Integrated drug profiling and CRISPR screening identify essential pathways for CAR T cell cytotoxicity.

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 of the potential for modulation using small-molecule drugs could improve current immunotherapies. Here, we systematically investigated druggable mechanisms of CAR T cell cytotoxicity using more than 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 (B-ALL) and diffuse large B cell lymphoma (DLBCL) 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.

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