In transcriptome profiling experiments that comprised 285,000 transcripts and isoforms, we did not observe a strong contribution of SOX6 to alternative splicing in EwS. 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 C a physiological driver of proliferation of osteo-chondrogenic progenitors 2-Atractylenolide C by binding to an intronic GGAA-microsatellite, which promotes EwS growth in vitro and in vivo. Through integration of 2-Atractylenolide 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. gene and variable members of the ETS-family of transcription factors, most commonly (85% of all cases)4,5. Prior studies demonstrated that Mouse monoclonal antibody to ATP Citrate Lyase. ATP citrate lyase is the primary enzyme responsible for the synthesis of cytosolic acetyl-CoA inmany tissues. The enzyme is a tetramer (relative molecular weight approximately 440,000) ofapparently identical subunits. It catalyzes the formation of acetyl-CoA and oxaloacetate fromcitrate and CoA with a concomitant hydrolysis of ATP to ADP and phosphate. The product,acetyl-CoA, serves several important biosynthetic pathways, including lipogenesis andcholesterogenesis. In nervous tissue, ATP citrate-lyase may be involved in the biosynthesis ofacetylcholine. Two transcript variants encoding distinct isoforms have been identified for thisgene acts as a pioneer transcription factor that massively rewires the tumor transcriptome ultimately promoting the malignant phenotype of EwS6,7. This is in part mediated through interference with and/or aberrant activation of developmental pathways3,8. Remarkably, EWSR1-FLI1 regulates ~40% of its target genes by binding to otherwise non-functional GGAA-microsatellites (mSats)9 that are thereby converted into potent de novo enhancers, whose activity increases with the number of consecutive GGAA-repeats7,10C12. Although EWSR1-FLI1 would in principle constitute a highly specific target for therapy, this fusion oncoprotein proved to be notoriously difficult to target due to its intranuclear localization, its activity as a transcription factor13,14, the absence of regulatory protein residues1, its low immunogenicity15, and the high and ubiquitous expression of its constituting genes in adult tissues1. Hence, we reasoned that developmental genes and pathways that are aberrantly activated by EWSR1-FLI1 and virtually inactive in normal adult tissues, could constitute druggable surrogate targets. As EwS most commonly arises in bone and possibly descends from osteo-chondrogenic progenitor cells3, we speculated that EWSR1-FLI1 might interfere with bone developmental pathways. The transcription and splicing factor SOX6 (SRY-box 6) plays an important role in endochondral ossification16. Interestingly, its transient high expression delineates cells along the osteo-chondrogenic lineage showing high rates of proliferation while maintaining an immature phenotype along this lineage17C19. In the current study, we show that 2-Atractylenolide EWSR1-FLI1 binds to an intronic GGAA-mSat within expression levels (Affymetrix microarrays) in EwS tumors, nine additional sarcoma or pediatric tumor entities, and 18 normal tissue types. Data are represented as dot plots, horizontal bars represent medians. The number of biologically independent samples per group (expression via an intronic GGAA-mSat The relatively high expression of in EwS compared to other sarcomas and pediatric cancers implied that there might be a regulatory relationship with the EwS specific fusion oncogene in A673/TR/shEF1 and SK-N-MC/TR/shEF1 cells harboring a doxycycline (Dox)-inducible short hairpin RNA (shRNA) against the fusion gene, strongly reduced expression in a time-dependent manner in vitro (Fig.?2a, Supplementary Fig.?2a) and in vivo (Fig.?2b). Conversely, ectopic expression of in human embryoid bodies strongly induced expression (Fig.?2c). Open in a separate window Fig. 2 EWSR1-FLI1 induces expression via an intronic GGAA-mSat.a and expression (qRT-PCR) in A673/TR/shEF1 cells after addition of Dox. Horizontal bars represent means, values determined by two-sided MannCWhitney test. 72?h (72?h (and expression (Affymetrix microarrays) in A673/TR/shEF1 xenografts after 96?h of Dox-treatment. Horizontal bars represent means, value determined via two-sided independent one-sample expression (Affymetrix microarrays) in embryoid bodies after ectopic expression. Horizontal bars represent means, value determined via unpaired two-sided value determined via two-sided MannCWhitney test (levels across eight EwS cell lines (TC-32 set as reference). The color code indicates the average number of consecutive GGAA-repeats of both alleles. value determined via two-tailed Pearson correlation test, knockdown (Fig.?2d). This EWSR1-FLI1 peak mapped to a GGAA-mSat located within a DNase 1 hypersensitivity site, indicating open chromatin, and showed EWSR1-FLI1-dependent acetylation of H3K27, which marks active enhancers (Fig.?2d). The EWSR1-FLI1-dependent enhancer activity of this GGAA-mSat was.