Abstract A10: Functional characterization of Ewing's sarcoma susceptibility loci
Authors: Grunewald Thomas , Aynaud Marie-Ming , Tirode Franck , Tomazou Eleni , Surdez Didier , Frio Thomas Rio , Bernard Virginie , Raynal Virginie , Lucchesi Carlo , Pierron Gaelle , Gilardi-Hebenstreit Pascale , Charnay Patrick , Kovar Heinrich , Delattre Olivier 
Published: May 2015

Background: We previously identified in a GWAS common variants near the genes TARDBP (chr1), EGR2 and ADO (both chr10), and BMF (chr15) that are associated with the susceptibility of Caucasians toward Ewing's sarcoma (ES). These variants lie in intergenic regions that appear to harbor expression quantitative trait loci (eQTLs) as they show strong modulatory effects on the expression of these “candidate genes”. While TARDBP is an mRNA and miRNA processing protein, EGR2 is a conserved transcription factor involved proliferation and differentiation of osteoprogenitors and Schwann cells. ADO, which shows a highly significant co-expression with EGR2, is the key enzyme converting cysteamine into hypotaurine. The BCL2-modifying factor BMF is involved in pro- and anti-apoptotic processes depending on the cellular context. As most SNPs identified by GWAS are usually not the causal variants but are expected to be in strong linkage disequilibrium with such variants, this project aims to comprehensively sequence and epigenetically profile the three ES susceptibility regions and to functionally characterize the role of the four candidate genes in ES.

Methods and Results: Fine-selection of the genomic regions (in total 1 Mb) was performed with own and HapMap CEU data. An initial pilot experiment (48 samples) using a custom Nextera DNA enrichment system followed by paired-end 150x150bp sequencing (Illumina HiSeq2500) yielded 95% coverage with at least 20 high-quality reads/nucleotide. Comparison with matched SNP arrays revealed that both the false positive and negative discovery rates were smaller than 1%. Currently, we sequence additional 576 cases and PCA-matched controls and perform a multilayered epigenetic profiling of ES cells with/without silencing of EWS/FLI1 for our susceptibility regions. Analysis of three comprehensive paired gene expression and genotype datasets for lymphoblastoid cell lines, medulloblastoma, and neuroblastoma, proved that none of our Ewing SNPs correlated with the expression the four candidate genes, suggesting a specific effect of these eQTLs in ES, possibly via direct or indirect interaction with EWS/FLI1. In accordance, siRNA-mediated knockdown of EWS/FLI1 in ES cell lines strongly down-regulates EGR2, while EWS/FLI1 expression in human mesenchymal stem cells (MSCs) induces EGR2. In contrast, only moderate modulatory effects of EWS/FLI1 were observed for TARDBP, ADO, and BMF. However, three independent ChIP-seq studies did not find evidence of EWS/FLI1 binding to the EGR2 promoter or nearby regions, arguing for an indirect mode of regulation. On a functional level, knockdown of TARDBP, EGR2, and BMF, but not ADO reduces proliferation and clonogenicity of ES cells by blocking cell cycle progression and inducing apoptosis, which is preceded by transcriptional changes in cell cycle regulators and pro-survival genes as measured by microarrays, RNA-seq and qRT-PCR. Interestingly, all candidate genes share a highly significant overlap in modulated genes with EWS/FLI1 and their transcriptional fingerprint is similar to that conferred by EWS/FLI1 to human MSCs, the most likely cells of origin of ES. Gene-set enrichment and Ingenuity pathway analyses revealed that the candidate genes are involved in cell growth and differentiation, albeit via diverse mechanisms. Additional experiments, including the generation of inducible shRNA expression systems, are ongoing to further characterize their role and their interplay with EWS/FLI1 in ES.

Conclusions: In synopsis, our data indicate that the previously identified ES susceptibility regions and candidate genes may play a prominent role in ES pathobiology.

Citation Format: Thomas Grunewald, Marie-Ming Aynaud, Franck Tirode, Eleni Tomazou, Didier Surdez, Thomas Rio Frio, Virginie Bernard, Virginie Raynal, Carlo Lucchesi, Gaelle Pierron, Pascale Gilardi-Hebenstreit, Patrick Charnay, Heinrich Kovar, Olivier Delattre. Functional characterization of Ewing's sarcoma susceptibility loci. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A10.