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

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

Supplementary Table 2 from EZH2 Inhibition Promotes Tumor Immunogenicity in Lung Squamous Cell Carcinomas

Supplementary Table 2 shows Gene Set Enrichment Analysis on genes with mRNA up-regulated and H3K27me3 peaks lost in combination treatment vs IFN-gamma alone in 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 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 5 from EZH2 Inhibition Promotes Tumor Immunogenicity in Lung Squamous Cell Carcinomas

EZH2 inhibition alone and combined with immunotherapy is effective at controlling tumor burden in mouse models of LSCC. A, Representative MRI scans of autochthonous mice from each treatment arm at baseline and after treatment. B, Waterfall plot showing change in tumor volume for each mouse on all treatment arms, ****, P P P P post hoc test on log2-transformed values. C, H&E and HALO nuclear phenotyper images showing the cells within an autochthonous Lkb1/Pten tumor and a syngeneic graft seeded from Lkb1/Pten tumoroids. D, Percentage tumor growth from the syngeneic mouse model during 14 days of indicated treatments. ***, P = 0.0004; ****, P post hoc test, *, P = 0.024 by two-tailed t test on log2-transformed values, Mice/tumors n are placebo = 4/8, EPZ6438 = 5/9, anti-PD1 = 6/8, combo = 5/9, mean ± SEM. is plotted. E, Flow cytometry analysis of dissociated tumors from the syngeneic grafts from the indicated treatment arms at day 14. Percentage of EpCAM+ cells expressing IA/IE or PD-L1 are graphed, mean ± SEM is plotted, placebo n = 7, EZH2 inhibitor n = 7, anti-PD1 n = 8, combo n = 7 with two experimental replicates each, *, P = 0.035; ***, P = 0.0008 by one-way ANOVA with multiple comparisons and Holm-Šídák post hoc test. F, From the same tumor grafts, MFI for HLA-A in the EpCAM+ cells was graphed, mean ± SEM is plotted, placebo n = 7, EZH2 inhibitor n = 7, anti-PD1 n = 8, combo n = 7 with 2 experimental replicates each, **, P = 0.0015 by one-way ANOVA with multiple comparisons and Holm-Šídák post hoc test. G, From tumor grafts, PD1+/CD3+/CD4+ cells and PD1+/CD3+/CD8+ were gated and percentage of cells bound to Rat-IgG2A antibody are graphed, mean ± SEM is plotted, placebo n = 6, EZH2 inhibitor n = 6, anti-PD1 n = 7, combo n = 7; **, P P = 0.0001; ****, P post hoc test. H, From the grafts, percentage of CD3+/SSC-low cells within the CD45+ fraction and percentage of CD8+ cells within the CD3+ fraction were graphed, please see Supplementary Fig. S5C for representative gates, placebo n = 8, EZH2 inhibitor n = 8, anti-PD1 n = 9, combo n = 7 with two experimental replicates each, *, P = 0.0197; ****, P post hoc test. See also Supplementary Fig. S5.</p

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

Supplementary Table 4 shows Gene Set Enrichment Analysis of differentially expressed mRNAs in combination-treated tumors vs. single agent-treated tumors for three major cell sub-clusters in the single cell RNA-sequencing.</p

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

Supplementary Figure 3 shows changes in gene expression and Gene Set Enrichment Analysis in lung squamous cell carcinoma tumoroids in response to EZH2 inhibitor and interferon-gamma treatment.</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