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Ewing sarcoma (EwS) is a highly metastatic bone cancer characterized by the ETS fusion oncoprotein EWS-FLI1. EwS cells are phenotypically highly plastic and switch between functionally distinct cell states dependent on EWS-FLI1 fluctuations. Whereas EWS-FLI1high cells proliferate, EWS-FLI1low cells are migratory and invasive. Recently, we reported activation of MRTFB and TEAD, effectors of RhoA and Hippo signalling, upon low EWS-FLI1, orchestrating key steps of the EwS migratory gene expression program. TEAD and its co-activators YAP and TAZ are commonly overexpressed in cancer, providing attractive therapeutic targets. We find TAZ levels to increase in the migratory EWS-FLI1low state and to associate with adverse prognosis in EwS patients. We tested the effects of the potent YAP/TAZ/TEAD complex inhibitor verteporfin on EwS cell migration in vitro and on metastasis in vivo. Verteporfin suppressed expression of EWS-FLI1 regulated cytoskeletal genes involved in actin signalling to the extracellular matrix, effectively blocked F-actin and focal-adhesion assembly and inhibited EwS cell migration at submicromolar concentrations. In a mouse EwS xenograft model, verteporfin treatment reduced relapses at the surgical site and delayed lung metastasis. These data suggest that YAP/TAZ pathway inhibition may prevent EwS cell dissemination and metastasis, justifying further preclinical development of YAP/TAZ inhibitors for EwS treatment.

Adult Schwann cells (SCs) possess an inherent plastic potential. This plasticity allows SCs to acquire repair-specific functions essential for peripheral nerve regeneration. Here, we investigate whether stromal SCs in benign-behaving peripheral neuroblastic tumors adopt a similar cellular state. We profile ganglioneuromas and neuroblastomas, rich and poor in SC stroma, respectively, and peripheral nerves after injury, rich in repair SCs. Indeed, stromal SCs in ganglioneuromas and repair SCs share the expression of nerve repair-associated genes. Neuroblastoma cells, derived from aggressive tumors, respond to primary repair-related SCs and their secretome with increased neuronal differentiation and reduced proliferation. Within the pool of secreted stromal and repair SC factors, we identify EGFL8, a matricellular protein with so far undescribed function, to act as neuritogen and to rewire cellular signaling by activating kinases involved in neurogenesis. In summary, we report that human SCs undergo a similar adaptive response in two patho-physiologically distinct situations, peripheral nerve injury and tumor development., Schwann cells (SCs) can acquire a repair phenotype following nerve injury. Here, the authors show that stromal SCs in ganglioneuromas express nerve-repair genes. Importantly, neuroblastoma cells respond to repair-related SCs increasing neuronal differentiation and reducing proliferation via EGFL8.

Langerhans cell histiocytosis (LCH) is a MAPK pathway-driven disease characterized by the accumulation of CD1a+langerin+ cells of unknown origin. We have previously reported that the Notch signaling pathway is active in LCH lesions and that the Notch ligand Jagged2 (JAG2) induces CD1a and langerin expression in monocytes in vitro. Here we show that Notch signaling induces monocytes to acquire an LCH gene signature and that Notch inhibition suppresses the LCH phenotype. In contrast, while also CD1c+ dendritic cells or IL-4-stimulated CD14+ monocytes acquire CD1a and langerin positivity in culture, their gene expression profiles and surface phenotypes are more different from primary LCH cells. We propose a model where CD14+ monocytes serve as LCH cell precursor and JAG2-mediated activation of the Notch signaling pathway initiates a differentiation of monocytes toward LCH cells in selected niches and thereby contributes to LCH pathogenesis. This study aims to identify the cell of origin of Langerhans Cell Histiocytosis and the molecular mechanisms of its pathogenesis.

Ewing sarcoma (EwS) is an aggressive pediatric bone cancer in need of more effective therapies than currently available. Most research into novel targeted therapeutic approaches is focused on the fusion oncogene EWSR1-FLI1, which is the genetic hallmark of this disease. In this study, a broad range of 3,325 experimental compounds, among them FDA approved drugs and natural products, were screened for their effect on EwS cell viability depending on EWS-FLI1 expression. In a network-based approach we integrated the results from drug perturbation screens and RNA sequencing, comparing EWS-FLI1-high (normal expression) with EWS-FLI1-low (knockdown) conditions, revealing novel interactions between compounds and EWS-FLI1 associated biological processes. The top candidate list of druggable EWS-FLI1 targets included genes involved in translation, histone modification, microtubule structure, topoisomerase activity as well as apoptosis regulation. We confirmed our in silico results using viability and apoptosis assays, underlining the applicability of our integrative and systemic approach. We identified differential sensitivities of Ewing sarcoma cells to BCL-2 family inhibitors dependent on the EWS-FLI1 regulome including altered MCL-1 expression and subcellular localization. This study facilitates the selection of effective targeted approaches for future combinatorial therapies of patients suffering from Ewing sarcoma.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological disease in which multiple genetic abnormalities cooperate in the malignant transformation of T-lymphoid progenitors. Although in pediatric T-ALL, CDKN1B deletions occur in about 12% of the cases and represent one of the most frequent copy number alterations, neither their association with other genetic alterations nor the clinical characteristics of these patients have been determined yet. In this study, we show that loss of CDKN1B increased the prevalence of cell cycle regulator defects in immature T-ALL, usually only ascribed to CDKN2A/B deletions, and that CDKN1B deletions frequently coincide with expression of MEF2C, considered as one of the driving oncogenes in immature early T-cell precursor (ETP) ALL. However, MEF2C-dysregulation was only partially associated with the immunophenotypic characteristics used to define ETP-ALL. Furthermore, MEF2C expression levels were significantly associated with or may even be predictive of the response to glucocorticoid treatment.

A translocation leading to the formation of an oncogenic EWS-ETS fusion protein defines Ewing sarcoma. The most frequent gene fusion, present in 85 percent of Ewing sarcomas, is EWS-FLI1. Here, a high-throughput RNA interference screen was performed to identify genes whose function is critical for EWS-FLI1 driven cell viability. In total, 6781 genes were targeted by siRNA molecules and the screen was performed both in presence and absence of doxycycline-inducible expression of the EWS-FLI1 shRNA in A673/TR/shEF Ewing sarcoma cells. The Leucine rich repeats and WD repeat Domain containing 1 (LRWD1) targeting siRNA pool was the strongest hit reducing cell viability only in EWS-FLI1 expressing Ewing sarcoma cells. LRWD1 had been previously described as a testis specific gene with only limited information on its function. Analysis of LRWD1 mRNA levels in patient samples indicated that high expression associated with poor overall survival in Ewing sarcoma. Gene ontology analysis of LRWD1 co-expressed genes in Ewing tumors revealed association with DNA replication and analysis of differentially expressed genes in LRWD1 depleted Ewing sarcoma cells indicated a role in connective tissue development and cellular morphogenesis. Moreover, EWS-FLI1 repressed genes with repressive H3K27me3 chromatin marks were highly enriched among LRWD1 target genes in A673/TR/shEF Ewing sarcoma cells, suggesting that LRWD1 contributes to EWS-FLI1 driven transcriptional regulation. Taken together, we have identified LRWD1 as a novel regulator of EWS-FLI1 driven cell viability in A673/TR/shEF Ewing sarcoma cells, shown association between high LRWD1 mRNA expression and aggressive disease and identified processes by which LRWD1 may promote oncogenesis in Ewing sarcoma.

Since publication of the article, the authors became aware that Figure 6(A,D) contained errors in the bands and loading controls. The newly compiled Figure 6A and 6D is given below.

Poor prognosis and frequent relapses are major challenges for patients with high-risk neuroblastoma (NB), especially when tumors show MYCN amplification. High-dose chemotherapy triggers apoptosis, necrosis and senescence, a cellular stress response leading to permanent proliferative arrest and a typical senescence-associated secretome (SASP). SASP components reinforce growth-arrest and act immune-stimulatory, while others are tumor-promoting. We evaluated whether metronomic, i.e. long-term, repetitive low-dose, drug treatment induces senescence in vitro and in vivo. And importantly, by using the secretome as a discriminator for beneficial versus adverse effects of senescence, drugs with a tumor-inhibiting SASP were identified. We demonstrate that metronomic application of chemotherapeutic drugs induces therapy-induced senescence, characterized by cell cycle arrest, p21WAF/CIP1 up-regulation and DNA double-strand breaks selectively in MYCN-amplified NB. Low-dose topotecan (TPT) was identified as an inducer of a favorable SASP while lacking NFKB1/p50 activation. In contrast, Bromo-deoxy-uridine induced senescent NB-cells secret a tumor-promoting SASP in a NFKB1/p50-dependent manner. Importantly, TPT-treated senescent tumor cells act growth-inhibitory in a dose-dependent manner on non-senescent tumor cells and MYCN expression is significantly reduced in vitro and in vivo. Furthermore, in a mouse xenotransplant-model for MYCN-amplified NB metronomic TPT leads to senescence selectively in tumor cells, complete or partial remission, prolonged survival and a favorable SASP. This new mode-of-action of metronomic TPT treatment, i.e. promoting a tumor-inhibiting type of senescence in MYCN-amplified tumors, is clinically relevant as metronomic regimens are increasingly implemented in therapy protocols of various cancer entities and are considered as a feasible maintenance treatment option with moderate adverse event profiles.

The ETV6/RUNX1 gene fusion defines the largest genetic subgroup of childhood ALL with overall rapid treatment response. However, up to 15% of cases relapse. Because an impaired glucocorticoid pathway is implicated in disease recurrence we studied the impact of genetic alterations by SNP array analysis in 31 relapsed cases. In 58% of samples, we found deletions in various glucocorticoid signaling pathway-associated genes, but only NR3C1 and ETV6 deletions prevailed in minimal residual disease poor responding and subsequently relapsing cases (p

P53 suppresses tumour growth via its diverse cellular activities, including induction of apoptosis, cell cycle arrest, differentiation and senescence (Vousden and Lu, 2002). Most of these effects reflect the transactivation of a number of genes by p53 acting as a transcription factor, but p53 also activates mitochondrial-dependent apoptotic pathways that are independent of p53 transcriptional activity (Green and Kroemer, 2009). Activation of p53 leads to apoptosis through either the death receptor pathway or the mitochondrial pathway (Selivanova, 2004). In the mitochondrial apoptotic pathway, p53 induces several genes including Bax, APAF-1, Puma and Noxa (Miyashita and Reed, 1995; Burns and El-Deiry, 1999; Lowe and Lin, 2000; Nakano and Vousden, 2001; Robles et al, 2001). TP53 mutations may be associated with an aggressive phenotype and poor prognosis, and some p53 mutants counteract the effects of anticancer agents that attack tumours (Bunz et al, 1999; Poeta et al, 2007). Given the high frequency of p53 mutations in human tumours, reactivation of the p53 pathway has been widely proposed as beneficial for cancer therapy (Selivanova, 2010). In addition, several reported structural studies have shown that mutant p53 core domain unfolding is reversible and, as mutant p53 is expressed at high levels in many tumours, it therefore serves as a potential target for novel cancer therapy (Lambert et al, 2010). Its reactivation will restore p53-dependent apoptosis, among others, resulting in efficient eradication of tumour cells (Tovar et al, 2006). Wild-type p53 can be induced by small organic molecules such as nutlin-3 inhibiting the p53/MDM2 interaction (Vassilev et al, 2004). PRIMA-1 (p53 reactivation and induction of massive apoptosis) and its potent methylated analog, APR-246/PRIMA-1Met, are small molecules that have the ability to convert mt-p53 to an active conformation, thereby restoring its sequence-specific DNA binding and transcriptional activation (Bykov et al, 2002b). It was reported that PRIMA-1 induces cell death through multiple pathways encompassing transcription-dependent and -independent signaling (Chipuk et al, 2003). In vitro and in vivo studies have shown that p53 reactivating small molecules are less toxic to normal cells than to cancer cells and have no significant adverse or genotoxic effects (Stuhmer et al, 2005; Tovar et al, 2006). Although about half of all human malignancies harbour dysfunctional, mutated p53 proteins, approximately 90% of all ES retain wild-type p53, and the downstream DNA damage cell cycle checkpoints and p53 pathways remain functionally intact (de Kovar et al, 1993; Alava et al, 2000; Kovar et al, 2003b; Huang et al, 2005). Exposure of ES cells to Nutlin-3a, a small organic molecule known to reactivate wild-type 53, resulted in a robust apoptotic phenotype that required the presence of wild-type p53 but did not affect the growth of mutant p53 expressing cells (Pishas et al, 2011). TP53 mutation alone rates high among variables, including p16/p14ARF alteration and tumour stage, predicting poorer overall survival in Ewing sarcoma (de Alava et al, 2000; Lopez-Guerrero et al, 2011). Multivariate analysis identified alterations of TP53 as an adverse prognostic factor defining a subset of ES with highly aggressive behaviour and poor chemoresponse (Huang et al, 2005). Therefore, novel treatment options specifically targeting mutant p53 are highly warranted. There is a prospective COG (Children's Oncology Group, USA) study ongoing to validate a retrospective study which strongly suggested that mutant p53 ES constitutes a particularly bad prognostic group (ClinicalTrials.gov identifier: NCT00898053). If data from the retrospective study is confirmed, this would strongly support the need for novel mutant p53 targeting therapeutic strategies in ES. In this study, we investigated whether the small pharmacological molecule APR-246 is able to reactivate mutant p53 in Ewing sarcoma cells in order to drive tumour cells into apoptosis. Using Ewing sarcoma patients' derived cell lines, we show that APR-246 is able to induce apoptosis to variable degrees independent of the p53 status. We observed that cell lines with similar p53 mutations as well as cell lines established from the same patient at different stages of the disease all exhibited variable responses to the drug. To interrogate the molecular basis for the differential responses to APR-246, we performed comparative transcriptomic analysis on the three STA-ET-7 cell lines established from the same patient. Data analysed revealed genes annotating to p53 and apoptosis pathways whose expression varies between the more APR-246-sensitive and the less APR-246-sensitive cell lines. We propose, therefore, that APR-246 will not be a suitable candidate to consider for targeting p53 mutant Ewing sarcoma.

Notch can act as an oncogene or as a tumour suppressor and thus can either promote or inhibit tumour cell growth. To establish Notch status in Ewing's sarcoma family of tumours (ESFT), we investigated the Notch pathway by gene expression profiling meta-analysis or immunohistochemistry in samples obtained from 96 and 24 ESFT patients, respectively. We found that although Notch receptors were highly expressed, Notch did not appear to be active, as evidenced by the absence of Notch receptors in cell nuclei. In contrast, we show that Notch receptors known to be active in colon adenocarcinoma, hepatocarcinoma, and pancreatic carcinoma stain cell nuclei in these tumours. High expression of the Notch effector HES1 transcription factor, usually used as a surrogate marker for active Notch, was also restricted to outside of the nucleus in the majority of ESFT, and analysis of HES1 gene targets indicated HES1 to be transcriptionally inactive. Neither forced activation nor pharmacological or genetic blocking of Notch affected HES1 expression in ESFT cells, indicating HES1 expression to be uncoupled from the Notch pathway. Additional functional studies in ESFT cell lines confirmed Notch to be switched off. Finally, unlike experiments in which HES1 expression was modulated, experimental activation of Notch in ESFT cell lines via several means blocked cell proliferation and reduced their clonogenic potential in soft agar. These indicate that HES1 is uncoupled from Notch in ESFT, that EWS-FLI1-mediated inhibition of Notch contributes to ESFT aggressive cell growth, and support a role for Notch in ESFT tumour suppression, at least partly through the Notch effector HEY1.

Approximately 25% of childhood acute lymphoblastic leukemias carry the ETV6/RUNX1 fusion gene. Despite their excellent initial treatment response, up to 20% of patients relapse. To gain insight into the relapse mechanisms, we analyzed single nucleotide polymorphism arrays for DNA copy number aberrations (CNAs) in 18 matched diagnosis and relapse leukemias. CNAs were more abundant at relapse than at diagnosis (mean 12.5 vs 7.5 per case; P = .01) with 5.3 shared on average. Their patterns revealed a direct clonal relationship with exclusively new aberrations at relapse in only 21.4%, whereas 78.6% shared a common ancestor and subsequently acquired distinct CNA. Moreover, we identified recurrent, mainly nonoverlapping deletions associated with glucocorticoid-mediated apoptosis targeting the Bcl2 modifying factor (BMF) (n = 3), glucocorticoid receptor NR3C1 (n = 4), and components of the mismatch repair pathways (n = 3). Fluorescence in situ hybridization screening of additional 24 relapsed and 72 nonrelapsed ETV6/RUNX1-positive cases demonstrated that BMF deletions were significantly more common in relapse cases (16.6% vs 2.8%; P = .02). Unlike BMF deletions, which were always already present at diagnosis, NR3C1 and mismatch repair aberrations prevailed at relapse. They were all associated with leukemias, which poorly responded to treatment. These findings implicate glucocorticoid-associated drug resistance in ETV6/RUNX1-positive relapse pathogenesis and therefore might help to guide future therapies.

Reversible transitions from a proliferative to a nonproliferative but highly migratory and invasive phenotype, widely known as epithelial-to-mesenchymal transition (EMT), is a prerequisite for wound healing and organ development but also a hallmark of cancer. Recently, phenotypic changes resembling EMT were described to occur in Ewing sarcoma upon modulation of expression of the driver oncogene EWS-FLI1. Evidence was provided for the presence of highly migratory EWS-FLI1-low cells in Ewing sarcoma cell lines as well as in about 20% of primary tumors at low frequency (Franzetti et al., 2017). Importantly, when injected into the tail vein of immunosuppressed mice, EWS-FLI1-low cells were more likely to result in lung metastases than EWS-FLI1-high cells upon re-expression of the oncogene. In our study, we identified a mechanism for the cytoskeletal reprograming underlying EWS-FLI1 dose-dependent phenotypic transitions in Ewing sarcoma. We provide evidence for EWS-FLI1 blocking access of the transcriptional coactivators MRTFB and TAZ to the YAP/TEAD transcription factor complex on enhancers of EWS-FLI1 anti-correlated genes. Upon modulation of EWS-FLI1 expression, TEADs get activated downstream of Rho signaling through recruitment of MRTFB/YAP/TAZ, leading to the expression of key cytoskeletal components and regulators involved in the reorganization of the actin cytoskeleton. Based on these findings, we hypothesized that small-molecule inhibitors of the YAP/TAZ/TEAD pathway should interfere with the migratory phenotype of EWS-FLI1-low cells. In fact, the YAP/TAZ inhibitor verteporfin (Visudyne®), which is clinically used as a photosensitive drug in the treatment of age-related macular degeneration, was able to block Ewing sarcoma cell migration at low nanomolar concentrations without affecting tumor cell proliferation. These results suggest that YAP/TAZ inhibitors may be considered as a promising add-on to conventional combination treatments to prohibit tumor cell dissemination and metastasis. Citation Format: Anna M. Katschnig, Maximilian O. Kauer, Raphaela Schwentner, Eleni M. Tomazou, Dave N.T. Aryee, Heinrich Kovar. Targeting EWS-FLI1 dose-dependent epithelial-to-mesenchymal transition [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A02.

Background: Poor prognosis and frequent relapses are major challenges for patients with high-risk neuroblastoma (NB), especially when tumors show MYCN amplification. High-dose chemotherapy triggers apoptosis, necrosis, and senescence, a cellular stress response leading to permanent proliferative arrest and a typical senescence-associated secretome (SASP). SASP components reinforce growth arrest and act in an immune-stimulatory fashion, while others are tumor promoting. Studies on low-dose long-term, i.e., metronomic, treatment schedules have demonstrated effects on the tumor microenvironment, but have not comprehensively analyzed cellular responses in tumor cells. Methods and Results: We evaluated efficacy and mode of action of low-dose metronomic treatment schedules in in vitro and in vivo NB models. Importantly, by using the secretome as a discriminator for beneficial versus adverse effects of senescence, drugs with a tumor-inhibiting SASP were identified. Our preclinical work has demonstrated that low-dose long-term topotecan (TPT) treatment (0.1 mg/kg/d, i.p., daily over 15 weeks) improves survival in mouse NB xenografts and leads to long-term cure in 40% of mice. TPT induces a tumor-inhibiting type of senescence, accompanied by changes in the tumor transcriptome and secretome, specifically in MYCN-amplified NB cells. As senescent NB cells showed a reduced MYCN copy number and strongly downregulated MYCN expression, we investigated the mechanism of MYCN inactivation. This showed that upon senescence MYCN is epigenetically silenced and gene copies are recruited to the nuclear periphery where changes in the nuclear lamina composition take place. Conclusion: This new mode of action of metronomic TPT treatment, i.e., promoting a tumor-inhibiting type of senescence selectively in MYCN-amplified neuroblastoma, is clinically relevant as metronomic regimens are increasingly implemented in therapy protocols of various cancer entities and are considered as a feasible maintenance treatment option with moderate adverse event profiles. Citation Format: Sabine Taschner-Mandl, Teresa Gerber, Magdalena Schwarz, Johanna Blaha, Maximilian Kauer, Florian Kromp, Nelli Frank, Fikret Rifatbegovic, Tamara Weiss, Ruth Ladenstein, Martin Hohenegger, Inge M. Ambros, Peter F. Ambros. Metronomic topotecan causes MYCN inactivation and impedes tumor growth selectively in MYCN-amplified neuroblastoma cells in vitro and in vivo by therapy-induced senescence [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A17.

Purpose: We report a novel analytic method, named intercohort co-analysis or Ican, which aids in the discovery of genes with predictive value for the progression or outcome of diseases from small-size cohorts. We tested this premise in Ewing's sarcoma (ES), a highly metastatic cancer of bone and soft tissues that lacks validated molecular metastasis and prognostic indicators. Experimental Design: To uncover genes significantly expressed in ES patient subsets, we first determined a nonarbitrary gene expression significance cutoff based on expression levels in validated expressing and nonexpressing tissues. We next searched for genes that were consistently significantly expressed in several ES cohort and cell line datasets. Significantly expressed genes were independently validated by quantitative reverse transcription-PCR in an additional ES cohort. Results: Analysis of ES cohorts revealed marked intercohort gene expression variability. After filtering out the intercohort variability, CXCR4 and CXCR7 were found to be consistently associated with specific ES subsets. Pairwise analyses showed CXCR4 to correlate with ES metastases, and CXCR4 and CXCR7 to patient survival, but not with several other clinicopathological variables. Conclusion: Ican is a powerful novel method to identifying genes consistently associated with particular disease states in cancers for which large cohorts are not available, currently the case of most cancers. We report for the first time that high CXCR4 expression preferentially associates with metastatic ES, and that of CXCR7 with poor patient survival. Clin Cancer Res; 16(14); 3769–78. ©2010 AACR.

Deregulated E2F transcription factor activity occurs in the vast majority of human tumors and has been solidly implicated in disturbances of cell cycle control, proliferation, and apoptosis. Aberrant E2F regulatory activity is often caused by impairment of control through pRB function, but little is known about the interplay of other oncoproteins with E2F. Here we show that ETS transcription factor fusions resulting from disease driving rearrangements in Ewing sarcoma (ES) and prostate cancer (PC) are one such class of oncoproteins. We performed an integrative study of genome-wide DNA-binding and transcription data in EWSR1/FLI1 expressing ES and TMPRSS2/ERG containing PC cells. Supported by promoter activity and mutation analyses, we demonstrate that a large fraction of E2F3 target genes are synergistically coregulated by these aberrant ETS proteins. We propose that the oncogenic effect of ETS fusion oncoproteins is in part mediated by the disruptive effect of the E2F–ETS interaction on cell cycle control. Additionally, a detailed analysis of the regulatory targets of the characteristic EWSR1/FLI1 fusion in ES identifies two functionally distinct gene sets. While synergistic regulation in concert with E2F in the promoter of target genes has a generally activating effect, EWSR1/FLI1 binding independent of E2F3 is predominantly associated with repressed differentiation genes. Thus, EWSR1/FLI1 appears to promote oncogenesis by simultaneously promoting cell proliferation and perturbing differentiation.

Ewing Sarcoma (ES) is the second most common bone cancer in children and adolescents with a high metastatic potential. Tumor development is driven by the specific t(11;22)(q24;q12) chromosomal translocation resulting in the generation of the chimeric transcription factor EWS-FLI1. NAD is a key metabolite of energy metabolism being involved in cellular redox reactions, DNA repair, and in the maintenance of genomic stability serving as a donor of ADP-ribose. This study describes targeting NAMPT (nicotinamide phosphoribosyltransferase), the rate-limiting enzyme in salvage generation of NAD, by FK866 in ES cells. Using FK866 has been proposed as a treatment option for various inflammatory diseases as well as cancer, rendering ES cells with high NAMPT expression especially susceptible to FK866-induced cytotoxicity. Here we report that NAMPT inhibition in ES cells leads to exhaustive NAD depletion, followed by a delayed reduction of ATP levels and concomitantly to apoptosis-mediated cell death. These effects can be reversed by nicotinic acid, a substrate for the NAD salvage generation. However, the use of a doxycycline-inducible shRNA against EWS-FLI1 revealed that the cytotoxic activity of NAMPT inhibition is significantly lowered in the absence of EWS-FLI1. EWS-FLI1-low ES cells have higher viability and lower rates of apoptosis throughout inhibitor treatment compared to cells with high EWS-FLI1 expression. Additionally, changes in mitochondrial respiration and glycolytic rate can be observed when comparing untreated versus EWS-FLI1 knockdown ES cells after NAMPT inhibition. Interestingly, loss of EWS-FLI1 leads to elevated NAD levels and results in alteration of RNA expression of some enzymes involved in the NAD synthesis pathway. These results might explain the high susceptibility of Ewing Sarcoma cells to FK866 treatment. Taken together, our data reveal evidence of an important role of the NAMPT-mediated NAD salvage pathway in the energy homeostasis of ES cells and suggests NAMPT inhibition as a potential new treatment approach for Ewing Sarcoma in combination with standard therapies. Supported by the Austrian Science fund, grant I1225-B19; and the Research Manitoba and CancerCare Manitoba Foundation. Citation Format: Cornelia N. Mutz, Raphaela Schwentner, Eric Bouchard, Edgard M. Mejia, Anna M. Katschnig, Maximilian O. Kauer, Dave N.T. Aryee, Antje Garten, Versha Banerji, Heinrich Kovar. Targeting NAMPT in Ewing's sarcoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1045.

Ewing sarcoma is the second most frequent bone cancer in Caucasian children and young adults, but hardly occurs in Africans. It is characterized by a relatively quiet genome with only one highly recurrent chromosomal aberration, a EWS-ETS (predominantly EWS-FLI1) gene rearrangement. The resulting fusion gene encodes for an aberrant ETS transcription factor which drives sarcomagenesis. In a large GWAS study, three Ewing sarcoma susceptibility loci were discovered on chromosomes 1, 10 and 15, but the molecular mechanisms, how these loci affect tumorigenesis, remained largely unknown. By epigenomic mapping, EWS-FLI1 ChIP-seq, and reporter gene assays we found one of the susceptibility loci to localize to an EWS-FLI1 bound enhancer in the intron of a long non-coding antisense RNA, which we termed lncESST1 (Ewing Sarcoma Susceptibility Transcript 1). Deletion of the ETS binding region in the enhancer by CRISPR/Cas9 mediated gene editing lead to a drastic decrease in lncESST1 expression. Modulation of lncESST expression by siRNA or enhancer deletion significantly reduced soft agar colony formation of Ewing sarcoma cells without affecting growth under adherent culture conditions. Detachment of Ewing sarcoma cells from the plastic surface and growth under spheroid culture conditions induced stress granule formation as an immediate response, and a steady increase in lncESST1 expression over a period of at least 72 hours. We report that lncESST1 binds to a stress granule component with which it shares the promoter, and that loss of lncESST1 expression due to enhancer deletion perturbs stress granule formation. Our study therefore unveils a novel mechanism of lncRNA mediated cancer susceptibility. Supported by FWF grant I1225-B19 Citation Format: Dave NT Aryee, Eleni Tomazou, Maximilian Kauer, Thomas Grünewald, Franck Tirode, Olivier Delattre, Heinrich Kovar. A long noncoding RNA-regulating enhancer links Ewing sarcoma susceptibility to stress response. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 973.

Hypoxia is an important condition in the tumor cell microenvironment and approximately 1% to 1.5% of the genome is transcriptionally responsive to hypoxia with hypoxia-inducible factor-1 (HIF-1) as a major mediator of transcriptional activation. Tumor hypoxia is associated with a more aggressive phenotype of many cancers in adults, but data on pediatric tumors are scarce. Because, by immunohistochemistry, HIF-1α expression was readily detectable in 18 of 28 primary Ewing's sarcoma family tumors (ESFT), a group of highly malignant bone-associated tumors in children and young adults, we studied the effect of hypoxia on ESFT cell lines in vitro. Intriguingly, we found that EWS-FLI1 protein expression, which characterizes ESFT, is upregulated by hypoxia in a HIF-1α–dependent manner. Hypoxia modulated the EWS-FLI1 transcriptional signature relative to normoxic conditions. Both synergistic as well as antagonistic transcriptional effects of EWS-FLI1 and of hypoxia were observed. Consistent with alterations in the expression of metastasis-related genes, hypoxia stimulated the invasiveness and soft agar colony formation of ESFT cells in vitro. Our data represent the first transcriptome analysis of hypoxic ESFT cells and identify hypoxia as an important microenvironmental factor modulating EWS-FLI1 expression and target gene activity with far-reaching consequences for the malignant properties of ESFT. Cancer Res; 70(10); 4015–23. ©2010 AACR.

Background EWS-FLI1 is a chimeric ETS transcription factor that is, due to a chromosomal rearrangement, specifically expressed in Ewing's sarcoma family tumors (ESFT) and is thought to initiate the development of the disease. Previous genomic profiling experiments have identified EWS-FLI1–regulated genes and genes that discriminate ESFT from other sarcomas, but so far a comprehensive analysis of EWS-FLI1–dependent molecular functions characterizing this aggressive cancer is lacking. Methodology/Principal Findings In this study, a molecular function map of ESFT was constructed based on an integrative analysis of gene expression profiling experiments following EWS-FLI1 knockdown in a panel of five ESFT cell lines, and on gene expression data from the same platform of 59 primary ESFT. Out of 80 normal tissues tested, mesenchymal progenitor cells (MPC) were found to fit the hypothesis that EWS-FLI1 is the driving transcriptional force in ESFT best and were therefore used as the reference tissue for the construction of the molecular function map. The interrelations of molecular pathways were visualized by measuring the similarity among annotated gene functions by gene sharing. The molecular function map highlighted distinct clusters of activities for EWS-FLI1 regulated genes in ESFT and revealed a striking difference between EWS-FLI1 up- and down-regulated genes: EWS-FLI1 induced genes mainly belong to cell cycle regulation, proliferation, and response to DNA damage, while repressed genes were associated with differentiation and cell communication. Conclusions/Significance This study revealed that EWS-FLI1 combines by distinct molecular mechanisms two important functions of cellular transformation in one protein, growth promotion and differentiation blockage. By taking MPC as a reference tissue, a significant EWS-FLI1 signature was discovered in ESFT that only partially overlapped with previously published EWS-FLI1–dependent gene expression patterns, identifying a series of novel targets for the chimeric protein in ESFT. Our results may guide target selection for future ESFT specific therapies.

The oncogene EWS-FLI1 encodes a chimeric transcription factor expressed in Ewing's sarcoma family tumors (ESFTs). EWS-FLI1 target gene expression is thought to drive ESFT pathogenesis and, therefore, inhibition of EWS-FLI1 activity holds high therapeutic promise. As the activity of many transcription factors is regulated by post-translational modifications, we studied the presence of modifications on EWS-FLI1. The immuno-purified fusion-protein was recognized by an antibody specific for O-linked beta-N-acetylglucosaminylation, and bound readily to a phosphoprotein-specific dye. Inhibition of Ser/Thr-specific phophatases increased EWS-FLI1 molecular weight and reduced its O-GlcNAc content, suggesting that phosphorylation and O-GlcNAcylation of EWS-FLI1 interact dynamically. By mutation analysis, O-GlcNAcylation was delineated to Ser/Thr residues of the amino-terminal EWS transcriptional-activation domain. Metabolic inhibition of the hexosamine biosynthetic pathway abrogated O-GlcNAcylation of EWS-FLI1 and interfered specifically with transcriptional activation of the EWS-FLI1 target Id2. These results suggest that drugs modulating glycosylation of EWS-FLI1 interfere functionally with its activity and might, therefore, constitute promising additions to the current ESFT chemotherapy.

Background Metastases in the bone marrow (BM) in form of disseminated tumor cells (DTCs) are frequent events at diagnosis and also at relapse in high-risk neuroblastoma patients. The frequently highly diluted occurrence of DTCs requires adequate enrichment strategies to enable their detailed characterization. However, to avoid methodical artifacts we tested whether pre-analytical processing steps—including transport duration, temperature and, importantly, tumor cell enrichment techniques—are confounding factors for gene expression analysis in DTCs. Methods LAN-1 neuroblastoma cells were spiked into tumor free BM and/or peripheral blood and: i) kept at room temperature or at 4°C for 24, 48 and 72 hours; ii) frozen down at -80°C and thawed; iii) enriched via magnetic beads. The effect on the gene expression signature of LAN-1 cells was analyzed by qPCR arrays and gene expression microarrays. Results Neither storage at –80°C in DMSO and subsequent thawing nor enrichment of spiked-in neuroblastoma cells changed the expression of the analyzed genes significantly. Whereas storage at 4°C altered the expression of analyzed genes (14.3%) only at the 72h-timepoint in comparison to the 0h-timepoint, storage at room temperature had a much more profound effect on gene expression by affecting 20% at 24h, 26% at 48h and 43% at 72h of the analyzed genes. Conclusion Using neuroblastoma as a model, we show that tumor cell enrichment by magnetic bead separation has virtually no effect on gene expression in DTCs. However, transport time and temperature can influence the expression profile remarkably. Thus, the expression profile of routinely collected BM samples can be analyzed without concern as long as the transport conditions are monitored.

Cell cycle deregulation and enhanced proliferation of cells is one of the hallmarks of oncogenesis. In Ewing Sarcoma, a highly aggressive pediatric cancer, the chimeric transcription factor EWS-FLI1 deregulates cell cycle by targeting several cell cycle regulators including the E2F family of transcription factors. ChIP-seq studies showed a significant overlap of EWS-FLI1 and E2F3/4 binding in Ewing sarcoma cells. We show that EWS-FLI1 is able to directly activate E2F3, followed by the combinatorial binding of EWS-FLI1 and E2F3 on their target genes synergistically activating their transcription. Furthermore we propose a model where EWS-FLI1 directly exchanges repressive E2F4/p130 by E2F3/pRB thereby driving cells into enhanced cell proliferation. However, so far we were unable to experimentally demonstrate a physical interaction of EWS-FLI1 with any E2F or pocket protein. As an alternative approach to the study of functional synergy between EWS-FLI1 and E2F3, we used time resolved RNA and protein expression data as a basis for mathematical modeling of EWS-FLI1 dependent E2F target gene regulation. By Bayesian model selection, we were able to postulate the formation of a complex between EWS-FLI1 and E2F3 as the most likely explanation for the observed ETS/E2F synergy. This study therefore provides an example of how computational systems approaches can complement experimental data in the discovery of disease mechanisms. This study was supported in part by the Austrian Science Fund (FWF), [grant 22328-B09], and by the 7th framework program of the European Commission, [grant 259348] (‘ASSET’). Citation Format: Raphaela Schwentner, Theodore Papamarkou, Maximilian Kauer, Vassilios Stathopoulos, Fan Yang, Sven Bilke, Paul S. Meltzer, Mark Girolami, Heinrich Kovar. Evidence for E2F/EWS-FLI1 oncoprotein synergism using systems biology. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2104. doi:10.1158/1538-7445.AM2015-2104

Metastasis is the major cause of disease-related death in Ewing sarcoma. Patients, who present with clinically overt disseminated disease at diagnosis and those who relapse early with distant metastases have a poor outcome despite multi-modal high-dose chemotherapy. Therefore, new treatment options are highly warranted. Ewing sarcoma pathogenesis is driven by the chimeric ETS oncogene EWS-FLI1. We here describe regulation of sirtuin SIRT1 by EWS-FLI1 and its role in metastasis. SIRT1 belongs to a family of NAD+-dependent group III deacetylases that target histone and non-histone proteins in response to metabolic stress resulting in widespread gene expression changes through epigenetic and direct transcriptional mechanisms. Among SIRT1 target proteins is the tumor suppressor p53. SIRT1 overexpression prevents p53 acetylation, which is otherwise required for the induction of its transcriptional activity. Also, SIRT1 is involved in negative regulation of several signaling pathways including NOTCH that we had previously described as being inactivated in Ewing sarcoma by the EWS-FLI1 fusion oncogene. Studying the molecular basis of EWS-FLI1 mediated functional p53 perturbation in Ewing sarcoma, we here describe a feed-back regulatory loop in which the NOTCH effector HEY1, which is epigenetically repressed by SIRT1, in turn suppresses SIRT1 expression leading to histone H4K16 and H3K56 acetylation and to p53 activation, and consequently to Ewing sarcoma cell death in vitro. By immunohistochemistry, the study of almost 400 Ewing sarcoma patient samples revealed high SIRT1 expression in about one third of primary tumors and a highly significant association of SIRT1 positivity with Ewing sarcoma metastases. The analysis of 18 paired primary tumor/metastasis samples indicated that SIRT1 was more frequently observed in lung metastases (88%) than in bone marrow metastases (55%), and was already detectable in the corresponding primary tumors in more than 60% of cases. In vitro treatment of Ewing sarcoma cell lines with a pharmacological SIRT1/2 inhibitor resulted in cell death with IC50 values between 0.8 and 3.5 μmol that depended on SIRT1 expression level and p53 status. The greatest sensitivity was obtained with cells that express high SIRT1 and are wildtype for p53. Consistent with this in vitro result, SIRT1 inhibition prohibited tumor cell migration and proliferation in a zebrafish yolk sac xenotransplantation model of a SIRT1 positive, wildtype p53 Ewing sarcoma cell line, but not of a mutant p53 cell line with low SIRT1 expression. Together, these results suggest that SIRT1 expression, which is enabled by EWS-FLI1 mediated suppression of the NOTCH signaling pathway resulting in p53 inactivation, may be therapeutically targeted to fight metastasis using pharmacological sirtuin inhibitors. Supported by EU-FP7 grant 259348 (“ASSET”) and by the Austrian Research Fund FWF, ERA-NET grant I1225-B19 (“PROVABES”). This abstract is also presented as Poster A38. Citation Format: Jozef Ban, Argyrou Fourtouna, Isidro Machado, Stephan Niedan, Ewa Snaar-Jagalska, Dave NT Aryee, Maximilian Kauer, Marco Alberghini, Adrienne Flanagan, Katia Scotlandi, Sandra J. Strauss, Elisabeth R. Lawlor, Antonio Llombart-Bosch, Heinrich Kovar. Inhibition of deacetylase SIRT1 offers a novel treatment option in metastatic Ewing sarcoma. [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 PR10.

Ewing Sarcoma is a malignant bone tumor in children and young adults. Upon current multimodal therapy only ∼60% of patients are long term survivors. Due to the rarity of the disease, however, only few novel agents can be tested in clinical trials. An animal model closely recapitulating the human disease would be highly needed to more rapidly explore new therapies. Ewing sarcoma is driven by the EWS/FLI1 (EF) chimeric oncogene, an aberrant Ets transcription factor that is toxic to many cell types, but tolerated by mesenchymal stem cells (MSC). Ewing sarcoma is postulated to develop from mesenchymal progenitor cells (MPC), but transgenic approaches tolerating EF expression lack behind. EF expression was shown to induce a differentiation block in MSC but does not drive full transformation. Thus, it was hypothesized that cooperating mutations are required for sarcomagenesis. We challenged this concept in a conditional mouse model in which EWS/FLI1 is activated early during endochondral bone formation by a Prx1-driven Cre recombinase (EFPrx1). EFPrx1 mice follows Mendelian ratio, but newborns suffocated due to a malformed rib cage. Bone formation was blocked at an early chondrocytic stage. EFPrx1 mice lacked limb bones, had reduced calvaria, craniofacial abnormalities and polydactyly. We found that EF expression blocked differentiation of MSC at the pre-hypertrophic chondrocyte stage due to blocked TGF-β and enhanced Hedgehog signaling (down regulated tgfβrI/II, smad1/5, dlx5 and upregulated gli1/2 and caspase 3 mRNA expression). Moreover, EF expression blocked sox9, runx2 and osterix expression. These genes are important for bone differentian. We established MSC-like cells (MSCL) from EFPrx1 mice that self-renewed, but full transformation was absent. We ought to test if the timing of EF induction plays a role, therefor we tested many time points later that the time point described before and we found that when Prx1Cre expression and consequently EWS/FLI1 induction was delayed to a time point after birth using a tamoxifen inducible system, a significant fraction of mice developed solid tumors up to 4 months after Tamoxifen induction. The histo-pathology of the tumors resembled PAS and VIMENTIN positive small round cell tumors, and genetically an EWS/FLI1 signature was seen similar to a patient-derived Ewing sarcoma gene expression pattern. These data suggest that EWS/FLI1 can drive oncogenesis without the need for additional genetic aberrations. We propose that this transgenic mouse model could resemble a faithful model for a solid EWS/FLI1 induced tumor phenocopying human Ewing sarcoma. Citation Format: Tahereh Javaheri, Barbara Sax, Harini Nivarthi, Eleni Tomazou, Mario Mikula, Jan Pencik, Zahra Kazemi, Maximilian Kauer, Marc Wiedner, Jan Tuckermann, Michaela Schlederer, Lukas Kenner, Reinhold Erben, Malkolm Logan, Christine Hartmann, Heinrich Kovar, Richard Moriggl. A mouse model for small round cell tumors induced by the Ewing sarcoma oncogene EWS/FLI1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 61. doi:10.1158/1538-7445.AM2014-61

Background ETV6/RUNX1 (E/R) (also known as TEL/AML1) is the most frequent gene fusion in childhood acute lymphoblastic leukemia (ALL) and also most likely the crucial factor for disease initiation; its role in leukemia propagation and maintenance, however, remains largely elusive. To address this issue we performed a shRNA-mediated knock-down (KD) of the E/R fusion gene and investigated the ensuing consequences on genome-wide gene expression patterns and deducible regulatory functions in two E/R-positive leukemic cell lines. Findings Microarray analyses identified 777 genes whose expression was substantially altered. Although approximately equal proportions were either up- (KD-UP) or down-regulated (KD-DOWN), the effects on biological processes and pathways differed considerably. The E/R KD-UP set was significantly enriched for genes included in the “cell activation”, “immune response”, “apoptosis”, “signal transduction” and “development and differentiation” categories, whereas in the E/R KD-DOWN set only the “PI3K/AKT/mTOR signaling” and “hematopoietic stem cells” categories became evident. Comparable expression signatures obtained from primary E/R-positive ALL samples underline the relevance of these pathways and molecular functions. We also validated six differentially expressed genes representing the categories “stem cell properties”, “B-cell differentiation”, “immune response”, “cell adhesion” and “DNA damage” with RT-qPCR. Conclusion Our analyses provide the first preliminary evidence that the continuous expression of the E/R fusion gene interferes with key regulatory functions that shape the biology of this leukemia subtype. E/R may thus indeed constitute the essential driving force for the propagation and maintenance of the leukemic process irrespective of potential consequences of associated secondary changes. Finally, these findings may also provide a valuable source of potentially attractive therapeutic targets.

Surveillance of DNA replication licensing is often perturbed in cancer cells allowing for genomic aberrations. Although this process contributes to cellular transformation, it is not well understood to what extent and how oncogenes might deregulate genes involved in DNA replication and in the control of replication licensing. Previous findings on the EWS-FLI1 fusion oncogene (which is the genetic hallmark Ewing's sarcoma), and our recent findings on MYC targets in this aggressive childhood tumor suggested that both these oncogenes constitute a regulatory feed-back loop. Our gene expression data from Ewing's cells in which MYC had been knocked down, we propose that MYC mediates at least part of the oncogenic EWS-FLI1 functions since it controls most essential biological functions required for cell proliferation. Gene expression correlation and gene set enrichment analysis in tumors and cell lines suggests the MYC/EWS-FLI1 loop as a central regulator in a regulatory network that controls DNA replication. To further narrow down the target gene repertoire and to identify direct transcriptional targets, we performed a genome wide chromatin-immunoprecipitation study using an anti MYC antibody in combindation with ChIP/CHIP analysis. We further extended our model whereby the core EWS-FLI1/MYC feedback loop could be linked to other candidate transcriptional regulators of DNA replication. Several replication licensing genes including MCM, ORC or licensing control genes such as CDT1 and CDC6 were found to be under direct and/or indirect control of the MYC/EWS-FLI1 loop and their associated transcription factor network. A subset of DNA replication genes is particularly deregulated in Ewing's cells when compared to mesenchymal progenitor cells that are considered the tissue of origin of Ewing's sarcoma. When comparing Ewing's sarcoma with other childhood tumors such as e.g. neuroblastoma, we found Ewing's-specific versus more general regulatory connections between the MYC/EWS-FLI1 loop and DNA replication genes. Together, our data provide first insight into a newly defined MYC/EWS-FLI1-driven regulatory network underlying transcriptional DNA replication control. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3413.

Ewing's sarcoma (ESFT) is a highly aggressive pediatric cancer of the bone and characterized by the chimeric ETS transcription factor EWS-FLI1 as a result of a chromosomal translocation. By whole genome gene expression and ChIP-seq analyses we obtained evidence that EWS-FLI1 directly regulates E2F target genes and possibly cooperates with E2F transcription factors. Furthermore, several E2F factors were themselves inferred to be direct targets of EWS-FLI1. To unravel the hierarchical structure of this EWS-FLI1 transcriptional network module we performed luciferase reporter gene assays in combination with DNA motif mutation analyses. The promoter regions of nine candidates for direct target genes of EWS-FLI1 and E2F factors were chosen for in-depth analysis. Corroborating the transcriptomic data, promoter activity of all 9 genes showed reduced levels upon knockdown of EWS-FLI1. The strongest modulatory effect of EWS-FLI1 silencing was observed for the E2F3 promoter, and mutation of a single ETS binding site lead to a comparable reduction of luciferase activity as knockdown of EWS-FLI1. These results validate E2F3 as a directly EWS-FLI1 regulated gene in Ewing's sarcoma. For the other EWS-FLI1 target candidates mutation analysis lead to more complex results. In the RAD51 promoter simultaneous mutation of two ETS binding sites was necessary to obtain a significant reduction of luciferase activity. Furthermore, mutation of a single E2F binding site abolished the effect of EWS-FLI1 knockdown. In promoters of two other candidates, GEMIN4 and ATAD2, mutation of the ETS site resulted in only a moderate reduction of reporter activity, and additional destruction of an E2F binding site was required to achieve a significant decrease in promoter activity. Our results imply that EWS-FLI1 uses several modes of interaction with E2F factors to control common target genes. First, EWS-FLI1 directly activates E2F3 and other E2F factors which, in turn, co-operate with EWS-FLi1 on target gene promoters in a feed-forward loop. Second, results for the RAD51 promoter are compatible with a mechanism where a repressive E2F factor is exchanged for an activating E2F factor via binding of EWS-FLI1. Finally, EWS-FLI1 might have a more indirect role in the regulation of ATAD2 and GEMIN4 where most of the transcriptional effect seems to be exerted by E2F factors. Acknowledgement: This study was funded by the 6th framework program of the European Commission, (“E.E.T. Pipeline” contract LSHC-CT-2006-037260) and by the Austrian genome research program “GEN-AU Ch.I.L.D.“ (GZ 200.136/1 - VI/1/2005). R. Schwentner is a recipient of a DOC-fFORTE-fellowship of the Austrian Academy of Sciences. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4968.

Cancer genomics can help uncover genes that may serve as indicators of tumor susceptibility, progression, or treatment outcome, as exemplified by studies of adult tumors for which large cohorts are readily available. Childhood cancers comprise a large number of low-incidence histogenetically and molecularly distinct neoplasms, rendering population-based genomic studies unrealistic for these diseases. In recent years, several small-size cohort childhood cancer genomic studies have been published. We report here a novel analytical approach, named intercohort co-analysis or Ican, whereby gene expression significance is inferred from gene expression levels in biologically relevant normal tissues, and gene-disease association significance is assessed in each individual cohort. Next, only genes found to be consistently significant in all cohorts are considered as putative markers of specific disease states. We tested Ican in Ewing's sarcoma (ESFT), a family of tumors that lacks a reliable molecular prognostic or metastasis indicator. We report here that this approach revealed a high intercohort gene expression significance variability, and identified CXCR4 and CXCR7, two chemokine receptors known to correlate to metastasis and/or to poor prognosis mostly in adult cancers, as significantly and consistently associated with ESFT subsets. Accordingly, we show for the first time that CXCR4 expression marks metastatic ESFT and that of CXCR7 serves as a predictor of adverse patient survival. We submit these findings as a proof-of-principle that Ican of publicly available gene expression profiles can yield genes with predictive value for the progression or outcome of low-incidence diseases. We further submit that this approach is poised to gain momentum as many journals now request the mandatory contribution of gene expression profiles to public repositories. These policies, along with the growing number of cancer gene expression profiling investigations, we predict, will swiftly drive upward the number of publicly available low-incidence disease expression datasets, and consequently, of Ican applications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3410.