GGAA Microsatellite Research Articles

Chimeric protein EWS::FLI1 drives cell proliferation in Ewing Sarcoma via aberrant expression of KCNN1/SK1 and dysregulation of calcium signaling.

Ewing sarcoma (ES) is characterized by EWS::FLI1 or EWS::ERG fusion proteins. Knowing that ion channels are involved in tumorigenesis, this work aimed to study the involvement of the KCNN1 gene, which encodes the SK1 potassium channel, in ES development. Bioinformatics analyses from databases were used to study KCNN1 expression in patients and cell lines. Molecular approaches and in vitro assays were used to study the transcriptional regulation of KCNN1 and its involvement in the regulation of ES cell proliferation. KCNN1 is overexpressed in ES patient biopsies, and its expression is inversely correlated with patient survival. EWS::FLI1, like EWS::ERG, promotes KCNN1 and SK1 expression, binding to GGAA microsatellites near the promoter of KCNN1 isoforms. KCNN1 is involved in the regulation of ES cell proliferation, with its silencing being associated with a slowing of the cell cycle, and its expression modulates membrane potential and therefore calcium flux. These results highlight that KCNN1 is a direct target of EWS::FLI1 and EWS::ERG and demonstrate that KCNN1 is involved in the regulation of intracellular calcium activity and ES cell proliferation, making it a promising therapeutic target in ES.

Abstract 6148: Multiple new Ewing sarcoma susceptibility loci expand knowledge of germline genetic etiology and nominate mechanisms of risk

Abstract Ewing sarcoma (EwS) is a rare and aggressive pediatric cancer with peak incidence in the second decade of life. EwS occurs primarily in children of European ancestry suggesting a genetic component to susceptibility. Prior investigations identified six susceptibility loci associated with EwS, with evidence for additional signals not reaching the commonly accepted threshold for genome-wide significance (i.e., p<5×10−8). At least two of these loci demonstrate evidence of tagging variation in length of nearby GGAA microsatellites, which alter binding of the pathognomonic EWSR1::FLI1 fusion resulting in downstream dysregulation of target genes promoting sarcomagenesis. To identify additional susceptibility loci, we performed the largest genome-wide association study (GWAS) to date consisting of 1,640 EwS cases and 8,457 cancer-free individuals of European ancestry from 8 studies and matched by principal components. Genotype imputation was carried out by study/array using TOPMed data as the reference and each set was analyzed using PLINK 1.9 adjusting for significant principal components. Variants were included in each study if minor allele count was greater than five in each set of cases. Individual study results were combined using fixed effects meta-analysis in Metasoft with variants demonstrating evidence for heterogeneity excluded.For previously reported EwS loci (i.e., 1p36.22, 6p25.1, 10q21.3, 15q15.1, 20p11.22 and 20p11.23), odds ratios (OR) remained high for GWAS (OR=1.5-2.4) and the -log10 p-values grew by several orders of magnitude, providing strong evidence for replication in the additional 907 EwS cases and 7,111 cancer-free controls. Our analysis identified five novel loci reaching genome-wide significance (P-valuemeta< 5×10−8) at 5q32, 7q32.3, 8q24.2, 13q32.3, and 17q23.2. Risk allele frequency of newly identified loci ranged from 0.001 to 0.75with estimated ORs ranging from 1.31-4.30. eQTL analyses nominated nearby biologically plausible candidate genes for future functional investigation including LARS1, a gene implicated in osteosarcoma and skeletal muscle dysgenesis, at the 5q32 locus and COX5BP6, a gene implicated in age at menarche and testosterone levels, at 13q32.3.Our results add to evidence supporting a strong inherited genetic component to EwS risk and particularly a genetic architecture harboring a substantial number of common variants with moderate effect. Ongoing work is focusing on enrichment analyses of GGAA microsatellite repeat sequences in identified risk loci to search for evidence for germline-somatic interactions with EWSR1::FLI1. Citation Format: Aubrey K. Hubbard, Weiyin Zhou, Carrie Boyce, Javed Khan, Melissa M. Hudson, Kirsten K. Ness, Zhaoming Wang, Katherine A. Janeway, Philip J. Lupo, Lindsay M. Morton, Stephen J. Chanock, Thomas G. Grünewald, Olivier Delattre, Mitchell J. Machiela, Ewing Sarcoma Germline Genetics Group. Multiple new Ewing sarcoma susceptibility loci expand knowledge of germline genetic etiology and nominate mechanisms of risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6148.

Targeted long-read sequencing of the Ewing sarcoma 6p25.1 susceptibility locus identifies germline-somatic interactions with EWSR1-FLI1 binding

Ewing sarcoma (EwS) is a rare bone and soft tissue malignancy driven by chromosomal translocations encoding chimeric transcription factors, such as EWSR1-FLI1, that bind GGAA motifs forming novel enhancers that alter nearby expression. We propose that germline microsatellite variation at the 6p25.1 EwS susceptibility locus could impact downstream gene expression and EwS biology. We performed targeted long-read sequencing of EwS blood DNA to characterize variation and genomic features important for EWSR1-FLI1 binding. We identified 50 microsatellite alleles at 6p25.1 and observed that EwS-affected individuals had longer alleles (>135bp) with more GGAA repeats. The 6p25.1 GGAA microsatellite showed chromatin features of an EWSR1-FLI1 enhancer and regulated expression of RREB1, a transcription factor associated with RAS/MAPK signaling. RREB1 knockdown reduced proliferation and clonogenic potential and reduced expression of cell cycle and DNA replication genes. Our integrative analysis at 6p25.1 details increased binding of longer GGAA microsatellite alleles with acquired EWSR-FLI1 to promote Ewing sarcomagenesis by RREB1-mediated proliferation.

Abstract IA019: Ectopic levels of low-complexity domain interactions repress endogenous oncogenic transcription

Abstract Mammalian transcription factors (TFs) ubiquitously contain intrinsically disordered low-complexity domains (LCDs) that perform transactivation functions. Recent studies have shown that dynamic, multivalent, and selective interactions between TF LCDs play an essential role in transactivation, but how such interactions mediate gene transcription remains unclear. It has been proposed that liquid-liquid phase separation (LLPS) of TFs driven by extensive LCD-LCD interactions is the mechanism underlying transactivation. Here, we investigate how tuning the level and localization of LCD-LCD interactions in vivo affects transcription of endogenous human genes. By quantitative single-cell imaging, we reveal that efficient gene activation by the oncogenic TF EWS/FLI1 requires narrow optimum of LCD-LCD interactions at its target genes associated with GGAA microsatellites. Increasing LCD-LCD interactions toward putative LLPS represses transcription of these genes in Ewing sarcoma cells. Likewise, ectopically creating LCD-LCD interactions to sequester EWS/FLI1 into a well-documented LLPS compartment, the nucleolus, represses EWS/FLI1 -driven transcription and inhibits malignant transformation of Ewing sarcoma. In addition, using live-cell single-molecule imaging, we show that a TF diffuses significantly more slowly in the nucleolus than in the nucleoplasm, demonstrating an important impact of LLPS on TF dynamic behaviors and single-molecule diffusion measurement as an effective approach to diagnose LLPS in vivo. Our findings show how altering the balance of LCD-LCD interactions can influence transcriptional regulation and suggest a potential therapeutic strategy for targeting disease-causing TFs. Citation Format: Shasha Chong. Ectopic levels of low-complexity domain interactions repress endogenous oncogenic transcription. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr IA019.

Systematic multi-omics cell line profiling uncovers principles of Ewing sarcoma fusion oncogene-mediated gene regulation.

Ewing sarcoma (EwS) is characterized by EWSR1-ETS fusion transcription factors converting polymorphic GGAA microsatellites (mSats) into potent neo-enhancers. Although the paucity of additional mutations makes EwS a genuine model to study principles of cooperation between dominant fusion oncogenes and neo-enhancers, this is impeded by the limited number of well-characterized models. Here we present the Ewing Sarcoma Cell Line Atlas (ESCLA), comprising whole-genome, DNA methylation, transcriptome, proteome, and chromatin immunoprecipitation sequencing (ChIP-seq) data of 18 cell lines with inducible EWSR1-ETS knockdown. The ESCLA shows hundreds of EWSR1-ETS-targets, the nature of EWSR1-ETS-preferred GGAA mSats, and putative indirect modes of EWSR1-ETS-mediated gene regulation, converging in the duality of a specific but plastic EwS signature. We identify heterogeneously regulated EWSR1-ETS-targets as potential prognostic EwS biomarkers. Our freely available ESCLA (http://r2platform.com/escla/) is a rich resource for EwS research and highlights the power of comprehensive datasets to unravel principles of heterogeneous gene regulation by chimeric transcription factors.

Abstract IA009: Oncogenic transcription factors drive highly tumor-specific LincRNA

Abstract Many cancers are characterized by gene fusions encoding oncogenic chimeric transcription factors (TFs) such as EWS::FLI1 in Ewing sarcoma (EwS). EWS::FLI1 induces a complete reprogramming of mesenchymal stem cells, the putative cell of origin of Ewing sarcoma, through binding and subsequent generation of thousands of neoenhancers at GGAA microsatellite repeats. We show that EWS::FLI1 induces the robust expression of a specific set of novel spliced and polyadenylated transcripts within otherwise transcriptionally silent regions of the genome. These neo-genes (NGs) are virtually undetectable in large collections of normal tissues or non-EwS tumors and can be silenced by CRISPR interference at regulatory EWS::FLI1-bound microsatellites. Ribosome profiling and proteomics further show that some NGs are translated into highly EwS-specific peptides. More generally, hundreds of NGs can be detected in diverse cancers characterized by chimeric TFs. Altogether, we identify the transcription, processing, and translation of novel, specific, highly expressed multi-exonic transcripts from otherwise silent regions of the genome as a new activity of aberrant TFs in cancer. These neotranscripts, which can be classified as LincRNAs, or neopeptides may have specific functions in the tumor cells. The latter may also constitute ideal neoepitopes as they are tumor-specific, clonally expressed in sarcoma cells, induced by the driver genetic alterations and public for a given sarcoma. Citation Format: Olivier Delattre, Julien Vibert, Olivier Saulnier, Joshua Waterfall. Oncogenic transcription factors drive highly tumor-specific LincRNA [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr IA009.

Human EWS-FLI protein recapitulates in Drosophila the neomorphic functions that induce Ewing sarcoma tumorigenesis.

Ewing sarcoma (EwS) is a human malignant tumor typically driven by the Ewing sarcoma-Friend leukemia integration (EWS-FLI) fusion protein. A paucity of genetically modified animal models, partially owed to the high toxicity of EWS-FLI, hinders research on EwS. Here, we report a spontaneous mutant variant, EWS-FLI1FS, that circumvents the toxicity issue in Drosophila. Through proteomic and genomic analyses, we show that human EWS-FLI1FS interacts with the Drosophila homologues of EWS-FLI human protein partners, including core subunits of chromatin remodeling complexes, the transcription machinery, and the spliceosome; brings about a massive dysregulation of transcription that affects a significant fraction of known targets of EWS-FLI in human cells; and modulates splicing. We also show that EWS-FLI1FS performs in Drosophila the two major neomorphic activities that it is known to have in human cells: activation of transcription from GGAA microsatellites and out competition of ETS transcription factors. We conclude that EWS-FLI1FS reproduces in Drosophila the known oncogenic activities of EWS-FLI that drive EwS tumorigenesis in humans. These results open up an unprecedented opportunity to investigate EWS-FLI's oncogenic pathways in vivo in a genetically tractable organism.

EWS::FLI1 and HOXD13 Control Tumor Cell Plasticity in Ewing Sarcoma.

Propagation of Ewing sarcoma requires precise regulation of EWS::FLI1 transcriptional activity. Determining the mechanisms of fusion regulation will advance our understanding of tumor progression. Here we investigated whether HOXD13, a developmental transcription factor that promotes Ewing sarcoma metastatic phenotypes, influences EWS::FLI1 transcriptional activity. Existing tumor and cell line datasets were used to define EWS::FLI1 binding sites and transcriptional targets. Chromatin immunoprecipitation and CRISPR interference were employed to identify enhancers. CUT&RUN and RNA sequencing defined binding sites and transcriptional targets of HOXD13. Transcriptional states were investigated using bulk and single-cell transcriptomic data from cell lines, patient-derived xenografts, and patient tumors. Mesenchymal phenotypes were assessed by gene set enrichment, flow cytometry, and migration assays. We found that EWS::FLI1 creates a de novo GGAA microsatellite enhancer in a developmentally conserved regulatory region of the HOXD locus. Knockdown of HOXD13 led to widespread changes in expression of developmental gene programs and EWS::FLI1 targets. HOXD13 binding was enriched at established EWS::FLI1 binding sites where it influenced expression of EWS::FLI1-activated genes. More strikingly, HOXD13 bound and activated EWS::FLI1-repressed genes, leading to adoption of mesenchymal and migratory cell states that are normally suppressed by the fusion. Single-cell analysis confirmed that direct transcriptional antagonism between HOXD13-mediated gene activation and EWS::FLI1-dependent gene repression defines the state of Ewing sarcoma cells along a mesenchymal axis. Ewing sarcoma tumors are comprised of tumor cells that exist along a mesenchymal transcriptional continuum. The identity of cells along this continuum is, in large part, determined by the competing activities of EWS::FLI1 and HOXD13. See related commentary by Weiss and Bailey, p. 4360.

Tuning levels of low-complexity domain interactions to modulate endogenous oncogenic transcription

Gene activation by mammalian transcription factors (TFs) requires multivalent interactions of their low-complexity domains (LCDs), but how such interactions regulate transcription remains unclear. It has been proposed that extensive LCD-LCD interactions culminating in liquid-liquid phase separation (LLPS) of TFs is the dominant mechanism underlying transactivation. Here, we investigated how tuning the amount and localization of LCD-LCD interactions invivo affects transcription of endogenous human genes. Quantitative single-cell and single-molecule imaging reveals that the oncogenic TF EWS::FLI1 requires a narrow optimum of LCD-LCD interactions to activate its target genes associated with GGAA microsatellites. Increasing LCD-LCD interactions toward putative LLPS represses transcription of these genes in patient-derived cells. Likewise, ectopically creating LCD-LCD interactions to sequester EWS::FLI1 into a well-documented LLPS compartment, the nucleolus, inhibits EWS::FLI1-driven transcription and oncogenic transformation. Our findings show how altering the balance of LCD-LCD interactions can influence transcriptional regulation and suggest a potential therapeutic strategy for targeting disease-causing TFs.

Genome-wide functional perturbation of human microsatellite repeats using engineered zinc finger transcription factors

SUMMARYRepeat elements can be dysregulated at a genome-wide scale in human diseases. For example, in Ewing sarcoma, hundreds of inert GGAA repeats can be converted into active enhancers when bound by EWS-FLI1. Here we show that fusions between EWS and GGAA-repeat-targeted engineered zinc finger arrays (ZFAs) can function at least as efficiently as EWS-FLI1 for converting hundreds of GGAA repeats into active enhancers in a Ewing sarcoma precursor cell model. Furthermore, a fusion of a KRAB domain to a ZFA can silence GGAA microsatellite enhancers genome wide in Ewing sarcoma cells, thereby reducing expression of EWS-FLI1-activated genes. Remarkably, this KRAB-ZFA fusion showed selective toxicity against Ewing sarcoma cells compared with non-Ewing cancer cells, consistent with its Ewing sarcoma-specific impact on the transcriptome. These findings demonstrate the value of ZFAs for functional annotation of repeats and illustrate how aberrant microsatellite activities might be regulated for potential therapeutic applications.

GGAA-Microsatellites of NR0B1 Promoter Region in Ewing’s Sarcoma Patients and Healthy Individuals of a Southern Brazilian Population

Introduction: The very aggressive soft tissue and bone pediatric tumor Ewing’s sarcoma (ES) is caused in most cases by the chromosomal translocation t(11;22)(q24;q12), which encodes an aberrant chimeric transcription factor (EWS-FLI1) that regulates target genes, including the critical oncogene NR0B1 (Xp21.2), via GGAA microsatellites. Objective: Our objective was to analyze the GGAA microsatellites of NR0B1 promoter region of ES patients and healthy subjects in our population. Methodology: Ten male ES patients and 71 adult healthy males from Rio Grande do Sul state, Brazil, were included in this study. Leukocyte DNA was extracted, amplified by PCR, sequenced by the Sanger method and analyzed by capillary electrophoresis. Total number of GGAA motifs, length of microsatellite in base pairs, number of segments separated by "A" insertions and greatest number of consecutive GGAA motifs were analyzed. Statistical analyses were performed in the SPSS® statistical software and p-value <0,05 was considered significant. Results: A total of 21 different alleles was identified in the 81 subjects, with 24.2 allele [(GGAA)7A(GGAA)7A(GGAA)10 sequence] being the most frequent, but when comparing the data between the two groups, no significant difference was found. Conclusion: We concluded that the studied sample was highly variable in terms microsatellite structure, including the presence of rare alleles, giving us the opportunity to describe our population which is a fundamental step on identifying genetic implications in ES tumorigenesis.

STAG2 mutations alter CTCF-anchored loop extrusion, reduce cis-regulatory interactions and EWSR1-FLI1 activity in Ewing sarcoma.

STAG2, a cohesin family gene, is among the most recurrently mutated genes in cancer. STAG2 loss of function (LOF) is associated with aggressive behavior in Ewing sarcoma, a childhood cancer driven by aberrant transcription induced by the EWSR1-FLI1 fusion oncogene. Here, using isogenic Ewing cells, we show that, while STAG2 LOF profoundly changes the transcriptome, it does not significantly impact EWSR1-FLI1, CTCF/cohesin, or acetylated H3K27 DNA binding patterns. In contrast, it strongly alters the anchored dynamic loop extrusion process at boundary CTCF sites and dramatically decreases promoter-enhancer interactions, particularly affecting the expression of genes regulated by EWSR1-FLI1 at GGAA microsatellite neo-enhancers. Down-modulation of cis-mediated EWSR1-FLI1 activity, observed in STAG2-LOF conditions, is associated with enhanced migration and invasion properties of Ewing cells previously observed in EWSR1-FLI1low cells. Our study illuminates a process whereby STAG2-LOF fine-tunes the activity of an oncogenic transcription factor through altered CTCF-anchored loop extrusion and cis-mediated enhancer mechanisms.

Abstract 2882: Mithramycin impedes EWS-FLI1 driven transcription by preventing target promoter clearance

Abstract Background: Ewing sarcoma (ES) is a primary bone tumor of adolescence with poor prognosis. ES is characterized by the presence of EWS-FLI1 which acts as an oncogenic transcription factor to promote and maintain a tumorigenic phenotype. All ES cells express EWS-FLI1, however, other actionable mutations are exceedingly rare. We previously identified mithramycin (MMA) as a potent and specific EWS-FLI1 inhibitor. MMA effectively decreases ES tumor cell viability and tumor volume in vivo by reversing the EWS-FLI1 transcriptional signature. Structural and biochemical data suggest that sub-micromolar MMA increases the stability of the DNA binding domain of EWS-FLI1 at GGAA repeats. These data suggest that MMA may prevent EWS-FLI1 from successfully promoting Pol II transcriptional elongation. Methods: We used ChIP-qPCR to detect EWS-FLI1 binding at the promoter of well characterized targets. We used a Pol II processivity assay to measure the efficiency of transcriptional elongation at EWS-FLI1 targets. We use RT-qPCR, western blotting, and RNAseq to confirm that MMA sensitizes ES cells to elongation inhibitors. Finally, we use GROseq to identify the changes to Pol II dynamics at EWS-FLI1 targets following MMA treatment. Results: Our ChIP-qPCR data demonstrate an increased fraction of EWS-FLI1 DNA binding at the promoters of target genes with GGAA microsatellite regions. MMA significantly inhibits the processivity of Pol II at EWS-FLI1 targets when added both into a run-on reaction or following ES cell pretreatment. Combining MMA with elongation inhibitors induces strong synergy as measured by Bliss-Independence, renders multiple ES cell lines unviable by inhibiting the effects of EWS-FLI1 at the mRNA and protein level, and is effective in vivo. MMA biases GROseq reads toward the 5’ end of genes, a result consistent with a promoter trapping mechanism. We are currently correlating GROseq data to global EWS-FLI1 DNA binding using ChIPseq. Conclusions: We characterized a mechanism of MMA-induced EWS-FLI1 inhibition that involves increased stabilization of the fusion protein at target promoter regions. This mechanism is supported by changes to EWS-FLI1 DNA-binding, EWS-FLI1 target transcriptional processivity, and a reversal of the EWS-FLI1 transcriptional signature by combining MMA with downstream elongation inhibition. We hope that this work will inform future endeavors to develop a targeted therapy for ES that inhibits the main driver of disease, EWS-FLI1. Citation Format: Guillermo Flores, Susan Kitchen-Goosen, Brandon Oswald, Elissa Boguslawski, Marie Adams, Ian Beddows, Zachary Madaj, Patrick Grohar. Mithramycin impedes EWS-FLI1 driven transcription by preventing target promoter clearance [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 2882.

Abstract IA02: Identity shifts in pediatric cancers

Abstract In contrast to adult cancers, most pediatric malignancies display limited genetic heterogeneity. In Ewing sarcoma, the major driver of tumor development is the expression of the chimeric EWSR1-FLI1 transcription factor, which results from a chromosome translocation. Additional recurrent genetic alterations include the CDKN2A, STAG2, and TP53 genes. Through its binding to specific elements including GGAA microsatellite sequences, EWSR1-FLI1 has a major impact on epigenetic landscape of the cancer cells and consequently, obscures the epigenetic makeup of the Ewing cell-of-origin. The Ewing cell is somehow a “neomorphic” cell, the biologic properties of which are tightly dependent upon the expression level of EWSR1-FLI1. When a cell harbors a low level of expression of EWSR1-FLI1, it loses this neomorphic identity and retrieve its cell-of-origin, mesenchymal phenotype. This leads us to propose a new model of phenotypic plasticity whereby the dynamic fluctuation of the expression level of a dominant oncogene is an intrinsic characteristic of its oncogenic potential. The causes of EWSR1-FLI1 expression fluctuations are presently unknown. They may be partly stochastic as they are observed in cell lines in vitro but also dependent upon signals from the surrounding microenvironment. The situation is very different in neuroblastoma where the blueprint of the cell-of-origin, most frequently a noradrenergic progenitor, can be clearly detected whatever the genetic abnormalities of the cancer cells. Our recent data nevertheless suggest that neuroblastoma cells may also exhibit some plasticity and transition between different epigenetic states that witness different cell identities. In conclusion, pediatric cancers constitute simplified cancer systems to investigate the relative and interdependent contributions of genetic and nongenetic factors and to document identity shifts. Citation Format: Olivier Delattre, G.A. Franzetti, K. Laud-Duval, A. Brisac, Valentina Boeva, Caroline Louis-Brennetot, Agathe Peltier, Simon Durand, Cécile Pierre-Eugène, Didier Surdez, Eve Lapouble, Gaelle Pierron, Sandrine Grossetête-Lalami, Isabelle Janoueix-Lerosey. Identity shifts in pediatric cancers [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 IA02.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Epigenome editing of microsatellite repeats defines tumor-specific enhancer functions and dependencies.

Various types of repetitive sequences are dysregulated in cancer. In Ewing sarcoma, the oncogenic fusion protein EWS-FLI1 induces chromatin features typical of active enhancers at GGAA microsatellite repeats, but the function of these sites has not been directly demonstrated. Here, by combining nascent transcription profiling with epigenome editing, we found that a subset of GGAA microsatellite repeats is transcriptionally active in Ewing sarcoma and that silencing individual repeats abolishes local nascent transcription and leads to markedly reduced expression of putative target genes. Epigenome silencing of these repeat sites does not affect gene expression in unrelated cells, can prevent the induction of gene expression by EWS-FLI1, and, in the case of a GGAA repeat that controls SOX2 expression from a distance of 470 kb, is sufficient to impair the growth of Ewing sarcoma xenografts. Using an experimental approach that is broadly applicable to testing different types of repetitive genomic elements, our study directly demonstrates that specific repeat microsatellites can have critical gene regulation functions in cancer and thus represent tumor-specific vulnerabilities that may be exploited to develop new therapies.

EWS-FLI1 regulates a transcriptional program in cooperation with Foxq1 in mouse Ewing sarcoma.

EWS‐FLI1 constitutes an oncogenic transcription factor that plays key roles in Ewing sarcoma development and maintenance. We have recently succeeded in generating an ex vivo mouse model for Ewing sarcoma by introducing EWS‐FLI1 into embryonic osteochondrogenic progenitors. The model well recapitulates the biological characteristics, small round cell morphology, and gene expression profiles of human Ewing sarcoma. Here, we clarified the global DNA binding properties of EWS‐FLI1 in mouse Ewing sarcoma. GGAA microsatellites were found to serve as binding sites of EWS‐FLI1 albeit with less frequency than that in human Ewing sarcoma; moreover, genomic distribution was not conserved between human and mouse. Nevertheless, EWS‐FLI1 binding sites within GGAA microsatellites were frequently associated with the histone H3K27Ac enhancer mark, suggesting that EWS‐FLI1 could affect global gene expression by binding its target sites. In particular, the Fox transcription factor binding motif was frequently observed within EWS‐FLI1 peaks and Foxq1 was identified as the cooperative partner that interacts with the EWS portion of EWS‐FLI1. Trib1 and Nrg1 were demonstrated as target genes that are co‐regulated by EWS‐FLI1 and Foxq1, and are important for cell proliferation and survival of Ewing sarcoma. Collectively, our findings present novel aspects of EWS‐FLI1 function as well as the importance of GGAA microsatellites.

Ewing sarcoma.

Ewing sarcoma is the second most frequent bone tumour of childhood and adolescence that can also arise in soft tissue. Ewing sarcoma is a highly aggressive cancer, with a survival of 70-80% for patients with standard-risk and localized disease and ~30% for those with metastatic disease. Treatment comprises local surgery, radiotherapy and polychemotherapy, which are associated with acute and chronic adverse effects that may compromise quality of life in survivors. Histologically, Ewing sarcomas are composed of small round cells expressing high levels of CD99. Genetically, they are characterized by balanced chromosomal translocations in which a member of the FET gene family is fused with an ETS transcription factor, with the most common fusion being EWSR1-FLI1 (85% of cases). Ewing sarcoma breakpoint region 1 protein (EWSR1)-Friend leukaemia integration 1 transcription factor (FLI1) is a tumour-specific chimeric transcription factor (EWSR1-FLI1) with neomorphic effects that massively rewires the transcriptome. Additionally, EWSR1-FLI1 reprogrammes the epigenome by inducing de novo enhancers at GGAA microsatellites and by altering the state of gene regulatory elements, creating a unique epigenetic signature. Additional mutations at diagnosis are rare and mainly involve STAG2, TP53 and CDKN2A deletions. Emerging studies on the molecular mechanisms of Ewing sarcoma hold promise for improvements in early detection, disease monitoring, lower treatment-related toxicity, overall survival and quality of life.

Protein phosphatase 1 regulatory subunit 1A in ewing sarcoma tumorigenesis and metastasis.

Protein phosphatase inhibitors are often considered as tumor promoters. Protein phosphatase 1 regulatory subunit 1A (PPP1R1A) is a potent protein phosphatase 1 (PP1) inhibitor; however, its role in tumor development is largely undefined. Here we characterize, for the first time, the functions of PPP1R1A in Ewing sarcoma (ES) pathogenesis. We found that PPP1R1A is one of the top ranked target genes of EWS/FLI, the master regulator of ES, and is upregulated by EWS/FLI via a GGAA microsatellite enhancer element. Depletion of PPP1R1A resulted in a significant decrease in oncogenic transformation and cell migration in vitro as well as xenograft tumor growth and metastasis in an orthotopic mouse model. RNA-sequencing and functional annotation analyses revealed that PPP1R1A regulates genes associated with various cellular functions including cell junction, adhesion and neurogenesis. Interestingly, we found a significant overlap of PPP1R1A-regulated gene set with that of ZEB2 and EWS, which regulates metastasis and neuronal differentiation in ES, respectively. Further studies for characterization of the molecular mechanisms revealed that activation of PPP1R1A by PKA phosphorylation at Thr35, and subsequent PP1 binding and inhibition, was required for PPP1R1A-mediated tumorigenesis and metastasis, likely by increasing the phosphorylation levels of various PP1 substrates. Furthermore, we found that a PKA inhibitor impaired ES cell proliferation, tumor growth and metastasis, which was rescued by the constitutively active PPP1R1A. Together, these results offered new insights into the role and mechanism of PPP1R1A in tumor development and identified an important kinase and phosphatase pathway, PKA/PPP1R1A/PP1, in ES pathogenesis. Our findings strongly suggest a potential therapeutic value of inhibition of the PKA/PPP1R1A/PP1 pathway in the treatment of primary and metastatic ES.

EWS–FLI1 Retargets BAF Chromatin Remodeling Complexes in Ewing Sarcoma

Abstract BAF recruitment to GGAA microsatellites activates the Ewing sarcoma transcriptional program.