Ana Teresa Amaral

Stable interference of EWS–FLI1 in an Ewing sarcoma cell line impairs IGF-1/IGF-1R signalling and reveals TOPK as a new target

BACKGROUND: Ewing sarcoma is a paradigm of solid tumour -bearing chromosomal translocations resulting in fusion proteins that act as deregulated transcription factors. Ewing sarcoma translocations fuse the EWS gene with an ETS transcription factor, mainly FLI1. Most of the EWS–FLI1 target genes still remain unknown and many have been identified in heterologous model systems.METHODS: We have developed a stable RNA interference model knocking down EWS–FLI1 in the Ewing sarcoma cell line TC71. Gene expression analyses were performed to study the effect of RNA interference on the genetic signature of EWS–FLI1 and to identify genes that could contribute to tumourigenesis.RESULTS: EWS–FLI1 inhibition induced apoptosis, reduced cell migratory and tumourigenic capacities, and caused reduction in tumour growth. IGF-1 was downregulated and the IGF-1/IGF-1R signalling pathway was impaired. PBK/TOPK (T-LAK cell-originated protein kinase) expression was decreased because of EWS–FLI1 inhibition. We showed that TOPK is a new target gene of EWS–FLI1. TOPK inhibition prompted a decrease in the proliferation rate and a dramatic change in the cells ability to grow in coalescence.CONCLUSION: This is the first report of TOPK activity in Ewing sarcoma and suggests a significant role of this MAPKK-like protein kinase in the Ewing sarcoma biology.

DNA methylation heterogeneity defines a disease spectrum in Ewing sarcoma

Developmental tumors in children and young adults carry few genetic alterations, yet they have diverse clinical presentation. Focusing on Ewing sarcoma, we sought to establish the prevalence and characteristics of epigenetic heterogeneity in genetically homogeneous cancers. We performed genome-scale DNA methylation sequencing for a large cohort of Ewing sarcoma tumors and analyzed epigenetic heterogeneity on three levels: between cancers, between tumors, and within tumors. We observed consistent DNA hypomethylation at enhancers regulated by the disease-defining EWS-FLI1 fusion protein, thus establishing epigenomic enhancer reprogramming as a ubiquitous and characteristic feature of Ewing sarcoma. DNA methylation differences between tumors identified a continuous disease spectrum underlying Ewing sarcoma, which reflected the strength of an EWS-FLI1 regulatory signature and a continuum between mesenchymal and stem cell signatures. There was substantial epigenetic heterogeneity within tumors, particularly in patients with metastatic disease. In summary, our study provides a comprehensive assessment of epigenetic heterogeneity in Ewing sarcoma and thereby highlights the importance of considering nongenetic aspects of tumor heterogeneity in the context of cancer biology and personalized medicine.

The PARP inhibitor olaparib enhances the sensitivity of Ewing sarcoma to trabectedin

Recent preclinical evidence has suggested that Ewing Sarcoma (ES) bearing EWSR1-ETS fusions could be particularly sensitive to PARP inhibitors (PARPinh) in combination with DNA damage repair (DDR) agents. Trabectedin is an antitumoral agent that modulates EWSR1-FLI1 transcriptional functions, causing DNA damage. Interestingly, PARP1 is also a transcriptional regulator of EWSR1-FLI1, and PARPinh disrupts the DDR machinery. Thus, given the impact and apparent specificity of both agents with regard to the DNA damage/DDR system and EWSR1-FLI1 activity in ES, we decided to explore the activity of combining PARPinh and Trabectedin in in vitro and in vivo experiments. The combination of Olaparib and Trabectedin was found to be highly synergistic, inhibiting cell proliferation, inducing apoptosis, and the accumulation of G2/M. The drug combination also enhanced γH2AX intranuclear accumulation as a result of DNA damage induction, DNA fragmentation and global DDR deregulation, while EWSR1-FLI1 target expression remained unaffected. The effect of the drug combination was corroborated in a mouse xenograft model of ES and, more importantly, in two ES patient-derived xenograft (PDX) models in which the tumors showed complete regression. In conclusion, the combination of the two agents leads to a biologically significant deregulation of the DDR machinery that elicits relevant antitumor activity in preclinical models and might represent a promising therapeutic tool that should be further explored for translation to the clinical setting.

The clinical relevance of molecular genetics in soft tissue sarcomas.

Bone and soft tissue sarcomas are an infrequent and heterogeneous group of mesenchymal tumors including more than a hundred different entities attending to histologic patterns. Research into the molecular aspects of sarcomas has increased greatly in the last few years. This enormous amount of knowledge has allowed, for instance, to refine the classification of sarcomas, improve the diagnosis, and increase the number of therapeutical targets available, most of them under preclinical evaluation. However, other important key issues, such as sarcomagenesis and the cell of origin of sarcomas, remain unresolved. From a molecular point of view, these neoplasias are grouped into 2 main types: (a) sarcomas showing relatively simple karyotypes and translocations, which originate gene fusions (eg, EWS-FLI1 in Ewing sarcoma) or point mutations (eg, c-kit in the gastrointestinal tumors) and (b) sarcomas showing unspecific gene alterations, very complex karyotypes, and no translocations. The discovery of the early mechanisms involved in the genesis of sarcomas, the more relevant signaling pathways, and the development of genetically engineered mouse models could also provide a new individualized therapeutic strategy against these tumors. This review describes the clinical application of some of the molecular alterations found in sarcomas, some advances in the field of sarcomagenesis, and the development of animal models.

Characterization of human mesenchymal stem cells from ewing sarcoma patients. Pathogenetic implications.

Background Ewing Sarcoma (EWS) is a mesenchymal-derived tumor that generally arises in bone and soft tissue. Intensive research regarding the pathogenesis of EWS has been insufficient to pinpoint the early events of Ewing sarcomagenesis. However, the Mesenchymal Stem Cell (MSC) is currently accepted as the most probable cell of origin. Materials and Methods In an initial study regarding a deep characterization of MSC obtained specifically from EWS patients (MSC-P), we compared them with MSC derived from healthy donors (MSC-HD) and EWS cell lines. We evaluated the presence of the EWS-FLI1 gene fusion and EWSR1 gene rearrangements in MSC-P. The presence of the EWS transcript was confirmed by q-RT-PCR. In order to determine early events possibly involved in malignant transformation, we used a multiparameter quantitative strategy that included both MSC immunophenotypic negative/positive markers, and EWS intrinsic phenotypical features. Markers CD105, CD90, CD34 and CD45 were confirmed in EWS samples. Results We determined that MSC-P lack the most prevalent gene fusion, EWSR1-FLI1 as well as EWSR1 gene rearrangements. Our study also revealed that MSC-P are more alike to MSC-HD than to EWS cells. Nonetheless, we also observed that EWS cells had a few overlapping features with MSC. As a relevant example, also MSC showed CD99 expression, hallmark of EWS diagnosis. However, we observed that, in contrast to EWS cells, MSC were not sensitive to the inhibition of CD99. Conclusions In conclusion, our results suggest that MSC from EWS patients behave like MSC-HD and are phenotypically different from EWS cells, thus raising important questions regarding MSC role in sarcomagenesis.

Trabectedin efficacy in Ewing sarcoma is greatly increased by combination with anti-IGF signaling agents.

Purpose: Goal of this study was to identify mechanisms that limit efficacy of trabectedin (ET-743, Yondelis) in Ewing sarcoma (EWS), so as to develop a clinical applicable combination therapy. Experimental Design: By chromatin immunoprecipitation, we analyzed EWS–FLI1 binding to the promoters of several target genes, such as TGFβR2, CD99, insulin-like growth factor receptor 1 (IGF1R), and IGF1, both in vitro and in xenografts treated with trabectedin or doxorubicin. Combined therapy with trabectedin and anti-IGF1R agents (AVE1642 HAb; OSI-906) was tested in vitro and in xenografts. Results: We confirm that both trabectedin and doxorubicin were able to strongly reduce EWS–FLI1 (both type I and type II) binding to two representative target genes (TGFβR2 and CD99), both in vitro and in xenografts. However, trabectedin, but not doxorubicin, was also able to increase the occupancy of EWS–FLI1 to IGF1R promoters, leading to IGF1R upregulation. Inhibition of IGF1R either by the specific AVE1642 human antibody or by the dual IGF1R/insulin receptor inhibitor OSI-906 (Linsitinib) greatly potentiate the efficacy of trabectedin in the 13 EWS cell lines here considered as well as in TC-71 and 6647 xenografts. Combined therapy induced synergistic cytotoxic effects. Trabectedin and OSI-906 deliver complementary messages that likely converge on DNA-damage response and repair pathways. Conclusions: We showed that trabectedin may not only inhibit but also enhance the binding of EWS–FLI1 to certain target genes, leading to upregulation of IGF1R. We here provide the rationale for combining trabectedin to anti-IGF1R inhibitors. Clin Cancer Res; 21(6); 1373–82. ©2015 AACR.

Ex vivo identification and characterization of a population of CD13high CD105+ CD45− mesenchymal stem cells in human bone marrow

IntroductionMesenchymal stem cells (MSCs) are multipotent cells capable of self-renewal and multilineage differentiation. Their multipotential capacity and immunomodulatory properties have led to an increasing interest in their biological properties and therapeutic applications. Currently, the definition of MSCs relies on a combination of phenotypic, morphological and functional characteristics which are typically evaluated upon in vitro expansion, a process that may ultimately lead to modulation of the immunophenotypic, functional and/or genetic features of these cells. Therefore, at present there is great interest in providing markers and phenotypes for direct in vivo and ex vivo identification and isolation of MSCs.MethodsMultiparameter flow cytometry immunophenotypic studies were performed on 65 bone marrow (BM) samples for characterization of CD13high CD105+ CD45– cells. Isolation and expansion of these cells was performed in a subset of samples in parallel to the expansion of MSCs from mononuclear cells following currently established procedures. The protein expression profile of these cells was further assessed on (paired) primary and in vitro expanded BM MSCs, and their adipogenic, chondrogenic and osteogenic differentiation potential was also determined.ResultsOur results show that the CD13high CD105+ CD45− immunophenotype defines a minor subset of cells that are systematically present ex vivo in normal/reactive BM (n = 65) and that display immunophenotypic features, plastic adherence ability, and osteogenic, adipogenic and chondrogenic differentiation capacities fully compatible with those of MSCs. In addition, we also show that in vitro expansion of these cells modulates their immunophenotypic characteristics, including changes in the expression of markers currently used for the definition of MSCs, such as CD105, CD146 and HLA-DR.ConclusionsBM MSCs can be identified ex vivo in normal/reactive BM, based on a robust CD13high CD105+ and CD45− immunophenotypic profile. Furthermore, in vitro expansion of these cells is associated with significant changes in the immunophenotypic profile of MSCs.

PO-459 Unravelling endoglin as a potential therapeutic target for the treatment of uveal melanoma

Introduction Uveal Melanoma(UM) is a rare deadly tumour where, despite highly aggressive management of the primary disease, approximately 50% of patients develop metastases. Therapies directed towards systemic/metastatic disease have failed in the clinic.Endoglin (ENG),a co-receptor of TGF-β,plays an important role in tissue remodelling and cancer.A soluble form is produced upon proteolytic cleavage by matrix metalloprotease MMP14.Our work focuses on the role of ENG/MMP14, how they influence UM metastasis, and their potential as therapeutic targets. Material and methods Normalised gene expression data with patient follow-up from 80 primary UM tumours was downloaded from The Cancer Genome Atlas (TCGA) NIH Data Portal.mRNA/protein from UM and Ewing Sarcoma cell lines were isolated, and ENG/MMP14 levels were evaluated by q-RT-PCR/western blot. Immunohistochemistry analysis of ENG/MMP14 expression was performed in 2 independent patient cohorts.MTT assays were performed to evaluate proliferation inhibition after exposure to OMTX503 anti-ENG Antibody Drug Conjugate (ADC).CRISPR technology with high fidelity Cas9 was used to knockout MMP14/ENG, which was validated by mismatch-cleavage assays and by the loss of protein expression. Results and discussions Bioinformatics analysis showed that a higher expression of ENG correlated with worst disease-free survival (DFS)(p=0.00023), whereas MMP14 showed a similar trend without reaching statistical significance(p=0.077).These results differ from skin melanoma DFS(p=0.74 for ENG,p=0.22 for MMP14).Pathway enrichment analysis showed that higher ENG expression is associated with a higher abundance of CD8+T-cells (p<0.002),endothelial cells(p<0.003) and fibroblasts(p<0.001).In the 2 patient cohorts, differential levels of ENG/MMP14 were detected.In vitro, cell line MUM2B expressed higher levels of ENG/MMP14.UM cell lines were highly sensitive to treatment with ADC OMTX503, with IC50 in the sub-nM range.Sensitivity was dependent on ENG expression.Knockout of MMP14 resulted in lack of protein expression in the single cell derived clones.The effect on ENG shedding and malignant features upon lack of MMP14 is currently being analysed. Conclusion ENG/MMP14 are expressed in UM cell lines and in patient tumour samples.Results suggest that higher levels of ENG are associated with a more aggressive phenotype.ENG is an attractive target, and ENG-targeting biologics such as OMTX503 show promising in vitro activity.In-depth evaluation of the role of ENG/MMP14 in UM is ongoing.

Abstract PR13: DNA methylation mapping and computational modeling in a large Ewing sarcoma cohort identifies principles of tumor heterogeneity and their impact on clinical phenotypes

Ewing sarcoma is an excellent model for studying the role of epigenetic deregulation and tumor heterogeneity, given its low mutation rates and the well-defined oncogenic driver. We have recently shown that the fusion oncogene EWS-FLI1 induces widespread epigenetic rewiring in proximal and distal enhancers (Tomazou et al. Cell Reports 2015). In the current study, we validate the clinical relevance of our results in a large cohort of primary tumors, and we explore the prevalence, characteristics, and clinical impact of epigenetic tumor heterogeneity in Ewing sarcoma. We used reduced representation bisulfite sequencing (RRBS) to generate genome-wide profiles of DNA methylation in 141 Ewing sarcoma primary tumors, 17 Ewing sarcoma cell lines, and 32 primary mesenchymal stem cell (MSC) samples. Deep sequencing resulted in DNA methylation measurements for an average of 3.5 million unique CpGs per sample with excellent data quality (>98% bisulfite conversion efficiency). In addition, for three primary tumors we generated comprehensive reference epigenome maps using whole genome bisulfite sequencing (WGBS) and ChIP-seq for seven histone marks (H3K4me3, H3K4me1, H3K27me3, H3K27ac, H3K56ac, H3K36me3, and H3K9me3). We show that DNA methylation data can be used to infer enhancer activity differences among tumors, allowing us to exploit our large primary tumor dataset to systematically compare the regulation of EWS-FLI1 correlated and anticorrelated enhancers. We also identified Ewing-specific DNA methylation patterns. For example, Ewing sarcoma samples consistently show higher DNA methylation than MSCs at AP-1 binding sites, but lower DNA methylation at EWS-FLI1 binding sites. To explore epigenetic heterogeneity within individual tumors, we developed a bioinformatic algorithm that quantifies DNA methylation disorder. Using individual reads containing multiple DNA methylation measurements from single cells, we assign scores at single-nucleotide resolution. This method uses a probabilistic model to account for overall methylation rate and expected disorder levels. By evaluating the likelihood of the data in a model that assumes that the DNA methylation status of a CpG is independent of the methylation status of a nearby CpG, we identify extremely heterogeneous as well as highly epigenetically conserved genomic elements. These different region types show distinct patterns of enrichment for regulatory modes and transcription factor binding. We also compared the observed DNA methylation disorder in 141 Ewing tumors to those observed in 17 Ewing sarcoma cell lines, 32 primary MSC samples, and several hundred additional tumor and normal samples that are unrelated to Ewing sarcoma. This analysis identified Ewing-specific patterns of DNA methylation heterogeneity and stratifies patients based on epigenetic heterogeneity. Our dataset constitutes the largest available resource of genome-scale DNA methylation maps in a solid pediatric tumor. It strongly confirms the relevance of enhancer reprogramming and tumor heterogeneity in Ewing sarcoma, and it constitutes a starting point to develop DNA methylation biomarkers for prognosis and patient stratification. This study is supported by the Austrian National Bank (OeNB project #15714) and the Kapsch group (https://www.kapsch.net/). This abstract is also presented as Poster A24. Citation Format: Nathan C. Sheffield, Franck Tirode, Sandrine Grossetete-Lalami, Paul Datlinger, Andreas Schonegger, Johanna Hadler, Diana Walder, Ingeborg M. Ambros, Ana Teresa Amaral, Enrique de Alava, Katharina Schallmoser, Dirk Strunk, Beate Rinner, Bernadette Liegl-Atzwanger, Berthold Huppertz, Andreas Leithner, Uta Dirksen, Peter Ambros, Olivier Delattre, Heinrich Kovar, Christoph Bock, Eleni M. Tomazou. DNA methylation mapping and computational modeling in a large Ewing sarcoma cohort identifies principles of tumor heterogeneity and their impact on clinical phenotypes. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr PR13.