ETV4 and ETV5 drive synovial sarcoma through cell cycle and DUX4 embryonic pathway control

Synovial sarcoma is an aggressive malignancy with no effective treatments for patients with metastasis. The synovial sarcoma fusion SS18-SSX, which recruits the SWI/SNF-BAF chromatin remodeling and polycomb repressive complexes, results in epigenetic activation of FGF receptor (FGFR) signaling. In genetic FGFR-knockout models, culture, and xenograft synovial sarcoma models treated with the FGFR inhibitor BGJ398, we show that FGFR1, FGFR2, and FGFR3 were crucial for tumor growth. Transcriptome analyses of BGJ398-treated cells and histological and expression analyses of mouse and human synovial sarcoma tumors revealed prevalent expression of two ETS factors and FGFR targets, ETV4 and ETV5. We further demonstrate that ETV4 and ETV5 acted as drivers of synovial sarcoma growth, most likely through control of the cell cycle. Upon ETV4 and ETV5 knockdown, we observed a striking upregulation of DUX4 and its transcriptional targets that activate the zygotic genome and drive the atrophy program in facioscapulohumeral dystrophy patients. In addition to demonstrating the importance of inhibiting all three FGFRs, the current findings reveal potential nodes of attack for the cancer with the discovery of ETV4 and ETV5 as appropriate biomarkers and molecular targets, and activation of the embryonic DUX4 pathway as a promising approach to block synovial sarcoma tumors

The Synovial Sarcoma-Associated SYT-SSX2 Oncogene Antagonizes the Polycomb Complex Protein Bmi1

This study demonstrates deregulation of polycomb activity by the synovial sarcoma-associated SYT-SSX2 oncogene, also known as SS18-SSX2. Synovial sarcoma is a soft tissue cancer associated with a recurrent t(X:18) translocation event that generates one of two fusion proteins, SYT-SSX1 or SYT-SSX2. The role of the translocation products in this disease is poorly understood. We present evidence that the SYT-SSX2 fusion protein interacts with the polycomb repressive complex and modulates its gene silencing activity. SYT-SSX2 causes destabilization of the polycomb subunit Bmi1, resulting in impairment of polycomb-associated histone H2A ubiquitination and reactivation of polycomb target genes. Silencing by polycomb complexes plays a vital role in numerous physiological processes. In recent years, numerous reports have implicated gain of polycomb silencing function in several cancers. This study provides evidence that, in the appropriate context, expression of the SYT-SSX2 oncogene leads to loss of polycomb function. It challenges the notion that cancer is solely associated with an increase in polycomb function and suggests that any imbalance in polycomb activity could drive the cell toward oncogenesis. These findings provide a mechanism by which the SYT-SSX2 chimera may contribute to synovial sarcoma pathogenesis

Optimized lentiviral vector transduction of adherent cells and analysis in sulforhodamine B proliferation and chromatin immunoprecipitation assays

Transduction with lentiviral vectors is a useful approach to study the molecular function of specific genes in mammalian cells. Here, we present a calcium phosphate-based transfection protocol that guarantees highly efficient production and delivery of lentiviral vectors in adherent cultured cells. We also describe in detail a direct lysis technique to measure protein expression, an optimized sulforhodamine B proliferation assay, and a step-by-step chromatin immunoprecipitation procedure to verify the binding of ETV5 to E2F1 first intron in SYO-1 sarcoma cells. For complete details on the use and execution of this protocol, please refer to Kingston et al. (2003), 1 Ireton et al. (2002), 2 Brown et al. (2009), 3 DeSalvo et al. (2021), 4 Vichai and Kirtikara (2006), 5 and Boyer et al. (2005). 6 • A high-efficiency calcium phosphate/BES approach to lentivirus transduction • Allows fast analysis of gene expression by direct lysis • Reproducible measurements of growth over several days in SRB assays • Includes an optimized protocol for ChIP and PCR validation Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Transduction with lentiviral vectors is a useful approach to study the molecular function of specific genes in mammalian cells. Here, we present a calcium phosphate-based transfection protocol that guarantees highly efficient production and delivery of lentiviral vectors in adherent cultured cells. We also describe in detail a direct lysis technique to measure protein expression, an optimized sulforhodamine B proliferation assay, and a step-by-step chromatin immunoprecipitation procedure to verify the binding of ETV5 to E2F1 first intron in SYO-1 sarcoma cells

Genome-wide recruitment to Polycomb-modified chromatin and activity regulation of the synovial sarcoma oncogene SYT-SSX2

Abstract Background SYT-SSX is the oncogene associated with synovial sarcoma (SS), a stem cell disease. SYT-SSX is thought to be responsible for sarcoma initiation and development. It interacts with components of Polycomb and SWI/SNF complexes, the two epigenetic controllers that maintain the heritable status of differentiation-specific genes in the stem/progenitor cell. Through these associations SYT-SSX is thought to alter gene expression programs by epigenetic mechanisms. Recently, we reported that SYT-SSX2 reprograms mesenchymal stem cells and myoblasts by dictating their commitment to the neural lineage while disrupting their normal differentiation. This reprogramming was due to the direct occupancy of proneural genes by the SYT-SSX2 nuclear complex. To gain a clear understanding of SYT-SSX2 control of gene expression networks, we conducted a thorough genome-wide analysis to determine the mechanism of its recruitment and identify signature sets of epigenetic markers that would predict its targeting and transcriptional activity. Results SYT-SSX2 was recruited to distinct loci across all chromosomes, and an overwhelming number of Polycomb-modified sites enriched with the trimethylated histone H3 on lysine 27 (H3K27me3) formed the main recruiting module for SYT-SSX2. Not all SYT-SSX2/H3K27me3-occupied genes had altered expression, denoting the requirement for additional signals upon oncogene binding. Differential binding and epigenetic patterns distinguished upregulated and downregulated genes. Most activated genes had SYT-SSX2 sites enriched with H3K27me3 within their body or near their transcription start site (TSS) whereas a majority of downregulated genes were characterized by SYT-SSX2/H3K27me3-rich regions at long-range, or by modifications associated with transcription activation within the gene body or near the TSS. Hierarchical and functional clustering identified H3K27me3 as the dominant epigenetic marker associated with SYT-SSX2 binding and gene expression. Notably, this analysis revealed a cluster of upregulated neuronal genes densely covered by H3K27me3, consistent with programming toward the neural lineage by SYT-SSX2 observed previously. Conclusions The data analysis revealed that Polycomb complexes or their modified chromatin and their stably silenced differentiation programs seem to be the main target for SYT-SSX2, suggesting that their perturbation is at the center of tumorigenesis driven by the oncogene. Further research into this mechanism is crucial to the full understanding of SS biology.</p

Metabolic Enzymes in Sarcomagenesis: Progress Toward Biology and Therapy

Cellular metabolism reprogramming is an emerging hallmark of cancer, which provides tumor cells with not only necessary energy but also crucial materials to support growth. Exploiting the unique features of cancer metabolism is promising in cancer therapies. The growing interest in this field has led to numerous inhibitors being developed against key molecules in metabolic pathways, though most of them are still in preclinical development. Potential targeted cancer cell metabolic pathways under investigation include glycolysis, tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), glutaminolysis, pentose phosphate pathway (PPP), lipid synthesis, amino acid and nucleotide metabolism. Sarcoma is a type of cancer that arises from transformed cells of mesenchymal origin, in contrast to carcinoma which originates from epithelial cells. Compared with carcinoma, progress towards harnessing the therapeutic potential of targeting sarcoma cell metabolism has been relatively slow. Recently however, with the discovery of cancer-specific mutations in metabolic enzymes such as isocitrate dehydrogenase (IDH) and succinate dehydrogenase (SDH) in certain sarcoma types, cancer cellular metabolism has been considered more as a source of new targets for treating sarcoma. In this article, we review metabolic enzymes currently tested for cancer therapies and describe the therapeutic potential of targeting IDH mutations and SDH deficiency in sarcomas

Abstract 864: Mutant IDH1 is essential for chondrosarcoma growth

Abstract Chondrosarcomas are malignant bone tumors that produce cartilaginous matrix. Mutations in isocitrate dehydrogenase enzymes (IDH1/2) were recently described in several cancers, including chondrosarcomas. IDH mutations detected in human cancers invariably are heterozygous missense substitutions. These mutations lead to the inability of IDH to convert isocitrate into α-ketoglutarate (α-KG). Instead, α-KG is reduced into 2-hydroxyglutarate (D-2HG), an oncometabolite. Due to the structural similarity between D-2HG and α-KG, it has been reported that high levels of D-2HG competitively inhibit α-KG-dependent dioxygenases such as TET, JHDM and PHD enzymes, thus contributing to tumorigenesis. We sought to determine the role of IDH1 mutations in the tumorigenesis of human chondrosarcomas by inactivating mutant IDH1 using pharmacological and genetic approaches. In our study, we employed two human chondrosarcoma cell lines, JJ012 and HT1080, that carry endogenous IDH1 mutations. IDH mutation analysis was performed by PCR-based DNA sequencing, and D-2HG levels were measured by tandem mass spectrometry. Mutant IDH1 was knocked down via siRNA and knocked out via CRISPR/Cas9. We analyzed the effect of mutant IDH1 on chondrosarcoma growth in murine xenograft models. We found that knockdown of mutant IDH1 via siRNA significantly reduced D-2HG production in chondrosarcoma cells. In addition, mutant IDH1 knockdown dramatically inhibited colony formation in the sarcoma cells. Consistently, genetic knockout of mutant IDH1 almost completely depleted D-2HG production and significantly inhibited colony formation in the chondrosarcoma cells. To assess the significance of these results in vivo, we implanted the mutant IDH1- knockout cells in nude mice and studied their capacity for tumor initiation and growth. In these models, we observed that loss of mutant IDH1 led to a marked attenuation of chondrosarcoma formation. Our findings clearly demonstrate that mutant IDH1 plays a vital role in chondrosarcoma tumor formation. By investigating the role of IDH mutations in the pathogenesis of chondrosarcomas, we aim to uncover the potential therapeutic targets against this aggressive cancer. Citation Format: Luyuan Li, Xiaoyu Hu, Josiane E. Eid, Joanna DeSalvo, Jonathan C. Trent. Mutant IDH1 is essential for chondrosarcoma growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 864

IDH1 Mutation Induces HIF-1α and Confers Angiogenic Properties in Chondrosarcoma JJ012 Cells

Chondrosarcoma is a group of primary bone cancers that arise from transformed cells of chondrocytic lineage. Tumor recurrence and metastasis are devastating for patients with chondrosarcoma since there are no effective treatment options. IDH mutations occur in over 50% of tumors from patients with conventional or dedifferentiated chondrosarcomas and represent an attractive target for therapy. However, their role in the pathogenesis of chondrosarcoma remains largely unknown. In this study, we sought to determine the association of IDH mutation and HIF-1α in chondrosarcoma. We used the chondrosarcoma JJ012 cell line and its derived CRISPR/Cas9 mutant IDH1 (IDH1mut) knockout (KO) cells. RNA-Seq data analysis revealed downregulation of several HIF-1α target genes upon loss of IDH1mut. This was associated with reduced HIF-1α levels in the IDH1mut KO cells and tumors. Loss of IDH1mut also attenuated the expression of angiogenic markers in tumor tissues and abrogated the angiogenic capacity of JJ012 cells. Moreover, we observed that exogenous expression of HIF-1α significantly promoted anchorage-independent colony-formation by IDH1mut KO cells. These results suggest IDH1 mutation confers angiogenic and tumorigenic properties of JJ012 cells by inducing HIF-1α. Thus, the HIF pathway represents a promising candidate for combinatorial regimens to target IDH1 mutated chondrosarcomas

Mutant IDH1 Depletion Downregulates Integrins and Impairs Chondrosarcoma Growth

Chondrosarcomas are a heterogeneous group of malignant bone tumors that produce hyaline cartilaginous matrix. Mutations in isocitrate dehydrogenase enzymes (IDH1/2) were recently described in several cancers, including conventional and dedifferentiated chondrosarcomas. These mutations lead to the inability of IDH to convert isocitrate into α-ketoglutarate (α-KG). Instead, α-KG is reduced into D-2-hydroxyglutarate (D-2HG), an oncometabolite. IDH mutations and D-2HG are thought to contribute to tumorigenesis due to the role of D-2HG as a competitive inhibitor of α-KG-dependent dioxygenases. However, the function of IDH mutations in chondrosarcomas has not been clearly defined. In this study, we knocked out mutant IDH1 (IDH1 ) in two chondrosarcoma cell lines using the CRISPR/Cas9 system. We observed that D-2HG production, anchorage-independent growth, and cell migration were significantly suppressed in the IDH1 knockout cells. Loss of IDH1 also led to a marked attenuation of chondrosarcoma formation and D-2HG production in a xenograft model. In addition, RNA-Seq analysis of IDH1 knockout cells revealed downregulation of several integrin genes, including those of integrin alpha 5 (ITGA5) and integrin beta 5 (ITGB5). We further demonstrated that deregulation of integrin-mediated processes contributed to the tumorigenicity of IDH1-mutant chondrosarcoma cells. Our findings showed that IDH1 knockout abrogates chondrosarcoma genesis through modulation of integrins. This suggests that integrin molecules are appealing candidates for combinatorial regimens with IDH1 inhibitors for chondrosarcomas that harbor this mutation