Towards elucidating disease-relevant states of neurons and glia by CRISPR-based functional genomics

Leng, Kun; Kampmann, Martin

doi:10.1186/s13073-022-01134-7

Cited by 3 publications

(5 citation statements)

References 133 publications

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“…The screens were performed in parallel with CAR T cells derived from two separate donors. sgRNA abundances were compared between the B7-H3 and CD19 groups to determine the phenotype and significance of each gene perturbation using the MAGeCK-iNC pipeline [ 58 ].…”

Section: Resultsmentioning

confidence: 99%

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

Li,

Sun,

Zhang

et al. 2024

J Exp Clin Cancer Res

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Background 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. Methods 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. Results 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. Conclusions 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.

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Section: Resultsmentioning

confidence: 99%

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

Li,

Sun,

Zhang

et al. 2024

J Exp Clin Cancer Res

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Section: A Co-culture Crispri Screen Identified Regulators Of B7-h3 C...mentioning

confidence: 99%

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

Li,

Sun,

Zhang

et al. 2024

Preprint

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Background 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. Methods We conducted large-scale CRISPR interference (CRISPRi) screens in GBM cell line U87 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 cells. Results 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. Conclusions 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.

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“… 9 Attributed to the programmable and multiplexable nature of CRISPR-based technologies, they have been widely applied to introduce massively parallel genetic perturbations in large-scale functional genomics screens. 10 , 11 , 12 Currently, three types of CRISPR-based perturbations are commonly used in such screens (as shown in Figure 1 ): (1) CRISPR knockout (CRISPRn), which utilizes the Cas9 nuclease to disrupt a target gene by introducing frameshift indels, (2) CRISPR interference (CRISPRi), which utilizes a catalytically dead Cas9 (dCas9) fused with a transcriptional repressor domain to silence the transcription of a target gene, and (3) CRISPR activation (CRISPRa), which utilizes dCas9 fused with transcriptional activator domains to activate the transcription of a target gene. Compared with CRISPRn, CRISPRi does not induce DNA double-strand breaks and thus is less toxic to cells that are sensitive to DNA damage, such as hPSCs.…”

Section: Introductionmentioning

confidence: 99%

“…Different screening strategies can be employed depending on phenotypes of interest 10 , 11 , 12 ( Figure 1 ). For example, survival/proliferation-based screens can be used to identify essential genes and genes that modify the sensitivity of cells to a certain insult, such as drug treatment.…”

Section: Introductionmentioning

confidence: 99%

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CRISPR-based functional genomics screening in human-pluripotent-stem-cell-derived cell types

Li¹,

Ouyang²,

Zhan³

et al. 2023

Cell Genomics

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Towards elucidating disease-relevant states of neurons and glia by CRISPR-based functional genomics

Cited by 3 publications

References 133 publications

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

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

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

CRISPR-based functional genomics screening in human-pluripotent-stem-cell-derived cell types

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