Identification of genetic modifiers enhancing B7-H3-targeting CAR T cell therapy against glioblastoma through large-scale CRISPRi screening

• Published in: Journal of experimental & clinical cancer research Vol. 43; no. 1; p. 95
• Main Authors: Li, Xing, Sun, Shiyu, Zhang, Wansong, Liang, Ziwei, Fang, Yitong, Sun, Tianhu, Wan, Yong, Ma, Xingcong, Zhang, Shuqun, Xu, Yang, Tian, Ruilin
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 01.04.2024; BioMed Central; BMC
• Subjects: Antigens; B7-H3; Blood-brain barrier; Brain cancer; Brain tumors; Cancer therapies; CAR T cell; Cell culture; Chemotherapy; Cloning; CRISPR; CRISPR screening; DNA methylation; Glioblastoma multiforme; Glioma; Immunotherapy; Kinases; Lymphocytes; Medical prognosis; Mutation; Penicillin; Plasmids; RNA; RNA sequencing; T cells; TNFSF15; Tumors; B7-H3; CAR T cell; TNFSF15; Glioblastoma multiforme; CRISPR screening
• ISSN: 1756-9966; 0392-9078; 1756-9966
• DOI: 10.1186/s13046-024-03027-6

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with a poor prognosis. Current treatment options are limited and often ineffective. CAR T cell therapy has shown success in treating hematologic malignancies, and there is growing interest in its potential application in solid tumors, including GBM. However, current CAR T therapy lacks clinical efficacy against GBM due to tumor-related resistance mechanisms and CAR T cell deficiencies. Therefore, there is a need to improve CAR T cell therapy efficacy in GBM. We conducted large-scale CRISPR interference (CRISPRi) screens in GBM cell line U87 MG cells co-cultured with B7-H3 targeting CAR T cells to identify genetic modifiers that can enhance CAR T cell-mediated tumor killing. Flow cytometry-based tumor killing assay and CAR T cell activation assay were performed to validate screening hits. Bioinformatic analyses on bulk and single-cell RNA sequencing data and the TCGA database were employed to elucidate the mechanism underlying enhanced CAR T efficacy upon knocking down the selected screening hits in U87 MG cells. We established B7-H3 as a targetable antigen for CAR T therapy in GBM. Through large-scale CRISPRi screening, we discovered genetic modifiers in GBM cells, including ARPC4, PI4KA, ATP6V1A, UBA1, and NDUFV1, that regulated the efficacy of CAR T cell-mediated tumor killing. Furthermore, we discovered that TNFSF15 was upregulated in both ARPC4 and NDUFV1 knockdown GBM cells and revealed an immunostimulatory role of TNFSF15 in modulating tumor-CAR T interaction to enhance CAR T cell efficacy. Our study highlights the power of CRISPR-based genetic screening in investigating tumor-CAR T interaction and identifies potential druggable targets in tumor cells that confer resistance to CAR T cell killing. Furthermore, we devised targeted strategies that synergize with CAR T therapy against GBM. These findings shed light on the development of novel combinatorial strategies for effective immunotherapy of GBM and other solid tumors.