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The Chromatin Landscape of Primary Synovial Sarcoma Organoids Is Linked to Specific Epigenetic Mechanisms and Dependencies

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The Chromatin Landscape of Primary Synovial Sarcoma Organoids Is Linked to Specific Epigenetic Mechanisms and Dependencies Published Online: 23 December, 2020 | Supp Info: http://doi.org/10.26508/lsa.202000808 Downloaded from life-science-alliance.org on 25 September, 2021 Research Article The chromatin landscape of primary synovial sarcoma organoids is linked to specific epigenetic mechanisms and dependencies Gaylor Boulay3,4,*, Luisa Cironi1,2,* , Sara P Garcia3,†, Shruthi Rengarajan3,†, Yu-Hang Xing3, Lukuo Lee3, Mary E Awad3, Beverly Naigles3 , Sowmya Iyer3, Liliane C Broye1,2, Tugba Keskin1,2, Alexandra Cauderay1,3, Carlo Fusco1,2, Igor Letovanec1, Ivan Chebib3, Petur Gunnalugur Nielsen3,Stephane´ Tercier6 ,Stephane´ Cherix5 , Tu Nguyen-Ngoc7 , Gregory Cote8 , Edwin Choy8 , Paolo Provero9,10 , Mario L Suva` 3,4, Miguel N Rivera3,4, Ivan Stamenkovic1,2 , Nicolo` Riggi1,2 Synovial sarcoma (SyS) is an aggressive mesenchymal malig- Introduction nancy invariably associated with the chromosomal transloca- tion t(X:18; p11:q11), which results in the in-frame fusion of the Synovial sarcoma (SyS) is the second most common soft tissue BAF complex gene SS18 to one of three SSX genes. Fusion of SS18 malignancy in the adolescent and young adult population, in which it to SSX generates an aberrant transcriptional regulator, which, in comprises 10–20% of all soft tissue sarcomas (Nielsen et al, 2015). Its permissive cells, drives tumor development by initiating major defining genetic feature is the chromosomal translocation t(X:18; p11: chromatin remodeling events that disrupt the balance between q11), which generates fusions between the nearly entire coding BAF-mediated gene activation and polycomb-dependent re- sequence of the SS18 gene and a portion of an SSX gene family pression. Here, we developed SyS organoids and performed member (SSX1 and SSX2 being the two most commonly implicated genome-wide epigenomic profiling of these models and mes- [Nielsen et al, 2015; Riggi et al, 2018]). As is often observed in pediatric enchymal precursors to define SyS-specificchromatinremod- malignancies, SyS displays few genetic mutations other than the eling mechanisms and dependencies. We show that SS18-SSX chromosomal translocation itself, suggesting that SS18-SSX bears the induces broad BAF domains at its binding sites, which oppose prime responsibility for SyS pathogenesis. Accordingly, a transgenic polycomb repressor complex (PRC) 2 activity, while facilitating mouse model of SyS was generated by expressing human SS18-SSX in recruitment of a non-canonical (nc)PRC1 variant. Along with the an early Myf5+ murine myoblast population (Haldar et al, 2007). uncoupling of polycomb complexes, we observed H3K27me3 Whereas this model pointed to Myf5+ myoblasts as a potential cell of eviction, H2AK119ub deposition and the establishment of de origin of SyS, experiments using conditional expression of SS18-SSX novo active regulatory elements that drive SyS identity. These produced tumors in atypical anatomic locations, suggesting that alterations are completely reversible upon SS18-SSX depletion other cell types may also be permissive for SS18-SSX–mediated and are associated with vulnerability to USP7 loss, a core transformation. Consistent with this notion, mouse and human member of ncPRC1.1. Using the power of primary tumor orga- mesenchymal stem cells (MSCs) have been shown to acquire a gene noids, our work helps define the mechanisms of epigenetic fi dysregulation on which SyS cells are dependent. expression pro le that resembles that of primary human SyS upon expression of SS18-SSX (Cironi et al, 2009, 2016). DOI 10.26508/lsa.202000808 | Received 5 June 2020 | Revised 1 December The oncogenic properties of SS18-SSX have been linked to its 2020 | Accepted 10 December 2020 | Published online 23 December 2020 ability to interact with proteins that control epigenetic states (Thaete 1Institute of Pathology, Centre Hospitalier Universitaire Vaudois, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland 2Swiss Cancer Center Leman, Centre Hospitalier Universitaire Vaudois, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland 3Department of Pathology and Center for Cancer Research, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA 4Broad Institute of Harvard and MIT, Cambridge, MA, USA 5Department of Orthopedics, Faculty of Biology and Medicine, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland 6Department of Woman-Mother Child, Centre Hospitalier Universitaire Vaudois, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland 7Department of Oncology, Centre Hospitalier Universitaire Vaudois, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland 8Division of Hematology and Oncology, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA 9Center for Translational Genomics and Bioinformatics, San Raffaele Scientific Institute, Milan, Italy 10Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy Correspondence: [email protected]; [email protected] *Gaylor Boulay and Luisa Cironi contributed equally to this work †Sara P Garcia and Shruthi Rengarajan contributed equally to this work Miguel N Rivera, Ivan Stamenkovic, and Nicolo Riggi are senior authors ©2020Boulay et al. https://doi.org/10.26508/lsa.202000808 vol 4 | no 2 | e202000808 1of17 et al, 1999; Nagai et al, 2001). SS18 is a component of the mamma- landscape of Sys organoids not only reflects the key pathogenic lian chromatin remodeling complex switch/sucrose non-fermentable mechanisms that lead to SyS but is also linked to specificepigenetic (SWI/SNF also known as BRG1/BRM-associated factor, BAF) (Wang et al, dependencies. 1996a, 1996b; Kadoch & Crabtree, 2013), which facilitates transcrip- tion by increasing chromatin accessibility at promoter and enhancer regions. In contrast, SSX1/2 belongs to a family of transcriptional re- pressors that co-localize with polycomb group proteins (PcG), including Results BMI1 and RING1B (Hale et al, 2019; Johansen & Gjerstorff, 2020). The Primary synovial sarcoma organoids have a distinctive pattern of two constituents of the fusion protein are thus associated with op- BAF complex distribution posing effects on chromatin regulation and the integration of their activities appears to provide the key toward establishing the oncogenic tTo mimic primary tumor features as closely as possible, we de- signaling network that generates SyS. Biochemical studies have shown veloped organoids from primary synovial sarcoma samples removed that SS18-SSX replaces wt SS18 in the BAF complex and alters its at surgery. Tumor organoids have consistently shown superiority to composition by ejecting the tumor suppressor SNF5/BAF47 (Kadoch & cell lines in providing adequate models for the study of tumor bi- Crabtree, 2013). The resulting effect is the redirection of the newly ology (Drost & Clevers, 2018) but have not previously been estab- constituted BAF complexes from enhancers to PcG domains to relieve lished from synovial sarcoma. We generated organoids from four polycomb repressor complex (PRC)2–mediated repression of bivalent primary SyS (SyS1-4) and expanded them in serum-free conditions target promoters (McBride et al, 2018). Recent functional genomic to avoid any serum-relatedepigeneticalteration(Dangles- experiments have also uncovered physical interaction between SS18- Marie et al, 2007)(Fig 1A). Expression of SS18-SSX in all four SSX and the histone demethylase KDM2B, a component of a non- organoids was confirmed by qRT-PCR (Fig 1B) and by chromatin canonical polycomb repressive complex 1 (ncPRC1.1), which leads to analyses using the H3K36me3 histone mark in the SSX1 39 terminal aberrant reactivation and expression of PcG target genes that are region as an indicator of transcription (Fig 1C). To determine SyS- required for transformation (Banito et al, 2018a). An additional re- specificity of the observed chromatin patterns, we used two sets of pressive role for SS18-SSX, which leads to PRC2 recruitment to ATF2 controls: two primary Ewing sarcoma organoids, established under target genes, has also been proposed (Su et al, 2012). conditions identical to those used to generate SyS organoids (Suva Given the importance of epigenetic events as drivers of its et al, 2009), and three different non-transformed human cell types, pathogenesis, chromatin regulators constitute potentially attractive including primary mesenchymal stem/stromal cells (MSCs), em- therapeutic targets for SyS. Accordingly, recent studies suggest that bryonic lung fibroblasts (MRC5) and mesothelial cells (MET5A). We small-molecule–mediated degradation of the BAF component BRD9 determined the epigenomic profiles of the primary organoids and can reverse oncogenic gene regulation in SyS (Brien et al, 2018; Michel non-transformed cells by genome-wide ChIP-seq analysis of the et al, 2018). However, the development of new anticancer therapies is histone marks H3K4me1, H3K4me3, H3K9ac, H3K27ac, H3K27me3, and hindered by the paucity of reliable pre-clinical models. Patient- H3K36me3. Because SS18-SSX becomes integrated into the BAF derived tumor organoids have emerged as a powerful system for complex (Kadoch & Crabtree, 2013), we also assessed BAF-binding modeling key biological properties of human carcinomas and their sites using antibodies against the core ATPase BAF members response to therapy (Drost & Clevers, 2018). Whereas organoids from SMARCA2/4. human sarcomas have generally been challenging to establish, we ChIP-seq analysis revealed BAF ATPases
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