Oncogenic hijacking of a developmental transcription factor evokes therapeutic vulnerability for ROS-induction in Ewing sarcoma

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 (mSats) as enhancers. Here, we show that EWSR1-FLI1 hijacks the developmental transcription factor SOX6 – a physiological driver of proliferation of osteo-chondrogenic progenitors – by binding to an intronic GGAA-mSat, which promotes EwS growth in vitro and in vivo. Through integration of transcriptome-profiling, published drug-screening data, and functional in vitro and in vivo experiments, we discovered that SOX6 interferes with the antioxidant system resulting in constitutively elevated reactive oxygen species (ROS) levels that create a therapeutic vulnerability toward the ROS-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.

microsatellites (mSats) 9 that are thereby converted into potent de novo enhancers, whose activity increases with the number of consecutive GGAA-repeats 7,10-14 .
Although EWSR1-FLI1 would in principle constitute a highly specific target for therapy, this fusion oncoprotein proved to be notoriously difficult to drug due to its intranuclear localization, its activity as a transcription factor 15,16 , the absence of regulatory protein residues 1 , its low immunogenicity 17 , and the high and ubiquitous expression of its constituting genes in adult tissues 1 . 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 arise in bone and possibly descend from osteo-chondrogenic progenitor cells, 3 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 ossification 18 . Interestingly, its transient high expression delineates cells along the osteo-chondrogenic lineage showing high rates of proliferation while maintaining an immature phenotype along this lineage [19][20][21][22] .
In the current study, we show that EWSR1-FLI1 binds to an intronic GGAA-mSat within SOX6, which acts as an EWSR1-FLI1-dependent enhancer that induces the high and constitutive overexpression of SOX6 in EwS tumors. Moreover, we report that SOX6 promotes proliferation and tumorigenicity of EwS cells, and confers a druggable, therapeutic vulnerability toward the reactive oxygen species (ROS)-inducing small molecule Elesclomol, through upregulation of cell intrinsic ROS by interference with the antioxidant system.

SOX6 is highly but variably expressed in EwS
To explore the expression pattern of SOX6, we took advantage of a well-curated set of >750 DNA microarrays, which we established previously 23,24 , comprising 18 representative normal tissues types and 10 cancer entities. Comparative analyses revealed that SOX6 is overexpressed in EwS relative to normal tissues and other cancers (Fig. 1a). These data were validated on the protein level in a tissue microarray 23,24 comprising the same normal tissue types and cancer entities (Fig. 1b,c). Both analyses showed that SOX6 is highly expressed in EwS tumors, albeit with substantial inter-tumor heterogeneity.
The generally high but variable expression of SOX6 was also observed in EwS cell line models compared to cell lines of three other pediatric cancer types including osteosarcoma (U2OS and SAOS-2), neuroblastoma (TGW and SK-N-AS) and rhabdomyosarcoma (Rh36 and Rh4) ( Supplementary Fig. 1).

EWSR1-FLI1 induces SOX6 expression via an intronic enhancer-like GGAA-mSat
The relatively high expression of SOX6 in EwS compared to other sarcomas and pediatric cancers implied that there might be a regulatory relationship with the EwS specific fusion oncogene EWSR1-FLI1. Indeed, knockdown of EWSR1-FLI1 in A673/TR/shEF1 and SK-N-MC/TR/shEF1 cells harboring a Dox-inducible short hairpin RNA (shRNA) against the fusion gene strongly reduced SOX6 expression in a time-dependent manner in vitro (Fig. 2a, b,   Supplementary Fig. 2a) and in vivo (Fig. 2b). Conversely, ectopic expression of EWSR1-FLI1 in human embryoid bodies strongly induced SOX6 expression (Fig. 2c).
To investigate the underlying mechanism of this regulatory relationship, we analyzed publicly available DNase-Seq and ChIP-Seq data of two EwS cell lines (A673 and SK-N-MC) and found a prominent EWSR1-FLI1 peak within intron 1 of SOX6, which was strongly reduced upon EWSR1-FLI1 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-FLI1dependent acetylation of H3K27, which marks active enhancers (Fig. 2d). The EWSR1-FLI1dependent enhancer activity of this GGAA-mSat was confirmed by luciferase reporter assays in A673/TR/shEF1 cells transfected with pGL3 reporter plasmids in which we cloned a 1-kb fragment containing this SOX6-associated GGAA-mSat from the human reference genome (Fig. 2e).
As prior studies showed that the enhancer activity at EWSR1-FLI1-bound GGAA-mSats positively correlates with the number of consecutive GGAA-repeats 12,25 , we hypothesized that the observed variability in SOX6 expression might be caused by differences in repeat numbers at the SOX6-associated GGAA-mSat. To test this possibility, we cloned both alleles for this mSat from six EwS cell lines with largely different SOX6 expression levels ( Supplementary   Fig. 1), determined their repeat number by Sanger sequencing, and measured their enhancer activity in reporter assays. We observed a positive correlation (P = 0.047) of the average SOX6 expression levels with the observed average enhancer activity across cell lines, which corresponded to the average repeat numbers of both alleles (Fig. 2f, Supplementary Table 1).
Interestingly, the inter-individual differences in SOX6 expression levels correlated neither with (minor) differences of SOX6 promoter methylation nor with copy number variations at the SOX6 locus in primary EwS tumors (Supplementary Fig. 2b, c).
Collectively, these data suggest that EWSR1-FLI1 induces SOX6 by binding to a polymorphic intronic GGAA-mSat, which exhibits length-dependent enhancer activity.

SOX6 promotes proliferation of EwS cells in vitro and in vivo
To explore the possible function of SOX6 in EwS, we generated two cell lines (RDES and TC-32) with doxycycline (Dox)-inducible shRNAs against SOX6 (shSOX6_2 and shSOX6_3) and corresponding controls with a Dox-inducible non-targeting control shRNA (shCtrl). In these transduced cells, addition of Dox (0.1 µg/ml) to the culture medium effectively silenced SOX6 expression at the mRNA and protein level (Fig. 3a, Supplementary Fig. 3a).
Since SOX6 acts -depending on the cellular context -as a splicing and/or transcription factor 26,27 , we explored the effect of SOX6 knockdown in RDES and TC-32 EwS cell lines using Affymetrix Clariom D arrays, which enable the simultaneous transcriptome-wide analysis of splicing events and differential gene expression. While the knockdown of SOX6 for 96h had little effect on splicing (Supplementary Table 2), we noted a strong effect on differential gene expression (Fig. 3b). In fact, SOX6 silencing induced a concordant up-or downregulation (FC<−0.5 and FC >+0.5; P<0.05) of 816 and 3,145 genes, respectively, across shRNAs and cell lines (Supplementary Table 3). Gene set enrichment analysis (GSEA) of these differentially expressed genes (DEGs) identified a strong depletion of proliferation-related gene signatures in SOX6 silenced EwS cells (Fig. 3b, Supplementary Table 4).
To validate the predicted role of SOX6 in EwS proliferation, we performed knockdown experiments using pooled short interfering RNAs (sipool) against SOX6 in three EwS cell lines (POE, RDES, TC-32). Each sipool consisted of 30 different siRNAs, which virtually eliminates off-target effects 28 , and which induced a 60−80% SOX6 knockdown as compared to a nontargeting control sipool (sipCtrl) after 96h. In these experiments, we noted a significant reduction of the viable cell count in all EwS cell lines in standardized cell counting experiments (including the supernatant) (Supplementary Fig. 3b). In accordance, Dox-induced long-term SOX6 knockdown significantly reduced the 2D clonogenic and 3D sphere formation capacities of EwS cell lines as compared to controls (Dox (−) and shCtrl) (Fig. 3c, Supplementary   Fig. 3c).
To test whether this effect was mediated via alteration of the cell cycle, we carried out flow cytometric assays with propidium iodine (PI). In serum-starved and thus G0-synchronized cells, we observed a significant delay in cell cycle progression 20h after re-addition of serum in SOX6-silenced cells (Supplementary Fig. 3d).
To assess the potential contribution of SOX6 to tumor growth of EwS cells in vivo, we performed xenograft experiments by injecting two different EwS cell lines with Dox-inducible shRNAs against SOX6 subcutaneously into the flanks of NSG mice. While no effect of Doxtreatment was apparent in EwS cell lines expressing the non-targeting control shRNA, we noted a strong and consistent reduction of tumor growth upon SOX6 knockdown in both shRNA constructs and both cell lines (Fig. 3d). The knockdown of SOX6 was confirmed ex vivo in xenografts by qRT-PCR (Fig. 3e, Supplementary Fig. 3e) and by immunohistochemistry (IHC) (not shown). Immunohistological assessment showed that SOX6 silencing was associated with a significant reduction of proliferation as indicated by numbers of mitotic cells per high-power filed (HPF) and Ki67 stains (Fig. 3f, Supplementary Fig. 3f). In contrast, no significant differences in cleaved caspase 3 and Annexin V staining were observed (Fig. 3g,   Supplementary Fig. 3g), suggesting that the apparent reduction of tumor growth was not mediated by apoptotic cell death.
Among the proliferation-associated genes downregulated after SOX6 knockdown (Fig. 3b), three genes (CDCA3, DEPDC1 and E2F8) appeared as plausible candidate genes to promote the pro-proliferative phenotype of SOX6, as they were previously shown to be involved in cell cycle progression [29][30][31][32] . In accordance, knockdown of any one of the genes with specific sipools in RDES and TC-32 EwS cells phenocopied, at least in part, the proliferative effect of SOX6 ( Supplementary Fig. 3h, i).
Collectively, these results highlight a contribution of SOX6 to proliferation, clonogenic growth and tumorigenicity of EwS cells.

High expression of SOX6 confers sensitivity toward the small-molecule Elesclomol in EwS
To explore whether high SOX6 levels could constitute a specific vulnerability of EwS that may be exploited therapeutically, we interrogated a published gene expression dataset with matched drug-response data comprising 22 EwS cell lines 33 . To this end, we calculated for all 264 tested drugs the Pearson correlation coefficient and its statistical significance of the corresponding IC50 values with the observed SOX6 expression levels across EwS cell lines (Fig. 4a).
Among the top 7 drugs, Elesclomol (N-malonyl-bis (N-methyl-N-thiobenzoyl hydrazide) (rPearson= −0.565; P=0.014) was the only drug, which effectively could inhibit EwS growth at a nanomolar range (IC50 ~20 nM) (Fig. 4b). Elesclomol is a potent oxidative stress inducer, which is believed to exert its pro-apoptotic effects in cancer cells via elevating ROS levels beyond a tolerable threshold 34 . Indeed, in validation drug-response assays, Elesclomol strongly decreased viability of EwS cells with high SOX6 levels while the osteosarcoma cell line SAOS-2 and non-transformed human primary MSC line MSC-52 that exhibit low SOX6 expression levels were relatively resistant (Fig. 4c, d). The high sensitivity of EwS cells toward Elesclomol appeared to be independent of proliferation under normal conditions, since the osteosarcoma cell line SAOS-2 proliferated even more than the tested EwS cells (Fig. 4e). Yet, knockdown of SOX6 in RDES and TC-32 EwS cells significantly diminished their sensitivity toward Elesclomol (Fig. 4f), pointing to a functional role of SOX6 in Elesclomol-sensitivity.
Consistent with a prior report in other cancer cell lines 34 , Elesclomol strongly induced apoptosis in EwS cell lines in vitro when treated with corresponding IC50 concentrations (Fig. 4g), without affecting SOX6 expression levels (Supplementary Fig. 4a). In accordance, intravenous administration of Elesclomol for 9 days reduced local tumor growth of TC-32 EwS xenografts in vivo (Fig. 4h), which was accompanied by induction of apoptosis and cell death as evidenced by significantly increased numbers of cells positive for cleaved caspase 3 ( Fig. 4i), and more necrotic tumor area (Fig. 4j). Of note, mice treated with Elesclomol did not exhibit overt adverse effects such as weight-loss (Supplementary Fig. 4b) or histo-morphological changes in the inner organs (not shown).
In sum, these results demonstrate that SOX6 expression confers a proliferation-independent sensitivity toward the ROS-inducing small-molecule Elesclomol to EwS cells.

SOX6 induces intracellular ROS through interference with the antioxidant system
Since Elesclomol can induce oxidative stress, we investigated whether Elesclomol treatment modulates ROS levels in EwS cell lines and why EwS cells are sensitive to Elesclomol. Indeed, treatment of RDES and TC-32 cells with Elesclomol (10 nM) significantly induced ROS in both EwS cell lines compared to control (DMSO) (Fig. 5a). To test whether ROS levels play a role in the capacity of Elesclomol to kill EwS cells, we carried out drug-response assays in the presence/absence of the antioxidant N-acetylcysteine (Nac), which is able to scavenge free radicals 35 . In both cell lines, Nac-treatment resulted in significantly increased IC50 values indicating that Elesclomol exerts its pro-apoptotic effect in EwS via ROS (Fig. 5b).
In line with this hypothesis, Dox-induced knockdown of SOX6 reduced ROS levels in both EwS cell lines (RDES and TC-32) and for both shRNA constructs, which was not observed in corresponding controls (shCtrl) (Fig. 5c). To functionally validate the ROS-dependent sensitivity toward Elesclomol conferred by SOX6 on EwS cells, we performed rescue experiments. In those we noted that addition of the potent ROS-inducer H2O2 on the SOX6 silenced EwS cells could fully restore the sensitivity of these cell lines toward Elesclomol ( Fig. 5d), while having no effect on viability of EwS cells that were not treated with Elesclomol (Supplementary Figure 5).
These data suggest that SOX6 is involved in ROS metabolism and prompt further analysis of our available microarray data obtained from EwS cells with/without SOX6 knockdown.
Although we did not find evidence for a systematic enrichment/depletion of ROS-associated pathways in our GSEA, we identified TXNIP (thioredoxin interacting protein) -a key inhibitor of the thioredoxin antioxidant system -among the top 10 downregulated genes after SOX6 Table 3). The downregulation of TXNIP after silencing of SOX6 was confirmed in independent experiments on mRNA and protein level (Fig. 5e). Interestingly, knockdown of TXNIP in EwS cells reduced intracellular ROS levels ( Fig. 5f).

silencing (Supplementary
Taken together, these data suggest that SOX6 interferes via TXNIP with the antioxidant system, which increases intracellular ROS levels and thus promotes Elesclomol sensitivity in EwS cells ( Fig. 5g).

DISCUSSION
EwS is a highly aggressive cancer, affecting bone or soft-tissue, possibly descending from chondro-/osteo-progenitors. Since the transcription factor SOX6 is crucial for endochondral ossification and thus for bone development 18,36 , we aimed at analyzing its role in EwS.
Our results show that SOX6 is a direct EWSR1-FLI1 target gene that is highly but variably overexpressed on the mRNA and protein level in EwS as compared to most normal tissues and other cancers. However, we found no correlation of copy number variations and differences in promoter methylation with SOX6 expression levels in EwS tumors. In contrast, we identified an intronic SOX6-associated GGAA-mSat that was bound by EWSR1-FLI1 in vivo and which exhibited strong length-and EWSR1-FLI1-dependent enhancer activity in EwS cell lines. Thus, it is tempting to speculate that the observed inter-tumor heterogeneity in SOX6 expression of EwS tumors and EwS cell lines is likely caused by inter-individual differences in the number of consecutive GGAA-repeats at this SOX6-associated GGAA-mSat. These findings are in line with recent observations for other EWSR1-FLI1 target genes such as EGR2 and NR0B1 whose variable expression in EwS tumors is caused by inter-individual differences in GGAA-repeat numbers of the corresponding enhancer-like GGAA-mSat 1,37 .
Depending on the cellular context, SOX6 may act as a splicing factor 26,27 or as transcriptional regulator 36 . 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.
Instead, we identified a broad deregulation of a large number of genes after SOX6 silencing, pointing to a more pronounced role of SOX6 as a transcription factor in EwS. Especially the downregulated DEGs after SOX6 knockdown were significantly enriched for gene sets involved in proliferation and cell cycle progression. These changes in the cellular transcriptome were mirrored in functional in vitro and in vivo experiments. The strong pro-tumorigenic function of SOX6 in EwS is intriguing in other cancer entities such as esophageal squamous cell carcinoma and hepatocellular carcinoma SOX6 was reported to act as a tumor suppressor 38,39 . However, in EwS, knockdown of SOX6 strongly reduced anchorageindependent growth and tumorigenicity, which was accompanied by delayed transition through cell cycle phases and reduced expression of the proliferation marker Ki67. Thus, our results suggest that SOX6 may also have oncogenic properties, and that its oncogenic or tumorsuppressive function may depend on the cellular context.
Since novel therapeutic options for EwS patients are urgently required 3 , we investigated whether the high expression of SOX6 in EwS may provide a vulnerability that could be exploited therapeutically. Indeed, we discovered that high expression of SOX6 confers hypersensitivity toward the small-molecule Elesclomol. While Elesclomol was shown to inhibit cancer cell growth in vitro at micro-molar concentrations in melanoma, breast cancer and leukemia cell lines [40][41][42][43] we noted a much higher sensitivity of EwS cells toward Elesclomol with IC50 values in the nano-molar range. These observations suggest that the higher sensitivity of EwS toward Elesclomol may be caused by the relatively higher expression of SOX6 in EwS compared to other cancers such as osteosarcoma and melanoma (Supplementary Fig. 4c). In support of this hypothesis, Elesclomol-treatment in combination with paclitaxel had only moderate effect on outcome of unselected patients affected by malignant melanoma in phase II and III clinical trials 44,45 , and may have shown higher efficacy when preselecting patients with higher SOX6 levels or higher intracellular ROS levels.
Since many cancer types including EwS display an oxidative stress phenotype characterized by higher ROS levels than normal tissues 46,47 , cancer cells tend to be more sensitive toward further increases in oxidative stress as nonmalignant cells 48 . Previous reports demonstrated that Elesclomol can induce ROS levels beyond a tolerable threshold triggering apoptosis 34,41,49 . In line with these findings, we observed an increase of ROS followed by apoptosis after The elevated intracellular ROS levels and associated hyper-sensitivity of SOX6 high expressing EwS cells toward Elesclomol can be explained, at least in part, by the SOX6-mediated upregulation of TXNIP, an inhibitor of the thioredoxin (TRX) antioxidant system that plays an essential role in buffering intracellular ROS levels 50,51 .
These data suggest that ROS-inducing drugs such as Elesclomol could offer a new therapeutic option for EwS patients with high SOX6 expression levels. Additionally, SOX6 may serve as a biomarker to predict the efficacy of Elesclomol treatment in EwS, and perhaps other cancer types. Interestingly, Elesclomol treatment has been shown to potentiate the pro-apoptotic effect of ROS-dependent chemotherapeutic drugs such as doxorubicin in breast cancer 42 . Since doxorubicin is part of current standard treatment regimens for EwS patients, it is tempting to speculate that Elesclomol treatment may also serve as an enhancer for doxorubicin treatment, even in patients with relatively low intratumoral SOX6 levels.
In synopsis, we discovered that EWSR1-FLI1 hijacks SOX6 in EwS, which promotes tumor growth, and interferes with the antioxidant system creating a therapeutic vulnerability toward ROS-inducing drugs. Our results exemplify how aberrant activation of a developmental transcription factor by a dominant oncogene can promote malignancy but provide opportunities for targeted therapy.

Cell lines and cell culture conditions
The neuroblastoma cell line SK-N-AS as well as HEK293T were purchased from ATCC. Cells were lysed after 72h and monitored with a dual luciferase assay system (Berthold, Germany). Firefly luciferase activity was normalized to Renilla luciferase activity.

Analysis of copy-number-variation (CNV) and promoter methylation in primary EwS
For CNV analysis, publicly available DNA copy number data for EwS tumors 53 with corresponding RNA expression data (GSE34620 and GSE37371, n=32) , were downloaded from the 'soft tissue cancer -Ewing sarcoma -FR' project from the International Cancer Genome Consortium (ICGC) Data Portal and Gene Expression Omnibus (GEO) of the NCBI, respectively. For the SOX6 locus, segment mean values were extracted from these data using Visual Basic for Applications (VBA). The segment mean values were correlated with the log2transformed expression of the candidate gene. For CpG methylation analysis, publicly available data on CpG methylation in 40 EwS tumors (GSE88826) 54 with corresponding RNA expression data (GSE34620) were downloaded from GEO. For the SOX6 locus, the ratio of methylated versus unmethylated reads was calculated for two CpG sites (CpG1 Hg19: chr11:15994482; CpG2 Hg19: chr11:15994519) in each sample (n=40) using VBA, which were covered by at least four reads.

Analysis of SOX6 expression levels in human embryoid bodies
Publicly available gene expression microarray data for ectopic EWSR1-FLI1 expression in human embryoid bodies generated on the Affymetrix HG-U133Plus2.0 array (GSE64686) 55 were normalized by Robust Multiarray Average (RMA) 56 using custom brainarray chip description files (CDF; ENTREZG, v19) yielding one optimized probe-set per gene 57 .

Analysis of published DNase sequencing (DNase-Seq) and chromatin immuneprecipitation followed by high-throughput DNA sequencing (ChIP-Seq) data
ENCODE SK-N-MC DNase-Seq (GSM736570) and ChIP-Seq data (GSE61944) were downloaded from the GEO, processed as previously described 25

Transcriptome and splicing analyses
To asses an impact of SOX6 on gene expression and on alternative splicing in EwS, microarray analysis was performed. To this end, 1.2×10 4 cells were seeded in wells of 6-well plates and treated with Dox for 96h (Dox-refreshment after 48h). Thereafter, total RNA was extracted with the ReliaPrep RNA Cell Miniprep System (Promega) and transcriptome profiled at IMGM laboratories (Germany). RNA quality was assessed with a Bioanalyzer and samples with RNA integrity numbers (RIN)>9 were hybridized to Human Affymetrix Clariom D microarrays. Data were log2-transformed quantile normalized with Affymetrix Expression Console Software (v1.4) using the SST-RMA algorithm as previously described 58 . Annotation of the data was However, none of the PSRs remained significant after Bonferroni correction for multiple testing. Gene expression data were deposited at the GEO (accession code GSE120576). (1:5000, R1364HRP, OriGene, Germany). Proteins were detected using chemiluminescence HRP substrate (Merck). Densitometric protein quantifications were carried out by ImageJ.

Proliferation assays
For proliferation assays, 2×10 5 EwS cells were seeded in wells of 6-well plates and treated with 0.1 µg/ml Dox every 48h for knockdown or transiently transfected with Lipofectamine RNAiMAX (Invitrogen, USA) and the respective sipool every 48h for a total period of 96h.

Cell viability was determined including the supernatant by counting the cells with Trypan-Blue
(Sigma-Aldrich) in standardized hemocytometers (C-Chip, Biochrom).

Clonogenic growth assays
For clonogenic growth assays, RDES and TC-32 harboring shRNAs against SOX6 were seeded at low density (200 cells) per well of a 12-well plate and grown for 21 days with renewal of Dox every 48h. The colonies were counted in three technical replicates and the colony area was measured with the ImageJ Plugin Colony area. The clonogenicity index was calculated by multiplying the counted colonies with the corresponding colony area.

Sphere formation assay
For the analysis of anchorage-independent growth, EwS cell lines RDES and TC-32 harboring Dox-inducible shRNAs against SOX6, were pre-treated with Dox for 48h before seeding. Then, 1×10 3 cells/96-well were seeded in Costar Ultra-low attachment plates (Corning, Germany) for 12 days. 20 µl of fresh medium with/without Dox was added every 48h. At day 12, wells were photographed and spheres larger than 500 µm in diameter were counted. The area was measured using ImageJ. The sphere volumes were calculated as follows: V = 4/3×π×r 3 . The sphere index was calculated by multiplying the counted colonies with the corresponding colony volume.

Cell cycle analysis
For cell cycle analysis, RDES and TC-32 cells harboring a Dox-inducible shRNA against SOX6 were seeded at 4×10 5 cells per 10 cm dish and subsequently starved for 56h. Stimulation of the cells was performed with 10% FCS for 20h. Cells were fixed with ice-cold 70% ethanol, treated with 100 µg/ml RNAse (ThermoFisher, USA) and stained with 50 µg/ml propidium iodide (Sigma Aldrich). Analysis of the cell cycle was performed with BD Accuri C6 Cytometer (BD Biosciences) by counting at least 1×10 5 events. An example for the gating strategy is provided in Supplementary Figure 6a.

Annexin V staining
For analysis of Annexin V positive cells, RDES and TC-32 cells harboring a Dox-inducible shRNA against SOX6 were seeded at 3×10 5 cells/10 cm dish and treated with 0.1 µg/ml Dox every 48h for knockdown. After 96h, cells were washed with PBS and cells were resuspended in 1xAnnexin V buffer (BD Biosciences) with 5µl of Annexin V and 5µl PI solution for further 15 minutes. Analysis of Annexin V positivity was performed with BD Accuri C6 Cytometer (BD Biosciences) by counting at least 1×10 5 events. An example for the gating strategy is provided in Supplementary Figure 6b.

Reactive oxygen species (ROS) detection via DCF-DA fluorescence
For detection of ROS changes after SOX6 knockdown, EwS cells were seeded at a density of 5×10 4 cells/2 ml per 6-well and directly treated for 96h with Dox to induce the knockdown. For the knockdown of TXNIP, TC-32 wild type cells were seeded at a density of 7×10 4 cells/2 ml per 6-well and reversely transfected with siRNA against TXNIP for 72h. At the day of analysis, cells were incubated in their medium with 2.5 µM DCF-DA (ThermoFisher) for 30 min at 37°C.
Afterwards, cells were harvested and resuspended in PBS for flow cytometry analysis with Accuri C6 Cytometer (BD Biosciences). Gating strategy is provided in Supplementary   Figure 6c.

Gene expression and drug response correlation
To identify drugs whose efficacy correlates with SOX6 expression in EwS cells, publicly available EwS cell line gene expression microarray data and drug-response values were downloaded from the EBI (E-MTAB-3610) and from www.cancerrxgene.org 33 . All CEL-files generated on Affymetrix Human Genome U219 arrays were simultaneously normalized using RMA 56 and a custom brainarray chip description file (v20, ENTREZG) yielding one optimized probe set for each gene 57 . For all drugs tested in EwS cell lines, the Pearson correlation coefficient and its significance between SOX6 expression and LN_IC50 values were calculated.
Besides a high negative correlation coefficient and significance level, low IC50 values were chosen as criteria for selection of plausible and potentially relevant gene expression-drug response dependencies.

Human samples and ethics approval
Human tissue samples were retrieved from the archives of the Institute of Pathology of the LMU Munich (Germany) with approval of the institutional review board. The ethics committee of the LMU Munich approved the current study (approval no. 18-481 UE).

Immunohistochemistry (IHC) and immunoreactivity scoring (IRS)
For IHC, 4-µm sections were cut and antigen retrieval was carried out by heat treatment with Target Retrieval Solution (S1699, Agilent Technologies, Germany). The slides were stained with either polyclonal anti-SOX6 antibody raised in rabbit (1:1,600; HPA003908, Atlas was carried out in analogy to scoring of hormone receptor Immune Reactive Score (IRS) ranging from 0-12 as previously described 23 . The percentage of cells with expression of the given antigen was scored and classified in five grades (grade 0 = 0−19%, grade 1 = 20−39%, grade 2 = 40−59%, grade 3 = 60−79% and grade 4 = 80−100%). In addition, the intensity of marker immunoreactivity was determined (grade 0 = none, grade 1 = low, grade 2 = moderate and grade 3 = strong). The product of these two grades defined the final IRS.

Statistical analysis
Statistical data analysis was performed using PRISM 5 (GraphPad Software Inc.) on the raw data. If not otherwise specified in the figure legends, comparison of two groups in functional in vitro experiments was carried out using a two-sided Mann-Whitney test. If not otherwise specified in the figure legends, data are presented as dot plots with horizontal bars representing means and whiskers representing the standard error of the mean (SEM). Sample size for all in vitro experiments were chosen empirically. For in vivo experiments, sample size was predetermined using power calculations with b=0.8 and a<0.05 based on preliminary data and in compliance with the 3R system (replacement, reduction, refinement).

Code availability
Custom code is available from the corresponding author upon reasonable request.

Data availability
The authors declare that all data supporting the findings of this study are available within the article, its extended data files, source data or from the corresponding author upon reasonable request. Original sequencing data that support the findings of this study were deposited at the National Center for Biotechnology Information (NCBI) GEO and are accessible through the series accession number GSE120576.  and SOX6 expression by Affymetrix microarrays in xenografts from A673/TR/shEF1 cells 96h after start of Dox-addition, Horizontal bars represent means and whiskers SEM, n=3. P value determined via independent one-sample t-test. Right: Representative immunohistological stains of xenografts stained for (EWSR1)FLI1 and SOX6. Scale bar=20µm. c) Analysis of SOX6 expression by Affymetrix microarrays in embryoid bodies after ectopic EWSR1-FLI1 expression. Horizontal bars represent means and whiskers SEM, n=3. P value determined via unpaired two-sided t-test with Welch's correction. d) Integrated genomic view of the SOX6 locus displaying tracks for DNAse 1 hypersensitivity (HS) and ChIP-Seq data for EWSR1-FLI1 and H3K27ac in A673 and SK-N-MC EwS cells transfected with shRNA against EWSR1-FLI1 (shEF1) or control shRNA (shGFP). e) Analysis of relative enhancer activity of the SOX6associated GGAA-mSat by dual luciferase reporter assays in A673/TR/shEF1 cells (−/+). Horizontal bars represent means and whiskers SEM, n=4. P value determined via two-sided Mann-Whitney test. f) Correlation of the average enhancer activity of both alleles of the SOX6associated GGAA-mSat and the average SOX6 mRNA expression levels across six EwS cell lines (TC-32 was set as reference). The color code indicates the average number of consecutive GGAA-repeats of both alleles. ***P<0.001, **P<0.01, *P<0.05 d) Analysis of tumor growth of xenografted RDES and TC-32 cells containing either Doxinducible specific shRNAs against SOX6 (shSOX6_2/shSOX6_3) or a non-targeting control shRNA (shCtrl). When tumors were palpable (arrow), mice were randomized and henceforth treated with Dox (+) or vehicle (-). Data are represented as means and SEM, n≥3 mice per condition. P values determined via two-sided Mann-Whitney test. e) Representative micrographs of xenografts from (d) showing IHC stains for SOX6, cleaved caspase 3 and Ki67. Scale bar=20µm. f) Quantification of the relative number of mitoses per high-power field (HPF) of xenografts shown in (d). Horizontal bars represent means and whiskers the SEM, n≥3. P values determined via two-sided Mann-Whitney test. g) Quantification of the relative number of cells positive for cleaved caspase 3 of xenografts shown in (d). Horizontal bars represent means and whiskers the SEM, n≥3. ***P<0.001, **P<0.01, *P<0.05.