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PurposeEwing sarcoma (EwS) is a highly aggressive bone- or soft tissue-associated malignancy mostly affecting children, adolescents, and young adults. Although multimodal therapies have strongly improved patients’ overall survival over the past decades, the development of prognostic biomarkers for risk-based patient stratification and more effective therapies with less adverse effects is stagnating. Thus, new personalized medicine approaches are urgently required.Experimental designGene expression data of EwS and normal tissues were crossed with survival data to identify highly overexpressed, prognostically relevant, and actionable potential targets. RNA-interference and dose-response assays as well as tissue-microarray analyses were carried out to explore the functional role and druggability of a prominent candidate gene in vitro and in vivo, and to validate its suitability as a prognostic biomarker.ResultsEmploying a multilayered screening approach, we discover ribonucleotide reductase regulatory subunit M2 (RRM2) as a promising therapeutic target and prognostic biomarker in EwS. Through analysis of two independent EwS patient cohorts, we show that RRM2 mRNA and protein overexpression is associated with an aggressive clinical phenotype and poor patients’ overall survival. In agreement, RRM2 silencing as well as pharmacological inhibition by the specific inhibitor triapine (3-AP) significantly reduces EwS growth in vitro and in vivo. Furthermore, we present evidence that pharmacological RRM2 inhibition by triapine can overcome chemoresistance against doxorubicin or gemcitabine, and synergize with cell cycle checkpoint inhibitors (CHEK1 or WEE1).ConclusionsBased on the aggressive phenotype mediated by and the druggability of RRM2 our results provide a translational rationale for exploiting RRM2 as a novel therapeutic target in EwS and prompt further clinical investigations.

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., In this study, the authors show that that oncogenic hijacking of PRC1 sensitizes genomically stable Ewing sarcoma cells for PLK1 inhibition alone or in synergy with a microtubule-destabilizing drug via induction of cytokinesis defects, rendering PRC1 a promising, broadly applicable predictive biomarker

BACKGROUND: Up to 30-40% of Ewing sarcoma (EwS) patients with non-metastatic disease develop local or metastatic relapse within a time span of 2-10 years. This is in part caused by the absence of prognostic biomarkers that can identify high-risk patients and thus assign them to risk-adapted monitoring and treatment regimens. Since cancer stemness has been associated with tumour relapse and poor patient outcomes, we investigated in the current study the prognostic potential SOX2 (sex determining region Y box 2) - a major transcription factor involved in development and stemness - which was previously described to contribute to the undifferentiated phenotype of EwS. METHODS: Two independent patient cohorts, one consisting of 189 retrospectively collected EwS tumours with corresponding mRNA expression data (test-cohort) and the other consisting of 141 prospectively collected formalin-fixed and paraffin-embedded resected tumours (validation and cohort), were employed to analyse SOX2 expression levels through DNA microarrays or immunohistochemistry, respectively, and to compare them with clinical parameters and patient outcomes. Two methods were employed to test the validity of the results at both the mRNA and protein levels. FINDINGS: Both cohorts showed that only a subset of EwS patients (16-20%) expressed high SOX2 mRNA or protein levels, which significantly correlated with poor overall survival. Multivariate analyses of our validation-cohort revealed that high SOX2 expression represents a major risk-factor for poor survival (HR = 3·19; 95%CI 1·74-5·84; p

Pediatric malignancies including Ewing sarcoma (EwS) feature a paucity of somatic alterations except for pathognomonic driver-mutations that cannot explain overt variations in clinical outcome. Here, we demonstrate in EwS how cooperation of dominant oncogenes and regulatory germline variants determine tumor growth, patient survival and drug response. Binding of the oncogenic EWSR1-FLI1 fusion transcription factor to a polymorphic enhancer-like DNA element controls expression of the transcription factor MYBL2 mediating these phenotypes. Whole-genome and RNA sequencing reveals that variability at this locus is inherited via the germline and is associated with variable inter-tumoral MYBL2 expression. High MYBL2 levels sensitize EwS cells for inhibition of its upstream activating kinase CDK2 in vitro and in vivo, suggesting MYBL2 as a putative biomarker for anti-CDK2-therapy. Collectively, we establish cooperation of somatic mutations and regulatory germline variants as a major determinant of tumor progression and highlight the importance of integrating the regulatory genome in precision medicine., Interactions between germline variants and somatic mutations is a relatively unexplored topic in cancer. Here, in Ewing sarcoma, the authors show that binding of the oncogenic EWSR1-FLI1 fusion transcription factor to a polymorphic enhancer-like DNA element controls MYBL2, whose high expression correlates with prognosis.

Introduction Treatment of patients with metastatic melanoma is hampered by drug-resistance and often requires combination with radiotherapy as last-resort option. However, also after radiotherapy, clinical relapses are common. Methods & results Our preclinical models indicated a higher rate of tumour relapse when melanoma cells were first treated with BRAFV600E inhibition (BRAFi) followed by radiotherapy as compared to the reverse sequence. Accordingly, retrospective follow-up data from 65 stage-IV melanoma patients with irradiated melanoma brain metastases confirmed a shortened duration of local response of mitogen-activated protein kinase (MAPK)-inhibitor–pretreated compared with MAPK-inhibitor–naive intracranial metastases. On the molecular level, we identified JARID1B/KDM5B as a cellular marker for cross-resistance between BRAFi and radiotherapy. JARID1Bhigh cells appeared more frequently under upfront BRAFi as compared with upfront radiation. JARID1B favours cell survival by transcriptional regulation of genes controlling cell cycle, DNA repair and cell death. Conclusion The level of cross-resistance between combined MAPK inhibition and radiotherapy is dependent on the treatment sequence. JARID1B may represent a novel therapy-overarching resistance marker.

Cell lines have been essential for major discoveries in cancer including Ewing sarcoma (EwS). EwS is a highly aggressive pediatric bone or soft-tissue cancer characterized by oncogenic EWSR1-ETS fusion transcription factors converting polymorphic GGAA-microsatellites (mSats) into neo-enhancers. However, further detailed mechanistic evaluation of gene regulation in EwS have been hindered by the limited number of well-characterized cell line models. Here, we present the Ewing Sarcoma Cell Line Atlas (ESCLA) comprising 18 EwS cell lines with inducible EWSR1-ETS knockdown that were profiled by whole-genome-sequencing, DNA methylation arrays, gene expression and splicing arrays, mass spectrometry-based proteomics, and ChIP-seq for EWSR1-ETS and histone marks. Systematic analysis of these multi-dimensional data illuminated hundreds of new potential EWSR1-ETS target genes, the nature of EWSR1-ETS-preferred GGAA-mSats, and potential indirect modes of EWSR1-ETS-mediated gene regulation. Moreover, we identified putative co-regulatory transcription factors and heterogeneously regulated EWSR1-ETS target genes that may have implications for the clinical heterogeneity of EwS. Collectively, our freely available ESCLA constitutes an extremely rich resource for EwS research and highlights the power of leveraging multidimensional and comprehensive datasets to unravel principles of heterogeneous gene regulation by dominant fusion oncogenes.

Identification of cancer stemness genes is crucial to understanding the underlying biology of therapy resistance, relapse, and metastasis. Ewing sarcoma (EwS) is the second most common bone tumor in children and adolescents. It is a highly aggressive cancer associated with a dismal survival rate (EWSR1-ETS). Thus, EwS constitutes an ideal model to study how perturbation of a transcriptional network by a dominant oncogene can mediate metastasis, even though canonical metastasis-associated genes are not mutated.Here, through the implementation of an integrative systems biology approach, we identified transcription factor 7 like 1 (TCF7L1, alias TCF3) as a prognostically-relevant and EWSR1-ETS suppressed determinant of metastasis in EwS. We demonstrated that conditional TCF7L1 re-expression significantly reduces EwS single-cell migration, invasion and anchorage-independent growth in 3D assays in vitro, and tumorigenesis in vivo mediated by its DNA binding domain. In primary EwS tumors as well as in functional orthotopic in vivo models, low TCF7L1 expression was associated with pro-metastatic gene signatures and a much higher migratory and metastatic capacity of EwS cells, which correlated with poor outcome of EwS patients.Collectively, our findings establish TCF7L1 as a major regulator of metastasis in EwS, which may be utilized as a prognostic biomarker and open inroads to future therapeutic intervention.

Western blot is an experimental method used to analyze protein expression. In Ewing sarcoma, as in many other diseases, Western blot provides information about the level of protein expression in different cell conditions, in comparison with other tissues or upon induced molecular changes. Based on the specific pattern of protein expression of the tissue, as well as on the characteristics of the protein of interest, the antibodies and protocol of Western blot may be modified according to different specifications. Here we describe some of these peculiarities in frame of Ewing sarcoma field.

Western blot is an experimental method used to analyze protein expression. In Ewing sarcoma, as in many other diseases, Western blot provides information about the level of protein expression in different cell conditions, in comparison with other tissues or upon induced molecular changes. Based on the specific pattern of protein expression of the tissue, as well as on the characteristics of the protein of interest, the antibodies and protocol of Western blot may be modified according to different specifications. Here we describe some of these peculiarities in frame of Ewing sarcoma field.

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.

Developmental pathways play an important role in cancer. We have recently demonstrated that the constitutive activation of the developmental transcription factor SOX6 via the fusion oncoproteinne EWSR1-FLI1 (Ewing sarcoma breakpoint region 1 - Friend leukemia virus integration 1) contributes to the aggressive phenotype of Ewing sarcoma but on another hand provides an opportunity for targeted therapy.

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.

Ewing sarcoma (EwS) is an aggressive cancer displaying an undifferentiated small-round-cell histomorphology that can be easily confused with a broad spectrum of differential diagnoses. Using comparative transcriptomics and immunohistochemistry (IHC), we previously identified BCL11B and GLG1 as potential specific auxiliary IHC markers for EWSR1-FLI1-positive EwS. Herein, we aimed at validating the specificity of both markers in a far larger and independent cohort of EwS (including EWSR1-ERG-positive cases) and differential diagnoses. Furthermore, we evaluated their intra-tumoral expression heterogeneity. Thus, we stained tissue microarrays from 133 molecularly confirmed EwS cases and 320 samples from morphological mimics, as well as a series of patient-derived xenograft (PDX) models for BCL11B, GLG1, and CD99, and systematically assessed the immunoreactivity and optimal cut-offs for each marker. These analyses demonstrated that high BCL11B and/or GLG1 immunoreactivity in CD99-positive cases had a specificity of 97.5% and an accuracy of 87.4% for diagnosing EwS solely by IHC, and that the markers were expressed by EWSR1-ERG-positive EwS. Only little intra-tumoral heterogeneity in immunoreactivity was observed for differential diagnoses. These results indicate that BCL11B and GLG1 may help as specific auxiliary IHC markers in diagnosing EwS in conjunction with CD99, especially if confirmatory molecular diagnostics are not available.

Soft-tissue sarcomas are rare, heterogeneous, and often aggressive mesenchymal cancers. Many of them are associated with poor outcome, partially because biomarkers that can identify high-risk patients are lacking. Studies on sarcomas are often limited by small sample-sizes rendering the identification of biomarkers difficult when focusing on individual cohorts. However, the increasing number of publicly available 'omics' data opens inroads to overcome this obstacle. Here, we combine transcriptome analyses, immunohistochemistry, and functional assays to show that high adenosine monophosphate deaminase 2 (AMPD2) is a robust prognostic biomarker for worse outcome in undifferentiated pleomorphic sarcoma (UPS). Gene expression and survival data for UPS from two independent studies were subjected to survival association-testing. Genes, whose high expression was significantly correlated with worse outcome in both cohorts, were considered as biomarker candidates. The best candidate, AMPD2, was validated in a tissue microarray. Analysis of DNA copy-number data and matched transcriptomes indicated that high AMPD2 expression is significantly correlated with gains at the AMPD2 locus. Gene set enrichment analyses of AMPD2 co-expressed genes in both transcriptome datasets suggested that AMPD2-high UPS are enriched in tumorigenic signatures. Consistently, knockdown of AMPD2 by RNA interference in an UPS cell line inhibited proliferation in vitro and tumorigenicity in vivo. Collectively, we provide evidence that AMPD2 may serve as a biomarker for outcome prediction in UPS. Our study exemplifies how the integration of 'omics' data, immunohistochemistry, and functional experiments can identify novel biomarkers even in a rare sarcoma, which may serve as a blueprint for biomarker identification for other rare cancers.

The epithelial-to-mesenchymal transition (EMT) is a reversible process comprised of various subprograms via which epithelial cells reduce their intercellular adhesions and proliferative capacity while gaining a mesenchymal phenotype with increased migratory and invasive properties. This process has been well described in several carcinomas, which are cancers of epithelial origin, and is crucial to metastatic tumor cell dissemination and drug resistance. In contrast, the precise role of EMT-related processes in tumors originating from mesenchymal tissues, such as bone and soft-tissues sarcomas, is still largely unclear. In fact, although the existence of the EMT in sarcomas appears paradoxical because these cancers are, by definition, mesenchymal ab initio, accumulating evidence suggests that many sarcomas can undergo EMT-related processes, which may be associated with aggressive clinical behavior. These processes may be especially operative in certain sarcoma subtypes, such as carcinosarcomas displaying a biphenotypic morphology with characteristics of both mesenchymal and epithelial tumors. In this review, we discuss findings regarding the potential existence of EMT-related processes in sarcomas and propose that sarcomas can reside in a metastable state, enabling them to become either more mesenchymal or epithelial under specific conditions, which likely has important clinical implications. Cancer Res; 77(17); 4556–61. ©2017 AACR.

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.

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 (mSats) 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-mSat, which promotes EwS growthin vitroandin vivo. Through integration of transcriptome-profiling, published drug-screening data, and functionalin vitroandin vivoexperiments, we discovered that SOX6 interferes with the antioxidant system resulting in constitutively elevated reactive oxygen species (ROS) levels that create a therapeutic vulnerability toward the ROS-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.

INTRODUCTORY PARAGRAPHDeciphering principles of inter-individual tumor heterogeneity is essential for refinement of personalized anti-cancer therapy. Unlike cancers of adulthood, pediatric malignancies including Ewing sarcoma (EwS) feature a striking paucity of somatic alterations except for pathognomonic driver-mutations that cannot explain overt variations in clinical outcome.Here we demonstrate in the EwS model how cooperation of a dominant oncogene and regulatory variants determine tumor growth, patient survival and drug response.We show that binding of the oncogenic EWSR1-FLI1 fusion transcription factor to a polymorphic enhancer-like DNA element controls expression of the transcription factor MYBL2, whose high expression promotes poor patient outcome via activation of pro-proliferative signatures. Analysis of paired germline and tumor whole-genome sequencing data revealed that regulatory variability at this locus is inherited via the germline. CRISPR-mediated interference with this regulatory element almost abolished MYBL2 transcription, and MYBL2 knockdown decreased cell proliferation, cell survival and tumorigenicity of EwS cells. Combined RNA- and ChIP-seq analyses as well as functional experiments and clinical data identified CCNF, BIRC5 and AURKB as direct MYBL2 targets and critical mediators of its phenotype. In drug-response experiments, high MYBL2 levels sensitized EwS cells for inhibition of its activating cyclin dependent kinase CDK2 in vitro and in vivo, suggesting MYBL2 as a predictive biomarker for targeted anti-CDK2-therapy.Collectively, our findings establish cooperation of somatic mutations and regulatory germline variants as a major determinant of tumor progression and indicate the importance of integrating the regulatory genome in the process of developing new diagnostic and/or therapeutic strategies to fully harness the potential of precision medicine.

Purpose: Up to 30-40% of Ewing sarcoma (EwS) patients with non-metastatic disease develop local or metastatic relapse within a time range of 2-10 years. This is in part caused by the absence of prognostic biomarkers that can identify high-risk patients to assign them to risk-adapted monitoring and treatment regimens. Since cancer stemness has been associated with tumor relapse and poor outcome, we investigated in the current study the prognostic potential SOX2 (sex determining region Y box 2) - a major transcription factor involved in development and stemness - previously described to contribute to the undifferentiated phenotype of EwS. Methods: Two independent patient cohorts, one consisting of 189 retrospectively collected EwS tumors with corresponding mRNA expression data (test cohort) and the other of 141 prospectively collected formalin-fixed and paraffin embedded resected tumors (validation cohort), were employed to analyze SOX2 expression levels by DNA microarrays or immunohistochemistry, respectively, and to compare them with clinical parameters and patient outcome. Two methods were employed to test the validity of the results at both mRNA and protein levels. Results: Both cohorts showed that only a subset of EwS patients (16-20%) express high SOX2 mRNA or protein levels, which significantly correlated with poor overall survival. Multivariate analyses of our validation cohort revealed that high SOX2 expression represents a major risk factor for survival (HR=3.19; P

Ewing sarcoma (EwS) is an aggressive cancer caused by chromosomal translocations generating fusions of theEWSR1gene withETStranscription factors (in 85%FLI1). EWSR1-FLI1 induces gene expression via binding to enhancer-like GGAA-microsatellites, whose activity increases with the number of consecutive GGAA-repeats.Herein, we investigate the role of the secretory neuropeptide CALCB (calcitonin related polypeptide β) in EwS, which signals via the CGRP-(calcitonin gene-related peptide) receptor complex, containing RAMP1 (receptor activity modifying protein 1) as crucial part for receptor specificity. Analysis of 2,678 gene expression microarrays comprising 50 tumor entities and 71 normal tissue types revealed thatCALCBis specifically and highly overexpressed in EwS. Time-course knockdown experiments showed thatCALCBexpression is tightly linked to that ofEWSR1-FLI1. Consistently, gene set enrichment analyses of genes whose expression in primary EwS is correlated to that ofCALCBindicated that it is co-expressed with other EWSR1-FLI1 target genes and associated with signatures involved in stemness and proliferation. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) data for EWSR1-FLI1 and histone marks from EwS cells demonstrated that EWSR1-FLI1 binds to a GGAA-microsatellite close toCALCB, which exhibits characteristics of an active enhancer. Reporter assays confirmed the strong EWSR1-FLI1- and length-dependent enhancer activity of this GGAA-microsatellite. Mass-spectrometry analyses of supernatants of EwS cell cultures demonstrated that CALCB is secreted by EwS cells. While short-term RNA interference-mediatedCALCBknockdown had no effect on proliferation and clonogenic growth of EwS cellsin vitro, its long-term knockdown decreased EwS growthin vitroandin vivo. Similarly, knockdown ofRAMP1reduced clonogenic/spheroidal growth and tumorigenicity, and small-molecule inhibitors directed against the CGRP-receptor comprising RAMP1 reduced growth of EwS.Collectively, our findings suggest thatCALCBis a direct EWSR1-FLI1 target and that targeting the CALCB/RAMP1-axis may offer a new therapeutic strategy for inhibition of EwS growth.

Soft-tissue sarcomas are rare, heterogeneous, and often aggressive mesenchymal cancers. Many of them are associated with poor outcome, partially because biomarkers that can identify high-risk patients are lacking. Studies on sarcomas are often limited by small sample-sizes rendering the identification of biomarkers difficult when focusing on individual cohorts. However, the increasing number of publicly available 'omics' data opens inroads to overcome this obstacle. Here, we combine transcriptome analyses, immunohistochemistry, and functional assays to show that high adenosine monophosphate deaminase 2 (AMPD2) is a robust prognostic biomarker for worse outcome in undifferentiated pleomorphic sarcoma (UPS). Gene expression and survival data for UPS from two independent studies were subjected to survival association-testing. Genes, whose high expression was significantly correlated with worse outcome in both cohorts, were considered as biomarker candidates. The best candidate, AMPD2, was validated in a tissue microarray. Analysis of DNA copy-number data and matched transcriptomes indicated that high AMPD2 expression is significantly correlated with gains at the AMPD2 locus. Gene set enrichment analyses of AMPD2 co-expressed genes in both transcriptome datasets suggested that AMPD2-high UPS are enriched in tumorigenic signatures. Consistently, knockdown of AMPD2 by RNA interference in an UPS cell line inhibited proliferation in vitro and tumorigenicity in vivo. Collectively, we provide evidence that AMPD2 may serve as a biomarker for outcome prediction in UPS. Our study exemplifies how the integration of 'omics' data, immunohistochemistry, and functional experiments can identify novel biomarkers even in a rare sarcoma, which may serve as a blueprint for biomarker identification for other rare cancers.

Soft-tissue sarcomas are rare, heterogeneous and often aggressive mesenchymal cancers. Many of them are associated with poor outcome, in part because biomarkers that can reliably identify high-risk patients are lacking. Studies on sarcomas often are limited by small sample sizes rendering the identification of novel biomarkers difficult when focusing only on individual cohorts. However, the increasing number of publicly available ‘omics’ data opens inroads to overcome this obstacle.Here, we combine high-throughput transcriptome analyses, immunohistochemistry, and functional assays to show that high adenosine monophosphate deaminase 2 (AMPD2) is a robust prognostic biomarker for worse patient outcome in undifferentiated pleomorphic sarcoma (UPS). Publicly available gene expression and survival data for UPS from two independent studies, The Cancer Genome Atlas (TCGA) and the CINSARC reference dataset, were subjected to survival association testing. Genes, whose high expression was significantly correlated with worse outcome in both cohorts (overall and metastasis-free survival), were considered as prognostic marker candidates. The best candidate, AMPD2, was validated on protein level in an independent tissue microarray. Analysis of DNA copy-number and matched gene expression data indicated that high AMPD2 expression is significantly correlated with copy-number gains at the AMPD2 locus. Gene-set enrichment analyses of AMPD2 co-expressed genes in both UPS gene expression datasets suggested that highly AMPD2 expressing tumors are enriched in gene signatures involved in tumorigenesis. Consistent with this prediction in primary tumors, knockdown of AMPD2 by RNA interference with pooled siRNAs or a doxycycline-inducible shRNA construct in the UPS cell line FPS-1 markedly inhibited proliferation in vitro and tumorigenicity in vivo.Collectively, these results provide evidence that AMPD2 may serve as a novel biomarker for outcome prediction in UPS. Our study exemplifies how the integration of available ‘omics’ data, immunohistochemical analyses, and functional experiments can identify novel biomarkers even in a rare sarcoma, which may serve as a blueprint for biomarker identification for other rare cancers.

Immunotherapy can revolutionize anti-cancer therapy if specific targets are available. Recurrent somatic mutations in the exome can create highly specific neo-antigens. However, especially pediatric cancers are oligo-mutated and hardly exhibit recurrent neo-antigens. Yet, immunogenic peptides encoded by cancer-specific genes (CSGs), which are virtually not expressed in normal tissues, may enable a targeted immunotherapy of such cancers. Here, we describe an algorithm and provide a user-friendly software named RAVEN (Rich Analysis of Variable gene Expressions in Numerous tissues), which automatizes the systematic and fast identification of CSG-encoded peptides highly affine to Major Histocompatibility Complexes (MHC) starting from publicly available gene expression data. We applied RAVEN to a dataset assembled from more than 2,700 simultaneously normalized gene expression microarrays comprising 50 tumor entities, with a focus on sarcomas and pediatric cancers, and 71 normal tissue types. RAVEN performed a transcriptome-wide scan in each cancer entity for gender-specific CSGs. As a proof-of-concept we identified several established CSGs, but also many novel candidates potentially suitable for targeting multiple cancer types. The specific expression of the most promising CSGs was validated by qRT-PCR in cancer cell lines and by immunohistochemistry in a comprehensive tissue-microarray comprising 412 samples. Subsequently, RAVEN identified likely immunogenic peptides encoded by these CSGs by predicting the affinity to MHCs. Putative highly affine peptides were automatically crosschecked with the UniProt protein-database to exclude sequence identity with abundantly expressed proteins. The predicted affinity of selected peptides was validated in T2-cell peptide-binding assays in which many showed similar kinetics to a very immunogenic influenza control peptide.Collectively, we provide a comprehensive, exquisitely curated and validated catalogue of cancer-specific and highly MHC-affine peptides across 50 cancer entities. In addition, we developed an intuitive and freely available software to easily apply our algorithm to any gene expression dataset (https://github.com/JSGerke/RAVENsoftware). We anticipate that our peptide libraries and software constitute a rich resource to accelerate the development of novel immunotherapies.

Ewing sarcoma is an undifferentiated small-round-cell sarcoma. Although molecular detection of pathognomonic EWSR1-ETS fusions such as EWSR1-FLI1 enables definitive diagnosis, substantial confusion can arise if molecular diagnostics are unavailable. Diagnosis based on the conventional immunohistochemical marker CD99 is unreliable due to its abundant expression in morphological mimics. To identify novel diagnostic immunohistochemical markers for Ewing sarcoma, we performed comparative expression analyses in 768 tumors representing 21 entities including Ewing-like sarcomas, which confirmed that CIC-DUX4-, BCOR-CCNB3-, EWSR1-NFATc2-, and EWSR1-ETS-translocated sarcomas are distinct entities, and revealed that ATP1A1, BCL11B, and GLG1 constitute specific markers for Ewing sarcoma. Their high expression was validated by immunohistochemistry and proved to depend on EWSR1-FLI1-binding to highly active proximal super-enhancers. Automated cut-off-finding and combination-testing in a tissue-microarray comprising 174 samples demonstrated that detection of high BCL11B and/or GLG1 expression is sufficient to reach 96% specificity for Ewing sarcoma. While 88% of tested Ewing-like sarcomas displayed strong CD99-immunoreactivity, none displayed combined strong BCL11B- and GLG1-immunoreactivity. Collectively, we show that ATP1A1, BCL11B, and GLG1 are EWSR1-FLI1 targets, of which BCL11B and GLG1 offer a fast, simple, and cost-efficient way to diagnose Ewing sarcoma by immunohistochemistry. These markers may significantly reduce the number of misdiagnosed patients, and thus improve patient care., This work was supported by the Kind-Philipp-Foundation (to J.M., M.C.B., M.F.O., M.D., A.M, and G.S.), the ‘Deutsche Stiftung für Junge Erwachsene mit Krebs’ (to M.D.), the German National Academic Foundation (to M.C.B. and M.M.L.K.), the ‘Verein zur Förderung von Wissenschaft und Forschung an der Medizinischen Fakultät der LMU München’ (WiFoMed; to T.G.P.G.), the Daimler and Benz Foundation in cooperation with the Reinhard Frank Foundation (to T.G.P.G.), the Friedrich-Baur-Stiftung (to T.G.P.G.) by LMU Munich’s Institutional Strategy LMUexcellent within the framework of the German Excellence Initiative (to T.G.P.G.), the ‘Mehr LEBEN für krebskranke Kinder - Bettina-Bräu-Stiftung’ (to T.G.P.G.), the Fritz-Thyssen Foundation (FTF-40.15.0.030MN, to T.G.P.G. and G.S.), the Wilhelm-Sander-Foundation (2016.167.1 to T.G.P.G.), and the German Cancer Aid (DKH-111886 and DKH-70112257 to T.G.P.G.; DKH-108128 to U.D.), EU FP7 and TRANSCAN EraNet - PROVABES [01KT1310], EU-FP7 EEC [602856-2] to U.D.

Introduction Ewing sarcoma (EwS) is an aggressive bone cancer of likely mesenchymal origin characterised by fusions of the EWSR1 gene and members of the ETS-family of transcription factors (most commonly FLI1). EWSR1-FLI1 diverts GGAA-microsatellites (GGAA-mSat) as enhancers, whose activity increases with the number of consecutive GGAA–repeats, to regulate many of its target genes. Specific EWSR1-FLI1 targets may contribute to maintenance of a highly proliferative and undifferentiated phenotype in EwS. Material and methods Using DNA microarrays, ChIP-Seq, qRT-PCR, immunohistochemistry (IHC) and RNA interference (RNAi) for in vitro and inducible shRNA constructs for in vivo experiments, we functionally characterised the role of SOX6 in EwS. Results and discussions Using microarray analysis of cancer and normal tissue samples as well as IHC, we show that the transcription factor SOX6 is strongly but variably overexpressed in EwS. Analysis of publicly available ChIP–Seq data revealed a prominent EWSR1-FLI1 peak in intron 1 of SOX6 that overlaid with regulatory histone marks and mapped to a GGAA-mSat, which showed EWSR1-FLI1-dependent enhancer activity in reporter assays. Notably, the number of consecutive GGAA-repeats at this microsatellite correlated with the SOX6 expression levels of EwS cell lines. These data prove that SOX6 is a direct EWSR1–FLI1 target gene controlled by an intronic GGAA-mSat. Functionally, RNAi-mediated SOX6 knockdown significantly reduced proliferation and clonogenic growth of different EwS cell lines through G1-phase arrest and induction of cell death. Inducible shRNA-mediated SOX6 knockdown markedly reduced tumour growth of EwS xenografts in vivo. Consistently, gene-set enrichment analysis of primary tumours and cell lines after SOX6 knockdown showed that SOX6 is associated with cell cycle progression. By crossing transcriptome profiles of SOX6-silenced EwS cells and ChIP-Seq data for SOX6, we identified three likely direct SOX6 target genes, which cooperate to promote its pro-proliferative effect. Through analysis of published drug-screen data and functional drug-response assays we found that SOX6 overexpression confers an exquisite sensitivity to EwS cells toward the small molecule Elesclomol. Conclusion Collectively, our data indicate that SOX6 is a direct EWSR1-FLI1 target gene contributing to proliferation, clonogenicity and tumour growth of EwS cells, and that high SOX6 expression may constitute a drugable vulnerability of EwS.