Expression patterns of HDAC6 in correlation to ARID1A status in different subtypes of endometriosis: A retrospective tissue microarray analysis.

Endometriosis is a disease affecting approximately 10% of reproductive age women. Loss of the tumor suppressor gene AT-rich interactive domain-containing protein 1A (ARID1A) occurs in some endometriosis cases. Histone deacetylase 6 (HDAC-6) is an enzyme with implication in several diseases including different cancer types and immunological disorders, where it is involved in protein trafficking and degradation, cell shape, and migration. In ARID1A-deficient ovarian cancer increased HDAC-6 expression lead to apoptosis-inhibiting post-translational modification of p53. It is not known if HDAC-6 expression is also altered in ARID1A-deficient endometriosis. The aim of this study was to assess HDAC-6 expression in endometriotic lesions in correlation to ARID1A-status. Two tissue-microarrays with 168 endometriotic lesions, including ovarian (64/168, 38 %), peritoneal (66/168, 39 %) and deep-infiltrating (38/168, 23 %) subtypes, and 73 endometrium of women without endometriosis were assessed. Mean ARID1A immunoreactivity score (IRS) in endometriosis group was 10.83 (±2.36) and 10.78 (±1.94) in the epithelium and stroma, respectively, while the respective mean HDAC6 IRS were 9.16 (±2.76) and 5.94 (±2.88). The comparison of the HDAC6 expression between endometriosis subtypes showed higher expression in deep-infiltrating endometriosis, in both, epithelium (p = 0.032) and stroma (p = 0.007). In ARID1A negative cases, epithelial expression of HDAC6 was higher in endometriosis compared to women without endometriosis (p = 0.031), and this was also specifically observed in the subset of ovarian endometriosis (p = 0.037). There were no significant differences in the stromal expression of HDAC6. In conclusion, our results demonstrate a complex expression pattern of HDAC6 depending on ARID1A status in different endometriosis subtypes. Further studies on HDAC6 and ARID1A are important to elucidate mechanisms involved in malignant transformation of endometriosis.

ARID1A-BAF coordinates ZIC2 genomic occupancy for epithelial-to-mesenchymal transition in cranial neural crest specification.

The BAF chromatin remodeler regulates lineage commitment including cranial neural crest cell (CNCC) specification. Variants in BAF subunits cause Coffin-Siris syndrome (CSS), a congenital disorder characterized by coarse craniofacial features and intellectual disability. Approximately 50% of individuals with CSS harbor variants in one of the mutually exclusive BAF subunits, ARID1A/ARID1B. While Arid1a deletion in mouse neural crest causes severe craniofacial phenotypes, little is known about the role of ARID1A in CNCC specification. Using CSS-patient-derived ARID1A+/- induced pluripotent stem cells to model CNCC specification, we discovered that ARID1A-haploinsufficiency impairs epithelial-to-mesenchymal transition (EMT), a process necessary for CNCC delamination and migration from the neural tube. Furthermore, wild-type ARID1A-BAF regulates enhancers associated with EMT genes. ARID1A-BAF binding at these enhancers is impaired in heterozygotes while binding at promoters is unaffected. At the sequence level, these EMT enhancers contain binding motifs for ZIC2, and ZIC2 binding at these sites is ARID1A-dependent. When excluded from EMT enhancers, ZIC2 relocates to neuronal enhancers, triggering aberrant neuronal gene activation. In mice, deletion of Zic2 impairs NCC delamination, while ZIC2 overexpression in chick embryos at post-migratory neural crest stages elicits ectopic delamination from the neural tube. These findings reveal an essential ARID1A-ZIC2 axis essential for EMT and CNCC delamination.

A mutational signature and ARID1A mutation associated with outcome in hepatocellular carcinoma.

OBJECTIVE: The prognosis of hepatocellular carcinoma (HCC) is poor and there is no stable and reliable molecular biomarker for evaluation. This study attempted to find reliable prognostic markers from tumor mutational profiles. METHODS: A total of 362 HCC samples with whole-exome sequencing were collected as discovery datasets, and 200 samples with targeted sequencing were used for validation of the relevant results. All HCC samples were obtained from previously published studies. Bayesian non-negative matrix factorization was used to extract mutational signatures, and multivariate Cox regression models were utilized to identify the prognostic role of mutational factors. Gene set enrichment analysis was employed to discover potential signaling pathways associated with specific mutational groups. RESULTS: In the HCC discovery dataset, a total of four mutational signatures (i.e., signatures 4, 6, 16, and 22) were extracted, of which signature 16 characterized by T>C mutations was observed to be associated with favorable HCC prognosis, and this correlation was also found in the validation dataset. Further analysis showed that patients with ARID1A mutations exhibited inferior survival outcomes in both discovery and validation datasets. Mechanistic exploration revealed that the presence of signature 16 was associated with better immune infiltration and tumor immunogenicity, while patients with ARID1A mutations were away from these favorable immunological features. CONCLUSION: By integrating somatic mutation data and clinical information of HCC, this study identified that signature 16 and ARID1A mutations were associated with better and worse outcomes respectively, providing a basis for prognosis prediction and clinical treatment strategies of HCC.

Medullary carcinomas of the nonampullary small intestine: association with coeliac disease, mismatch repair deficiency, PD-L1 expression, and favourable prognosis.

AIM: Gastrointestinal medullary carcinoma is a rare histologic subtype of adenocarcinoma. As nonampullary small bowel medullary carcinomas (SB-MCs) are poorly characterized, we aimed to analyse their clinicopathologic and immunohistochemical features and to compare them with nonmedullary small bowel adenocarcinomas (NM-SBAs). METHODS AND RESULTS: Surgically resected SBAs collected through the Small Bowel Cancer Italian Consortium were classified as SB-MCs (carcinomas with ≥50% of tumour fulfilling the typical histologic criteria of MC) or NM-SBAs. Immunohistochemistry for cytokeratin (CK)7, CK20, CDX2, programmed death-ligand 1 (PD-L1) and mismatch repair proteins was performed in both SB-MCs and NM-SBAs. SB-MCs were also tested for CK8/18, synaptophysin, SMARCB1, SMARCA2, SMARCA4, and ARID1A and for Epstein-Barr virus (EBV)-encoded RNAs by in-situ hybridization. MLH1 promoter methylation status was evaluated in MLH1-deficient cases. Eleven SB-MCs and 149 NM-SBAs were identified. One (9%) SB-MC was EBV-positive, while 10 (91%) harboured mismatch repair deficiency (dMMR). MLH1 promoter hypermethylation was found in all eight dMMR SB-MCs tested. Switch/sucrose nonfermentable deficiency was seen in two (18%) SB-MCs, both with isolated loss of ARID1A. Compared with NM-SBAs, SB-MCs exhibited an association with coeliac disease (P < 0.001), higher rates of dMMR (P < 0.001), and PD-L1 positivity by both tumour proportion score and combined positive score (P < 0.001 for both), and a lower rate of CK20 expression (P = 0.024). Survival analysis revealed a better prognosis of SB-MC patients compared to NM-SBA cases (P = 0.02). CONCLUSION: SB-MCs represent a distinct histologic subtype, with peculiar features compared to NM-SBAs, including association with coeliac disease, dMMR, PD-L1 expression, and better prognosis.

Arid1a Loss Enhances Disease Progression in a Murine Model of Osteosarcoma.

Osteosarcoma is an aggressive bone malignancy, molecularly characterized by acquired genome complexity and frequent loss of TP53 and RB1. Obtaining a molecular understanding of the initiating mutations of osteosarcomagenesis has been challenged by the difficulty of parsing between passenger and driver mutations in genes. Here, a forward genetic screen in a genetic mouse model of osteosarcomagenesis initiated by Trp53 and Rb1 conditional loss in pre-osteoblasts identified that Arid1a loss contributes to OS progression. Arid1a is a member of the canonical BAF (SWI/SNF) complex and a known tumor suppressor gene in other cancers. We hypothesized that the loss of Arid1a increases the rate of tumor progression and metastasis. Phenotypic evaluation upon in vitro and in vivo deletion of Arid1a validated this hypothesis. Gene expression and pathway analysis revealed a correlation between Arid1a loss and genomic instability, and the subsequent dysregulation of genes involved in DNA DSB or SSB repair pathways. The most significant of these transcriptional changes was a concomitant decrease in DCLRE1C. Our findings suggest that Arid1a plays a role in genomic instability in aggressive osteosarcoma and a better understanding of this correlation can help with clinical prognoses and personalized patient care.

Pathogenesis of Endometriosis and Endometriosis-Associated Cancers.

Endometriosis is a hormone-dependent, chronic inflammatory condition that affects 5-10% of reproductive-aged women. It is a complex disorder characterized by the growth of endometrial-like tissue outside the uterus, which can cause chronic pelvic pain and infertility. Despite its prevalence, the underlying molecular mechanisms of this disease remain poorly understood. Current treatment options are limited and focus mainly on suppressing lesion activity rather than eliminating it entirely. Although endometriosis is generally considered a benign condition, substantial evidence suggests that it increases the risk of developing specific subtypes of ovarian cancer. The discovery of cancer driver mutations in endometriotic lesions indicates that endometriosis may share molecular pathways with cancer. Moreover, the application of single-cell and spatial genomics, along with the development of organoid models, has started to illuminate the molecular mechanisms underlying disease etiology. This review aims to summarize the key genetic mutations and alterations that drive the development and progression of endometriosis to malignancy. We also review the significant recent advances in the understanding of the molecular basis of the disorder, as well as novel approaches and in vitro models that offer new avenues for improving our understanding of disease pathology and for developing new targeted therapies.

Loss of ARID1A 'loops' in STING.

In a recent article, Maxwell et al. report that loss of tumor cell-specific AT-rich interaction domain 1A (ARID1A), a component of the chromatin remodeling SWI/SNF complex, triggers antitumor immunity via R-loop-mediated upregulation of the type-I interferon (IFN) pathway. These recent findings uncover a molecular mechanism underlying improved responses to immune checkpoint therapy (ICT) seen in patients harboring an ARID1A loss-of-function mutation.

ARID1A is involved in DNA double-strand break repair in gastric cancer.

Background: Defects in DNA damage repair can cause genetic mutations, which in turn can cause different types of cancers. Chromatin remodeling complexes, which help repair damaged DNA, can cause the chromatin structure to change as a result of DNA damage. ARID1A may play a role in the process of DNA damage repair, and arid1a may be related to the occurrence and development of gastric cancer (GC). This study aimed to investigate the mechanism of ARID1A regulating the DNA damage repair of gastric adenocarcinoma cell lines AGS and SGC-7901 and its effect on migration, proliferation and apoptosis. Methods: The expression of ARID1A plasmid was detected by Western blot and real-time polymerase chain reaction (PCR). The effect of etoposide (ETO) on the survival rate of AGS and SGC-7901 gastric adenocarcinoma cell lines was detected by MTT assay. The DNA double-strand break model was established by ETO and then passed through the comet assay and immunofluorescence co-localization to observe DNA damage; western blot method was used to detect the effect of ARID1A on the expression of related proteins in DNA damage repair pathway in gastric adenocarcinoma cells; scratch test and colony formation experiments were used to observe ARID1A migration and proliferation of gastric adenocarcinoma cells. The flow cytometry was used to detect the effect of ARID1A on apoptosis of gastric adenocarcinoma cells. Results: The expression of mRNA and protein was increased after transfection of ARID1A plasmid. ETO was confirmed by MTT assay to inhibit cell survival in a dose-dependent manner. After the DNA double-strand break model was established by ETO, the expression levels of phospho-ataxia telangiectasia mutated (p-ATM) protein increased in the overexpressed ARID1A group. Meanwhile, the overexpressed ARID1A group had a shortened tail moment, and γ-H2AX and ARID1A co-localized in the DNA damage site of the nucleus. The over-expressed ARID1A group had weaker wound healing ability, reduced number of clone formation, and increased apoptosis rate. Conclusions: ARID1A may repair DNA double-strand breaks caused by ETO by p-ATM pathway; ARID1A can inhibit the migration and proliferation of gastric adenocarcinoma cells and promote apoptosis. Our findings indicate that ARID1A could serve as a therapeutic target and biomarker for GC patients.

Genomic events stratifying prognosis of early gastric cancer.

BACKGROUND: The purpose of the study was to conduct a comprehensive genomic characterization of gene alterations, microsatellite instability (MSI), and tumor mutational burden (TMB) in submucosal-penetrating (Pen) early gastric cancers (EGCs) with varying prognoses. METHODS: Samples from EGC patients undergoing surgery and with 10-year follow-up data available were collected. Tissue genomic alterations were characterized using Trusight Oncology panel (TSO500). Pathway instability (PI) scores for a selection of 218 GC-related pathways were calculated both for the present case series and EGCs from the TCGA cohort. RESULTS: Higher age and tumor location in the upper-middle tract are significantly associated with an increased hazard of relapse or death from any cause (p = 0.006 and p = 0.032). Even if not reaching a statistical significance, Pen A tumors more frequently present higher TMB values, higher frequency of MSI-subtypes and an overall increase in PI scores, along with an enrichment in immune pathways. ARID1A gene was observed to be significantly more frequently mutated in Pen A tumors (p = 0.006), as well as in patients with high TMB (p = 0.027). Tumors harboring LRP1B alterations seem to have a higher hazard of relapse or death from any cause (p = 0.089), being mutated mainly in relapsed patients (p = 0.093). CONCLUSIONS: We found that the most aggressive subtype Pen A is characterized by a higher frequency of ARID1A mutations and a higher genetic instability, while LRP1B alterations seem to be related to a lower disease-free survival. Further investigations are needed to provide a rationale for the use of these markers to stratify prognosis in EGC patients.

Restoration of ARID1A Protein in ARID1A-deficient Clear Cell Carcinoma of the Ovary Attenuates Reactivity to Cytotoxic T Lymphocytes.

BACKGROUND/AIM: Clear cell carcinoma is a prevalent histological type of ovarian cancer in East Asia, particularly in Japan, known for its resistance to chemotherapeutic agents and poor prognosis. ARID1A gene mutations, commonly found in ovarian clear cell carcinoma (OCCC), contribute to its pathogenesis. Recent data revealed that the ARID1A mutation is related to better outcomes of cancer immunotherapy. Thus, this study aimed to investigate the immunotherapy treatment susceptibility of OCCC bearing ARID1A mutations. MATERIALS AND METHODS: Expression of ARID1A was analyzed using western blotting in ovarian cancer cell lines. OCCC cell lines JHOC-9 and RMG-V were engineered to overexpress NY-ESO-1, HLA-A*02:01, and ARID1A. Sensitivity to chemotherapy and T cell receptor-transduced T (TCR-T) cells specific for NY-ESO-1 was assessed in ARID1A-restored cells compared to ARID1A-deficient wild-type cells. RESULTS: JHOC-9 cells and RMG-V cells showed no expression of ARID1A protein. Overexpression of ARID1A in JHOC-9 and RMG-V cells did not impact sensitivity to gemcitabine. While ARID1A overexpression decreased sensitivity to cisplatin in RMG-V cells, it had no such effect in JHOC-9 cells. ARID1A overexpression reduced the reactivity of NY-ESO-1-specific TCR-T cells, as observed by the IFNγ ESLIPOT assay. CONCLUSION: Cancer immunotherapy is an effective approach to target ARID1A-deficient clear cell carcinoma of the ovary.

IRF4 requires ARID1A to establish plasma cell identity in multiple myeloma.

Multiple myeloma (MM) is an incurable plasma cell malignancy that exploits transcriptional networks driven by IRF4. We employ a multi-omics approach to discover IRF4 vulnerabilities, integrating functional genomics screening, spatial proteomics, and global chromatin mapping. ARID1A, a member of the SWI/SNF chromatin remodeling complex, is required for IRF4 expression and functionally associates with IRF4 protein on chromatin. Deleting Arid1a in activated murine B cells disrupts IRF4-dependent transcriptional networks and blocks plasma cell differentiation. Targeting SWI/SNF activity leads to rapid loss of IRF4-target gene expression and quenches global amplification of oncogenic gene expression by MYC, resulting in profound toxicity to MM cells. Notably, MM patients with aggressive disease bear the signature of SWI/SNF activity, and SMARCA2/4 inhibitors remain effective in immunomodulatory drug (IMiD)-resistant MM cells. Moreover, combinations of SWI/SNF and MEK inhibitors demonstrate synergistic toxicity to MM cells, providing a promising strategy for relapsed/refractory disease.

Efficacy of immunotherapy in ARID1A-mutant solid tumors: a single-center retrospective study.

BACKGROUND: Immune checkpoint inhibitors (ICIs), especially those targeting programmed cell death-1 (PD-1) and programmed cell death ligand-1 (PD-L1), have introduced a new treatment landscape for many types of tumors. However, they only achieve a limited therapeutic response. Hence, identifying patients who may benefit from ICIs is currently a challenge. METHODS: 47 tumor patients harboring ARID1A mutations were retrospectively studied. The genomic profiling data through next-generation sequencing (NGS) and relevant clinical information were collected and analyzed. Additionally, bioinformatics analysis of the expression of immune checkpoints and immune cell infiltration levels was conducted in ARID1A-mutant gastric cancer (GC). RESULTS: ARID1A mutations frequently co-occur with mutations in DNA damage repair (DDR)-associated genes. Among the 35 ARID1A-mutant patients who received immunotherapy, 27 were evaluable., with the objective response rate (ORR) was 48.15% (13/27), and the disease control rate (DCR) was 92.59% (25/27). Moreover, survival assays revealed that ARID1A-mutant patients had longer median overall survival (mOS) after immunotherapy. In ARID1A-mutated GC patients, receiving ICIs treatment indicated longer progressive-free survival (PFS). Additionally, the incidence of microsatellite instability-high (MSI-H), high tumor mutation burden (TMB-H) and Epstein‒Barr virus (EBV) infection was elevated. Bioinformatic analysis showed significant enrichment of immune response and T cell activation pathway within differentially expressed genes in ARID1A-mutant GC group. Finally, ARID1A mutations status was considered to be highly correlated with the level of tumor infiltrating lymphocytes (TILs) and high expression of immune checkpoints. CONCLUSIONS: Patients with tumors harboring ARID1A mutations may achieve better clinical outcomes from immunotherapy, especially in GC. ARID1A mutations can lead to genomic instability and reshape the tumor immune microenvironment (TIME), which can be used as a biomarker for immunotherapy.

Molecular landscape of borderline ovarian tumours: A systematic review.

Borderline ovarian tumours (BOTs) show intriguing characteristics distinguishing them from other ovarian tumours. The aim of the systematic review was to analyse the spectrum of molecular changes found in BOTs and discuss their significance in the context of the overall therapeutic approach. The systematic review included articles published between 2000 and 2023 in the databases: PubMed, EMBASE, and Cochrane. After a detailed analysis of the available publications, we qualified for the systematic review: 28 publications on proto-oncogenes: BRAF, KRAS, NRAS, ERBB2, and PIK3CA, 20 publications on tumour suppressor genes: BRCA1/2, ARID1A, CHEK2, PTEN, 4 on adhesion molecules: CADM1, 8 on proteins: B-catenin, claudin-1, and 5 on glycoproteins: E-Cadherin. In addition, in the further part of the systematic review, we included eight publications on microsatellite instability and three describing loss of heterozygosity in BOT. Molecular changes found in BOTs can vary on a case-by-case basis, identifying carcinogenic mutations through molecular analysis and developing targeted therapies represent significant advancements in the diagnosis and treatment of ovarian malignancies. Molecular studies have contributed significantly to our understanding of BOT pathogenesis, but substantial research is still required to elucidate the relationship between ovarian neoplasms and extraneous disease, identify accurate prognostic indicators, and develop targeted therapeutic approaches.

ARID1A loss promotes RNA editing of CDK13 in an ADAR1-dependent manner.

BACKGROUND: ARID1A, a subunit of the SWI/SNF chromatin remodeling complex, is thought to play a significant role both in tumor suppression and tumor initiation, which is highly dependent upon context. Previous studies have suggested that ARID1A deficiency may contribute to cancer development. The specific mechanisms of whether ARID1A loss affects tumorigenesis by RNA editing remain unclear. RESULTS: Our findings indicate that the deficiency of ARID1A leads to an increase in RNA editing levels and alterations in RNA editing categories mediated by adenosine deaminases acting on RNA 1 (ADAR1). ADAR1 edits the CDK13 gene at two previously unidentified sites, namely Q113R and K117R. Given the crucial role of CDK13 as a cyclin-dependent kinase, we further observed that ADAR1 deficiency results in changes in the cell cycle. Importantly, the sensitivity of ARID1A-deficient tumor cells to SR-4835, a CDK12/CDK13 inhibitor, suggests a promising therapeutic approach for individuals with ARID1A-mutant tumors. Knockdown of ADAR1 restored the sensitivity of ARID1A deficient cells to SR-4835 treatment. CONCLUSIONS: ARID1A deficiency promotes RNA editing of CDK13 by regulating ADAR1.

Evaluation of ARID1A as a Potential Biomarker for Predicting Response to Immune Checkpoint Inhibitors in Patients with Endometrial Cancer.

BACKGROUND: AT-rich interaction domain 1A (ARID1A) has been proposed as a new biomarker for predicting response to immune checkpoint inhibitors (ICIs). The predictive value of ARID1A for predicting ICI effectiveness has not been reported for endometrial cancer. Therefore, we investigated whether ARID1A negativity predicts ICI effectiveness for endometrial cancer treatment. METHODS: We evaluated ARID1A expression, tumor-infiltrating lymphocytes (CD8+), and immune checkpoint molecules (PD-L1/PD-1) by immunostaining endometrial samples from patients with endometrial cancer. Samples in which any of the four mismatch repair proteins (MLH1, MSH2, MSH6, and PMS2) were determined to be negative via immunostaining were excluded. In the ARID1A-negative group, microsatellite instability (MSI) status was confirmed via MSI analysis. RESULTS: Of the 102 samples investigated, 25 (24.5%) were ARID1A-negative. CD8 and PD-1 expression did not differ significantly between the ARID1A-negative group and the ARID1A-positive group; however, the ARID1A-negative group showed significantly lower PD-L1 expression. Only three samples (14.2%) in the ARID1A-negative group showed high MSI. Sanger sequencing detected three cases of pathological mutation in the MSH2-binding regions. We also established an ARID1A-knockout human ovarian endometriotic epithelial cell line (HMOsisEC7 ARID1A KO), which remained microsatellite-stable after passage. CONCLUSION: ARID1A negativity is not suitable as a biomarker for ICI effectiveness in treating endometrial cancer.

ARID1A Mutations in Gastric Cancer: A Review with Focus on Clinicopathological Features, Molecular Background and Diagnostic Interpretation.

AT-rich interaction domain 1 (ARID1A) is a pivotal gene with a significant role in gastrointestinal tumors which encodes a protein referred to as BAF250a or SMARCF1, an integral component of the SWI/SNF (SWItch/sucrose non-fermentable) chromatin remodeling complex. This complex is instrumental in regulating gene expression by modifying the structure of chromatin to affect the accessibility of DNA. Mutations in ARID1A have been identified in various gastrointestinal cancers, including colorectal, gastric, and pancreatic cancers. These mutations have the potential to disrupt normal SWI/SNF complex function, resulting in aberrant gene expression and potentially contributing to the initiation and progression of these malignancies. ARID1A mutations are relatively common in gastric cancer, particularly in specific adenocarcinoma subtypes. Moreover, such mutations are more frequently observed in specific molecular subtypes, such as microsatellite stable (MSS) cancers and those with a diffuse histological subtype. Understanding the presence and implications of ARID1A mutations in GC is of paramount importance for tailoring personalized treatment strategies and assessing prognosis, particularly given their potential in predicting patient response to novel treatment strategies including immunotherapy, poly(ADP) ribose polymerase (PARP) inhibitors, mammalian target of rapamycin (mTOR) inhibitors, and enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) inhibitors.

Genome-wide DNA methylation in relation to ARID1A deficiency in ovarian clear cell carcinoma.

BACKGROUND: The poor chemo-response and high DNA methylation of ovarian clear cell carcinoma (OCCC) have attracted extensive attentions. Recently, we revealed the mutational landscape of the human kinome and additional cancer-related genes and found deleterious mutations in ARID1A, a component of the SWI/SNF chromatin-remodeling complex, in 46% of OCCC patients. The present study aims to comprehensively investigate whether ARID1A loss and genome-wide DNA methylation are co-regulated in OCCC and identify putative therapeutic targets epigenetically regulated by ARID1A. METHODS: DNA methylation of ARID1Amt/ko and ARID1Awt OCCC tumors and cell lines were analyzed by Infinium MethylationEPIC BeadChip. The clustering of OCCC tumors in relation to clinical and mutational status of tumors were analyzed by hierarchical clustering analysis of genome-wide methylation. GEO expression profiles were used to identify differentially methylated (DM) genes and their expression level in ARID1Amt/ko vs ARID1Awt OCCCs. Combining three pre-ranked GSEAs, pathways and leading-edge genes epigenetically regulated by ARID1A were revealed. The leading-edge genes that passed the in-silico validation and showed consistent ARID1A-related methylation change in tumors and cell lines were regarded as candidate genes and finally verified by bisulfite sequencing and RT-qPCR. RESULTS: Hierarchical clustering analysis of genome-wide methylation showed two clusters of OCCC tumors. Tumor stage, ARID1A/PIK3CA mutations and TP53 mutations were significantly different between the two clusters. ARID1A mutations in OCCC did not cause global DNA methylation changes but were related to DM promoter or gene-body CpG islands of 2004 genes. Three pre-ranked GSEAs collectively revealed the significant enrichment of EZH2- and H3K27me3-related gene-sets by the ARID1A-related DM genes. 13 Leading-edge DM genes extracted from the enriched gene-sets passed the expression-based in-silico validation and showed consistent ARID1A-related methylation change in tumors and cell lines. Bisulfite sequencing and RT-qPCR analysis showed promoter hypermethylation and lower expression of IRX1, TMEM101 and TRIP6 in ARID1Amt compared to ARID1Awt OCCC cells, which was reversed by 5-aza-2'-deoxycytidine treatment. CONCLUSIONS: Our study shows that ARID1A loss is related to the differential methylation of a number of genes in OCCC. ARID1A-dependent DM genes have been identified as key genes of many cancer-related pathways that may provide new candidates for OCCC targeted treatment.

Collaboration between distinct SWI/SNF chromatin remodeling complexes directs enhancer selection and activation of macrophage inflammatory genes.

Macrophages elicit immune responses to pathogens through induction of inflammatory genes. Here, we examined the role of three variants of the SWI/SNF nucleosome remodeling complex-cBAF, ncBAF, and PBAF-in the macrophage response to bacterial endotoxin (lipid A). All three SWI/SNF variants were prebound in macrophages and retargeted to genomic sites undergoing changes in chromatin accessibility following stimulation. Cooperative binding of all three variants associated with de novo chromatin opening and latent enhancer activation. Isolated binding of ncBAF and PBAF, in contrast, associated with activation and repression of active enhancers, respectively. Chemical and genetic perturbations of variant-specific subunits revealed pathway-specific regulation in the activation of lipid A response genes, corresponding to requirement for cBAF and ncBAF in inflammatory and interferon-stimulated gene (ISG) activation, respectively, consistent with differential engagement of SWI/SNF variants by signal-responsive transcription factors. Thus, functional diversity among SWI/SNF variants enables increased regulatory control of innate immune transcriptional programs, with potential for specific therapeutic targeting.

DNA methylation analysis in patients with neurodevelopmental disorders improves variant interpretation and reveals complexity.

Analysis of genomic DNA methylation by generating epigenetic signature profiles (episignatures) is increasingly being implemented in genetic diagnosis. Here we report our experience using episignature analysis to resolve both uncomplicated and complex cases of neurodevelopmental disorders (NDDs). We analyzed 97 NDDs divided into (1) a validation cohort of 59 patients with likely pathogenic/pathogenic variants characterized by a known episignature and (2) a test cohort of 38 patients harboring variants of unknown significance or unidentified variants. The expected episignature was obtained in most cases with likely pathogenic/pathogenic variants (53/59 [90%]), a revealing exception being the overlapping profile of two SMARCB1 pathogenic variants with ARID1A/B:c.6200, confirmed by the overlapping clinical features. In the test cohort, five cases showed the expected episignature, including (1) novel pathogenic variants in ARID1B and BRWD3; (2) a deletion in ATRX causing MRXFH1 X-linked mental retardation; and (3) confirmed the clinical diagnosis of Cornelia de Lange (CdL) syndrome in mutation-negative CdL patients. Episignatures analysis of the in BAF complex components revealed novel functional protein interactions and common episignatures affecting homologous residues in highly conserved paralogous proteins (SMARCA2 M856V and SMARCA4 M866V). Finally, we also found sex-dependent episignatures in X-linked disorders. Implementation of episignature profiling is still in its early days, but with increasing utilization comes increasing awareness of the capacity of this methodology to help resolve the complex challenges of genetic diagnoses.

ARID1A in Gynecologic Precancers and Cancers.

The highest frequency of genetic alterations in the tumor suppressor ARID1A occurs in malignancies of the female reproductive tract. The prevalence of ARID1A alterations in gynecologic precancers and cancers is summarized from the literature, and the putative mechanisms of tumor suppressive action examined both in benign/precursor lesions including endometriosis and atypical hyperplasia and in malignancies of the ovary, uterus, cervix and vagina. ARID1A alterations in gynecologic cancers are usually loss-of-function mutations, resulting in diminished or absent protein expression. ARID1A deficiency results in pleiotropic downstream effects related not only to its role in transcriptional regulation as a SWI/SNF complex subunit, but also related to the functions of ARID1A in DNA replication and repair, immune modulation, cell cycle progression, endoplasmic reticulum (ER) stress and oxidative stress. The most promising actionable signaling pathway interactions and therapeutic vulnerabilities of ARID1A mutated cancers are presented with a critical review of the currently available experimental and clinical evidence. The role of ARID1A in response to chemotherapeutic agents, radiation therapy and immunotherapy is also addressed. In summary, the multi-faceted role of ARID1A mutation in precancer and cancer is examined through a clinical lens focused on development of novel preventive and therapeutic interventions for gynecological cancers.