PO-047 SOX6 is a direct EWSR1-FLI1 target gene contributing to tumour growth of ewing sarcoma

Abstract

IntroductionEwing 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 methodsUsing 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 discussionsUsing 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.ConclusionCollectively, 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.