Journal: Nature Communications

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

doi: 10.1038/s41467-023-35784-x

Figure Lengend Snippet: a Schematic for LoxP-mediated deletion of Ezh2 to generate EZH2 WT, heterozygous, and homozygous null KRAS /Trp53-null GEMMs. b Representative H&E-stained cross sections from lungs of closely related mice that were induced and sacrificed at the same timepoint, scale bar = 5 mm. c Tumor burden relative to the whole lung at the different timepoints post Adeno-Cre graphed as mean values +/− SEM. * indicates P = 0.0461 Ezh2 WT vs. null Bin 2, P = 0.028 Bin 3, P = 0.0437 Bin 4, ** indicates P = 0.005, *** indicates P = 0.0001 Ezh2 heterozygous vs. null by one-way ANOVA with multiple comparisons. Bin 1 n = 4, 9, 6, Bin 2 n = 10, 10, 9, Bin 3 n = 11, 14, 6, Bin 4 n = 9, 11, 9 for Ezh2 WT, heterozygous, null individual mice, respectively. d Percentage survival of mice of indicated genotypes. * indicates P = 0.0376 Ezh2 WT vs. Ezh2 heterozygous, *** indicates P = 0.0003 Ezh2 heterozygous vs. Ezh2 null, and * indicates P = 0.0397 Ezh2 WT vs. Ezh2 null by log-rank test. n = 8, 18, 18 for Ezh2 WT, heterozygous, null individual mice, respectively. e Nuclear grade was scored on sectioned tissue by a blinded pathologist and graphed as mean values +/− SEM. * indicates P < 0.042 by one-way ANOVA with multiple comparisons, n = 6, 9, 8 for Ezh2 WT, heterozygous, null individual mice, respectively. f Mice of indicated genotypes were scored as having metastasis (Met) or not by gross examination and histology at days 91–133 post tumor induction and percentage of each are graphed. * indicates P = 0.012 and ** indicates P = 0.0024 by Fisher’s exact test. n = 30, 36, 25 for Ezh2 WT, heterozygous, null individual mice, respectively. g Representative images of tumor sections stained with H&E, EZH2 or H3K27me3, scale bar = 100 µm. n = 26 or greater individual mouse tumor sections were stained and data are summarized in Supplementary Fig. . Genotypes are signified as: WT, +/+; heterozygous, ∆/+; null, ∆/∆. Source Data are provided for this figure.

Article Snippet: The murine Ezh2 expression vectors plasmids #24927 and plasmid # 24926 were purchased from Addgene .

Techniques: Staining

Journal: Nature Communications

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

doi: 10.1038/s41467-023-35784-x

Figure Lengend Snippet: a Representative images of in vivo tumor histology, in vitro 3D tumor spheroid histology and 2D cell cultures of the indicated Ezh2 genotypes, scale bar = 100 µm. n = 3 or greater individual mouse tumor-derived cultures were examined for each genotype and culture system. b Heatmap of sample-to-sample correlations using highest loading genes from PC3 analysis, where the Pearson correlations are weighted by sample-sample information and sample-sample consistency (see “Methods”). The 1 − correlation value was then used as a pseudo distance for hierarchical clustering and ordered using default argument of dendsort. n = 17, 13, 20, 21 for total tumor, sorted tumor, 2D cell cultures, 3D tumoroid individual mouse tumor-derived samples, respectively. c Rank-ordered gene lists were queried against the MSigDB databases and enrichment scores of selected gene signatures for 2D, 3D, or in vivo sorted tumors were plotted. Dot size reflect FDR estimates. Data are shown in Supplementary Table . d Percentages of cells in each cell cycle phase were measured by 7AAD cell cycle flow cytometry in 2D cells and 3D tumoroids of the indicated Ezh2 genotypes plotted as mean values +/− SEM. * indicates P < 0.035, ** indicates P < 0.009, *** indicates P < 0.0009 by one-way ANOVA with multiple comparisons and Holm–Šídák’s post hoc test for comparing genotypes within culture system, and by two-tailed t test for comparing the same genotype in the two culture systems. 2D: n = 4, 3, 4; 3D: n = 3, 4, 3 for Ezh2 WT, heterozygous, null individual mouse tumor-derived cultures, respectively. e 2D cells and 3D tumoroids were examined for expression of the indicated proteins by immunoblot. f Normalized protein abundance of the indicated proteins in 2D cells and 3D cultures by immunoblot. **** indicates P = 0.0000086 for pH3 in 2D vs. 3D and * P = 0.024 for PCNA in 2D vs. 3D for all Ezh2 genotypes combined by two-tailed t test. n = 2 individual mouse tumor-derived cultures per genotype/culture system. Genotypes are signified as: WT, +/+; heterozygous, ∆/+; null, ∆/∆. Source Data are provided for this figure.

Article Snippet: The murine Ezh2 expression vectors plasmids #24927 and plasmid # 24926 were purchased from Addgene .

Techniques: In Vivo, In Vitro, Derivative Assay, Flow Cytometry, Two Tailed Test, Expressing, Western Blot, Quantitative Proteomics

Journal: Nature Communications

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

doi: 10.1038/s41467-023-35784-x

Figure Lengend Snippet: a Rank-ordered gene lists were queried against the MSigDB databases and enrichment scores of selected gene signatures enriched in the Ezh2 null or Ezh2 heterozygous compared to Ezh2 WT for the indicated models were plotted. Dots size estimates FDR. Data are summarized in Supplementary Table . b Venn diagram and heatmaps depict significant differentially expressed genes in Ezh2 null vs. WT cells for the indicated culture systems, with log-fold change LFC > 0.5 or LFC < −0.5, FDR < 0.05. Genes higher in Ezh2 null are in red, genes lower in Ezh2 null are in blue. Heatmaps depict LFC of selected genes identified by the indicated contrasts. c Heatmap representation of EZH2 and H3K27me3-bound chromatin peaks centered across a ± 5-kb window that showed decreased occupancy of Ezh2 heterozygous, Ezh2 null, and EPZ6438-treated Ezh2 WT mouse tumor spheroids compared to Ezh2 WT. d Venn diagram showing EZH2 and H3K27me3 peaks significantly called by SICER at FDR < E-10 for genes in the indicated Ezh2 genotypes or EZH2 inhibitor-treated organoids. e GREAT analysis heatmaps depicting the H3K27me3 peaks of mouse 3D organoid ChIP-seq samples enriched in the indicated gene sets. Data are summarized in Supplementary Table . f Venn diagram showing overlap of shared DEGs from ( b ) with H3K27me3 ChIP peaks found in Ezh2 WT tumoroids that were decreased by more than twofold or absent in Ezh2 null tumoroids, with Foxp2 indicated as a shared gene. Genotypes are signified as: WT, +/+; heterozygous, ∆/+; null, ∆/∆. Source Data are provided for this figure.

Article Snippet: The murine Ezh2 expression vectors plasmids #24927 and plasmid # 24926 were purchased from Addgene .

Techniques: ChIP-sequencing

Journal: Nature Communications

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

doi: 10.1038/s41467-023-35784-x

Figure Lengend Snippet: a Relative expression of Foxp2 mRNA in mouse primary 2D tumor cells and 3D tumor spheroids with the indicated Ezh2 genotypes graphed as mean values +/− SEM. * indicates P < 0.043 by one-way ANOVA with multiple comparisons and Holm–Šídák’s post hoc test between genotypes and two-tailed t test between conditions. n = 3 individual mouse tumor-derived cultures per genotype/culture system. b FOXP2 protein expression in 2D cells and 3D tumoroids of the indicated Ezh2 genotypes was examined by immunoblot. Total histone H3 is the same as Fig. . ǂ indicates the non-specific band seen with FOXP2 antibody from Cell Signaling Technologies. c Immunoblot and d the normalized abundance of the indicated proteins in Ezh2 WT, Ezh2 null, Ezh2 null with WT Ezh2 re-expression (WT), and Ezh2 null with F667I-mutant Ezh2 re-expression (F6771). Quantification data are plotted as mean values +/− SEM. * indicates P < 0.0261 by one-way ANOVA with multiple comparisons and Holm–Šídák’s post hoc test. n = 3 individual blotting experiments from two cell cultures. e Percentages of positively stained tumor nuclei for FOXP2 in the tumors of the indicated genotypes plotted as box-and-whisker plots, error bars are min–max, box bounds are 25th and 75th percentiles and center line is median. * indicates P = 0.0233 by one-way ANOVA with multiple comparisons and Holm–Šídák’s post hoc test for both comparisons. n = 29, 38, 23 for Ezh2 WT, heterozygous, null individual mice, respectively. f Visualization of H3K27me3 ChIP-seq peaks enriched in the enhancer and promoter regions of Foxp2 in the indicated 3D tumor spheroid samples. g Expression of FOXP2 mRNA in human lines HBEC3KT, BEAS2B, BEAS-KP, H460, H2030, A549, and H2009 with or without 5 µM EPZ6438 treatment for 10 days plotted as mean values +/− SEM. * indicates P < 0.043, ** indicates P < 0.009, *** indicates P = 0.0003 by two-tailed t test. n = 3 individual cell cultures per cell line/condition. h Immunoblot and i the normalized abundance of the indicated proteins in human lines HBEC3KT, BEAS2B, BEAS-KP, H460, H2030, A549, and H2009 with or without 5 µM EPZ6438 treatment for 10 days. Top FOXP2 panel is SIGMA antibody, bottom FOXP2 panel is Cell Signaling Technologies antibody. Quantification data are plotted as mean values +/− SEM. * indicates P < 0.032, *** indicates P = 0.000778 with two-tailed t test. n = 4 with two individual blotting experiments derived from one cell culture per cell line/condition each probed with two distinct FOXP2 antibodies. Genotypes are signified as: WT, +/+; heterozygous, ∆/+; null, ∆/∆. Source Data are provided for this figure.

Article Snippet: The murine Ezh2 expression vectors plasmids #24927 and plasmid # 24926 were purchased from Addgene .

Techniques: Expressing, Two Tailed Test, Derivative Assay, Western Blot, Mutagenesis, Staining, Whisker Assay, ChIP-sequencing, Cell Culture

Journal: Nature Communications

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

doi: 10.1038/s41467-023-35784-x

Figure Lengend Snippet: a Schematic depicting the mechanisms to restore epigenetic states lost in PRC2-deficient tumors. b , c GSK-J4 (upper) and JQ1 (lower) percentage survival of ( b ) 2D cultures after 4 days culture with drugs plotted as mean values +/− SEM. GSK-J4: n = 7, 7, 7; JQ1; n = 5, 6, 5 for Ezh2 WT, heterozygous, null individual mouse tumor-derived cultures, respectively. c 3D tumoroids after 12–14 days culture with drugs plotted as mean values +/− SEM. GSK-J4: * indicates P = 0.0158 Ezh2 WT vs. Ezh2 null, *** P = 0.0006 Ezh2 WT or heterozygous vs. Ezh2 null; JQ1: * indicates P < 0.039 and ** P = 0.0084 for Ezh2 WT vs. Ezh2 null (lower) or Ezh2 WT vs. heterozygous (upper) by one-way ANOVA with multiple comparisons and Holm-Šídák’s post hoc. For both drugs, n = 7, 5, 5 for Ezh2 WT, heterozygous, null individual mouse tumor-derived cultures, respectively. d Changes in volumes of grafts in mice treated with GSK-J4 and JQ1 relative to changes in placebo-treated mice plotted over time as mean values +/− SEM. *** indicates P < 0.0008, ** P < 0.0091, * P < 0.0472 for Ezh2 WT vs. Ezh2 null (lower) or Ezh2 heterozygous vs. Ezh2 null by one-way ANOVA with repeated measures and multiple comparisons. GSK-J4: n = 5, 5, 4; JQ1: n = 5, 5, 6 for Ezh2 WT, heterozygous, null individual mouse tumor-derived grafts, respectively. e Quantification of relative tumor volume change after 1 week and 2 weeks of placebo or JQ1 treatment of Ezh2 null or Ezh2 heterozygous tumor-bearing mice, ** indicates P = 0.0021 for week 1, P = 0.0003 for weak 2 by two-sided t test on log2-transformed values. f Representative magnetic resonance images of mouse lungs at baseline (week 0) and week 1 of JQ1 treatment for an Ezh2 null mouse. g Heatmap of Bliss synergy scores for EPZ6438 combined with JQ1 in H2009 and BEAS-KP cultures. The most synergistic areas had Bliss scores of 10.15 (H2009) and 10.99 (BEAS-KP), indicating synergy. n = 3, 4 for H2009 and BEAS-KP individual cell cultures, respectively. Genotypes are signified as: WT, +/+; heterozygous, ∆/+; null, ∆/∆. Source Data are provided for this figure.

Article Snippet: The murine Ezh2 expression vectors plasmids #24927 and plasmid # 24926 were purchased from Addgene .

Techniques: Derivative Assay, Transformation Assay

Journal: Nature Communications

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

doi: 10.1038/s41467-023-35784-x

Figure Lengend Snippet: a Positively stained tumor nuclei in ADC, ADSCC, poorly differentiated tumor and SCC specimens for the markers EZH2, H3K27me3 and FOXP2 plotted as box-and-whisker plots, error bars are min–max, box bounds are 25th and 75th percentiles and center line is median. * indicates P = 0.0164, ** indicates P < 0.0084, *** indicates P < 0.0006 by one-way ANOVA with multiple comparisons and Holm–Šídák’s post hoc test. n = 92, 24, 17, 104 for ADC, ADSCC, poorly differentiated and SCC individual tumors, respectively. b The percentage of EZH2 and H3K27me3 positive nuclei in lung adenocarcinoma and poorly differentiated cancer specimens. * indicates P < 0.0425, ** indicates P < 0.0086 by one-way ANOVA with multiple comparisons and Holm–Šídák’s post hoc test. n = 13, 51, 97 for well, moderately, poorly differentiated individual tumors, respectively. c Representative images of lung adenocarcinoma specimens defined as well, moderately, and poorly differentiated stained for the indicated markers, scale bar = 100 µm. The number of tumors stained are summarized in ( b ). d Correlation between EZH2 and FOXP2 based on the percentage of positively stained tumor nuclei in primary lung cancer specimens. Pearson’s correlation coefficients and p values are shown. n = 161 individual tumors. e Kaplan–Meier relapse-free survival curves for FOXP2 -high and FOXP2 -low tumors as measured by RNA-sequencing 250 months post diagnosis; positive and negative groups were determined by best cut-off. The P value was calculated by log-rank test. n = 366 individual tumors. f , g Immunoblot in 2D ( f ) H460, A549, BEAS-KP, and g Ezh2 WT UK803 cells treated with or without 500 μM SAM for 6 days. h Normalized protein abundance in H460, A549, and BEAS-KP cells treated with or without 500 μM SAM for 6 days plotted as mean values +/− SEM. * indicates P = 0.012, ** indicates P = 0.009 for EZH2 and P = 0.0045 for FOXP2 by two-tailed t test. n = 3 different cell lines. Genotypes are signified as: WT, +/+; heterozygous, ∆/+; null, ∆/∆. SAM, S-adenosyl methionine. Source Data are provided for this figure.

Article Snippet: The murine Ezh2 expression vectors plasmids #24927 and plasmid # 24926 were purchased from Addgene .

Techniques: Staining, Whisker Assay, RNA Sequencing, Biomarker Discovery, Western Blot, Quantitative Proteomics, Two Tailed Test