Polycomb deficiency drives a FOXP2-high aggressive state targetable by epigenetic inhibitors

Delineating the specific role of Polycomb Repressive Complex 2 (PRC2) in various cancer systems is desirable as inhibitors for EZH2 inhibitors are approved for some cancers. Here the authors show haplo- and full-insufficiency of EZH2 drive divergent phenotypes in lung cancer. 3D tumoroids recapitulate transcriptional profiles, including FOXP2 derepression, and drug responses of in vivo tumors

FIGURE 1 from EZH2 Inhibition Promotes Tumor Immunogenicity in Lung Squamous Cell Carcinomas

EZH2 inhibition allows upregulation of MHCI and MHCII in 2D human LSCC cell lines. A, Schematic for proposed mechanism: Inhibition of EZH2 methyltransferase activity by the drugs GSK126 or EPZ6438 will lead to derepression of antigen presentation genes that can then be more effectively activated by IFNγ. B, qRT-PCR in the indicated four human lung cancer cell lines treated for 7 days with vehicle or 5 µmol/L EZH2 inhibition with 20 ng/mL IFNγ added on day 5 for the genes B2M, HLA-A, CIITA, and HLA-DRA, mean ± SEM is graphed, n = 4 individual cultures, *, P P P P post hoc test. C, Flow cytometry analysis of indicated four human lung cancer cell lines treated for 7 days with vehicle or 5 µmol/L EZH2 inhibition with 20 ng/mL IFNγ added on day 5 for the cell surface proteins HLA-A,B,C and HLA-DR; mean ± SEM is graphed; n = 4 individual cultures; *, P P P P post hoc test. Representative histograms from HCC15 cell lines are shown, G = GSK126, E = EPZ6438, I = IFNγ, I+G = IFNγ+GSK126, and I+E = IFNγ+EPZ6438. D, Western blotting of A549 and HCC15 cell lines treated for 7 days with vehicle or 5 µmol/L EZH2 inhibition with 20 ng/mL IFNγ added on day 5 for the proteins B2M, HLA-DR, DQ, DP, EZH2, H3K27me3 and total histone H3. Data are representative of two individual cultures. See also Supplementary Fig. S1.</p

FIGURE 4 from EZH2 Inhibition Promotes Tumor Immunogenicity in Lung Squamous Cell Carcinomas

ChIP-seq of human patient-derived organoids confirms direct regulation of MHC and pro-T cell cytokines by EZH2. A, Peaks called at FDR 1E-7 for ChIP-seq using the chromatin marks H3K27me3, H3K27ac, and H3K4me3 in PDTs from the indicated treatment groups. Wiggle plots for H3K27me3, H3K27ac, and H3K4me3 histone mark enrichments, and matched RNA-seq tracks in PDTs from the indicated treatment groups for the genes: HLA-DRA (B), CXCL9/10/11 (C), ALOX15 (D), IL1B (E), H3K27me3 (F) peaks were called for each treatment group and GREAT was used to identify associated genes, which were then depicted by Venn diagram. G, H3K27me3 peaks that were gained or increased more than 2-fold with IFNγ treatment, and lost with EPZ6438 treatment were linked to associated genes by GREAT. The Venn diagram shows the overlap of these genes with those significantly upregulated in combination treated versus IFNγ-treated tumoroids. See also Supplementary Fig. S4.</p

FIGURE 6 from EZH2 Inhibition Promotes Tumor Immunogenicity in Lung Squamous Cell Carcinomas

scRNA-seq highlights neutrophil heterogeneity shifts in response to EZH2 inhibition combined with immunotherapy. A, Annotated UMAP plot showing the 16 different populations within lung tumors of the Lkb1/Pten model of LSCC after treatment with placebo, GSK126, anti-PD1, or combined GSK126 with anti-PD1. B, Percentage of cells per treatment group graphed for all populations, # indicates adjusted P C, GSEA depicting gene sets that are enriched or depleted in Tumor, Macrophage/Dendritic Cells, or Neutrophils in mice treated with EZH2 inhibitor and anti-PD1 contrasted with either treatment alone. Normalized enrichment scores are plotted and bubble sizes estimate FDR. See also Supplementary Table S2. D, Heat maps showing DEGs among tumor, macrophages, and dendritic cells, and neutrophils between GSK126, anti-PD1, and combination treated mice compared with placebo. E, UMAP of five neutrophil populations showing selected genes that are highly expressed in each cluster. See also Supplementary Fig. S6.</p

Supplementary Figure 6 from EZH2 Inhibition Promotes Tumor Immunogenicity in Lung Squamous Cell Carcinomas

Supplementary Figure 6 shows analysis of bone marrow from mice treated with EZH2 inhibition and anti-PD1.</p

Supplementary Figure 5 from EZH2 Inhibition Promotes Tumor Immunogenicity in Lung Squamous Cell Carcinomas

Supplementary Figure 5 shows additional analysis of tumors from EZH2 inhibition and anti-PD1 treated mice.</p

Supplementary Figure 1 from EZH2 Inhibition Promotes Tumor Immunogenicity in Lung Squamous Cell Carcinomas

Supplementary Figure 1 shows changes in NGFR and CD274 (PD-L1) gene and protein expression in human lung cancer cell lines in response to EZH2 inhibitor and interferon-gamma treatment.</p

Supplementary Figure 4 from EZH2 Inhibition Promotes Tumor Immunogenicity in Lung Squamous Cell Carcinomas

Supplementary Figure 4 shows additional ChIP-sequencing data from human lung squamous cell carcinoma tumoroids.</p

FIGURE 3 from EZH2 Inhibition Promotes Tumor Immunogenicity in Lung Squamous Cell Carcinomas

Murine LSCC organoids share derepression of MHC and pro-T cell cytokines with human models. A, Schematic: Generation of murine tumoroids in air-liquid interface from tumor induced in Lkb1/Pten mice by adenoCre administration, showing H&E stain of tumoroids, scale bar = 100 µm, and brightfield microscopy, scale bar = 200 µm. B, Flow cytometry analysis of two separate murine tumoroid models treated for 11 days with 5 µmol/L EZH2 inhibition with 20 ng/mL IFNγ added on day 9 stained for cell surface expression of NGFR, PD-L1, H2Kd,Dd, and I-A/I-E, n = 5 individual experiments except mouse 2 I-A/I-E and PD-L1; n = 4 individual experiments; *, P P P = 0.0002; ****, P post hoc test. C, Heat maps of log2 fold change in expression level from patient-derived and murine tumoroids treated for 11 days with 5 µmol/L EZH2 inhibition with 20 ng/mL IFNγ added in on day 9, G = GSK126, E = EPZ6438, I = IFNγ, I+G = IFNγ+GSK126, and I+E = IFNγ+EPZ6438. For each map, the first columns are sample 1, the second columns are sample 2. Expression relative to vehicle control (left) and relative to IFNγ only (right) are depicted. See also Supplementary Fig. S3.</p

Supplementary Table 3 from EZH2 Inhibition Promotes Tumor Immunogenicity in Lung Squamous Cell Carcinomas

Supplementary Table 3 shows gene highly expressed in each of the 16 clusters called in the single cell RNA-sequencing from murine lung and lung squamous cell carcinoma samples.</p