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

in Journal of experimental & clinical cancer research : CR by Xing Li, Shiyu Sun, Wansong Zhang, Ziwei Liang, Yitong Fang, Tianhu Sun, Yong Wan, Xingcong Ma, Shuqun Zhang, Yang Xu, Ruilin Tian

TLDR

  • The study looked at how CAR T cell therapy, which is used to treat some types of cancer, might be improved for a type of brain tumor called Glioblastoma multiforme (GBM). The study used a special tool called CRISPRi to find out which genes in GBM cells might help CAR T cells kill the tumor better. The study found that some genes in GBM cells were making it harder for CAR T cells to kill the tumor. The study also found that a protein called TNFSF15 was helping CAR T cells kill the tumor better. The study's findings suggest that targeted strategies that work with CAR T cells to kill GBM tumors could be developed.

Abstract

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.

Overview

  • The study aims to investigate the potential of CRISPR interference (CRISPRi) in enhancing the efficacy of CAR T cell therapy against Glioblastoma multiforme (GBM).
  • The study used large-scale 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. The study established B7-H3 as a targetable antigen for CAR T therapy in GBM.

Comparative Analysis & Findings

  • The study identified genetic modifiers in GBM cells, including ARPC4, PI4KA, ATP6V1A, UBA1, and NDUFV1, that regulated the efficacy of CAR T cell-mediated tumor killing. Furthermore, the study 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. The 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. The findings suggest that targeted strategies that synergize with CAR T therapy against GBM could be developed.

Implications and Future Directions

  • The study's findings have significant implications for the development of novel combinatorial strategies for effective immunotherapy of GBM and other solid tumors. The study's findings suggest that targeted strategies that synergize with CAR T therapy against GBM could be developed. The study's findings also highlight the power of CRISPR-based genetic screening in investigating tumor-CAR T interaction and identifying potential druggable targets in tumor cells that confer resistance to CAR T cell killing. Future research could focus on developing targeted strategies that synergize with CAR T therapy against GBM and other solid tumors, as well as further investigating the mechanisms underlying tumor-CAR T interaction and identifying additional potential druggable targets in tumor cells.
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