限。本研究通过分别对CAR T细胞与患者来源的胶质母细胞瘤干细胞开展全基因组CRISPR筛选，系统探究了CAR T细胞介导胶质母细胞瘤杀伤的分子决定机制。针对CAR T细胞的CRISPR筛选鉴定出其效应功能所依赖的核心基因，包括TLE4与IKZF2。在CAR T细胞中对上述基因进行靶向敲除，可显著增强其对抗患者来源胶质母细胞瘤异种移植瘤（patient-derived xenografts, PDXs）的抗肿瘤活性。对编辑后的CAR T细胞进行批量RNA测序与单细胞RNA测序分析，结果揭示了其具备更优异的效应功能转录特征，并可抑制免疫细胞耗竭相关应答。针对胶质母细胞瘤干细胞的反向筛选则鉴定出其对CAR介导杀伤敏感性所必需的关键基因，包括RELA与NPLOC4；敲除此类基因可重塑肿瘤-免疫信号轴，并提升CAR疗法的临床应答敏感性。综上，对CAR T细胞与胶质母细胞瘤干细胞进行CRISPR筛选，是发掘潜在治疗路径、为优化CAR T细胞对抗胶质母细胞瘤的治疗效果提供联合策略依据的有效手段，且该研究框架可推广至揭示实体瘤中免疫治疗应答的关键介导因子。