Hippo pathway effectors YAP1/TAZ induce an EWS–FLI1‐opposing gene signature and associate with disease progression in Ewing sarcoma

YAP1 and TAZ (WWTR1) oncoproteins are the final transducers of the Hippo tumor suppressor pathway. Deregulation of the pathway leads to YAP1/TAZ activation fostering tumorigenesis in multiple malignant tumor types, including sarcoma. However, oncogenic mutations within the core components of the Hippo pathway are uncommon. Ewing sarcoma (EwS), a pediatric cancer with low mutation rate, is characterized by a canonical fusion involving the gene EWSR1 and FLI1 as the most common partner. The fusion protein is a potent driver of oncogenesis, but secondary alterations are scarce, and little is known about other biological factors that determine the risk of relapse or progression. We have observed YAP1/TAZ expression and transcriptional activity in EwS cell lines. Analyses of 55 primary human EwS samples revealed that high YAP1/TAZ expression was associated with progression of the disease and predicted poorer outcome. We did not observe recurrent SNV or copy number gains/losses in Hippo pathway‐related loci. However, differential CpG methylation of the RASSF1 locus (a regulator of the Hippo pathway) was observed in EwS cell lines compared with mesenchymal stem cells, the putative cell of origin of EwS. Hypermethylation of RASSF1 correlated with the transcriptional silencing of the tumor suppressor isoform RASFF1A, and transcriptional activation of the pro‐tumorigenic isoform RASSF1C, which promotes YAP1/TAZ activation. Knockdown of YAP1/TAZ decreased proliferation and invasion abilities of EwS cells and revealed that YAP1/TAZ transcription activity is inversely correlated with the EWS–FLI1 transcriptional signature. This transcriptional antagonism could be explained partly by EWS–FLI1‐mediated transcriptional repression of TAZ. Thus, YAP1/TAZ may override the transcriptional program induced by the fusion protein, contributing to the phenotypic plasticity determined by dynamic fluctuation of the fusion protein, a recently proposed model for disease dissemination in EwS. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.


Introduction
Ewing sarcoma (EwS) represents the second most common primary malignant bone tumor in children and chance to survive, with a 3-year event-free survival of less than 25% [2,3]. While clinical prognostic markers such as the presence of metastases or tumor volume are established, little is known about the biological factors determining the risk of progression, thus precluding risk-adapted therapeutic approaches. EwS was the first solid malignancy defined by the presence of tumorspecific EWSR1-ETS gene fusions [4], mainly EWSR1-FLI1 translocations, which are considered the main driver of the disease, but fusion type itself does not have any impact on disease progression [5]. As in most developmental cancers, additional recurrent mutations are scarce. The most common somatic mutations have been detected in STAG2, CDKN2A, and TP53, associated with poor prognosis [6,7]. Copy number variation studies by the PROVABES Consortium using samples derived from the EURO-E.W.I.N. G. 99 (EE99) and EWING 2008 trials showed that chromosome 1q gain and possibly chromosome 16q loss define patients with a poor clinical outcome (Díaz-Martín et al, unpublished data), supporting previous retrospective studies [7,8]. However, these secondary alterations occur with a frequency that does not account for the large proportion of patients who relapse.
The Hippo tumor suppressor pathway plays a critical role in tissue and organ size regulation by restraining cell proliferation and apoptosis under homeostatic conditions [9]. Central to the Hippo pathway is a conserved cascade of adaptor proteins and inhibitory kinases that regulate the activity of the oncoproteins YAP1 and TAZ, the final effectors of this pathway in mammals. YAP1/TAZ do not directly bind to DNA, but act as transcriptional coactivators of target genes involved in cell proliferation and survival through their interaction with transcriptional regulators such as TEAD factors [10]. The role of YAP1 and TAZ as important drivers in tumorigenesis has been extensively reported in carcinomas, and they also contribute to malignancies of mesenchymal origin [11][12][13]. In fact, given its key function in developmental processes, an important role has been inferred for Hippo signaling in pediatric cancer [14]. Despite this, somatic or germline mutations in Hippo pathway genes are uncommon, in comparison to other welldefined signaling pathways that are commonly disrupted in cancer [13,15]. Since secondary genetic alterations are scarce in EwS and given the established role of YAP1 and TAZ in cancer without engaging mutation, we aimed to explore the contribution of these factors to oncogenesis in EwS. Herein, we evaluated a series of 55 EwS patients by immunohistochemistry (IHC) for expression/activation of YAP1 and TAZ. We observed a significant association of YAP1/TAZ nuclear expression and disease progression, as well as a potential mechanism of dysregulation involving epigenetic regulation of the RASSF1 locus. Moreover, we demonstrated an interesting interplay between TAZ/YAP1 function with the fusion protein, which fits into a recent model concept for metastatic spreading in EwS based on fluctuations of the expression of the fusion protein [16].

Materials and methods
Tumor samples, TMA construction, and immunohistochemistry (IHC) Tissue samples were obtained from the HUVR-IBiS Biobank (Universitary Hospital Virgen del Rocio-Institute of Biomedicine of Seville Biobank, Andalusian Public Health System Biobank). This study was performed following standard Spanish ethical regulations and was approved by the corresponding ethics committee of the Hospital Virgen del Rocío de Sevilla and the Fundación Pública Andaluza para la Gestión de la Investigación en Salud de Sevilla (FISEVI), Spain. Written informed consent was obtained from all patients and all clinical analyses were conducted in accordance with the principles of the Declaration of Helsinki.
In this study, we analyzed 88 formalin-fixed, paraffinembedded (FFPE) samples from 68 different EwS patients (55 samples corresponding to primary tumor). We also analyzed a subset of 21 frozen samples from the same series (17 primary tumors and four metastasis). Clinical diagnosis of all the samples was performed according to the World Health Organization (WHO) classification [17], performing fluorescence in situ hybridization (FISH) to assess the presence of EwS translocation in tissue sections. The only selection criteria were the availability of pathological data and tissue for tissue microarray (TMA) construction. Medical records were retrospectively reviewed and clinicopathologic information for 55 patients with primary tumor material was retrieved for further analyses (summarized in Table 1).
Representative tumor areas of EwS samples were selected on H&E-stained sections and two 1-mm diameter tissue cores were obtained from each specimen to set up four different TMAs. IHC was carried out on TMA sections using the Envision method (Dako, CA, USA) with a step of heat-induced antigen retrieval and using a primary antibody against YAP1 and TAZ (supplementary material, Table S1). YAP1/TAZ nuclear staining was separately evaluated by two pathologists. Tissue was given a score which resulted from multiplying the nuclear staining intensity from 0 (no staining) to 3 (strong staining) by the extension based on the percentage from positive cells (from 0 to 3). Samples were grouped as negative or weak positive (score 0-2), and strong positive (3)(4)(5)(6)(7)(8)(9). EwS cell lines SKNMC, TTC-466, TC32, A4573, A673,  CADO-ES, RD-ES, RM82, SKES1, STAET10, TC71, and WE68 were obtained from the EuroBoNet cell line panel [18]. MDA-MB-231, MCF7, RH30, SA-OS-2, and PC3 cell lines were purchased from the ATCC YAP1/TAZ expression associates with disease progression in EwS 375

Cell culture and transfection
(Manassas, VA, USA). All tumor cell lines were authenticated by short tandem repeat analyses (CLS, Germany). Primary human bone marrow mesenchymal cells (hMSCs), immortalized with telomerase reverse transcriptase, were provided by D Campana [19]. The A673 cell line engineered to express a doxycyclineinducible shRNA against EWS-FLI1 was kindly provided by J Alonso [20].

Methylation array
Methylation data were generated as described in Puerto-Camacho et al [21]. Data analyses of GSE118872 were performed using the Bioconductor lumi package [22].

Transcriptome analysis
SK-N-MC cells were transfected with control or a combination of YAP1/TAZ siRNAs for 72 h. Whole transcript expression analysis was conducted in four biological replicates of each sample. A 100 ng aliquot of total RNA was amplified using the GeneChip ® WT PLUS reagent kit (Thermo Fisher Scientific) following the manufacturer's instructions. The amplified cDNA was quantified, fragmented, and labeled in preparation for hybridization to GeneChip ® Human Transcriptome 2.0 Array (Thermo Fisher Scientific) using 5.5 μg of single-stranded cDNA product and following protocols outlined in the user manual. CHP files were analyzed by Transcriptome Analysis Console (TAC) 4.0 software (Thermo Fisher Scientific), which performs statistical analysis and provides a list of differentially expressed genes. Gene set enrichment analysis (GSEA v3.0) was performed to identify targets of YAP1/TAZ that were over-represented in previous defined gene sets [23,24].

Statistical analyses
Correlation between immunohistochemical YAP1/TAZ expression and clinicopathological characteristics was assessed by chi-squared test for the categorical variables. The Mann-Whitney test was used for the analysis of differences of the continuous variable age. EwS-specific survival was defined as the time from surgery to the time of death from EwS, with deaths from other causes being censored, whereas in time to relapse analysis, the end point was EwS recurrence, either local or distant. Survival curves were estimated using the Kaplan-Meier method, and the differences in survival were evaluated using the long-rank test. Cox's proportional hazards modeling of parameters potentially related to survival was conducted to calculate hazard ratios (HRs), in both univariate and multivariate analyses. All of these statistical analyses were performed using SPSS v20 (SPSS Inc, Chicago, IL, USA) and JMP10 software (SAS Institute Inc, Cary, NC, USA). p < 0.05 was considered statistically significant. Statistical analysis of in vitro functional assays was performed by using SPSS v20.

Results
YAP1/TAZ are expressed in EwS cell lines and tumor specimens, and are associated with the presence of metastasis and poor prognosis First, we examined YAP1/TAZ expression by western blotting (WB) in 13 EwS cell lines with different pathognomonic gene fusions ( Figure 1A). We observed heterogeneous expression of both proteins across the cell  [25]. YAP1/TAZ expression was also detected in human mesenchymal stem cells (hMSCs) derived from bone marrow, a proposed cell of origin of EwS. Importantly, nuclear expression was observed by subcellular fractionation and immunofluorescence ( Figure 1B,C and supplementary material, Figure S1), suggesting functional transcriptional activity which was confirmed with luciferase reporter assays ( Figure 1D). To test whether YAP1/TAZ abundance was associated with clinical variables in EwS, we analyzed their expression by IHC in a retrospective series of 55 primary tumors (Table 1). YAP1/TAZ-strong expressing tumor cells exhibited intense nuclear staining with a variable signal in the cytoplasm (Figure 2A). YAP1/TAZ expression was also observed in endothelial cells in negative samples, providing an internal positive control for the IHC determination ( Figure 2B). YAP1/TAZ strong expression was associated with disease progression (chi-squared test, p < 0.0054), whereas no significant association was observed with age at surgery or location (Table 1). We also observed increased YAP1/TAZ positivity in metastatic or relapsed tumors in 11 patients with paired primary tumor samples ( Figure 2C-I, paired t-test, p = 0.0204). Additional non-paired metastatic or relapsed tumor samples showed preferential strong expression as well ( Figure 2J, Fisher's exact test, p = 0.006).
We retrieved follow-up data for the EwS patients with primary tumor biopsies to evaluate prognosis (median duration of follow-up 35.23 months), but only 45 had a known relapse date (median duration of follow-up 41.43 months). YAP1/TAZ expression influenced significantly the time to relapse, which was shorter in strong positive patients than in weak/negative patients (mean 127.4 versus 50.66 months, p = 0.011, Figure 2K). Similarly, Kaplan-Meier estimates of EwS-specific survival were shorter (but not significantly) for the YAP1/TAZ strong positive group compared with the YAP1/TAZ weak/negative group (mean 129.32 versus 73.61 months, p = 0.159, Figure 2L). Accordingly, Cox regression   (Table 2). A significant correlation with survival and time to relapse was also observed for metastasis (Table 2). These variables were all included simultaneously, to assess the independent prognostic significance based on multivariate analysis. The adjusted HR of YAP1/TAZ strong expression for relapse did not reach significant confidence after controlling the Cox's regression model for the effects of age, tumor location, and metastasis. However, a significant HR for YAP1/TAZ was obtained in the multivariate analysis regarding overall survival ( Table 2).

Activation of YAP1/TAZ in Ewing sarcoma
We tried to determine the mechanisms that contribute to YAP1/TAZ activation in EwS. We did not find any recurrent somatic mutation in Hippo pathway-related genes in public datasets (supplementary material, Figure S2). Next, we analyzed copy number alterations in a series of 24 EwS using SNP arrays (Figure 3). Gross chromosomal alterations were similar to previous reports, i.e. gains of whole chromosomes 8 and 12 [7]. Copy number gain in the WWTR1 locus, with complete gain of chromosome 3, was detected in a single case. Gain at the YAP1 locus was detected in another case with an almost tetraploid genotype. Regarding the core regulatory kinases of the Hippo pathway and other negative regulators of YAP1/TAZ, no significant copy-loss events were observed ( Figure 3). Focal copy number aberration events in Hippo-related loci were also precluded after inspecting the data with the STAC algorithm (supplementary material, Table S2). Similarly, Hippo-related loci were unaffected in a retrospective series of 165 cases of EwS, which was analyzed within the PROVABES Consortium for validation of biomarkers in EwS (https://www.medizin.uni-muenster. de/provabes/network, Díaz-Martín J, unpublished data). Deregulation of the Hippo pathway leading to YAP1/ TAZ activation could be the consequence of epigenetic silencing of tumor suppressor genes through DNA hypermethylation [11,15,26]. We inspected previous results of the group comparing CpG methylation in EwS cell lines versus hMSCs from EwS patients and healthy donors (GSE118872) [21]. RASSF1 was the only Hippo-related locus showing differential methylation ( Figure 4A). Hypermethylation of RASSF1 accounts for the silencing of RASSF1A transcript expression, but promotes switching to an alternative gene promoter driving the expression of the isoform RASSF1C. RASSF1A contributes to Hippo pathway-mediated repression of YAP1/TAZ, whereas RASSF1C promotes Src family kinase (SFK)-mediated activation of YAP1 [27]. We confirmed the expression of the alternative isoform RASSF1C in EwS cell lines, whereas RASSF1A Figure 3. Summary of the copy number aberrations detected in 24 EwS samples. Frequencies of copy number gain (above axis, blue) and copy number loss (below axis, red) across the human genome. Hippo-related loci are indicated: Tumor suppressor genes such as core kinases of the pathway are marked in black, and oncogenes WWTR1 and YAP1 in red.

YAP1/TAZ expression associates with disease progression in EwS 379
expression was absent or reduced (with the exception of the STAET-10 and TC-32 cell lines) compared with hMSCs ( Figure 4B). Moreover, expression of YAP1/ TAZ target genes positively correlated with RASSF1C expression in the cell line panel, as well as in EwS tumor specimens ( Figure 4B,C). Interestingly, TAZ, but not YAP1, seems to be transcriptionally regulated as CTGF expression correlated with TAZ mRNA expression ( Figure 4C). Correlation of TAZ mRNA levels with Hippo target genes was also observed in larger EwS series in public repository expression data (supplementary material, Figure S3). There is extensive evidence that SFKs can directly phosphorylate YAP1 and TAZ, promoting their activity and stability [28]. Therefore, since RASSF1C activates SFKs in RASSF1-methylated cells [27], we blocked SFK activity by exposing EwS cells to dasatinib. Inhibition of SFKs, monitored as SRC phosphorylation, resulted in reduced cell viability (SK-N-MC IC 50 = 6.55 μM; TTC-466 IC 50 = 2.11 μM) and downregulation of YAP1/TAZ target genes ( Figure 5A). Upon dasatinib treatment, the mRNA levels of YAP1 and TAZ remained unaffected, but TAZ protein expression was decreased and YAP1 inactivating phosphorylation S127 increased in both cell lines ( Figure 5A). As an alternative approach of pharmacologic blockade of YAP1/TAZ activity, we tested pitavastatin. Statins prevent nuclear localization of YAP1/TAZ via inhibition of the enzyme HMG-CoA reductase, ultimately affecting the metabolic control of YAP1/TAZ by the mevalonate pathway [29]. We also observed an antiproliferative effect upon pitavastatin treatment (SK-N-MC IC 50 = 1.83 μM; TTC-466 IC 50 = 1.86 μM), with mild reduction of YAP1/TAZ target genes and TAZ protein downregulation ( Figure 5A). Neither dasatinib nor pitavastatin treatments affected EWS-FLI1 expression in the SK-N-MC cell line, thus precluding that the antiproliferative effect of these drugs was mediated by the fusion protein.

YAP1/TAZ loss-of-function affects cell proliferation and invasion capacity in EwS cells
To assess the oncogenic properties of YAP1 and TAZ in EwS cells, we induced transient knockdown of YAP1, TAZ or simultaneous depletion of both factors (supplementary material, Figure S4), and evaluated cell proliferation, invasion, and migration capacity of the silenced cells. We observed inhibition of proliferation for every individual or combined siRNA transfection. Individual depletion of YAP1 inhibited cell growth more efficiently than TAZ silencing (supplementary material, Figure 5B). Accordingly, Crispr-mediated knockout (KO) of YAP1 reduced colony formation in vitro and impaired tumor growth in a subcutaneous xenograft model ( Figure 5D,E). YAP1/TAZ silenced cells showed a significantly reduced invasive capacity as well ( Figure 5C). The migration capacity of EwS cells upon YAP1/TAZ silencing was not significantly altered compared with the control, but a slight trend toward diminished migration was observed in the double-silenced cells (supplementary material, Figure S5).

YAP1/TAZ-driven transcription activity is inversely correlated with the EWS-FLI1 transcriptional signature
To evaluate the transcriptome modulation by YAP1/ TAZ, we conducted gene expression profiling using Affymetrix microarrays in SK-N-MC cells upon simultaneous silencing of both factors. The microarray data have been deposited in the NCBI Gene Expression Omnibus (accession code GSE120512; https://www.ncbi.nlm.nih. gov/geo/query/acc.cgi?acc=GSE120512). We observed differential expression of 938 coding genes (supplementary material, Table S3) including well-established YAP1/TAZ target genes, such as CYR61, CTGF, and AMOTL2, which were confirmed by RT-qPCR analyses in two EwS cell lines with different gene fusions ( Figure 6A). Similar results were obtained with individual silencing of each factor (supplementary material, Figure S6). Of note, the expression levels of EWS-FLI1 were not affected in SK-N-MC ( Figure 6A) or other EwS cell lines tested (supplementary material, Figure S6).
Next, we collated this transcriptional profile with previously published curated gene sets. Interestingly, we found significant enrichment for several EwSrelated gene signatures both in YAP1/TAZ-correlated and in YAP1/TAZ-inversely correlated genes (Table 3 and Figure 6B). YAP1/TAZ-inversely correlated genes were significantly over-represented among EwS induced gene sets, and YAP1/TAZ-correlated genes overlapped with EwS repressed genes, thus suggesting opposite transcriptional activity of EWS-FLI1 chimeric protein and YAP1/TAZ factors. Accordingly, depletion of the EWS-FLI1 protein in the A673 EwS cell line resulted in the induction of YAP1/TAZ-regulated genes, as well as the TAZ but not the YAP1 factor, the latter showing a slight increase in the phosphorylated fraction ( Figure 6C, left and center panels). TAZ protein upregulation upon EWS-FLI1 silencing could be observed in both nuclear and cytoplasmic compartments ( Figure 6C, right panel). Therefore, transcriptional antagonism may be explained partially by EWS-FLI1-mediated downregulation of TAZ. We confirmed these observations in public datasets for EWS-FLI1 silencing in five EwS cell lines [30], and for ectopic expression of EWS-FLI1 in embryonic stem cells [31] (supplementary material, Figure S7). These observations are in accordance with recent reports describing that several genes are inversely regulated by TEAD factors and EWS-FLI1 [32,33]. TEADs are the main transcription factor partners of YAP1 and TAZ, and usually associate with AP-1 transcription factors at distal enhancers [25,34]. Both TEAD and AP-1 conserved binding motifs are present in EWS-FLI1 regulated genes [32]. Furthermore, EWS-FLI1 binding at the WWTR1 locus coding for TAZ correlates with a decrease of TAZ mRNA expression, suggesting direct repression of TAZ by EWS-FLI1 (supplementary material, Figure S8).

Discussion
In the present study, we have shown that YAP1/TAZ nuclear expression is associated with disease progression and poor prognosis in a large retrospective series of EwS patients. Few reports have addressed this issue so far, and the reported series were smaller, i.e. Ahmed et al [35] observed that YAP1 expression can be detected in 47% of samples (in a series of 32 cases) without an association with survival, whereas in another study with only five EwS cases, 60% and 80% showed YAP1 and TAZ expression, respectively [36]. Other pediatric sarcomas such as rhabdomyosarcoma, osteosarcoma, and neuroblastoma have been reported to express YAP1 and TAZ, with an impact on patient prognosis and conferring resistance to current therapies [37][38][39][40][41]. Moreover, recent studies have revealed that YAP1/TAZ are key signaling mediators of the oncogenic fusion genes in synovial sarcoma, myxoid liposarcoma, and alveolar rhabdomyosarcoma [42][43][44]. This positive functional cooperation in translocated sarcomas contrasts with the antagonism between YAP1/TAZ and EWS-FLI1 that we have observed in EwS.
Another fact that supports the relevance of YAP1/TAZ and other Hippo signaling effectors in sarcomas is their involvement in recurrent fusion genes in certain histological types [45,46]. Notwithstanding, aberrant activation of YAP1/TAZ in cancer is often promoted by mechanisms not involving somatic alterations. We have observed that epigenetic regulation of the RASSF1 locus could affect the expression of YAP1/TAZ target genes in EwS cell lines (Figure 4). This result may explain previous observations describing a correlation of hypermethylation of RASSF1

YAP1/TAZ expression associates with disease progression in EwS 381
and RASSF2 with worse clinical outcome in EwS [47,48]. Moreover, Src kinase activation of invadopodia in response to stress in EwS [49] could be related to SFKmediated activation of YAP1/TAZ by RASSF1C ( Figure 5A). However, YAP1/TAZ activation does not seem to rely on RASSF1 hypermethylation in hMSC (Figure 4), the putative cell of origin of EwS, which exhibits high expression levels of YAP1 and TAZ ( Figure 1). Unaffected expression of total levels of YAP1 and derepression of TAZ upon EWS-FLI1 silencing ( Figure 6E) also support the notion that both factors are maybe expressed in the cell of origin, as proposed for ZEB2, an EMT (epithelial-mesenchymal transition) inducer like YAP1 and TAZ [50]. The association of YAP1/TAZ with metastatic spread could arguably be related to the relative levels of the fusion protein, recently reported to promote phenotypic plasticity of EwS cells [16]. In this scenario, YAP1/TAZ may promote a mesenchymal phenotype in EWS-FLI1-depleted EwS cells together with Wnt/beta-catenin [51], since it is well established that the crosstalk between Hippo and Wnt signaling is essential for tumor progression in several types of cancer [52]. As has been described for Wnt/beta-catenin [51], the opposing transcriptional signature between YAP1/TAZ and EWS-FLI1 could partly contribute to the metastatic process; i.e. we found strong downregulation or upregulation of LOX (a mediator of metastasis [16]) in YAP1/TAZ-silenced or EWS-FLI1-silenced cells, respectively. These results suggest that LOX expression in EwS could be explained by derepression in a low-level state of the fusion protein and/or inducer mechanisms involving YAP1 or TAZ. However, we did not observe differences in LOX expression by IHC in metastatic tumors of eight matched samples, neither did we find differences in FLI1 or NKX2.2 (EWS-FLI induced target) staining in the same matched samples (supplementary Figure S8). Nevertheless, a larger series of paired samples would be necessary to reach a definite conclusion.

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P Rodríguez-Núñez, L Romero-Pérez et al ChIP-seq data from Bilke et al [53] reveal that EWSR1-FLI1 binds at regulatory elements of some of the well-established TAZ/YAP1 target genes [33]. Furthermore, an inverse correlation between AP-1-induced genes and the EWSR1-FLI1 transcriptional signature was observed in the same cell model that we used in this work: inducible silencing of EWSR1-FLI1 in the A673 cell line [33]. It is well established that YAP1/TAZ/ TEAD transcriptional complexes usually cooperate with AP-1 at regulatory DNA modules to synergistically activate target genes [25,34]. Therefore, the transcriptional antagonism might be a consequence of some interference between YAP1/TAZ/TEAD-AP1 complexes and the fusion protein, as demonstrated by Katschnig et al [32]. Another mechanism contributing to the opposing gene signatures might involve Ewing sarcoma-associated transcript 1 (EWSAT1), which we found to be significantly induced in YAP1/TAZsilenced SK-N-MC cells (supplementary material, Table S3). EWSAT1 is a long noncoding RNA that mediates EWS-FLI1 gene repression via interaction with a heterogeneous nuclear ribonucleoprotein [54]. In addition, we have observed inhibition of TAZ expression associated with the presence of EWS-FLI1, which also

YAP1/TAZ expression associates with disease progression in EwS 383
binds DNA at the WWTR1 locus (supplementary material, Figure S9). Indeed, regulation of TAZ seems to occur at the transcriptional level, whereas YAP1 activity is not correlated with mRNA levels ( Figures 4C and 6E). In summary, our study reveals that the interplay between the Hippo pathway effectors YAP1/TAZ and the function of the gene fusion is relevant to shape the transcriptional program in EwS. The transcriptional output elicited by these factors deserves further characterization as our observations provide clinical evidence that YAP1/TAZ expression is associated with disease progression in EwS patients. Studies with larger prospective series are needed in order to corroborate our observations and to establish whether YAP1/ TAZ could serve as reliable biomarkers to stratify and identify patients who could benefit from targeted therapies.

SUPPLEMENTARY MATERIAL ONLINE
Supplementary materials and methods Figure S1. Immunofluorescence microscopy with the indicated antibodies (60× objective magnification) Figure S2. Hippo pathway-associated genes do not harbor recurrent mutations in EwS cell lines and patients  Table S1. Antibodies, suppliers and dilutions, siRNAs, and qPCR primers