Enhanced T cell effector activity by targeting the Mediator kinase module

Abstract

T cells are the major arm of the immune system responsible for controlling and regressing cancers. To identify genes limiting T cell function, we conducted genome-wide CRISPR knockout screens in human chimeric antigen receptor (CAR) T cells. Top hits were MED12 and CCNC, components of the Mediator kinase module. Targeted MED12 deletion enhanced antitumor activity and sustained the effector phenotype in CAR- and T cell receptor-engineered T cells, and inhibition of CDK8/19 kinase activity increased expansion of nonengineered T cells. MED12-deficient T cells manifested increased core Meditator chromatin occupancy at transcriptionally active enhancers-most notably for STAT and AP-1 transcription factors-and increased IL2RA expression and interleukin-2 sensitivity. These results implicate Mediator in T cell effector programming and identify the kinase module as a target for enhancing potency of antitumor T cell responses.

Fig. 1.. Genome-wide CRISPR screen identifies subunits of the Mediator kinase module as regulators of CAR-T cell effector function.

A) Schematic depicting CRISPR knockout screen for regulators of cytokine production and CAR-T cell expansion using a tonic signaling model of CAR-T cell exhaustion. B) Enrichment of gene knockouts in replicate expansion screens. CRISPR-edited HA-28ζ CAR-T cells were generated from two donors, cultured in vitro for 15 days, and then co-cultured with GD2⁺ tumor cells until day 23. C) Enrichment of gene knockouts in replicate cytokine production screens. CRISPR-edited HA-28ζ CAR-T cells were generated from two donors, cultured in vitro for 15 days, stimulated with GD2⁺ tumor cells, and the top 10% of TNFα and IL-2 expressing cells were isolated by FACS. D) Predicted Cryo-EM structure of yeast Mediator complex (top) showing the effect that depletion of Cyclin C (CCNC deficient) or MED12 (MED12 deficient) would have on assembly of other subunits. Core Mediator is shown in grey, CDK8 kinase module is colored. Representations were created with Chimera using Protein Data Bank accessions 7KPX and 5U0P. E) Bar graphs depicting enrichment of sgRNA targeting all Mediator complex subunits in the expansion screen. Data are mean ± s.d. (n = 4–10 guides per genes). Colors indicate module of the Mediator complex assigned to each subunit. B, C, E) Data are mean of n = 4–10 guides per gene. Data are pooled from two independent experiments (n = 2 donors). Gene-level statistical significance was determined by the MAGeCK algorithm. *FDR < 0.05.

Fig. 2.. Disruption of Mediator kinase module in CAR-T cells enhances T cell effector function and tumor clearance.

A) In vitro T cell expansion of CCNC and MED12 deficient HA-28ζ (left) and CD19–28ζ (right) CAR-T cells. Fold change in total cell count after 23 days in culture relative to control CAR-T cells edited at the safe harbor AAVS1 locus. Two unique sgRNAs were used to validate each candidate gene. Data are mean ± s.d. of n = 3 donors. Ratio paired t-test. *P < 0.05, **P < 0.01. B) In vitro expansion of human primary T cells with duals inhibitors of CDK8 and CDK19 over 15 days in culture. Inhibitors were supplemented to the media every 48 hours. Data are mean ± s.d of n = 2 replicate wells. Representative of 3 independent experiments. C) IL-2 (top), IFNγ (middle), and TNFα (bottom) cytokine release after 24-hour co-culture with tumor cells from non-transduced (NT) and CAR-T cells edited with sgRNAs targeting MED12, CCNC, or safe-harbor control. HA-28ζ, CD19–28ζ and HER2–4-1BBζ CAR-T cells were stimulated 1:1 with NALM6-GD2, NALM6, or 143B cells respectively. Data are mean ± s.d. from duplicate or triplicate wells. Representative results of n = 4 donors (HA-28ζ, CD19–28ζ) or n = 2 donors (HER2–4-1BBζ). Non-transduced T cells were activated with CD3/28 stimulation, but not transduced with retrovirus or gene-edited. D) Heatmap of 38 cytokines produced by control, CCNC, or MED12 deficient CD19–28ζ CAR-T cells following 24-hour co-culture with NALM6 leukemia cells. Data are mean from duplicate wells in a multiplex bead-based assay. Two unique sgRNAs were used to validate each candidate gene. E and F) Metabolic rate as measured by Seahorse analysis of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of control or MED12 deficient CD19–28ζ CAR-T cells under resting and challenge conditions. Data are mean of n = 12 replicate wells. Representative results from two independent experiments. G) Analysis of tumor clearance. NSG mice were injected intravenously with 1.0 ×10⁶ NALM6-GD2 leukemia cells and treated with 2.0 × 10⁵ non-transduced or CCNC or MED12 deficient HA-28ζ CAR-T cells 9 days after tumor infusion (n = 5 mice). H) Analysis of tumor clearance. NSG mice were injected intravenously with 1.0 ×10⁶ NALM6-Luc leukemia and treated with 2.5 × 10⁵ non-transduced or CCNC or MED12 deficient CD19–28ζ CAR-T cells 3 days after tumor infusion (n = 5 mice). I) Analysis of tumor clearance. Tumor area of NSG mice injected intramuscular with 1 × 10⁶ 143B osteosarcoma cells and treated 4 days later with 5 × 10⁶ non-transduced or CCNC or MED12 deficient HER2–4-1BBζ CAR-T cells. Data are mean ± s.d of n = 5 mice (non-transduced, MED12 and CCNC deficient) or n = 4 mice (Control). Two-way ANOVA test with Dunnett’s multiple comparison test. *P < 0.01. G, H, and I) Representative experiment from two independent experiments (n = 2 donors). B, C and F) Two-tailed unpaired Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 3.. Loss of MED12 sustains effector function during chronic stimulation.

A) Antigen-driven in vitro expansion of Control and MED12 deficient HA-28ζ CAR-T cells. CAR-T cells were serially stimulated with GD2⁺ tumor cells in the absence of IL-2 at 48hr intervals at 1:1 effector to target cell ratio. Data are mean ± s.d. of n = 3 donors. B) Cytotoxicity of Control and MED12 deficient HA-28ζ CAR-T cells against GFP⁺ NALM6-GD2 leukemia following serial stimulation beginning 10 days after T cell activation. Cells were counted and replated at a 1:1 ratio of T cells to tumor cells at 48–72-hour intervals in media without IL-2. Data are mean ± s.d. of n = 3 replicate cultures. Representative of 3 independent experiments. C) In vitro expansion of Control and MED12 deficient HA-28ζ CAR-T cells cultured with IL-2. Two unique sgRNAs were used to validate each candidate gene. Data are mean ± s.d. of n = 2 replicate cultures. D) IL-2 (left) and IFNγ (right) release after 24-hour co-culture with tumor cells 45 days after T cell activation. Control and MED12 deficient HA-28ζ CAR-T cells were stimulated 1:1 with NALM6-GD2. Data are mean ± s.d. from n = 3 replicate cultures. E and F) Flow cytometric analysis of CD39 expression in non-transduced or HA-28ζ CAR-T cells. Mean fluorescence intensity is normalized to the Control at each timepoint. Data are mean ± s.d. of n = 3 replicate wells. Statistical comparison is between Control and MED12 deficient CAR-T cells. G and I) Analysis of tumor clearance. Bioluminescent imaging (BLI) of NSG mice injected intravenously with 1 × 10⁶ Nalm6-GD2 leukemia and treated with 4 × 10⁵ non-transduced or HA-28ζ CAR-T cells 7 days after tumor infusion and rechallenged 26 days later with Nalm6 or Nalm6-GD2 cells (n = 5 mice). H) Survival of CAR-treated mice shown in (G). Survival curves were compared with the Log-rank Mantel-Cox test. *P < 0.01. A-D) Two-tailed unpaired Student’s t-test. *P < 0.05, **P < 0.01. E and G) Two-way ANOVA test with Dunnett’s multiple comparison test. *P < 0.01. C - H) Representative of two independent experiments.

Fig. 4.. Loss of MED12 in T cells utilizing a TCR for tumor recognition enhances anti-tumor activity and effector function.

A and B) Frequency of T effector memory cells (CD45RO⁺, CCR7^-) and stem cell memory (CCR7⁺, CD45RO^-) in NY-ESO-1 TCR⁺ cells 15 day after T cell activation. Data are mean ± of n = 3 donors. Two-tailed paired Student’s t-test. **P < 0.01, ***P < 0.001. C-E) UMAP analysis of control and MED12 deficient NY-ESO-1 TCR⁺ cells. Expression of 34 markers was analyzed by CyTOF. Control and MED12 deficient samples are combined and colored C) by genotype, D) by donor, or E) by marker intensity. Each dot represents a single cell (n = 8319 cells). Data are pooled from two donors. F) In vitro expansion of Control and MED12 deficient NY-ESO-1 T cells cultured with IL-2. Data are mean ± s.d. of n = 3 donors. G) IL-2 (left) and IFNγ (right) release after 24-hour co-culture of NY-ESO-1 T cells with A375 melanoma cells. Data are mean ± s.d. from n = 3 cultures. Representative of 3 independent experiments. H and I) Analysis of tumor clearance. Tumor area of NSG mice injected subcutaneously with 3 × 10⁶ A375 melanoma cells and treated 7 days later with 3 × 10⁶ NY-ESO-1 TCR⁺ cells. Tumor area was measured by caliper. H) One-way ANOVA test with Dunnett’s multiple comparison test, *P < 0.01. I) Two-tailed unpaired Student’s t-test, **P < 0.01. n = 9 mice pooled from two independent experiments. J) scRNA-seq profiles of NY-ESO-1 TCR⁺ T cells isolated from A375 melanoma tumors by FACS. T cells were administered 12 days following tumor engraftment, and tumors were harvested 6 days after treatment. Each dot represents a single cell and is colored by gene expression of the indicated genes or by genotype. Data are n = 1542 single cells pooled from 4 tumors from each genotype. K) Violin plots depicting transcript expression level of selected genes in NY-ESO-1⁺ tumor infiltrating T cells. n = 669 Control sg1 cells and 855 MED12 sg49 cells. Boxes indicate median and interquartile ranges.

Fig. 5.. MED12 deficient CD19–28ζ CAR-T cells have an effector-like phenotype and an activated transcriptional program.

A) Flow cytometry analysis of T cell subsets as assessed by CD45RO and CCR7 expression in control or MED12 deficient CD8⁺ CD19–28ζ CAR-T cells 23 days after T cell activation. Representative result of n = 3 donors. Gating and subtyping strategy shown (SCM, Stem Central Memory; CM, Central Memory, TE, Terminal Effector; EM, Effector Memory) B) Frequency of T effector memory cells (CD45RO⁺, CCR7^-) in CD8⁺ and CD4⁺ CAR-T cells. n = 3 donors. Two-tailed paired Student’s t-test. **P < 0.01, ***P < 0.001. C and D) Uniform Manifold Approximation and Projection (UMAP) analysis of control and MED12 deficient CD19–28ζ CAR-T cells 15 days after T cell activation. Expression of 34 markers was analyzed by CyTOF. Control and MED12 deficient samples are combined and colored C) by genotype or D) by marker intensity. Representative donor of n = 3 donors. Each dot represents a single cell (n = 30,000 cells). E) Heatmap of differentially expressed genes in control or MED12 deficient CD19–28ζ CAR-T cells detected by bulk RNA-seq 15 days after T cell activation. Cells were collected on day 15 and CAR stimulated for 3 hours with plate-bound anti-idiotype antibody. Adjusted P < 0.01. n = 3 donors. F and G) Gene Set Enrichment Analysis of unstimulated MED12 deficient CAR-T cells compared to control cells using the Hallmarks gene collection. Normalized Enrichment Scores (NES) and FDR q values are shown. A positive NES indicates the gene set was enriched in MED12 deficient cells.

Fig. 6.. Loss of MED12 increases MED1 chromatin occupancy at transcriptionally active enhancers regulating T cell differentiation.

A) Venn diagram depicting number of sites bound by MED1 and/or MED12 detected by ChIP-seq in CD19–28ζ control CAR-T cells. B) Number of genomic regions with significant change in MED1 occupancy detected by ChIP-seq between MED12 deficient and control cells. Adjusted P < 0.05. C) Mean normalized ChIP-seq signal at regions with significant differences in MED1 occupancy. D) Western blot analysis of MED1 protein present in soluble and chromatin-bound cellular fractions from control and MED12 deficient CD19–28ζ CAR-T cells 15 days after T cell activation. GAPDH and Histone 3 are used as markers for each cellular fraction. Representative blot from three independent experiments. E) Densitometric analysis of western blot shown in (D). MED1 staining in the chromatin-bound fraction was normalized to HIST3 staining. Data are mean ± s.d. of n = 3 donors. Two-tailed unpaired Student’s t-test. ***P < 0.001. F) Heatmap showing ATAC-seq or ChIP-seq coverage at sites with differential MED12 occupancy as defined in (B). G) DAVID functional annotation of 842 genes nearest to sites with increased MED1 occupancy in MED12 deficient cells compared with control cells. 12 selected terms of 22 significant terms are shown (FDR < 0.10). Full results table including MED1 ChIP-seq coverage at genes corresponding to GO terms is included in Supplemental Table 5. H) ATAC-seq and ChIP-seq tracks at the IFNG, IL2RA, and TOX loci. A-C) Pooled data from n = 3 donors. F and H) One representative donor of n = 3 donors.

Fig. 7.. Loss of MED12 increases STAT5 activity in CD19–28ζ CAR-T cells

A and B) Transcription factor binding motif enrichment at sites with increased chromatin accessibility by ATAC-seq in resting MED12 deficient CD19–28ζ CAR-T cells 15 days post T cell activation. n = 6035 sites with Log2FC > 1 and adj. P < 0.05. C) Transcription factor binding motif enrichment at sites with increased MED1 occupancy by ChIP-seq in resting MED12 deficient CD19–28ζ CAR-T cells. n = 842 sites with adjusted P < 0.05. D) Flow cytometry analysis of STAT5 phosphorylation in control and MED12 deficient CD19–28ζ CAR-T cells 15 days post T cell activation. Cells were cultured in vitro with continuous IL-2. The cells shown in light grey were rested without IL-2 for 24 hours prior to staining. E and F) Flow cytometry analysis of IL2RA expression in control and MED12 deficient F) CD19–28ζ CAR-T cells or G) non-transduced T cells 15 days post activation. Control staining with isotype antibody is shown in light grey. G) Flow cytometry analysis of IL2RA expression in non-transduced T cells cultured with dual inhibitors of CDK8 and CDK19 for 15 days. Inhibitors were supplemented to the culture medium every 48 hours. H) Heatmaps of genomic loci with significantly increased chromatin accessibility by ATAC-seq in MED12 deficient CD19–28ζ CAR-T cells. Genomic loci are overlaid with ATAC-seq and MED1 ChIP-seq signal from resting CD19–28ζ CAR-T cells 15 days post T cell activation. STAT5A and STAT5B ChIP-seq data was obtained from human CD4⁺ T cells stimulated with IL-2 (GEO accessions GSM671400, GSM671402). I) Mean ChIP-seq signal intensities at n = 6035 sites corresponding to panel H. A-C) Pooled data from n = 3 donors. Homer motif enrichment was performed with a set of all A and B) ATAC-seq or C) MED1 ChIP-seq peaks detected in CAR-T cells as the background. D-G) Data are mean ± s.d. from n = 2–3 wells. Representative results from two independent experiments. Two-tailed unpaired Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 H-I) One representative donor of n = 3 donors.