Co-existing TP53 and ARID1A mutations promote aggressive endometrial tumorigenesis

TP53 and ARID1A are frequently mutated across cancer but rarely in the same primary tumor. Endometrial cancer has the highest TP53-ARID1A mutual exclusivity rate. However, the functional relationship between TP53 and ARID1A mutations in the endometrium has not been elucidated. We used genetically engineered mice and in vivo genomic approaches to discern both unique and overlapping roles of TP53 and ARID1A in the endometrium. TP53 loss with oncogenic PIK3CAH1047R in the endometrial epithelium results in features of endometrial hyperplasia, adenocarcinoma, and intraepithelial carcinoma. Mutant endometrial epithelial cells were transcriptome profiled and compared to control cells and ARID1A/PIK3CA mutant endometrium. In the context of either TP53 or ARID1A loss, PIK3CA mutant endometrium exhibited inflammatory pathway activation, but other gene expression programs differed based on TP53 or ARID1A status, such as epithelial-to-mesenchymal transition. Gene expression patterns observed in the genetic mouse models are reflective of human tumors with each respective genetic alteration. Consistent with TP53-ARID1A mutual exclusivity, the p53 pathway is activated following ARID1A loss in the endometrial epithelium, where ARID1A normally directly represses p53 pathway genes in vivo, including the stress-inducible transcription factor, ATF3. However, co-existing TP53-ARID1A mutations led to invasive adenocarcinoma associated with mutant ARID1A-driven ATF3 induction, reduced apoptosis, TP63+ squamous differentiation and invasion. These data suggest TP53 and ARID1A mutations drive shared and distinct tumorigenic programs in the endometrium and promote invasive endometrial cancer when existing simultaneously. Hence, TP53 and ARID1A mutations may co-occur in a subset of aggressive or metastatic endometrial cancers, with ARID1A loss promoting squamous differentiation and the acquisition of invasive properties.

TP53 and ARID1A are frequently mutated across cancer but rarely in the same primary tumor. Endometrial cancer has the highest TP53-ARID1A mutual exclusivity rate. However, the functional relationship between TP53 and ARID1A mutations in the endometrium has not been elucidated. We used genetically engineered mice and in vivo genomic approaches to discern both unique and overlapping roles of TP53 and ARID1A in the endometrium. TP53 loss with oncogenic PIK3CA H1047R in the endometrial epithelium results in features of endometrial hyperplasia, adenocarcinoma, and intraepithelial carcinoma. Mutant endometrial epithelial cells were transcriptome profiled and compared to control cells and ARID1A/ PIK3CA mutant endometrium. In the context of either TP53 or ARID1A loss, PIK3CA mutant endometrium exhibited inflammatory pathway activation, but other gene expression programs differed based on TP53 or ARID1A status, such as epithelial-to-mesenchymal transition. Gene expression patterns observed in the genetic mouse models are reflective of human tumors with each respective genetic alteration. Consistent with TP53-ARID1A mutual exclusivity, the p53 pathway is activated following ARID1A loss in the endometrial epithelium, where ARID1A normally directly represses p53 pathway genes in vivo, including the stress-inducible transcription factor, ATF3. However, co-existing TP53-ARID1A mutations led to invasive adenocarcinoma associated with mutant ARID1A-driven ATF3 induction, reduced apoptosis, TP63+ squamous differentiation and invasion. These data suggest TP53 and ARID1A mutations drive shared and distinct tumorigenic programs in the endometrium and promote invasive endometrial cancer when existing simultaneously. Hence, TP53 and ARID1A mutations may co-occur in a subset of aggressive or metastatic endometrial cancers, with ARID1A loss promoting squamous differentiation and the acquisition of invasive properties. Introduction TP53 (p53) and ARID1A are among the most frequently mutated tumor suppressor genes across cancer [1]. The historic tumor suppressor roles of TP53 have been well characterized in numerous reports since its discovery, when it was found interacting with the transforming agent SV40 large T antigen [2,3]. Meanwhile, the functions of ARID1A in cellular homeostasis and carcinogenesis have only recently been described since exome studies revealed widespread mutation prevalence in disease [4,5]. Both proteins serve roles in transcriptional regulation-ARID1A is a SWI/SNF chromatin remodeling complex subunit [5], while TP53 is a transcription factor [6]. Evidence supports TP53 and ARID1A also have other important nuclear functions including DNA repair and cell cycle regulation [7][8][9][10]. TP53 and ARID1A mutations are frequent among gynecologic cancers [11][12][13][14][15][16]. Both genes are commonly mutated in ovarian and uterine cancers, and there are mutation-defining subtypes within each cancer [13,[17][18][19][20][21]. However, an early mechanistic study showed biochemical and functional evidence linking ARID1A and TP53 regulation and mutant ARID1A-TP53 mutual exclusivity in a cohort of 77 ovarian clear cell and uterine endometrioid carcinomas, where all ARID1A mutant tumors were TP53 wild-type, and vice versa [22]. Since then, numerous reports have observed ARID1A and TP53 alterations co-occur less frequently than expected by chance in other human cancer types, including gastric, breast, and esophageal [23][24][25][26]. Among gynecologic cancers, loss of ARID1A expression by immunohistochemical staining was significantly associated with wild-type TP53 expression in high-grade endometrial tumors [27]. Within the endometrioid subtype of endometrial cancer, one study observed that tumors marked by high TP53 expression, indicative of TP53 mutation, almost never displayed low/absent ARID1A expression [28].
In this study, we show that endometrial cancer displays the highest mutual exclusivity rate for TP53 and ARID1A mutations, irrespective of histological subtype, across over 10,000 human tumors profiled by The Cancer Genome Atlas (TCGA). We develop a genetically engineered mouse model with co-existent TP53 loss of function and oncogenic PIK3CA H1047R activation specifically in the endometrial epithelium. To discern both overlapping and distinct molecular features associated with TP53 or ARID1A mutation, endometrial epithelial cells were isolated from this model, profiled by RNA-seq, and compared to control cells and ARID1A/PIK3CA mutant cells. These mouse model data were compared with human tumor ovarian serous cystadenocarcinoma, OV) display significant mutual exclusivity (two-tailed Fisher's exact test, p < 0.05), indicating these mutations co-occur less frequently than expected by chance. One tumor type, cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), indicated the opposite, that mutations in these two genes were occurring more frequently than expected. Uterine corpus endometrial carcinoma (UCEC) [18] displayed the highest rate of TP53-ARID1A mutual exclusivity out of all profiled cancer types (two-tailed Fisher's exact test, OR = 0.155, p < 10 −20 ).
We further investigated TP53 and ARID1A genetic alteration status in UCEC via the TCGA Pan-Cancer Atlas data set [30], which includes 509 primary tumor samples with both mutation and copy number alteration (CNA) data ( Fig 1B). Tumors were considered "altered" for a gene if they displayed either a mutation or CNA event at each locus. In the combined data set of all disease subtypes, TP53 and ARID1A mutations were co-altered in 7.5% of tumor samples, while the independent alteration frequencies for each gene were above 30% (Fig 1B). The UCEC data set is comprised of three distinct histological disease subtypes-endometrioid, serous, and mixed type morphology-which have different incidence rates and are strongly associated with TP53 and ARID1A status [15,18,31,32]. The original TCGA-UCEC report also classified four molecular subtypes based on integrative multi-omic analyses: POLE ultramutated, copy-number alteration high (CN high), copy-number alteration low (CN low), and microsatellite instable (MSI) [33]. Therefore, we sought to determine if TP53-ARID1A genetic mutual exclusivity could be attributed to sampling error by investigating alterations within each histological and molecular subtype independently. In the two predominant endometrial cancer histological subtypes, endometrioid and serous, TP53 and ARID1A alterations cooccurred less frequently than expected by chance in primary tumors (two-tailed Fisher's exact test, Fig 1C). Across the molecular subtypes, only CN low tumors displayed significant ARI-D1A-TP53 mutual exclusivity (OR = 0.12, Fig 1D). The other molecular subtypes are characterized by heightened genomic instability, which are more likely to harbor passenger mutations in those tumor subtypes. Supporting this, POLE ultra-mutated primary tumors are more associated with TP53/ARID1A co-alterations compared to other subtypes (OR = 4.6, two-tailed Fisher's exact test, Fig 1E). Overall, these analyses suggest that mutually exclusive TP53 and ARID1A alterations are observed in primary uterine endometrial tumors of both endometrioid and serous subtypes and are notable in CN low tumors.

TP53 loss in the presence of PIK3CA H1047R drives hyperplasia and endometrial intraepithelial carcinoma
We previously reported that ARID1A loss paired with PI3K activation through constitutive expression of oncogenic PIK3CA H1047R drives endometrial hyperplasia and myometrial invasion in mice [34,35]. In addition, we also showed that ARID1A and PIK3CA mutations frequently co-occur in UCEC tumor samples [34]. Upon further examination of TCGA-UCEC data, we found that roughly half of TP53 mutant tumors also harbor PIK3CA mutations (S1A Fig). Since TP53 and PIK3CA mutations are frequently observed together, we tested whether TP53 mutations could also promote endometrial tumorigenesis in the presence of PIK3-CA H1047R in mice.
TCGA-UCEC data indicates that roughly 19% of TP53 mutations in uterine serous carcinoma putatively result in direct TP53 protein truncation through frameshift or splice site alteration, and missense vs. truncating TP53 mutations are not associated with differences in survival or tumor grading (S1B-S1E Fig). Therefore, we modeled the effects of TP53 loss in combination with PIK3CA mutation in the endometrium by crossing the Trp53 fl allele [36] with (Gt)R26 Pik3ca � H1047R [37] in LtfCre + mice (Fig 2A). The LtfCre allele results in tissue-specific Cre recombinase expression in the endometrial epithelium at onset of puberty [38]. Vaginal bleeding, indicating endometrial dysfunction, was observed with biallelic loss of TP53 in the presence of PIK3CA H1047R at a median of 76 days (Fig 2B). Compared to control mice (Figs 2C and S2A), histological analysis of LtfCre 0/+ ; (Gt)R26 Pik3ca � H1047R ; Trp53 fl/fl mice (henceforth referred to as TP53/PIK3CA mutant mice) revealed features of hyperplasia, adenocarcinoma, and endometrial intraepithelial carcinoma (EIC) within luminal and glandular areas (Figs 2D and S2B). Mutant endometrial epithelial cells expressed KRT8, a marker of endometrial epithelium, and phospho-S6, a marker of PI3K pathway activity (S2C Fig). EIC is typically considered a precursor lesion to uterine serous carcinoma, a subtype of endometrial cancer dominated by TP53 mutations (see Fig 1C) Fig 3A). TRP53 gene expression was significantly decreased in TP53/PIK3CA mutant cells compared to controls (FDR < 10 −37 ) ( Fig 3B). Interestingly, TRP53 expression was significantly upregulated in ARID1A/PIK3CA mutant cells (FDR = 0.0025) (Fig 3B). Directly comparing TP53/ PIK3CA mutants to ARID1A/PIK3CA mutant cells resulted in 1799 significant differentially expressed (DE) genes at an FDR < 0.05 significance threshold (Fig 3C). Upon comparing each genetic model to control cells, 1514 genes were significantly affected in TP53/PIK3CA mutant cells, and 3455 genes were affected in ARID1A/PIK3CA mutant cells (Fig 3D). Of these gene sets, 470 DE genes overlapped between the genetic mouse models (hypergeometric enrichment, p < 10 −55 ) ( Fig 3D). Among overlapping DE genes, 92.3% of genes were affected in the same direction between both genetic models, which could be attributed to the PIK3CA H1047R tumorigenic mechanisms shared in both models ( Fig 3E).
Next, we asked what biological processes and pathways were affected in each genetic model. We performed Broad Gene Set Enrichment Analysis (GSEA) [44] for MSigDB Hallmark pathways [45] on human orthologs from each model compared to control cells. Comparing the GSEA normalized enrichment scores (NES) revealed that certain pathways were upregulated or downregulated in both genetic models, such as interferon responses (upregulated) and estrogen response (downregulated) (Fig 3F). However, many pathways were upregulated in ARID1A/PIK3CA mutant cells and downregulated in TP53/PIK3CA mutant cells (Fig 3F). These include Notch signaling, p53, epithelial-to-mesenchymal transition (EMT) and Myc targets. We previously reported that ARID1A transcriptionally represses mesenchymal fates through integrative genomic and cellular assays in vivo and in vitro [34].
Further investigation into the Hallmark EMT pathway revealed a cluster of 49 genes which are upregulated in both models as well as a cluster of 60 genes which are upregulated in ARID1A/PIK3CA mutant cells but mostly downregulated in TP53/PIK3CA mutants (S4A- S4D Fig). Among the affected genes, the EMT master regulator SNAI2 is highly upregulated in ARID1A/PIK3CA mutants (4.54 log 2 FC vs. controls) but unaffected in TP53/PIK3CA mutant endometrial epithelial cells (S4E Fig). SNAI2/SLUG is a conserved transcription factor that directly represses epithelial gene transcription to regulate cellular processes like adhesion, polarity, migration, and invasion [46]. The observation that EMT-promoting factors are aberrantly upregulated in ARID1A mutant cells, but not in TP53 mutants, may explain the lack of collective invasion in TP53/PIK3CA mutant endometrial epithelia. Enrichment for Gene Ontology (GO) Biological Process gene sets provided further insight into cellular processes affected in the genetic models ( Fig 3G). As was observed in the GSEA analysis, interferon and immune pathways were enriched in genes commonly upregulated in both models, though various extracellular matrix pathways were uniquely enriched among upregulated genes in ARID1A/PIK3CA mutant cells (Fig 3G). Interestingly, no pathways were enriched among genes uniquely upregulated in the TP53/PIK3CA mutant model. Further, direct comparison of the two genetic models showed there were no significantly enriched gene sets among 603 human ortholog genes more highly expressed in TP53/PIK3CA mutant cells among the Hallmark, GO, and Oncogenic signature MSigDB Collections (S5 Fig). Among downregulated pathways, ARID1A/PIK3CA mutant cells downregulate ER stress response and glycosylation processes, while TP53/PIK3CA mutant cells downregulate G1/S mitotic transition transcriptional programs, apoptotic signaling pathways, and cellular differentiation pathways ( Fig 3G). Notably, regulation of epithelial cell differentiation was enriched among genes downregulated in TP53/ PIK3CA mutants but also genes upregulated in ARID1A/PIK3CA mutants. Further investigation into genes involved in this process showed that some appear to be oppositely affected following ARID1A or TP53 loss (DLL1), while others are uniquely affected by TP53 loss (CD109, HES1), uniquely affected by ARID1A loss (MYCN), or affected by loss of both TP53 and ARID1A (OVOL2, ROCK2) ( Fig 3H). Altogether, these results highlight transcriptional programs with shared and unique regulation by ARID1A and TP53.

Gene expression programs in mouse models reflect human tumor genetics
The molecular profiling data generated by TCGA serve as an excellent resource to support the human disease relevance of mouse model observations. We segregated UCEC bulk primary tumor samples by ARID1A and TP53 genetic mutation status and histological subtype, then performed Broad GSEA for the MSigDB collection of Hallmark pathways and GO Biological Process gene sets using RNA-seq data from each sample. The various UCEC comparisons included TP53mut/ARID1Awt vs. wt/wt, ARID1Amut/TP53wt vs. wt/wt, ARID1Amut/ TP53wt vs. TP53mut/ARID1Awt, and endometrioid vs. serous. The same GSEA genetic comparison framework was also applied to the transcriptomic data generated from isolated mutant mouse cells compared to control cells and each other. GSEA results were then contrasted between the human and mouse comparisons, which were labeled as enriched by an absolute NES >1 threshold.
Hallmark pathway GSEA results for UCEC tumors corroborated certain observations in our genetic mouse models, such as upregulation of EMT, apoptosis, and p53 pathway in ARID1A mutant tumors, but downregulation of these pathways in TP53 mutant tumors (S6A Fig). As TP53 mutations are a hallmark of uterine serous carcinoma, while ARID1A mutations comprise roughly half of uterine endometrioid adenocarcinoma, we also observed that both Hallmark pathway and GO Biological Process GSEA results significantly correlated in a comparison between ARID1A mutant vs. TP53 mutant tumors and endometrioid vs. serous histological subtype (S6B and S6C Fig). We further confirmed the observed GSEA correlations were stronger than may be expected by chance due to sampling dependency (S6D Fig),

PLOS GENETICS
Endometrial TP53-ARID1A co-mutations From 3653 total GO Biological Process gene sets queried in all comparisons, 225 were mutually upregulated and 142 were mutually downregulated between TP53mut/ARID1Awt vs. wt/wt UCEC tumors and TP53/PIK3CA mutant vs. control mouse cells (Fig 4A). Upregulated TP53 mutant gene sets include response to type I interferon (NES 1.80 and 1.82, respectively) and double strand break repair (NES 1.93 and 1.11, respectively). Downregulated gene sets include intrinsic apoptotic signaling pathway by p53 (NES -1.59 and -1.49) and regulation of response to extracellular stimulus (NES -1.59 and -1.02). With regard to ARID1A mutant comparisons, 225 gene sets were mutually upregulated and 156 were mutually downregulated between ARID1Amut/TP53wt vs. wt/wt UCEC tumors and ARID1A/PIK3CA mutant vs. control mouse cells (Fig 4B). Upregulated ARID1A mutant gene sets include response to hyperoxia (NES 1.81 and 1.39, respectively) and collagen fibril organization (NES 1.75 and 1.42). Curiously, in both the ARID1A and TP53 human-mouse disease comparisons, mutually downregulated gene sets (compared to wild-types or controls) overlapped more than expected by chance, while upregulated gene sets did not (hypergeometric enrichment, S7 Fig). Direct comparison of ARID1A mutant vs. TP53 mutant human tumors and mouse models furthered that many related processes appear to be distinctly affected in TP53 vs. ARID1A mutants, such as extracellular matrix assembly and EMT ( Fig 4C). Notably, gene sets that were expressed higher in ARID1A mutants compared to TP53 mutants strongly overlapped between UCEC tumors and mouse models (p < 10 −8 , hypergeometric enrichment), while downregulated gene sets did not display significant overlap (S7 Fig). At the gene level, 81 genes were significantly more highly expressed in TP53 mutant mice and UCEC tumors as compared to ARID1A mutants, including ESRRB, MAL, WNT7A, RASAL1, USP51, PLCXD3, and AIF1L (S8 Fig). In contrast, 149 genes were significantly more highly expressed in ARID1A mutants, including COL17A1, KRT5, TP63, SNAI2, ZNF750, HAS3, ANKK1, WDR38, C6, and IL33 (S8 Fig).
In order to characterize the affected pathways in TP53 vs. ARID1A mutant disease in an unbiased manner, we identified over-represented terms among enriched GO Biological Process gene sets. Upregulated TP53 mutant gene sets were enriched for terms such as "type I interferon" and "response to virus", and downregulated sets involved terms "endoplasmic reticulum" and "epithelial" (Fig 4D). Upregulated ARID1A mutant gene sets were enriched for terms "cell cycle", "cell migration", "oxidative stress", and "p53" (Fig 4D). Human-human and mouse-mouse genetic comparisons also showed that p53 pathway-related processes are consistently upregulated in ARID1A mutant tumors and downregulated in TP53 mutant tumors (S9 Fig). ARID1A mutant tumors display p53 pathway activation p53 pathway gene signatures were upregulated in both ARID1A mutant UCEC tumors and the ARID1A/PIK3CA mutant genetic mouse model. This result suggests tumor cell dependencies on the p53 pathway itself could be a potential mechanism underpinning mutual exclusivity of TP53 and ARID1A mutations. Mouse model analysis of gene expression within the Hallmark p53 pathway showed that certain canonical members of the p53 pathway were upregulated in ARID1A/PIK3CA mutant mice, such as TP63, TP53, MDM2, DDIT3 (CHOP), and CDKN1A ( Fig 5A). As TP53 and ARID1A both regulate transcription, we proceeded further with an unbiased investigation to determine which aspects of TP53 directed transcriptional regulation are co-regulated by ARID1A in the endometrium. We interrogated a recently reported gene set composed of 103 high-confidence TP53 target genes that are transcriptionally regulated by TP53 in multiple cell lines, known as the core TP53 transcriptional program, which were further categorized based on known functions [47]. These genes were enriched for expression alterations in diseased endometrial epithelia from both ARID1A/PIK3CA and TP53/PIK3CA mutant mouse models (hypergeometric enrichment, p = 0.019 and 0.0063, respectively) (S10 Fig). We analyzed expression alterations of orthologous genes in TP53/ PIK3CA mutant and ARID1A/PIK3CA mutant mice compared to controls ( Fig 5B). Examples of opposing regulation by ARID1A and TP53 emerged, such as the pro-apoptotic Akt repressor PHLDA3 [48] which is upregulated in ARID1A/PIK3CA mutant mice but downregulated in TP53/PIK3CA mutant mice ( Fig 5B).
Next, we tested whether p53 pathway activation is a hallmark feature of ARID1A mutant tumors across human cancer. We leveraged PARADIGM [49] pathway activity data produced by a recent pan-cancer TCGA study [30], which infers protein and pathway regulatory activity from both gene expression and copy-number data across 9829 tumors. In analysis of all TP53 wild-type primary tumors across cancer, we computed the mean difference in PARADIGM scores between ARID1A mutant and ARID1A wild-type tumors (n = 492 and 4832, respectively) to determine pathway alterations associated with ARID1A mutation (Fig 5C). Overall, the 36 PARADIGM pathways with keyword "p53" were more highly activated in ARID1A mutant tumors than ARID1A wild-type tumors across cancer (Fig 5C-5E, permutation test, p = 0). This result was also recapitulated specifically in UCEC tumors (S11 Fig), corroborating the GSEA results. Altogether, these data implicate aberrant p53-mediated transcriptional regulation as a hallmark feature of ARID1A mutant tumors. However, the functional mechanism underlying this activation remains unclear.

p53 pathway target genes are directly regulated by ARID1A
We have previously shown that ARID1A normally represses key genes involved in endometrial pathologies through chromatin mechanisms [34,50]. We hypothesized that p53 pathway activation following ARID1A loss could result from the derepression of ARID1A target genes. To ARID1A mutation is associated with p53 pathway activation. a, k-means clustering and heatmap of genetic mouse model RNA-seq relative log 2 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 FDRadjusted, two-tailed, unpaired Wilcoxon test.
As an orthogonal experimental control supporting the validity of these ARID1A binding data, we leveraged our previously reported chromatin accessibility data (ATAC-seq) in this same cell population [34]. We previously found that ARID1A binding is associated with accessible chromatin genome-wide in vitro 12Z human endometriotic epithelial cells [34]. In agreement, 89% of ARID1A bound regions in vivo overlapped with accessible chromatin (Fig 6E  and 6F). We further observed that accessible chromatin regions bound by ARID1A were more highly accessible than those without detectable ARID1A binding (p < 10 −61 , two-tailed, unpaired Wilcoxon test, Fig 6G). These data support detection of ARID1A binding at active chromatin in mouse endometrial epithelial cells in vivo.
We next investigated genes regulated by ARID1A interactions at promoter chromatin in the endometrial epithelium. 587 annotated mouse genes had ARID1A binding detected within 3 kilobases of the primary TSS, indicating highly likely regulation of associated gene expression activity. In agreement with our expression data, we observed significant overlap between expressed genes with ARID1A promoter binding and DE genes in the ARID1A/PIK3CA mutant mouse model ( Fig 6H). Similarly, we also observed significant overlap between the ARID1A/PIK3CA mutant DE genes and a more relaxed set of 3065 genes with ARID1A binding <50 kb from the gene TSS (S12C Fig). Functionally, 494 human orthologs of the ARID1A promoter bound genes were significantly enriched for gene sets including TNFα signaling via NFκB, apoptosis, hypoxia, and response to endoplasmic reticulum stress (Fig 6I). Overall similar pathways were enriched in the larger set of genes with ARID1A binding detected within 50 kb of the TSS (S12D Fig). Among the Hallmark p53 pathway, 11 genes had ARID1A binding detected within the ±3 kb promoter region in endometrial epithelia ( � p = 0.025, hypergeometric enrichment) among those including Atf3, Plk3, Jun, Ndrg1, Rab40c, Hbegf, and Mxd1 ( Fig  6J). These data indicate that ARID1A regulates numerous target genes within the p53 pathway through direct chromatin interactions, and ARID1A loss may lead to aberrant gene expression by disrupting local chromatin regulation.

Simultaneous TP53 and ARID1A loss promotes aggressive tumorigenesis
Despite the implication of p53 pathway activation as a hallmark of ARID1A mutant tumors, rare co-mutant patient tumors indeed exist, but the etiology and characteristics of which have not been elucidated. Intriguingly, the 38 UCEC primary tumors with alterations in both TP53 and ARID1A were associated with higher histologic tumor grading (S13A Fig), suggesting coaltered tumors may be more aggressive. We also found that TP53/ARID1A co-altered primary tumors were enriched for mixed morphology subtype (hypergeometric enrichment, p = 0.015) (S13B Fig). To determine if TP53 and ARID1A co-alterations were associated with metastasis, we examined the MSK-IMPACT Clinical Sequencing Cohort of 10,501 primary or metastatic tumor samples with targeted mutation data [52]. We observed that TP53/ARID1A co- When controlling for TP53 mutation status, we observed a similar, minor but insignificant association trend between ARID1A mutations and metastatic TP53 mutant tumors (OR = 1.10, p = 0.21) (S13D Fig). As in the TCGA-UCEC cohort, POLE mutant tumors in the MSK-IMPACT cohort were also enriched for TP53/ARID1A co-mutations (S13E Fig). To rule out the possibility of tumor heterogeneity contributing to TP53-ARID1A co-dependencies, Cancer Dependency Map (DepMap [53]) data suggested that TP53 mutant cancer cell lines were not more genetically dependent on ARID1A than TP53 wild-type lines (Fig 7A). On the contrary, ARID1A loss had a significantly lesser effect on cellular health in TP53 mutant lines (two-tailed, unpaired Wilcoxon test, p = 0.0066).
As ARID1A is an established metastasis gene in numerous tumor contexts [24,54-58], including endometrial cancer [59], we hypothesized that TP53 mutant primary tumors are not dependent on ARID1A function, and ARID1A mutations may promote metastasis in this genetic context. Therefore, we established and interrogated the tumorigenic potential of LtfCre 0/+ ; (Gt)R26 Pik3ca � H1047R ; Arid1a fl/fl ; Trp53 fl/fl mice (henceforth referred to as TP53/ ARID1A/PIK3CA mutant mice), simultaneously harboring endometrial epithelial PIK3-CA H1047R , ARID1A loss, and TP53 loss (Fig 7B). By gross observation and histopathology, TP53/ARID1A/PIK3CA mutant mice had visibly greater tumor burden as compared to TP53/ PIK3CA mutant mice (Fig 7C and 7D). Vaginal bleeding was observed at a median of 98 days (S14A Fig). This latency period is significantly longer than TP53/PIK3CA mutant mice (S14A Fig), indicating that the humane survival endpoint of vaginal bleeding is not predictive of tumor burden in our mouse models, as we have previously observed [34]. Uterine histology of TP53/ARID1A/PIK3CA mutant mice showed invasive adenocarcinoma with areas of squamous differentiation (Fig 7D and 7E). Endometrial epithelial tumor origin was confirmed through staining for KRT8, phospho-S6, and loss of ARID1A (Figs 7D and S14B). Proliferation and caspase-mediated cell death indices of TP53/ARID1A/PIK3CA mutant endometrial epithelial cells were characterized through IHC staining for Ki67 and Cleaved Caspase-3, respectively. Compared to TP53/PIK3CA mutants, endometrial epithelia with additional ARID1A loss displayed markedly fewer caspase-3-positive foci while proliferation was not dramatically affected (Figs 7F and S15). These data suggest that co-occurring TP53-ARID1A mutations promote tumor cell progression towards invasive adenocarcinoma and metastatic disease.

ARID1A loss results in ATF3 activation and squamous differentiation
We observed that endometrial epithelia harboring loss of both TP53 and ARID1A in addition to PIK3CA activation formed invasive tumors marked by high proliferation and minimal cell death. Compared to TP53/PIK3CA mice, which displayed frequent cell death marked by cleaved caspase-3-positive foci, the addition of ARID1A loss in TP53/ARID1A/PIK3CA mutant mice drastically reduced the number of apoptotic cells. This result suggests that ARID1A may normally promote cell death and stress-related programs in the absence of 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. https://doi.org/10.1371/journal.pgen.1009986.g006
We observed that Atf3 gene expression is directly repressed by ARID1A chromatin regulation in endometrial epithelia in vivo (Figs 6J and S16A). Uterine ATF3 staining revealed focal nuclear staining in a subset of endometrial epithelia in ARID1A/PIK3CA mutant and TP53/ ARID1A/PIK3CA mutant mice, but not controls or TP53/PIK3CA mutants (Fig 8A and 8B). Among other roles, ATF3 is also dysregulated in some squamous tumor types [62,67,76,77], and ATF3 has been shown to promote epithelial squamous differentiation in vivo [68,78]. Areas of squamous-like cells were noted during histopathological analysis of TP53/ARID1A/ PIK3CA mutant uteri, as well as induction of squamous marker TP63 in ARID1A/PIK3CA mutant mouse RNA-seq data (see Fig 5A), so we further examined markers of squamous differentiation. Nuclear TP63 staining was observed in both TP53/ARID1A/PIK3CA and ARID1A/PIK3CA mutant endometrial epithelia that neighbored basement membrane and those cells collectively invading (Figs 8C and S17A and S17B), confirming squamous differentiation, while TP63 expression was not observed in TP53/PIK3CA mutants or controls (Figs 8C and S17A and S17B). We also observed in our RNA-seq data that expression of Trp63 (mouse TP63) correlated with pro-EMT transcription factor Snai2 (S16B Fig), supporting an association between squamous differentiation and invasion phenotypes. Using a recently reported set of direct and functional TP63 target genes [79], we identified 30 genes through our data predicted as TP63 target genes associated with endometrial ARID1A mutation (p < 10 −4 , hypergeometric enrichment), including COL17A1 (S16C-S16F Fig). In TP53/ ARID1A/PIK3CA and ARID1A/PIK3CA mutant endometrial epithelia, COL17A1 expression patterns were similar to TP63 (Figs 8D and S17C). As further evidence that squamous phenotypes are associated with ATF3 expression, we observed expression of ATF3, TP63, and COL17A1 in normal vaginal pseudostratified squamous epithelia (S17D Fig). Collectively, these results suggest that ARID1A mediated repression of ATF3 promotes apoptosis in the absence of TP53, and derepression of ATF3 following ARID1A loss suppresses proapoptotic genes and promotes squamous differentiation (Fig 9). In ARID1A-deficient cells with wildtype TP53, enhanced TP53 signaling blocks proliferation and induces senescence.

Discussion
Here, we develop new models of TP53 mutant endometrial dysfunction and provide genetic and molecular evidence in support of unique and overlapping roles for TP53 and ARID1A. We demonstrate that concurrent TP53 and oncogenic PIK3CA mutations in the endometrial epithelium led to the development of features of hyperplasia, adenocarcinoma, and endometrial intraepithelial carcinoma in the mouse, and additional ARID1A loss promotes invasive adenocarcinoma with squamous differentiation or metaplasia. Endometrial intraepithelial carcinoma is typically considered a precursor lesion to high-grade uterine serous carcinoma [43], an aggressive histologic presentation dominated by TP53 mutations. However, we cannot evaluate if the TP53/PIK3CA mutant mouse model develops uterine serous carcinoma due to the limitation of vaginal bleeding in early tumorigenesis. EIC lesions are non-invasive but frequently associate with extrauterine spread (e.g. to the peritoneum) [80]. Myometrial invasion in endometrioid tumors is associated with metastasis and poor outcomes. Acquisition of ARID1A mutation in EIC confined to the uterus may promote extrauterine dissemination. If TP53/PIK3CA and TP53/ARID1A/PIK3CA mutant mice were able to progress past the vaginal bleeding endpoint, it is possible that extrauterine dissemination could be observed.
Gene expression changes associated with TP53 and ARID1A mutations in the endometrium of humans and mice described here support mutation-specific programs that differentially affect cellular function, though commonalities are also found. Extensive pathway analysis in genetic mouse models and human tumors summarized distinct hallmarks of TP53 and ARID1A mutant tumors: TP53 mutations are associated with epithelial dedifferentiation and loss of intrinsic apoptosis and other p53-mediated cellular processes, while ARID1A mutations are associated with gene expression signatures related to EMT, cell cycle, cell migration, and the p53 pathway. PI3K pathway mutations are highly prevalent in endometrial cancer [33],

PLOS GENETICS
Endometrial TP53-ARID1A co-mutations and the aforementioned gene expression observations were noted in the context of oncogenic PIK3CA H1047R in our genetic mouse models. In the endometrium, ARID1A directly regulates promoter chromatin at p53 target genes, which notably includes repression of Atf3. As reviewed, stress-induced ATF3 has numerous characterized roles in p53 signaling, cell death, and senescence, and our data support it as a marker of squamous epithelium. We hypothesize that ATF3 induction is associated with squamous differentiation and anti-apoptotic mechanisms in the endometrium.
Despite accounts of mutual exclusivity observed in primary tumors, our genetic mouse model experiments indicate co-existing TP53 and ARID1A mutations are tolerated in vivo and promote more aggressive cancer phenotypes than either mutation independently. Myometrial invasion is suggested to be driven at least partially through squamous differentiation following ARID1A loss, since the invasive front cells were TP63/COL17A1+. Given the invasiveness observed in TP53/ARID1A/PIK3CA co-mutant mice, we hypothesize that a human TP53 mutant endometrial cancer cell that acquires an ARID1A mutation will gain metastatic properties. This hypothesis may explain the rarity of TP53/ARID1A co-mutant samples in clinical cohorts like TCGA, which have mostly focused on sequencing primary tumors. The observation that TP53/ARID1A co-alterations are enriched among POLE mutant ultra-mutators could be interpreted possibly such that these are often non-functional passenger mutations. However, our data suggest that functional deleterious ARID1A mutation in a TP53 mutant primary may promote metastatic dissemination. In addition, it is worth noting that 3.3% of POLE wild-type tumors profiled by MSK-IMPACT are TP53/ARID1A co-mutant, which could be functional mutations. Outside of POLE ultra-mutators, our analysis of the TCGA molecular subtype framework suggests that TP53/ARID1A co-mutated primary UCEC tumors are most likely to present as microsatellite instable (MSI).
Intriguingly, 5 out of 57 primary tumors (8.8%) profiled in the TCGA uterine carcinosarcoma (UCS) data set harbored mutations in both TP53 and ARID1A in the absence of hypermutator signatures [20]. A reported mouse model of uterine Fbxw7 and Pten loss developed invasive endometrioid intraepithelial neoplasia followed by uterine carcinosarcomas at late stage confirmed as endometrial epithelial origin [81]. If our mouse models were able to age past the point of vaginal bleeding, it remains possible that a subset of TP53/ARID1A/PIK3CA mutant mice could progress toward uterine carcinosarcoma phenotype.
Currently, one large-scale sequencing study has focused specifically on endometrial cancer metastasis, and frequent, subclonal ARID1A mutations were observed in metastatic lesions [59]. In that study by Gibson et al., ARID1A mutations were more associated with late-occurring metastatic endometrial cancer clones as compared to other frequently mutated driver genes like TP53 associated with early tumorigenesis. These results further support a role for ARID1A mutations as metastatic drivers in endometrial cancer. Another study examined cases of synchronous endometrial and epithelial ovarian cancers and found 4 out of 7 TP53 mutant tumors also harbored ARID1A mutations [82]. Additional clinical sequencing of matched primary and metastases from endometrial cancer patients would provide additional support for these findings.
Numerous reports have now shown in various tissue contexts that deleterious ARID1A alterations promote metastatic molecular and cellular signatures, such as EMT induction [34, [83][84][85][86]. We also previously reported that ARID1A directly represses mesenchymal gene expression, and ARID1A loss promotes enhanced migration and invasion in the 12Z human endometriotic epithelial cell model [34]. However, TP53 has also long been considered a metastasis gene in addition to displaying general tumor suppressor properties. Early studies discovered that de-differentiation and high-grade status of some tumor types were associated with TP53 loss and highly malignant behavior [87]. TP53 has been demonstrated to promote epithelialization through cell-cell adhesion and maintenance of the extracellular matrix, and it also regulates cell migration and invasion, stemness, EMT, and anoikis phenotypes [88]. Classically, uterine endometrioid adenocarcinomas, which are most associated with ARID1A mutations, are often indolent, low-grade tumors not generally associated with metastatic risk [31]. On the other hand, uterine serous carcinomas associated with TP53 mutations are generally higher grade and more aggressive [31]. Numerous genetically engineered mouse model studies have demonstrated that TP53 functions as a tumor suppressor in the endometrium [89][90][91][92]. Gene expression pathway analysis in TP53 mutant lesions here supports endometrial TP53 loss as leading to epithelial de-differentiation. This phenotype is distinct from ARID1A deficient lesions, which also lose epithelial features like cell adhesion and cell-cell junctions but acquire invasive, mesenchymal characteristics akin to canonical EMT or transdifferentiation [34], in addition to a subset of cells undergoing squamous metaplasia in our ARID1A mutant mouse model. In TP53 mutant serous primary tumor cells, it is hypothesized that further ARID1A mutation may trigger metastatic progression. In ARID1A mutant cells with intact TP53 signaling, we suspect that TP53 suppresses proliferation and induces senescence, given the phenotypes observed and upregulation of TP53 target genes, such as p21 (CDKN1A) and CHOP (DDIT3). In addition, senescence-associated secretory phenotypes (SASP) are associated with metastatic invasion [93]. Further experiments will be required to fully understand the phenotypic effects of ARID1A mutations in TP53 mutant cells, and vice versa.
Lack of invading features in TP53/PIK3CA mutant endometrial epithelia could be attributed to the use of a Trp53 fl allele, which may not completely model non-deleterious, gain-of-function TP53 mutations. In mice, Trp53 missense alleles corresponding to known gain-offunction TP53 clinical mutations are associated with enhanced metastasis [94]. Using a gainof-function mutation model may elicit different phenotypes in the endometrial epithelium than those observed with loss-of-function alleles, and these features could be cell type specific. In support of a nullizygous approach in the endometrium, Trp53 null mutations in the mouse endometrium have been previously demonstrated to promote tumorigenesis [90,92]. In TCGA-UCEC samples, we show there is no significant difference in tumor grading or overall survival between TP53 missense vs. truncating mutations, further supporting the use of either strategy to study TP53 mutant tumors. Further, the type of TP53 mutation in endometrial cancer is not associated with genomic or histologic subtype [95]. Although a clinical sign of endometrial cancer in women, vaginal bleeding is a humane survival endpoint in mice, requiring euthanasia at early stages of the disease. In the absence of vaginal bleeding, it remains possible that endometrial invasion would be detected in TP53/PIK3CA mutant endometrium at later stages of the disease.
Mechanistically, ARID1A directly represses Atf3 promoter chromatin in the endometrial epithelium, and ARID1A loss induces ATF3 expression associated with TP63+ squamous differentiation independent of TP53 mutation status. Similar to our findings, ARID1A downregulation in testicular germ cell tumors also leads to ATF3 upregulation [96]. We also showed ATF3 expression in the vaginal squamous epithelium is strongest near basal epithelial cells marked by TP63 and COL17A1, indicating that ATF3 may be linked to squamous differentiation or metaplasia observed in ARID1A-deficient endometrial epithelia. Interestingly, previous reports have shown that ATF3 functions are context-dependent, and ATF3 may promote apoptosis or suppresses proapoptotic genes depending on whether it is activated in healthy versus tumor cells [71,72].
TP63 is a classical marker of squamous differentiation in cancer [97,98], and our studies suggest ATF3 induction is associated with TP63 expression in invading endometrial epithelial cells following ARID1A loss. Previous studies have identified roles for ATF3 in squamous tumors [62,67,76,77], and ATF3 promotes epithelial squamous differentiation in vivo [68,78]. Squamous differentiation is observed in 25% of human endometrial cancer and was recently associated with disease recurrence, and it has been observed in mouse models [99][100][101]. ARID1A and SWI/SNF mutations have been shown to promote carcinogenesis of various squamous tumor types [102,103]. We observed that ARID1A mutant UCEC tumors have higher TP63 expression than TP53 mutant tumors. A recent mouse model of ARID1A deletion paired with oncogenic KRAS G12D developed invasive vaginal squamous cell carcinoma [104]. TP63+ squamous differentiation in invasive endometrial cancer was also recently reported in a subset of Fbxw7/Pten knockout mice [81]. Another mouse model with uterine-specific β-catenin deletion showed increased TP63 expression and squamous differentiation in endometrial epithelia [105]. TP63 marks basal endometrial epithelial cells during fetal life [106], and TP63 + basal cells are reactivated in GATA2 knockout and SOX17 knockout mouse uteri [107,108]. Transcriptional regulation by TP63 has been previously linked to cellular migration and invasion [109]. A subset of direct TP63 target genes are activated following ARID1A mutation in both genetic mouse models and human tumors, including COL17A1. Given the reported links between ATF3 and TP53 post-translational regulation, stability, and target gene regulation, it remains possible that ATF3 regulates TP63. Additional work will be required to determine the functional relationship between ATF3 and TP63 in endometrial squamous differentiation.
Regulation of apoptotic processes, normally induced by DNA damage and stress, is an important TP53 tumor suppressor mechanism [110]. In addition to EMT and invasion, ARID1A and SWI/SNF are implicated in regulating genomic stability, such that ARID1A inactivation is associated with increased DNA damage, but the roles of ARID1A in apoptotic signaling are not well characterized and may be context dependent [8,50,[111][112][113]. Genetic experiments here suggest that TP53 mutant endometrial epithelia display increased caspasemediated apoptosis, and further ARID1A loss in TP53-ARID1A mutants suppresses cell death. ATF3 has well established ties to apoptosis and cell death regulation despite context-dependency [71,72], and ATF3 activation in ARID1A-deficient endometrial epithelia may suppress pro-apoptotic genes in the absence of TP53. TP63 has also been previously shown to antagonize BCL-2-related, pro-apoptotic transcriptional programs [114]. ATF3 induction and associated TP63 could be a mechanism of how ARID1A loss causes cells to bypass cell death, perhaps explaining why ARID1A mutant tumors are dependent on p53 pathway function, but not TP53 itself. The TNF-mediated extrinsic apoptotic pathway is also regulated by the p53 pathway to some extent but is less characterized [115]. Endoplasmic reticulum (ER) stress related gene sets were the most highly enriched GO terms among ARID1A promoter bound genes in vivo endometrial epithelia, and ER stress crosstalk between ARID1A and TP53 could contribute to interrelated transcriptional and cell death regulation [116,117].
The root of mutation mutual exclusivity could be attributed to interconnected transcriptional regulation by ARID1A and TP53. Our in vivo binding data revealed that ARID1A interacts with chromatin near TP53 target genes, notably including direct repression of stressinduced transcription factor Atf3. ATF3 also has been shown to bind 20-40% of TP53 genomic targets [72,73], suggesting ARID1A loss probably affects multiple aspects of TP53 regulated chromatin. Recently, integrative transcriptomic analyses in mouse tumors and human cellbased models of ARID1A mutant endometrial tumorigenesis have identified the p53 pathway as a key ARID1A-mediated signaling network [118]. Our analyses suggest that ARID1A mutant tumors are dependent on p53 pathway function. In TP53 mutant tumors, ATF3 induction and TP63+ squamous differentiation following ARID1A loss could be a compensatory mechanism that partially restores p53 pathway function. It is worth noting that these genetic mechanisms are difficult to explore in cell culture models, as the majority of immortalized cell lines have impaired p53 signaling, whether that be through inherent mutation [119], selection during culture [120], or immortalization techniques like SV40 large T antigen [121]. In fact, half of the 30 endometrial cancer cell lines profiled by CCLE are TP53/ARID1A co-mutant [122]. This places further emphasis on in vivo characterization of TP53-ARID1A functional and genetic relationships.

Ethics statement
Mice were housed at the Van Andel Research Institute (VARI) Animal Facility and the Michigan State University Grand Rapids Research Center in accordance with protocols approved by the Michigan State University Institutional Animal Care and Use Committee (IACUC).

Mice and animal husbandry
All mice were maintained on an outbred genetic background using CD-1 mice (Charles River). (Gt)R26 Pik3ca � H1047R , Trp53 fl , and LtfCre (Tg(Ltf-iCre)14Mmul) alleles were purchased from The Jackson Laboratory and confirmed by PCR using published methods [36][37][38]. Agematched, CRE-negative (LtfCre 0/0 ) littermates were used as controls. Endpoints were vaginal bleeding, severe abdominal distension, and signs of severe illness, such as dehydration, hunching, jaundice, ruffled fur, signs of infection, or non-responsiveness. Sample sizes within each genotype were chosen based on the proportions of animals with vaginal bleeding between each experimental group or a Kaplan-Meyer log-rank test for survival differences.

Endometrial epithelial cell isolation and RNA-seq
Approximately 76-day old mouse uteri were surgically removed, digested, enriched for EPCAM-positive epithelial cells by magnetic sorting, and purified for RNA as previously described [34]. Mouse libraries (n = 3 biological replicates) were prepared and sequenced by the Van Andel Genomics Core from 100 ng of isolated mouse cell total RNA as previously described [34]. For analysis, briefly, raw reads were trimmed and aligned to mm10 assembly and indexed to GENCODE (vM16) via STAR [123]. Low count genes with less than one count per sample on average were filtered prior to count normalization and differential gene expression (DGE) analysis by DESeq2 [124], using a single term model matrix. Differential expression probabilities were corrected for multiple testing by independent hypothesis weighting (IHW) [125] for downstream analyses. Comparisons between TP53/PIK3CA mutant and ARID1A/PIK3CA mutant mouse DGE results and gene sets were initially filtered for genes with transcripts commonly detected in both cell populations.

Construction and sequencing of CUT&RUN libraries
Libraries for CUT&RUN samples were prepared by the Van Andel Genomics Core from 0.1-0.3 ng of IP material, using the KAPA Hyper Prep Kit (v8.2) (Kapa Biosystems). Prior to PCR amplification, end-repaired and A-tailed DNA fragments were ligated to IDT for Illumina Unique Dual Index adapters (IDT, Coralville, IA USA) at a concentration of 500 nM. Quality and quantity of the finished libraries were assessed using a combination of Agilent DNA High Sensitivity chip (Agilent Technologies, Inc.), QuantiFluor dsDNA System (Promega Corp.), and Kapa Illumina Library Quantification qPCR assays (Kapa Biosystems). Individually indexed libraries were pooled, and 50 bp, paired-end sequencing was performed on an Illumina NovaSeq6000 sequencer using a 100 cycle sequencing kit (Illumina Inc.). Each library was sequenced to an average depth of 50 million reads. Base calling was done by Illumina RTA3 and output was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v1.9.0.

CUT&RUN bioinformatic analysis
Raw paired-end reads for anti-ARID1A or IgG CUT&RUN were trimmed with cutadapt [126] and Trim Galore! and analyzed for quality via FastQC [127]. Trimmed reads were aligned to mm10 genome assembly with bowtie2 [128] using flag '-very-sensitive'and filtered for only properly-paired reads with samtools [129] using flag '-f 3'. Picard MarkDuplicates (http:// broadinstitute.github.io/picard/) was used to remove PCR duplicates. For each biological replicate, MACS2 [130] was used to call ARID1A broad peaks against the respective IgG negative control as input, with FDR < 0.25 threshold. The resulting peaks were repeat-masked by ENCODE blacklist filtering and filtered for non-standard contigs [131]. A naïve replicateoverlapping peak set was constructed by calling peaks on pooled replicates followed by bedtools intersect [132] to select for peaks of at least 50% overlap with each biological replicate. HOMER [133] was used to annotate peaks, test genomic enrichment, compute motif enrichment, recenter peaks on motifs, and quantify signal profiles across regions of interest. csaw [134] was used to count sequencing reads in genomic loci of interest. IGV [135] was used for visualizing CUT&RUN and ATAC signal as MACS2 enrichment log-likelihood (logLR) at mm10 genomic loci.

Clinical and public cancer data analysis
The Cancer Genome Atlas (TCGA) Pan-Cancer Atlas [30] and UCEC cohort-specific [18] data were utilized in this study. TCGA Pan-Cancer Atlas somatic mutation data were extracted from the MC3 Public MAF (v0.2.8) analysis data set [29]. Clinical data and ARID1A and TP53 alteration incidence rates specifically in endometrial cancer were extracted from the TCGA Pan-Can UCEC cohort (n = 509) retrieved from cBioPortal [136,137]. MSK-IMPACT 2017 data [52] were also retrieved from cBioPortal. Cancer dependency map CRISPR knockout screen data from Achilles (DepMap Public 21Q1) were retrieved from the DepMap portal [53, 138,139]. PARADIGM pathway activity inference [49] data for TCGA Pan-Cancer Atlas were retrieved from NCI GDC [140]. For TCGA-UCEC specific molecular analyses, data were retrieved from the 28th January, 2016 release of Broad GDAC Firehose (https://doi.org/10. 7908/C11G0KM9). RNASeqV2 RSEM [141] normalized gene counts were further quantile normalized prior to filtering low-count genes (less than one normalized count per sample on average) and fitting linear models via limma [142] for differential expression analysis in subsets of patients using a single term model matrix. Empirical Bayes moderated statistics were computed via limma::eBayes with arguments 'trend = TRUE' and 'robust = TRUE', and probabilities were adjusted for multiple testing by Benjamini-Hochberg FDR [143] correction. Only non-silent frameshift, nonsense, or splice site mutations were considered functional ARID1A mutations in UCEC-specific molecular analyses, e.g. GSEA and DGE comparisons. All nonsynonymous or non-silent variants were considered as mutations for TP53, PIK3CA, POLE, and all pan-cancer molecular analyses. "Wild-type" samples included synonymous or silent mutations.

Gene set enrichment analysis
For MSigDB Hallmark pathways [45] and GO Biological Process [144,145] gene set collections (v6.2), Broad GSEA [44] was performed via GenePattern [146] on ortholog-converted DESeq2 [124] normalized counts from experimental mouse data and RNASeqV2 RSEM [141] normalized counts from TCGA-UCEC data. Manual hypergeometric enrichment tests or clusterProfiler enrichment functions were computed on gene sets of interest compared to respective expressed gene universes [147]. Identifying significantly over-represented terms in enriched GO Biological Process gene sets was achieved with a hypergeometric enrichment test framework. Briefly, universes of gene set terms were constructed for single words, word doublets, and word triplets within the 3653 measured gene sets. The number of gene sets containing a given term was then computed for the list of enriched gene sets as well as the respective gene set universe. A hypergeometric enrichment test was then employed to determine if the specific term is over-represented within the respective gene set universe, and this framework was applied to all single word, word doublet, and word triplet terms. For simplified visualization in Fig 4D, results were manually curated to omit some duplicate terms, such as "response to virus" and "to virus".

Bioinformatics and statistics
biomaRt was used for all gene nomenclature and mouse-human ortholog conversions [148]. The cumulative hypergeometric distribution was calculated in R for enrichment tests. Hierarchical clustering by Euclidean distance and heatmaps were generated by ComplexHeatmap [149]. Mouse mm10 genome sequence conservation across vertebrates, computed via PHAST [150,151], was extracted from the UCSC browser. ggplot2 was used for some plots in this study [152]. The statistical language R was used for various computing functions throughout this study [153].   Expression is quantified as log 2 (normalized counts + 1). Statistics are Pearson (r) and Spearman (r s ) coefficients. c, Workflow for identification of the TP63 target gene network induced by ARID1A mutation (n = 30 genes), beginning with 180 high confidence TP63 target genes defined by Riege et al. [79]. d, RNA-seq relative expression heatmap for genetic mouse models and UCEC primary tumor samples for the TP63 target gene network induced by ARID1A mutation. Red, bolded genes display similar expression patterns in genetic mouse models and human UCEC tumors. e, Top GO Biological Process gene sets enriched (hypergeometric enrichment p < 0.001) in the TP63 target gene network induced by ARID1A mutation. # represents the number of target genes found within each gene set. f, Broad GSEA results for the TP63 target gene network induced by ARID1A mutation among human UCEC primary tumors segregated by ARID1A and TP53 genetic status. Significant enrichment was observed in ARID1Amut/TP53wt vs. wt/wt tumors.