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. ### Competing Interest Statement K.A.F., E.S., and C.L.M. are coinventors on a patent for the use of T cells deficient in MED12 or CCNC for therapeutic use. C.L.M. holds multiple patents in the arena of CAR T cell therapeutics. C.L.M. is a cofounder and holds equity in Lyell Immunopharma and Syncopation Life Sciences, which are developing CAR-based therapies, and consults for Lyell, Syncopation, NeoImmune Tech, Apricity, Nektar, Immatics, Mammoth and Ensoma. A.T.S. is a cofounder of Immunai and Cartography Bio. H.Y.C. is an inventor on patents for the use of ATAC-seq, H.Y.C. is a co-founder of Accent Therapeutics, Boundless Bio, and an advisor for 10x Genomics, Arsenal Biosciences, Cartography Bio and Spring Discovery. E.W.W. consults for and holds equity in Lyell Immunopharma and VISTAN Health. E.S. consults for and holds equity in Lyell Immunopharma. R.G.M. is a co-founder of and holds equity in Syncopation Life Sciences. RGM is a consultant for Lyell Immunopharma, Syncopation, Zai Lab, NKarta, and Aptorum Group. J.A.B. is a consultant to Immunai.