Deletion Of Arid1a Research Articles

Abstract B19: ARID1A is a 1p36 tumor suppressor that collaborates with N-Myc in neuroblastoma

Abstract Neuroblastoma (NB) is an embryonal cancer of the sympathetic nervous system (SNS) that causes 15% of pediatric cancer deaths. A major barrier to advancing targeted therapy in NB is identifying the unknown oncogenes and tumor suppressors that are buried within large segmental chromosomal gains and losses that characterize high-risk NB. To assist in identifying them, we have established a novel system for studying NB based on the transformation of neural crest cells (NCCs), the progenitor cells of the SNS, isolated from mouse embryonic day 9.5 trunk neural tube explants. Based on pathology and gene expression analysis, we have demonstrated that the enforced expression of N-Myc in wild-type NCCs generates phenotypically and molecularly accurate NB that most closely models MYCN-amplified NB. Using this system we next addressed a longstanding problem in the NB field. Though 70% of MYCN-amplified NBs have 1p36 deletions, representing ~28% of high-risk NBs, the 1p36 tumor suppressor that collaborates with N-Myc is unknown. Using CRISPR/Cas9 technology in primary NCCs, we screened multiple 1p36 candidate tumor suppressors in a tumorsphere-forming assay. We found tumorspheres only formed when two chromatin-remodeling factors, CHD5 and ARID1A, were both lost, though their loss was not sufficient to generate tumors in vivo. Therefore, we next tested whether deletion of Chd5 and Arid1a, Arid1a alone, or deletion of the entire syntenic 1p36 region, as validated by FISH and karyotype analysis, is selected for during N-Myc driven transformation of primary NCCs. To achieve this, we started with low-efficiency deletion of each target in three independently derived NCC lines and then allowed clonal evolution during tumorigenesis to identify the favored deletion for N-Myc overexpressing NCCs. Tumors with CRISPR/Cas9 targeted 1p36 genes formed with a reduced latency of ~50 days over control (p<0.05), indicating that there was loss of an N-Myc collaborating tumor suppressor. From whole-exome sequencing we observed clonal selection from parental lines to tumors for both indel mutations in Arid1a and for larger deletions that encompassed Arid1a and surrounding genes. Arid1a mutations went from below level of detection in the parental NCC lines to >80% of the reads in the daughter tumors. We also saw selection for indel mutations and deletions of CHD5, but not in every tumor analyzed and no selection for loss of the entire 1p36 locus in any tumor. Using ex vivo adeno-cre mediated deletion of one or both floxed Arid1a alleles in NCCs, we found that loss of one Arid1a allele significantly (p=0.02) decreased latency by ~40 days over control in collaboration with N-Myc. Loss of both alleles conferred no advantage, indicating that when combined with N-Myc, ARID1A is an obligate haplo-insufficient tumor suppressor. In sum, these tumor studies support a model in which loss of one Arid1a, but not the entire syntenic 1p36 locus, is strongly selected for during N-Myc driven NB tumorigenesis, suggesting that Arid1a is the critical 1p36 tumor suppressor. The next step in this longstanding problem is to understand why loss of ARID1A collaborates with MYCN amplification and to exploit this discovery to improve treatment for MYCN amplified/1p36 deleted NBs. Citation Format: Jesus Garcia-Lopez, Kirby Wallace, Joel Otero, Rachelle Olsen, David Finkelstein, Jerold Rehg, Yongdong Wang, Kevin Freeman. ARID1A is a 1p36 tumor suppressor that collaborates with N-Myc in neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B19.

ARID1A, a SWI/SNF subunit, is critical to acinar cell homeostasis and regeneration and is a barrier to transformation and epithelial-mesenchymal transition in the pancreas

ObjectiveHere, we evaluate the contribution of AT-rich interaction domain-containing protein 1A (ARID1A), the most frequently mutated member of the SWItch/sucrose non-fermentable (SWI/SNF) complex, in pancreatic homeostasis and pancreatic ductal adenocarcinoma...

Abstract 2493: Inactivation of ARID1A-SWI/SNF complex alters chromatin compactness at enhancer regions and affects transcription of key tumor signaling circuitry

Abstract Somatic mutations in ARID1A, a SWI/SNF chromatin remodeling gene, are prevalent in human cancer and represent an emerging mechanism by which a molecular genetic alteration reprograms epigenomes and propels tumorigenesis. Through comprehensive sequencing of chromatin immunoprecipitation and chromatin accessibility, we found chromatin binding regions for ARID1A/BRG1-containing SWI/SNF remodeling complexes, which were enriched at enhancers and corresponded to euchromatin state. ARID1A deletion caused global re-distribution of BRG1-containing complexes in chromatin. Integrative analyses of ATAC-seq, ChIP-seq, and transcriptome data obtained from endometrial epithelium and human endometrioid carcinomas identified high-confidence ARID1A-regulated genes that participate in myriad key tumor and developmental signaling circuitry tissue. Deletion of Arid1a was found to inactivate the TGF-β pathway and to accelerate tumor progression from pre-cancerous lesions to endometrioid carcinomas. This study provides compelling evidence that Arid1a loss is required for developing endometrial cancer from its precursor. Collectively, targeting epigenomic modifications may hold promise for suppressing invasive behaviors of neoplasms carrying ARID1A mutations. Citation Format: Yohan Suryo Rahmanto, Xu Shi, Wenjing Shen, Tsutomu Miyamoto, Yu Yu, Xi Chen, Meng-Horng Lee, Vivek Singh, Michele I. Vitolo, Denis Wirtz, Ronny Drapkin, Jianhua Xuan, Ie-Ming Shih, Tian-Li Wang. Inactivation of ARID1A-SWI/SNF complex alters chromatin compactness at enhancer regions and affects transcription of key tumor signaling circuitry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2493.

Abstract PR02: The SWI/SNF component Arid1a regulates regenerative capacity and carcinogenesis in a dose-dependent fashion

Abstract It is unclear if regenerative capacity can be dissociated from cancer risk, a question that is highly relevant for liver carcinogenesis. Frequent SWI/SNF and Arid1a loss-of-function mutations suggest tumor suppressive roles, but the functional impact of these mutations in tissue homeostasis and cancer is unclear. Mice without Arid1a possessed improved healing abilities after an array of injuries. After liver resection, chemical hepatocyte injury, and toxic bile duct injury, Arid1a deficient livers exhibited increased regeneration, reduced tissue damage, and improved organ function. Moreover, global homozygous Arid1a loss potentiated soft tissue healing after ear hole punch and hematopoietic recovery after irradiation. The chromatin state as reprogrammed by Arid1a loss restricted access to promoters by lineage-specific transcription factors that ordinarily suppress cell cycle reentry, thus increasing regeneration after injury. Intriguingly, the homozygous Arid1a deficient state protected mice from chemical injury induced hepatocellular carcinoma (HCC) and extended survival in a MYC-driven hepatoblastoma model. Since Arid1a mutations are most frequently heterozygous in human HCC genome studies, we examined the haploinsufficient state in these mouse models and observed accelerated carcinogenesis and metastasis. Transcriptome analysis identified dysregulated differentiation, proliferation, and metastasis programs. These models show that full deletion of Arid1a can enhance mammalian regeneration without increasing cancer risk, but that Arid1a’s tumor suppressor functions are highly sensitive to dose. This abstract is also presented as Poster A16. Citation Format: Xuxu Sun, Sam Wang, Thomas Maples, Lin Li, Liem Nguyen, Jen-Chieh Chuang, Mahsa Sorouri, Shuyuan Zhang, Linwei Wu, Cemre Celen, Hao Zhu. The SWI/SNF component Arid1a regulates regenerative capacity and carcinogenesis in a dose-dependent fashion. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr PR02.

Suppression of the SWI/SNF Component Arid1a Promotes Mammalian Regeneration

Mammals have partially lost the extensive regenerative capabilities of some vertebrates, possibly as a result of chromatin-remodeling mechanisms that enforce terminal differentiation. Here, we show that deleting the SWI/SNF component Arid1a substantially improves mammalian regeneration. Arid1a expression is suppressed in regenerating tissues, and genetic deletion of Arid1a increases tissue repair following an array of injuries. Arid1a deficiency in the liver increases proliferation, reduces tissue damage and fibrosis, and improves organ function following surgical resection and chemical injuries. Hepatocyte-specific deletion is also sufficient to increase proliferation and regeneration without excessive overgrowth, and global Arid1a disruption potentiates soft tissue healing in the ear. We show that Arid1a loss reprograms chromatin to restrict promoter access by transcription factors such as C/ebpα, which enforces differentiation, and E2F4, which suppresses cell-cycle re-entry. Thus, epigenetic reprogramming mediated by deletion of a single gene improves mammalian regeneration and suggests strategies to promote tissue repair after injury.

Deletion of Arid1a in Reproductive Tract Mesenchymal Cells Reduces Fertility in Female Mice.

Women with endometriosis can suffer from decreased fecundity or complete infertility via abnormal oocyte function or impaired placental-uterine interactions required for normal pregnancy establishment and maintenance. Although AT-rich interactive domain 1A (SWI-like) (ARID1A) is a putative tumor suppressor in human endometrial cancers and endometriosis-associated ovarian cancers, little is known about its role in normal uterine function. To study the potential function of ARID1A in the female reproductive tract, we generated mice with a conditional knockout of Arid1a using anti-Müllerian hormone receptor 2-Cre. Female Arid1a conditional knockout mice exhibited a progressive decrease in number of pups per litter, with a precipitous decline after the second litter. We observed no tumors in virgin mice, although one knockout mouse developed a uterine tumor after pregnancy. Unstimulated virgin female knockout mice showed normal oviductal, ovarian, and uterine histology. Uteri of Arid1a knockout mice showed a normal decidualization response and appropriate responses to estradiol and progesterone stimulation. In vitro studies using primary cultures of human endometrial stromal fibroblasts revealed that small interfering RNA knockdown of ARID1A did not affect decidualization in vitro. Timed pregnancy studies revealed the significant resorption of embryos at Embryonic Day 16.5 in knockout mice in the third pregnancy. In addition to evidence of implantation site hemorrhage, pregnant Arid1a knockout mice showed abnormal placental morphology. These results suggest that Arid1a supports successful pregnancy through its role in placental function.

Abstract IA16: Endometriosis-related ovarian cancer.

Abstract Endometriosis-related ovarian neoplasms (ERONs) include clear cell carcinoma, endometrioid carcinoma, and, least frequently, seromucinous carcinomas. They belong to a unique group of ovarian tumors in that unlike other histotypes of ovarian cancer, these tumors are frequently associated with endometriosis, especially the lesions presenting as ovarian endometriotic cysts or endometriomas. ERONs usually present as large, unilateral, cystic neoplasms. In general, ERONs tend to behave in an indolent fashion because many of the diseases present at early clinical stages. When confined to the ovary, they have an excellent prognosis, but advanced stage tumors have a poor outcome. It has been demonstrated that ERONs can develop from endometriotic cyst epithelium through sequential stages of tumor progression. The microenvironment of an endometriotic cyst is highly inflammatory, and the cyst contains abundant iron-induced reactive oxygen species that are thought to be genotoxic and mutagenic. Chronic exposure of cystic epithelium to this microenvironment likely facilitates the accumulation of somatic molecular genetic changes that ultimately transform the cells and lead to tumor development. Molecular analyses of ERONs, including genome-wide analysis, have identified several alterations associated with aberrant activation or inactivation of pathways involving ARID1A, PI3K, Wnt, PP2A and mismatch repair. Specifically, canonical Wnt signaling pathway defects and microsatellite instability are more frequently detected in endometrioid carcinomas than in clear cell carcinomas. Among all of the molecular genetic changes identified to date, inactivating mutations of the ARID1A tumor suppressor gene are most common in ERON. Interestingly, ARID1A mutations are rarely observed in other types of ovarian cancer. ARID1A participates in the SWI/SNF chromatin remodeling complex, which plays a fundamental role in a variety of cellular processes, including DNA damage repair, telomere maintenance, transcription and DNA synthesis and methylation. The molecular events that dictate the development of different ERONs remain elusive, but studies using genetically engineered mouse models demonstrate that co-deletion of ARID1A and PTEN results in ovarian endometrioid carcinoma, while co-deletion of ARID1A and PIK3CA leads to formation of clear cell-like ovarian neoplasms in mice. However, deletion of ARID1A by itself is insufficient to drive ovarian tumor formation. Understanding the molecular changes and pathogenesis involved in the development of ERON is fundamental for future translational studies aimed at designing new diagnostic tests for outcome prediction and at identifying critical molecular features for target-based therapy. Citation Format: Ie-Ming Shih. Endometriosis-related ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr IA16.

Disruptive ARID1A Mutations in Follicular Lymphoma Impair DNA Repair Efficiency and Are Associated with Favorable Outcome in Patients Receiving First-Line Immunochemotherapy

Follicular lymphoma (FL) is among the most common lymphomas worldwide and considered incurable for the majority of patients who present with advanced disease. FL is a clinically and molecularly heterogeneous disease. We have recently established a clinicogenetic risk model (m7-FLIPI) that integrates the mutation status of 7 genes, including ARID1A, into a predictive algorithm for improved risk stratification for failure-free survival in patients receiving first-line immunochemotherapy (Pastore et al., Lancet Oncology 2015). When adjusted for FLIPI and ECOG performance status, the presence of ARID1A mutations correlated with longer failure-free survival (HR 2.85, 95% confidence interval (CI) 1.12-7.27; p=0.049) in patients receiving first-line R-CHOP. The underlying molecular mechanism for better treatment outcome of patients with ARID1A mutated FL is unclear.ARID1A is a member of SWI/SNF nucleosome remodeling complex. Evolving evidence indicates that the SWI/SNF complex functions as a tumor suppressor and is implicated in DNA damage repair. ARID1Ais the most commonly mutated SWI/SNF complex member in FL: in our series, we identified 44 ARID1A mutations in 304 FL cases (14%). These mutations were primarily heterozygous and disruptive (66%), another 6 cases harbored splice site mutations. Primary patient samples and lymphoma cell lines that harbored ARID1A mutations (e.g., SU-DHL5, WSU-FSCCL, Namalwa) were had lower ARID1A protein expression as compared to ARID1A wild type (wt) t(14;18)-positive lymphoma cell lines (OCI-Ly1, -Ly8, DB).Non-homologous end joining (NHEJ) has been reported to be the predominant pathway of DNA double strand break (DSB) repair in FL (Koues et al., Immunity 2015) and is indispensible for successful VDJ rearrangement during early B-cell maturation.To analyze the impact of Arid1a disruption on B-cell maturation in vivo, we used a conditional Arid1a knock-out mouse model (Arid1af/f; Gao, PNAS 2008). 8-10 week old Mx1-Cre+Arid1af/f mice and control (Mx1-Cre-Arid1af/f) mice were treated with poly(IC). Bone marrow (BM) were harvested from these animals 14 days later. Conditionally deletion of Arid1a (n=3) resulted in severely impaired B-cell maturation in BM cells affecting Hardy fractions B/C though F. E.g., flow cytometric analysis showed that the frequency of fraction C (B220+ CD43+ HSA+ BP-1+) was greatly reduced in BM from Mx1-Cre+Arid1af/f vs in the control group (0.1 % vs 1.2 %, n=3), consistent with the inability to successfully complete VDJ recombination.As a proof-of-principle experiment we assayed the impact of Arid1a loss on DSB repair efficiency using mouse embryo fibroblasts (MEFs) from either wt or conditional Arid1a knock-out mice. MEFs were irradiated with 2 Gy 36 hrs after retroviral transduction with Cre and stained for γH2A.X and 53BP1 to assay for NHEJ. In three independent and blinded experiments, evaluation of >100 cells per condition demonstrated significantly more double-positive cells (>6 foci/cell) in Arid1a-/- MEFs (43+/-14%) compared to both Arid1af/f MEFs (9+/-1%) and Cre-exposed wt MEFs (15+/-5%) at 16 hrs post irradiation, whereas there was no significant difference for Arid1af/f or wt MEFs with or without Cre at baseline (all <10%), and 1 hr post irradiation (all >50%).ARID1A haplodeficient lymphoma cell lines (SU-DHL5, WSU-FSCCL, Namalwa) were 10-100 fold more sensitive to the DSB inducing doxorubicin treatment compared to ARID1A wt lymphoma cell lines (OCI-Ly1, -Ly8, DB). Tet-induced shRNA knock-down (via pTRIPZ vectors) of endogenous ARID1A in two t(14;18)-positive lymphoma cell lines with wt ARID1A (OCI-Ly1 and OCI-Ly8) doubled the number of NHEJ foci by 53-BP1 staining in two independent experiments. Furthermore, shRNA knock-down of ARID1A resulted in increased sensitivity to doxorubicine (IC50 <10 nM) in OCI-Ly8 cell lines as compared to 25 nM for scrambled shRNA.We conclude that ARID1A is recurrently and significantly mutated in FL. Most mutations are disruptive and result in functionally relevant ARID1A protein loss. ARID1A disruption slows the repair kinetics of NHEJ and delays the clearance of DSB both in a genetically clean MEF model and in t(14;18)-positive lymphoma cell lines. As a result, ARID1A loss might underlie the genomic instability of FL, but -at the same time- sensitize tumors to DNA damaging agents and irradiation. DisclosuresNo relevant conflicts of interest to declare.

Global gene expression profiling of a mouse model of ovarian clear cell carcinoma caused by ARID1A and PIK3CA mutations implicates a role for inflammatory cytokine signaling

Ovarian clear-cell carcinoma (OCCC) is an aggressive form of epithelial ovarian cancer (EOC). OCCC represents 5–25% of all EOC incidences and is the second leading cause of death from ovarian cancer (Glasspool and McNeish, 2013) [1]. A recent publication by Chandler et al. reported the first mouse model of OCCC that resembles human OCCC both genetically and histologically by inducing a localized deletion of ARID1A and the expression of the PIK3CAH1047R substitution mutation (Chandler et al., 2015) [2]. We utilized Affymetrix Mouse Gene 2.1 ST arrays for the global gene expression profiling of mouse primary OCCC tumor samples and animal-matched normal ovaries to identify cancer-dependent gene expression. We describe the approach used to generate the differentially expressed genes from the publicly available data deposited at the Gene Expression Omnibus (GEO) database under the accession number GSE57380. These data were used in cross-species comparisons to publically available human OCCC gene expression data and allowed the identification of coordinately regulated genes in both mouse and human OCCC and supportive of a role for inflammatory cytokine signaling in OCCC pathogenesis (Chandler et al., 2015) [2].

Roles of deletion of Arid1a, a tumor suppressor, in mouse ovarian tumorigenesis.

The chromatin remodeling gene, ARID1A, has been implied as a tumor suppressor, and its somatic inactivating mutations occur in a wide variety of human cancers, most frequently in ovarian and uterine endometrioid and ovarian clear cell carcinomas. Tumors with ARID1A mutations also frequently harbor PTEN or PIK3CA mutations, suggesting their collaboration in tumorigenesis. Here, we used a conditional knockout mouse model in which Arid1a and Pten were deleted either individually or in combination in the mouse ovarian surface epithelium. After 6 months, 59.1% of mice with Arid1a and Pten double knockout developed ovarian endometrioid or undifferentiated carcinoma, whereas the remaining mice showed hyperplasia of ovarian surface epithelium. In contrast, 52 mice with homozygous or heterozygous deletion in either Arid1a or Pten did not develop ovarian lesions. These results demonstrate that inactivation of Arid1a alone is insufficient for tumor initiation but it requires additional genetic alteration(s) such as Pten deletion to drive tumorigenesis.