Related Experiment Video
Updated: Jan 2, 2026
Performing an In Vitro Genome-Wide CRISPR Knockout Screen in Chimeric Antigen Receptor T Cells
Published on: January 31, 2025
Integrated drug profiling and CRISPR screening identify essential pathways for CAR T-cell cytotoxicity
Olli Dufva1,2,3,4, Jan Koski5, Pilvi Maliniemi5
1Hematology Research Unit Helsinki, Helsinki University Hospital Comprehensive Cancer Center, Helsinki, Finland.
Abstract:
Chimeric antigen receptor (CAR) T-cell therapy has proven effective in relapsed and refractory B-cell malignancies, but resistance and relapses still occur. Better understanding of mechanisms influencing CAR T-cell cytotoxicity and the potential for modulation using small-molecule drugs could improve current immunotherapies. Here, we systematically investigated druggable mechanisms of CAR T-cell cytotoxicity using >500 small-molecule drugs and genome-scale CRISPR-Cas9 loss-of-function screens. We identified several tyrosine kinase inhibitors that inhibit CAR T-cell cytotoxicity by impairing T-cell signaling transcriptional activity. In contrast, the apoptotic modulator drugs SMAC mimetics sensitized B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma cells to anti-CD19 CAR T cells. CRISPR screens identified death receptor signaling through FADD and TNFRSF10B (TRAIL-R2) as a key mediator of CAR T-cell cytotoxicity and elucidated the RIPK1-dependent mechanism of sensitization by SMAC mimetics. Death receptor expression varied across genetic subtypes of B-cell malignancies, suggesting a link between mechanisms of CAR T-cell cytotoxicity and cancer genetics. These results implicate death receptor signaling as an important mediator of cancer cell sensitivity to CAR T-cell cytotoxicity, with potential for pharmacological targeting to enhance cancer immunotherapy. The screening data provide a resource of immunomodulatory properties of cancer drugs and genetic mechanisms influencing CAR T-cell cytotoxicity.
Insights
Chimeric antigen receptor (CAR) T-cell therapy shows promise but faces resistance. This study reveals that targeting death receptor signaling can enhance CAR T-cell effectiveness against B-cell malignancies.
Area of Science:
- Immunology
- Oncology
- Pharmacology
Background:
- Chimeric antigen receptor (CAR) T-cell therapy is effective for B-cell malignancies but can be limited by resistance and relapse.
- Understanding the mechanisms that regulate CAR T-cell activity and identifying ways to modulate them are crucial for improving immunotherapy outcomes.
Purpose of the Study:
- To systematically investigate druggable mechanisms that influence CAR T-cell cytotoxicity.
- To identify small-molecule drugs and genetic factors that can enhance CAR T-cell efficacy in B-cell malignancies.
Main Methods:
- Utilized a large-scale screening approach with over 500 small-molecule drugs.
- Employed genome-scale CRISPR-Cas9 loss-of-function screens to identify genetic mediators of CAR T-cell function.
- Investigated the role of death receptor signaling pathways in CAR T-cell-mediated killing.
Main Results:
- Identified tyrosine kinase inhibitors that reduce CAR T-cell cytotoxicity by affecting T-cell signaling.
- Demonstrated that SMAC mimetics sensitize B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma cells to anti-CD19 CAR T cells.
- Discovered that death receptor signaling, involving FADD and TRAIL-R2, is critical for CAR T-cell cytotoxicity, with SMAC mimetic sensitization mediated by RIPK1.
Conclusions:
- Death receptor signaling is a key determinant of cancer cell sensitivity to CAR T-cell therapy.
- Pharmacological targeting of death receptor pathways, potentially using SMAC mimetics, can enhance CAR T-cell immunotherapy.
- Cancer genetics may influence the effectiveness of CAR T-cell therapy through variations in death receptor expression.
