Fig 1.
TP53 and ARID1A mutations rarely co-occur in endometrial cancer.
a, Pan-cancer analysis of TP53 and ARID1A mutation rates across 33 TCGA tumor types, only considerate of somatic single nucleotide variants. For heatmap, darker color indicates a greater proportion of sequenced tumor samples. Odds ratio (OR) and statistics for each tumor type accompany two-tailed Fisher’s exact tests performed on TP53 and ARID1A mutation contingency tables. Asterisks indicate significant associations between TP53 and ARID1A mutations, either co-occurring or mutually exclusive: * p < 0.05; ** p < 0.01; *** p < 0.001. b, Details within the Uterine Corpus Endometrial Carcinoma (UCEC) cohort (n = 509), further inclusive of copy number alteration (CNA) events. TP53 and ARID1A mutation classes (left) and distribution of histological subtypes (right) across the UCEC cohort. c, Left, distribution of UCEC histological subtypes: endometrioid, serous, and mixed. Right, TP53 and ARID1A alteration rates and association of co-occurrence for primary tumors within each histological subtype. d, As in c but for UCEC molecular subtypes: POLE mutant, copy-number alteration high (CN high), CN low, and microsatellite instable (MSI). e, Association between POLE mutant molecular subtype tumors and TP53/ARID1A co-alterations. Statistic is two-tailed Fisher’s exact test.
Fig 2.
TP53 loss with oncogenic PIK3CA activation results in endometrial intraepithelial carcinoma.
a, Diagram of mouse alleles used in this study. b, Survival data of LtfCre0/+; (Gt)R26Pik3ca*H1047R; Trp53fl/fl (TP53/PIK3CA mutant) mice. Survival is measured as days to vaginal bleeding, requiring euthanasia. c, Representative H&E histology of control mouse endometrium from CRE-negative littermates. d, Representative H&E histology of endometrial intraepithelial carcinomas and hyperplastic epithelia in TP53/PIK3CA mutant uterus. Arrowheads denote dyplastic endometrial epithelia.
Fig 3.
Endometrial epithelial TP53 and ARID1A loss results in overlapping and distinct gene expression programs.
a, Unsupervised hierarchical clustering of gene-level RNA-seq data from sorted endometrial epithelial cells of TP53/PIK3CA mutant mice compared to ARID1A/PIK3CA mutant and control cells. Relative Z-score expression of targeted genes are displayed below clustering result. b, Relative linear Trp53 expression in endometrial epithelial cell transcriptomes. c, Volcano plot depicting differential gene expression between TP53/PIK3CA mutant and ARID1A/PIK3CA mutant cells. d, Overlap of DE genes between TP53/PIK3CA mutants and ARID1A/PIK3CA mutant cells vs. controls. Statistic is hypergeometric enrichment. e, Heatmap of 470 shared dysregulated genes in endometrial epithelial cells from each genetic model. 92.3% of intersecting DE genes are affected in the same direction. f, Overview of Broad GSEA results for MSigDB Hallmark pathways in endometrial epithelial cells from each genetic model. Axes display gene set normalized enrichment score (NES) for each model compared to control cells. g, Enrichment for Gene Ontology (GO) Biological Process gene sets among genetic model DE genes separated by directionality. h, Examples of DE genes within the GO regulation of epithelial cell differentiation gene set: DLL1, CD109, HES1, MYCN, OVOL2, ROCK2. Statistic is FDR as reported by DESeq2: * FDR < 0.05; ** FDR < 0.01; *** FDR < 0.001.
Fig 4.
Pathway analysis of TP53 and ARID1A regulated expression programs in human disease and mouse models.
a-c, Various Broad GSEA results for GO Biological Process gene sets (n = 3653) comparing TP53 and ARID1A mutant human UCEC tumors and genetically engineered mouse models: (a) TP53 mutant, ARID1A wild-type vs. wild-type/wild-type UCEC tumors compared to mouse endometrial epithelial cells from TP53/PIK3CA mutants vs. controls; (b) ARID1A mutant, TP53 wild-type vs. wild-type/wild-type UCEC tumors compared to mouse endometrial epithelial cells from ARID1A/PIK3CA mutants vs. controls; (c) ARID1A mutant, TP53 wild-type vs. TP53 mutant, ARID1A wild-type UCEC tumors compared to mouse endometrial epithelial cells from ARID1A/PIK3CA mutants vs. TP53/PIK3CA mutants. Presented are the overview of GSEA results (left) with zooms into shared upregulated (NES > 1, center) and shared downregulated (NES < -1, right) gene sets. Representative examples of highly enriched gene sets are labeled. d, Significantly over-represented terms in enriched gene sets (|NES| > 1) highlighted in a-c. Statistic is hypergeometric enrichment. See Materials and Methods for enrichment analysis framework.
Fig 5.
ARID1A mutation is associated with p53 pathway activation.
a, k-means clustering and heatmap of genetic mouse model RNA-seq relative log2 gene expression data for MSigDB Hallmark p53 pathway genes (n = 181 expressed orthologs). Representative genes are highlighted on the right. b, Differential expression of core TP53 transcriptional program gene orthologs, segregated by function, in mouse endometrial epithelial cells from ARID1A/PIK3CA mutants (green) and TP53/PIK3CA mutants (blue) compared to controls. Significant DE genes (FDR < 0.05) in each model are labeled and respectively colored. c, Distribution of PARADIGM score differences between ARID1A mutant (n = 492) vs. wild-type (n = 4832) TCGA Pan-Cancer Atlas tumors, considerate of only TP53 wild-type tumors. Top, all 19,503 measured pathways; bottom, the 36 pathways with keyword “p53”. d, Empirical distribution of mean differences between ARID1A mutant vs. wild-type PARADIGM scores, based on 50,000 samples of 36 random PARADIGM pathways. The blue line represents the mean score difference for the 36 pathways with keyword “p53” with associated permutation statistic. e, Example violin plot for the top p53 PARADIGM pathway significantly different between ARID1A mutant vs. wild-type tumors. Statistic is FDR-adjusted, two-tailed, unpaired Wilcoxon test.
Fig 6.
Analysis of ARID1A chromatin interactions in mouse endometrial epithelia in vivo.
a, Diagram of experimental workflow for measuring ARID1A chromatin interactions in vivo. Endometrial epithelial cells are purified from mouse uterus by positive enrichment with labeling and magnetic beads. Purified cells were then subject to CUT&RUN for ARID1A or IgG negative control. b, Correlation of ARID1A binding signal (compared to IgG) in vivo across 2146 genomic regions with significant binding detected (FDR < 0.25) in two independent experiments. c, Genomic annotation of 2146 ARID1A bound genomic regions in vivo. d, ARID1A binding and accessibility profiles at 3842 bound AP-1/bZIP motifs, using the top de novo enriched motif among ARID1A genome-wide binding sites in vivo, TGA(G/C)TCA. e, Heatmap of ARID1A binding and chromatin accessibility signal across 2146 genomic regions with significant ARID1A binding detected, segregated by accessibility. f, Overlap of ARID1A bound regions and accessible chromatin regions. g, Chromatin accessibility quantified at accessible regions with significantly detected ARID1A binding vs. not. Statistic is two-tailed, unpaired Wilcoxon test. h, Significant overlap of genes with ARID1A promoter binding (within 3kb of TSS) and DE genes from ARID1A/PIK3CA mutant endometrial epithelia. i, Enrichment statistics for top 10 (left) GO Biological Process gene sets and (right) Hallmark pathways among 494 human gene orthologs with ARID1A promoter binding in vivo mouse endometrial epithelia. j, Examples of ARID1A chromatin interactions and accessibility (ATAC) at Hallmark p53 pathway genes in vivo. y-axis is log-likelihood ratio of signal compared to background. Bars underneath signal tracks represent significant (FDR < 0.25) and reproducible (n = 2) signal detection i.e. peaks. phyloP track represents sequence conservation across vertebrates.
Fig 7.
Co-existing TP53 and ARID1A mutations promote aggressive endometrial tumorigenesis.
a, Cancer dependency map (DepMap) data for ARID1A wild-type cell lines, measuring ARID1A knockout viability effect on TP53 wild-type vs. mutant lines. Statistic is unpaired, two-tailed Wilcoxon test. b, Schematic of genetically engineered mice harboring endometrial epithelial specific PIK3CAH1047R, TP53 loss, and ARID1A loss. c, Example gross necropsy images in TP53/PIK3CA mutant (top) and LtfCre0/+; (Gt)R26Pik3ca*H1047R; Trp53fl/fl; Arid1afl/fl (TP53/ARID1A/PIK3CA mutant, bottom) mice. Arrowheads denote uterine abnormalities. d, Immunohistochemical staining of KRT8 staining in TP53/PIK3CA and TP53/ARID1A/PIK3CA mutant uterus. Arrowheads denote mutant endometrial epithelial cells. In TP53/ARID1A/PIK3CA mutant image. e, Representative H&E uterine histology images of TP53/ARID1A/PIK3CA mutant mice. Arrowheads depict mutant tumor cells with squamous differentiation. f, Uterine IHC staining for Cleaved Caspase-3 (cell death, left) and Ki67 (proliferation, right) in TP53/PIK3CA mutant (top) and TP53/ARID1A/PIK3CA mutant (bottom) mice. Arrowheads denote endometrial epithelial cells.
Fig 8.
ARID1A loss relieves Atf3 repression associated with squamous differentiation.
a, Representative H&E histology of (from left to right) control mice, TP53/PIK3CA mutants, ARID1A/PIK3CA mutants, and TP53/ARID1A/PIK3CA mutants. Arrowheads depict endometrial epithelia. b-d, Immunohistochemical staining of ATF3 (b), TP63 (GeneTex) (c), and COL17A1 (d) of mouse sections as in a.
Fig 9.
Model of independent and co-existing TP53 and ARID1A mutations in endometrial epithelia.
Summary of endometrial disease features and hypothesized molecular mechanisms in genetically engineered mouse models from this study.