Enhanced Effector Activity of Mediator Kinase Module Deficient CAR-T Cells

Adoptive T cell immune therapies mediate impressive clinical benefit in a fraction of patients, but anti-tumor effects are often limited by inadequate T cell potency. To identify genes limiting T cell effector function, we conducted genome-wide CRISPR knock-out screens in human primary CAR-T cells. The top hits were MED12 and CCNC , components of the cyclin-dependent kinase (CDK) module of the Mediator complex, an evolutionarily conserved regulator of gene transcription. MED12 or CCNC deficient CAR-T cells manifest increased expansion, cytokine production, metabolic fitness, effector function, anti-tumor activity and reduced terminal effector differentiation. Chemical inhibition of CDK8/19 kinase activity recapitulated some features of genetic loss of MED12 , including increased T cell expansion. MED12 deficient CAR-T cells showed widespread but selective increases in chromatin accessibility, MED1 chromatin occupancy, and H3K27 acetylation at enhancers used by transcription factors playing a critical role in T cell fate, including several STAT and AP1 family members. The most pronounced enhancement was observed for STAT5 which manifested as increased sensitivity to IL-2 in MED12 deficient T cells. These results link Mediator induced transcriptional coactivation with T cell effector programming and identify the CDK module as a target for enhancing the potency of anti-tumor T cell responses. One Sentence Summary The Mediator kinase module is a primary regulator of T cell differentiation, and genetic or small molecule-based inhibition of this module enhances effector T cell potency.