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

Abstract

Glioblastoma (GBM) contains self-renewing GBM stem cells (GSC) potentially amenable to immunologic targeting, but chimeric antigen receptor (CAR) T-cell therapy has demonstrated limited clinical responses in GBM. Here, we interrogated molecular determinants of CAR-mediated GBM killing through whole-genome CRISPR screens in both CAR T cells and patient-derived GSCs. Screening of CAR T cells identified dependencies for effector functions, including TLE4 and IKZF2. Targeted knockout of these genes enhanced CAR antitumor efficacy. 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 susceptibility to CAR-mediated killing, including RELA and NPLOC4, the knockout of which altered tumor–immune signaling and increased responsiveness of CAR therapy. Overall, CRISPR screening of CAR T cells and GSCs discovered avenues for enhancing CAR therapeutic efficacy against GBM, with the potential to be extended to other solid tumors. Significance: Reciprocal CRISPR screening identified genes in both CAR T cells and tumor cells regulating the potency of CAR T-cell cytotoxicity, informing molecular targeting strategies to potentiate CAR T-cell antitumor efficacy and elucidate genetic modifications of tumor cells in combination with CAR T cells to advance immuno-oncotherapy. This article is highlighted in the In This Issue feature, p. 995

Adoptive cellular therapy in solid tumor malignancies: review of the literature and challenges ahead

“…More recently, CRISPR technologies have been used to identify genes that are critical to effector T-cell function. 92 These technologies have the ability to create T cells resistant to multiple inhibitory pathways. For example, the generation of ablated Fas receptor CAR T cells using CRISPR allowed for a resistance to Fas ligand activation induced cell death both in vivo and in vitro when challenged with tumor cells.…”

“…One such study with CAR T and GBM stem cells using CRISPR screening found Ikaros family zinc finger protein 2 and TLE4 as critical factors in effector T-cell function, and subsequently that targeted knockouts of these genes increased CAR T-cell activity. 92 CRISPR screening may prove to serve a critical role for the development of enhanced CAR T cells that can reliably reverse exhaustion and inhibitory mechanisms of the proinflammatory tumor microenvironment. Figure 2 summarizes the challenges of ACT in solid tumor malignancies.…”

Gene editing to enhance the efficacy of cancer cell therapies

“…Recently, such efforts in CAR T cells identified increased potency upon deletion of TLE4, a transcriptional corepressor of multiple genes encoding inflammatory cytokines, 62 and IKZF2, which is upregulated in exhausted CAR T cells. [63][64][65] Recently, CRISPR-Cas9 transfection of primary human T cells without TCR stimulation to enable study of genes involved in T cell activation has been shown to result in a knockout efficiency >90%. 66 We anticipate significant additional data in the near term identifying additional candidates for knockout to confer enhanced potency in T cells.…”

Innovative and Promising Strategies to Enhance Effectiveness of Immunotherapy for CNS Tumors: Where Are We?

“…IL-13Rα2 T cell antitumor activity toward self-renewing GBM stem cells (GSCs), has been recently discovered by the application of whole-genome clustered randomly interspersed short palindromic repeats (CRISPR)-knockout screen applied to both CAR T cells and GSCs ( 97 ). In particular, TLE4 or IKZF2 gene targeting were associated to an increased and long-term efficacy of CAR T cells in this model, with a significant reduction in exhaustion signal of the T cells, whereas knockout of RELA or NPLOC4 GSCs were associated to the increased responsiveness to CAR T cell control ( 97 ). Confirmation papers are highly desirable to assess whether the same gene editing could potentially optimize the activity of CAR T lymphocytes characterized by antigenic specificity other than IL-13Rα2, in a CNS tumor context other than GSC.…”