Papel de SPRY1 en la patogénesis del sarcoma de Ewing: implicaciones pronósticas y terapeúticas

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 24-10-2016El sarcoma de Ewing se caracteriza por la presencia de translocaciones cromosómicas que originan genes de fusión entre el gen EWSR1 y diferentes miembros de la familia de factores de transcripción ETS, principalmente FLI1. El gen de fusión resultante, EWS-FLI1, es un factor de transcripción aberrante que desempeña un papel central en el origen del sarcoma de Ewing a través de la regulación transcripcional de otros genes diana. En esta Tesis mostramos que Sprouty 1 (SPRY1), un inhibidor downstream de los receptores del factor de crecimiento fibroblástico (FGFRs) y otros receptores activadores de Ras, es un gen regulado negativamente por EWS-FLI1 en células de sarcoma de Ewing A673. El análisis de la expresión de SPRY1 en una selección de células de sarcoma de Ewing mostró que SPRY1 no se expresa en estas líneas celulares, lo que sugiere que SPRY1 podría actuar como un supresor tumoral en células de sarcoma de Ewing. En concordancia con estos resultados, la re-expresión de SPRY1 inhibió la proliferación, el crecimiento clonogénico y la migración en tres líneas celulares diferentes de sarcoma de Ewing. Además, la re-expresión de SPRY1 inhibió la ruta de señalización de Ras/MAPK/ERK inducida por suero o FGFb. Por otro lado, al tratar las células de sarcoma de Ewing con el inhibidor de FGFR PD173074, se redujo la proliferación inducida por FGFb, la formación de colonias y el crecimiento de tumores in vivo de forma dependiente de dosis. Aunque la expresión de SPRY1 en células de sarcoma de Ewing es baja, SPRY1 se expresa de forma variable en las muestras de tumores primarios. El análisis de una cohorte grande de pacientes indica que los pacientes con tumores que presentaron una expresión mayor de SPRY1 tuvieron un mejor pronóstico. En resumen, los resultados mostrados en esta Tesis indican que la regulación negativa de SPRY1 mediada por EWS-FLI1 produce una desregulación de la proliferación celular inducida por FGFb, lo que sugiere que la ruta de señalización de FGFR/Ras/MAPK/ERK podría ser una diana interesante para el desarrollo de nuevas aproximaciones terapéuticas para esta devastadora enfermedad.Ewing sarcoma is characterized by chromosomal translocations fusing the EWSR1 gene with various members of the ETS family of transcription factors, most commonly FLI1. EWS-FLI1 is an aberrant transcription factor driving Ewing sarcoma tumorigenesis by either transcriptionally inducing or repressing specific target genes. Herein, we showed that Sprouty 1 (SPRY1), which is a physiological negative feedback inhibitor downstream of fibroblast growth factor (FGF) receptors (FGFRs) and other Ras-activating receptors, is an EWS-FLI1 repressed gene. EWS-FLI1 knock-down specifically increased the expression of SPRY1 while other Sprouty family members remained unaffected. Analysis of SPRY1 expression in a panel of Ewing sarcoma cells showed that SPRY1 was not expressed in Ewing sarcoma cell lines, suggesting that it could act as a tumor suppressor gene in these cells. In agreement, induction of SPRY1 in three different Ewing sarcoma cell lines functionally impaired proliferation, clonogenic growth and migration. In addition, SPRY1 expression inhibited ERK/MAPK signalling induced by serum and basic FGF (bFGF). Moreover, treatment of Ewing sarcoma cells with the potent FGFR inhibitor PD173074 reduced bFGF-induced proliferation, colony formation and in vivo tumor growth in a dose-dependent manner, thus mimicking SPRY1 activity in Ewing sarcoma cells. Although the expression of SPRY1 was low when compared to other tumors, SPRY1 was variably expressed in primary Ewing sarcoma tumors and higher expression levels were significantly associated with improved outcome in a large patient cohort. Taken together, our data indicate that EWS-FLI1-mediated repression of SPRY1 leads to unrestrained bFGF-induced cell proliferation, suggesting that targeting the FGFR/Ras/MAPK/ERK pathway can constitute a promising therapeutic approach for this devastating disease.A la Red Temática de Investigación Cooperativa en Cáncer (RTICC) por la financiación concedida (RD12/0036/0027). Me gustaría agradecer en especial a la Asociación Pablo Ugarte por su fantástica labor, por haber depositado su confianza en nosotros y por haber financiado de forma sostenida este proyecto (TPY-M 1149/13

Lysyl oxidase is downregulated by the EWS/FLI1 oncoprotein and its propeptide domain displays tumor supressor activities in Ewing sarcoma cells

This study was funded by grants of the Ministerio de Ciencia e Innovación (SAF2009-10158), the Fundación Científica de la Científica Española Contra el Cáncer and the Fundación María Francisca de Roviralta. N. Agra was supported by the Fundación General de la U.A.M. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

The second European interdisciplinary Ewing sarcoma research summit – A joint effort to deconstructing the multiple layers of a complex disease

Despite multimodal treatment, long term outcome for patients with Ewing sarcoma is still poor. The second “European interdisciplinary Ewing sarcoma research summit” assembled a large group of scientific experts in the field to discuss their latest unpublished findings on the way to the identification of novel therapeutic targets and strategies. Ewing sarcoma is characterized by a quiet genome with presence of an EWSR1-ETS gene rearrangement as the only and defining genetic aberration. RNA sequencing of recently described Ewing-like sarcomas with variant translocations identified them as biologically distinct diseases. Various presentations adressed mechanisms of EWS-ETS fusion protein activities with a focus on EWS-FLI1. Data were presented shedding light on the molecular underpinnings of genetic permissiveness to this disease uncovering interaction of EWS-FLI1 with recently discovered susceptibility loci. Epigenetic context as a consequence of the interaction between the oncoprotein, cell type, developmental stage, and tissue microenvironment emerged as dominant theme in the discussion of the molecular pathogenesis and inter- and intratumor heterogeneity of Ewing sarcoma, and the difficulty to generate animal models faithfully recapitulating the human disease. The problem of preclinical development of biologically targeted therapeutics was discussed and promising perspectives were offered from the study of novel in vitro models. Finally, it was concluded that in order to facilitate rapid pre-clinical and clinical development of novel therapies in Ewing sarcoma, the community needs a platform to maintain knowledge of unpublished results, systems and models used in drug testing and to continue the open dialogue initiated at the first two Ewing sarcoma summits.Unión Europea 261743(ENCCA)Unión Europea 259348 (ASSET

Targeting the undruggable: exploiting neomorphic features of fusion oncoproteins in childhood sarcomas for innovative therapies

While sarcomas account for approximately 1% of malignant tumors of adults, they are particularly more common in children and adolescents affected by cancer. In contrast to malignancies that occur in later stages of life, childhood tumors, including sarcoma, are characterized by a striking paucity of somatic mutations. However, entity-defining fusion oncogenes acting as the main oncogenic driver mutations are frequently found in pediatric bone and soft-tissue sarcomas such as Ewing sarcoma (EWSR1-FLI1), alveolar rhabdomyosarcoma (PAX3/7-FOXO1), and synovial sarcoma (SS18-SSX1/2/4). Since strong oncogene-dependency has been demonstrated in these entities, direct pharmacological targeting of these fusion oncogenes has been excessively attempted, thus far, with limited success. Despite apparent challenges, our increasing understanding of the neomorphic features of these fusion oncogenes in conjunction with rapid technological advances will likely enable the development of new strategies to therapeutically exploit these neomorphic features and to ultimately turn the \textquotedblundruggable\textquotedbl into first-line target structures. In this review, we provide a broad overview of the current literature on targeting neomorphic features of fusion oncogenes found in Ewing sarcoma, alveolar rhabdomyosarcoma, and synovial sarcoma, and give a perspective for future developments. Graphical abstract Scheme depicting the different targeting strategies of fusion oncogenes in pediatric fusion-driven sarcomas. Fusion oncogenes can be targeted on their DNA level (1), RNA level (2), protein level (3), and by targeting downstream functions and interaction partners (4)

Correction to: ABCA6 affects the malignancy of Ewing sarcoma cells via cholesterol-guided inhibition of the IGF1R/AKT/MDM2 axis

In this article an incorrect affiliation had inadvertently been added. The original article has been updated

Integrative clinical transcriptome analysis reveals TMPRSS2‐ERG dependency of prognostic biomarkers in prostate adenocarcinoma

In prostate adenocarcinoma (PCa), distinction between indolent and aggressive disease is challenging. Around 50% of PCa are characterized by TMPRSS2‐ERG (T2E)‐fusion oncoproteins defining two molecular subtypes (T2E‐positive/negative). However, current prognostic tests do not differ between both molecular subtypes, which might affect outcome prediction. To investigate gene‐signatures associated with metastasis in T2E‐positive and T2E‐negative PCa independently, we integrated tumor transcriptomes and clinicopathological data of two cohorts (total n = 783), and analyzed metastasis‐associated gene‐signatures regarding the T2E‐status. Here, we show that the prognostic value of biomarkers in PCa critically depends on the T2E‐status. Using gene‐set enrichment analyses, we uncovered that metastatic T2E‐positive and T2E‐negative PCa are characterized by distinct gene‐signatures. In addition, by testing genes shared by several functional gene‐signatures for their association with event‐free survival in a validation cohort (n = 272), we identified five genes (ASPN, BGN, COL1A1, RRM2 and TYMS)—three of which are included in commercially available prognostic tests—whose high expression was significantly associated with worse outcome exclusively in T2E‐negative PCa. Among these genes, RRM2 and TYMS were validated by immunohistochemistry in another validation cohort (n = 135), and several of them proved to add prognostic information to current clinicopathological predictors, such as Gleason score, exclusively for T2E‐negative patients. No prognostic biomarkers were identified exclusively for T2E‐positive tumors. Collectively, our study discovers that the T2E‐status, which is per se not a strong prognostic biomarker, crucially determines the prognostic value of other biomarkers. Our data suggest that the molecular subtype needs to be considered when applying prognostic biomarkers for outcome prediction in PCa

Therapeutic targeting of the PLK1-PRC1-axis triggers cell death in genomically silent childhood cancer.

Chromosomal instability (CIN) is a hallmark of cancer1. Yet, many childhood cancers, such as Ewing sarcoma (EwS), feature remarkably 'silent' genomes with minimal CIN2. Here, we show in the EwS model how uncoupling of mitosis and cytokinesis via targeting protein regulator of cytokinesis 1 (PRC1) or its activating polo-like kinase 1 (PLK1) can be employed to induce fatal genomic instability and tumor regression. We find that the EwS-specific oncogenic transcription factor EWSR1-FLI1 hijacks PRC1, which physiologically safeguards controlled cell division, through binding to a proximal enhancer-like GGAA-microsatellite, thereby promoting tumor growth and poor clinical outcome. Via integration of transcriptome-profiling and functional in vitro and in vivo experiments including CRISPR-mediated enhancer editing, we discover that high PRC1 expression creates a therapeutic vulnerability toward PLK1 inhibition that can repress even chemo-resistant EwS cells by triggering mitotic catastrophe.Collectively, our results exemplify how aberrant PRC1 activation by a dominant oncogene can confer malignancy but provide opportunities for targeted therapy, and identify PRC1 expression as an important determinant to predict the efficacy of PLK1 inhibitors being used in clinical trials.This work was mainly supported by a grant from the German Cancer Aid (DKH-70114111). In addition, the laboratory of T.G.P.G. was supported by the LMU Munich’s Institutional Strategy LMUexcellent within the framework of the German Excellence Initiative, the ‘Mehr LEBEN für krebskranke Kinder—Bettina-Bräu-Stiftung’, the Matthias-Lackas Foundation, the Dr. Leopold and Carmen Ellinger Foundation, the Boehringer-Ingelheim Foundation, the Wilhelm Sander-Foundation (2016.167.1), the Barbara and Hubertus Trettner Foundation, the Dr. Rolf M. Schwiete Foundation, the Friedrich-Baur Foundation, the German Cancer Aid (DKH-70112257 and DKH-111886), the Gert und Susanna Mayer Foundation, the Barbara und Wilfried Mohr Foundation, the SMARCB1 association, and the Deutsche Forschungsgemeinschaft (DFG-391665916). J.L. was supported by a scholarship of the Chinese Scholarship Council (CSC), and a grant of the German Cancer Aid (DKH-70114111). M.D. was by a scholarship of the ‘Deutsche Stiftung für junge Erwachsene mit Krebs‘, J.M. by a scholarship of the Kind-Philipp-Foundation, and C.M.F., M.K. and T.L.B.H. by scholarships from the German Cancer Aid. The laboratory of J.A. was supported by grants from the Instituto de Salud Carlos III (PI16CIII/00026; DTS18CIII/00005), Asociación Pablo Ugarte, ASION, Fundación Sonrisa de Alex, Asociación Todos somos Iván y Asociación Candela Riera. Freely available clipart used for design of parts of figures was kindly provided by Servier Medical Art (https://smart.servier.com/).S

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

Oncogenic hijacking of a developmental transcription factor evokes vulnerability toward oxidative stress in Ewing sarcoma

Ewing sarcoma (EwS) is an aggressive childhood cancer likely originating from mesenchymal stem cells or osteo-chondrogenic progenitors. It is characterized by fusion oncoproteins involving EWSR1 and variable members of the ETS-family of transcription factors (in 85% FLI1). EWSR1-FLI1 can induce target genes by using GGAA-microsatellites as enhancers.Here, we show that EWSR1-FLI1 hijacks the developmental transcription factor SOX6 - a physiological driver of proliferation of osteo-chondrogenic progenitors - by binding to an intronic GGAA-microsatellite, which promotes EwS growth in vitro and in vivo. Through integration of transcriptome-profiling, published drug-screening data, and functional in vitro and in vivo experiments including 3D and PDX models, we discover that constitutively high SOX6 expression promotes elevated levels of oxidative stress that create a therapeutic vulnerability toward the oxidative stress-inducing drug Elesclomol.Collectively, our results exemplify how aberrant activation of a developmental transcription factor by a dominant oncogene can promote malignancy, but provide opportunities for targeted therapy. Ewing sarcoma is characterized by the fusion of EWSR1 and FLI1. Here, the authors show that EWSR1-FLI1 increases the activity of the developmental transcription factor SOX6, which promotes tumor growth but also increases sensitivity to oxidative stress