Prospective Validation of Biomarkers in Ewing Sarcoma for Personalised Treatment (PROVABES)

Ewing sarcoma is a big challenge for oncologists since about 40% of patients cannot be cured with current multimodal treatment regimens. Several putative prognostic markers have been described in retrospective studies of small patient cohorts, but only one (EWS-FLI1 fusion type) has so far been prospectively tested, with negative results. The "PROspective VAlidation of Biomarkers in Ewing Sarcoma for personalised translational medicine" (PROVABES) consortium was therefore formed to validate previously described candidate molecular biomarkers of good/bad prognosis, alone and in combination. Biomarkers associated with poor or good prognosis are either themselves involved in disease pathogenesis and/or treatment response, or surrogates or products of pathways essential for disease progression. Drivers of disease progression are likely to show the highest stability as biomarkers and may even serve as potential therapeutic targets. Thus, the functional characterization of prognostically relevant molecules aids the prospective validation in patients by supplying mechanistic explanations for the clinical correlations under study. While for several of the PROVABES molecules (CXCR4, STEAP1, EZH2, PARP1) ample preclinical functional data already exist, this is not the case for candidate targets of recently described Ewing sarcoma predisposition loci (Postel-Vinay et al., 2012) (WP1.2), and microRNA hsa-mir-34a (Nakatani et al., 2012) (WP 2). As an added value to the PROVABES consortium, WP4 is an exploratory work package aiming at the definition of the relevant molecular targets of these epigenetic (miRNA, and SNP) biomarkers. In close collaboration with our partners at Rizzoli Institute Bologna, Katia Scotlandi/Piero Picci (hsa- mir-34a), and at Institut Curie Paris, Franck Tirode/Olivier Delattre (predisposition SNPs on chromosomes 10 and 15), we will investigate the functional relevance of previously identified candidate target genes. These targets have been selected because of their proximity to the marker SNPs (BMF and SIRT1), respectively based on physical interaction of their mRNA products with hsa-mir-34a-containing RISC complexes in a Ewing sarcoma cell line (TBL1XR1, XBP1, ARL6IP1, CAMK1D, TMEM189, RBMX). To this end we will establish an in vitro functional analysis pipeline (Deliverable D4.2), which is based on differential expression of these markers in 4 sets of paired Ewing sarcoma cell lines from localized tumors and corresponding metastases (test panel), and 6 cell lines each of localized tumors and unrelated metastases (validation panel). First, the test cell line panel will be analysed by Affymetrix OncoScanTM (WP1.1) and RNA next generation sequencing (RNA-seq, WP1.2) to characterize gene copy number, expression and sequence variations between localized and corresponding metastasis derived cell lines (Deliverable D4.1). Cell line results will be compared to primary tumor results from WP1.2. We will introduce hsa- mir-34a mimics, respectively knockdown endogenous hsa-mir-34a in selected cell lines from the validation panel to functionally confirm the 6 chosen candidate targets as hsa-mir-34a regulated genes. Subsequently, we will quantify the effect of experimental modulation of the candidate targets on cell growth under adherent and anchorage independent conditions, migration and invasion. These parameters will be analysed under normoxic and hypoxic growth conditions, as we described previously (Aryee et al., 2010). We expect that this in vitro functional validation of presumed target genes for chromosomes 10 and 15-associated Ewing sarcoma predisposition SNPs (SIRT1, BMF; Deliverable D4.3), and for hsa-mir-34a (TBL1XR1, XBP1, ARL6IP1, CAMK1D, TMEM189, RBMX; Deliverable D4.4) will identify the biological mediators of differential clinical course observed to be associated with these epigenetic markers.

Ewing sarcoma is a highly malignant bone tumor in children and adolescents. The causes of and the criteria predicting aggressiveness in this disease are largely unknown. The European research groups assembled by the ERA-NET project PROspective Validation of Biomarkers in Ewing sarcoma (PROVABES) have previously defined a number of potential biomarkers of prognosis based on the retrospective analysis of small patient cohorts. PROVABES aimed at systematically study and prospectively validate these candidate markers. This sub-project supported by the FWF was designed to functionally analyze some of these candidates in vitro to define their role for Ewing sarcoma aggressiveness. We focused on the study of microRNAs deregulated in Ewing sarcoma and on sequence variations in three chromosomal regions, which correlate with susceptibility to Ewing sarcomagenesis. We developed a method to identify the full complement of genes regulated by microRNAs within a given cell type, and applied it to the study of a microRNA cluster (hsa-miR-17/92) upregulated in Ewing sarcoma. We demonstrated that about a quarter of genes targeted by this microRNA family are involved in the switching between two related signal transduction pathways, which regulate the stemness of the tumor cells. In addition, we identified a relationship between these signaling pathways and a gene associated specifically with Ewing sarcoma metastases (SIRT1), which localizes in the vicinity of one of the Ewing sarcoma susceptibility regions. The gene product is an enzyme that requires NAD for its activity. We showed that Ewing sarcoma is specifically sensitive to pharmacological inhibition of NAD biosynthesis hinting to a potential new therapeutic avenue. For the remaining two disease associated chromosomal regions we discovered that they contain genes coding for components of the immediate cellular stress response. This response occurs at the level of protein synthesis and includes the formation of cytoplasmic granules (stress granules). We found that inhibition of these gene products leads to altered stress granule dynamics and loss of the ability of tumor cells to grow under surface independent conditions. Finally, together with our PROVABES partners, we performed a comprehensive epigenome analysis of about 140 primary Ewing sarcomas. Based on the results, we defined three levels of tumor heterogeneity, which allowed us to draw conclusions about diagnosis, phenotype and prognosis of individual tumors. In summary, this project resulted in several novel insights into the biology of Ewing sarcoma and the mechanisms, which are responsible for the aggressiveness of the disease.