Phenotype Of Ewing Sarcoma Research Articles

Abstract 1787: The β3-adrenergic receptor as novel target of metabolic phenotype in Ewing sarcoma

Abstract Purpose of study: Metabolic targeted therapies may represent an innovative strategy in Ewing sarcoma (ES). Introduction: Ewing sarcoma (ES) is highly aggressive round cell mesenchymal neoplasm, most often occurring in children and young adults. Metabolic peculiarities of ES cells are indeed involved in tumor growth, survival and resistance to therapy. We have further investigated apoptotic role of 𝜷;;3-adrenergic receptor (𝜷;;3-AR) SR59230A (SR) antagonist for its ability to reduce cells viability due increased oxidative stress, now we underline the metabolic role of SR. Materials & Methods: The metabolic profile after SR administration in A673 ES cell model was studied using the Seahorse XF Analyzer. Viability was evaluated with MTS. Changes in gene expression was evaluated with RNA sequencing. Western blot analysis and Immunofluorescence was performed for redox signature and metabolic alteration. Ros production and cell death was assessed by flow cytometry. Results: Metabolic analysis reveals glucose and fatty acid dependency in A673 cells using the Fuel Flex Test of Seahorse XFe Analyzer. Quantification of transcript abundance using Salmon in A673 after SR treatment showed decrease in response to reactive oxygen species, increase of Thioredoxin Interacting Protein (TXNIP) inhibits the antioxidative function of thioredoxin, moreover reduced glucose uptake, consequently, decrease NADPH/NADP resulting in the accumulation of ROS coincident with a decrease of GLUT1, SOD2, TrDX an increase of LC3IIA protein expression. SR enhances A673 pyruvate dependency, and decrease glyco and mito ATP production rate, more over observed under glucose deprivation. Furthermore, apoptotic effect was significantly amplified when cells were under nutrient starvation, revealing a 56% apoptotic rate after 24 hours of SR treatment. Conclusion: SR antagonist of 𝜷;;3-AR and combination of SR with BUF could be an innovative therapeutic option to raise awareness ES. Citation Format: Cristina Banella, Francesco Carrozzo, Laura Zocca, Amada Pasha, Gianluca Mattei, Marina Mola, Lara Ballerini, Nicla Lorito, Annalisa Tondo, Claudio Favre, Maura Calvani. The β3-adrenergic receptor as novel target of metabolic phenotype in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1787.

A logical model of Ewing sarcoma cell epithelial-to-mesenchymal transition supports the existence of hybrid cellular phenotypes.

Ewing sarcoma (ES) is a highly aggressive pediatric tumor driven by the RNA-binding protein EWS (EWS)/friend leukemia integration 1 transcription factor (FLI1) chimeric transcription factor, which is involved in epithelial-mesenchymal transition (EMT). EMT stabilizes a hybrid cell state, boosting metastatic potential and drug resistance. Nevertheless, the mechanisms underlying the maintenance of this hybrid phenotype in ES remain elusive. Our study proposes a logical EMT model for ES, highlighting zinc finger E-box-binding homeobox 2 (ZEB2), miR-145, and miR-200 circuits that maintain hybrid states. The model aligns with experimental findings and reveals a previously unknown circuit supporting the mesenchymal phenotype. These insights emphasize the role of ZEB2 in the maintenance of the hybrid state in ES.

LncRNA FOXP4-AS1 Promotes Progression of Ewing Sarcoma and Is Associated With Immune Infiltrates.

Ewing sarcoma (ES) is a highly malignant primary bone tumor with poor prognosis. Studies have shown that abnormal expression of lncRNA influences the prognosis of tumor patients. Herein, we established that FOXP4-AS1 was up-regulated in ES and this correlated with poor prognosis. Further analysis illustrated that FOXP4-AS1 down-regulation repression growth, migration, along with invasion of ES. On the contrary, up-regulation of FOXP4-AS1 promoted the growth, migration, as well as invasion of ES. To explore the mechanism of FOXP4-AS1, Spearman correlation analysis was carried out to determine genes that were remarkably linked to FOXP4-AS1 expression. The potential functions and pathways involving FOXP4-AS1 were identified by GO analysis, Hallmark gene set enrichment analysis, GSEA, and GSVA. The subcellular fractionation results illustrated that FOXP4-AS1 was primarily located in the cytoplasm of ES cells. Then a ceRNA network of FOXP4-AS1 was constructed. Analysis of the ceRNA network and GSEA yielded two candidate mRNAs for FOXP4-AS1. Results of the combined survival analysis led us to speculate that FOXP4-AS1 may affect the expression of TMPO by sponging miR-298, thereby regulating the malignant phenotype of ES. Finally, we found that FOXP4-AS1 may modulates the tumor immune microenvironment in an extracellular vesicle-mediated manner. In summary, FOXP4-AS1 correlates with poor prognosis of ES. It promotes the growth, migration, as well as invasion of ES cells and may modulate the tumor immune microenvironment.

SOX6: a double-edged sword for Ewing sarcoma

ABSTRACT Developmental pathways play an important role in cancer. We have recently demonstrated that the constitutive activation of the developmental transcription factor SOX6 via the fusion oncoprotein EWSR1-FLI1 (Ewing sarcoma breakpoint region 1 – Friend leukemia virus integration 1) contributes to the aggressive phenotype of Ewing sarcoma but on another hand provides an opportunity for targeted therapy.

Abstract 3181: Class I histone deacetylases (HDAC) critically contribute to Ewing sarcoma pathogenesis

Abstract Introduction: Recent evidence demonstrated that a low mutation rate seems a general feature of pediatric cancers. Ewing sarcoma (EwS) is defined by balanced chromosomal EWS/ETS translocations, which give rise to oncogenic chimeric proteins (EWS-ETS). Previously, we identified the histone methyl-transferase enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), the enzymatic subunit of the polycomb PRC2 complex, to be over-expressed in EwS. RNA interference of EZH2 suppressed tumor development and metastasis in vivo and microarray analysis of EZH2 knock down revealed an EZH2-maintained, undifferentiated, reversible phenotype in EwS. Microarray analysis further revealed that treatment with MS-275 or TSA resulted in the induction of a similar pattern of differentiation genes as was previously observed after EZH2 blockade and seems to be dependent on histone deacetylase (HDAC) activity. Experimental procedures: The role of class I histone deacetylases (HDACs) was determined using different potent inhibitors like Trichostatin A (TSA), Romidepsin (FK228), Entinostat (MS-275) and PCI-34051 as well as a CRISPR/Cas9 approach to knock out class I HDACs. To analyze resulting changes microarray analysis, gene set enrichment analysis (GSEA), qRT-PCR, western blotting, proliferation, apoptosis and invasion assays as well as immunofluorescence experiments were deployed. Results: Interestingly, treatment with enzymatic inhibitors blocking EZH2 activity could not mimic the results observed after EZH2 RNA interference indicating that EZH2-containing PRC2 complexes may serve as a building block of class I HDAC activity in ES. Consequently, in proliferation assays EwS cells were more susceptible to treatment with HDAC inhibitors than other small round blue cell tumors such as neuroblastoma or pediatric cALL cells. Microarray and GSEA analysis demonstrated that treatment with Romidepsin resulted in an inhibition of an EwS specific expression profile while Entinostat suppressed metastasis relevant genes. In addition, EwS cells demonstrated increased susceptibility to treatment with chemotherapeutics such as Doxorubicin and Vincristine in the presence of HDACi. Consequently, CRISPR/Cas9 knock outs of HDAC1 to HDAC3 in EwS cell lines resulted in a reduction of their proliferative ability and greater sensitivity to treatment with chemotherapeutics. Conclusion: Class I HDAC proteins seem to be important mediators of the pathognomonic EWS-ETS-mediated transcription program in EwS and thus interesting new treatment opportunities for this malignant disease. Citation Format: Oxana Schmidt, Nadja Nehls, Kristina von Heyking, Tim Hensel, Stefan Burdach, Günther H. Richter. Class I histone deacetylases (HDAC) critically contribute to Ewing sarcoma pathogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3181.

Abstract LB-323: A novel function of ETS transcription factors in alternative splicing is altered in oncogenic FET-ETS fusions

Abstract Ewing sarcomas are characterized by fusions proteins between FET (FUS, EWS and TAF15) family genes encoding RNA binding proteins and ETS (ERG, FLI1 and FEV) family genes encoding transcription factors. FET-ETS fusions regulate both transcription and alternative splicing programs in Ewing sarcoma cells. To date, the function of FET-ETS fusions in splicing is mostly attributed to the FET N-terminal region which is preserved in the fusions. We recently demonstrated that wild type ERG associates with poly-adenylated mRNA and control mRNAs stability in the cytoplasm (Rambout et al., 2016). This therefore indicates that ERG proteins might not be considered only as transcription factors, but can also regulate post-transcriptional processes. On this ground, we hypothesize that ERG proteins might control other post-transcriptional processes, including splicing. In this study, we show that the ERG transcription factor interacts with several spliceosome components and is associated with nascent RNA. Tethered ERG proteins controlled alternative splicing of a minigene reporter in an orientation-specific manner. Deep RNA-seq identified hundreds of alternatively spliced exons regulated by ERG depletion, with an enrichment in RNA motifs bound by RBFOX2 splicing factor. RBFOX2 similarly regulated alternative splicing of common targets and interacted together via the C-terminal domain of ERG which is present in FET-ERG fusions. In Ewing sarcoma cells, RNA-seq identified hundreds of alternatively spliced exons regulated by EWS-FLI1 depletion, with an enrichment in RNA motifs bound by RBFOX2. The oncogenic EWS-FLI1 protein binds RBFOX2 protein but has an antagonist effect on RBFOX2 function. Cross-linking immunoprecipitation (CLIP) experiments show that the EWS-FLI1 fusion inhibits RBFOX2 function by repressing its binding to target pre-mRNAs. We hypothesize that the low complexity prion-like domain of EWS included in the fusion protein may be responsible for sequestration of RBFOX2 outside of the splicing machinery. Importantly, we also show that some of the splicing events induced by the fusion protein may play a critical role in FET-ETS induced cell phenotype. Specific splicing switch partially recapitulate the phenotypic changes that participate in FET-ETS induced cell transformation. These data reveal a new role for ERG family proteins in alternative splicing that is altered in FET-ETS oncogenic proteins, thereby contributing to alternative splicing mis-regulation and cell phenotype in Ewing sarcoma. Citation Format: Olivier Saulnier, Katia Guerdi, Marie-Ming Aynaud, Alina Chakraborty, Josephine Pineau, Christina O Grady, Martin Dutertre, Franck Dequiedt, Olivier Delattre. A novel function of ETS transcription factors in alternative splicing is altered in oncogenic FET-ETS fusions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-323.

Beclin-1 knockdown decreases proliferation, invasion and migration of Ewing sarcoma SK-ES-1 cells via inhibition of MMP-9.

Although Beclin-1, a well-known key regulator of autophagy, has been demonstrated to serve a function in a number of disorders, including cancer, aging and degenerative diseases, its biological function in Ewing sarcoma (ES) remains unresolved. The objective of the present study was to determine the in vitro effect of Beclin-1 knockdown on the growth and malignant phenotype of ES SK-ES-1 cells, which have increased endogenous expression of Beclin-1 compared with RD-ES cells, and to investigate the underlying molecular mechanism. Cell proliferation, invasion and migration were investigated using CCK-8, Boyden chamber Transwell, and wound healing assays, respectively. Western blot analysis was used to detect expression levels of matrix metalloproteinase (MMP)-2 and MMP-9, which are associated with the malignant phenotype. Beclin-1 knockdown significantly inhibited proliferation, invasion and migration of SK-ES-1 cells. Western blot analysis revealed that Beclin-1 knockdown caused a significant reduction in the expression of MMP-9; no marked changes in MMP-2 expression were observed in the si-Beclin-1 group compared with the control group. The results of the present study suggest that Beclin-1 serves a function in proliferation, tumor progression and inhibition of autophagy in ES, and demonstrates it's potential as a target to increase the efficacy of anticancer agents.

Abstract 3866: Investigating the role of class I HDACs in Ewing sarcoma pathology

Abstract Introduction: Recent evidence demonstrated that a low mutation rate seems a general feature of pediatric cancers. Ewing sarcoma (ES) is defined by balanced chromosomal EWS/ETS translocations, which give rise to oncogenic chimeric proteins (EWS-ETS). Other contributing somatic mutations involved in disease development have only been observed at low frequency. Thus, cancer in children is not solely a genetic disease and can neither be understood nor cured presumably without epigenetics. Previously, we identified the histone methyl-transferase enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), the enzymatic subunit of the polycomb PRC2 complex, to be over-expressed in ES. RNA interference of EZH2 suppressed tumor development and metastasis in vivo and microarray analysis of EZH2 knock down revealed an EZH2-maintained, undifferentiated, reversible phenotype in ES. Experimental procedures: The role of class I histone deacetylases (HDACs) was determined using different potent inhibitors like Trichostatin A (TSA), Romidepsin (FK22), Entinostat (MS-275) and PCI-34051 as well as a CRISPR/Cas9 approach to knock out specific HDACs. To analyze resulting changes qRT-PCR, Western Blotting, proliferation, apoptosis and invasion assays as well as immunofluorescence experiments were deployed. Results: Interestingly, the effects of gene silencing after RNA interference of EZH2 could be mimicked by treatment of ES with TSA or MS-275. Microarray analysis revealed that treatment with MS-275 or TSA resulted in the induction of a similar pattern of differentiation genes as was previously observed after EZH2 blockade and seems to be dependent on histone deacetylase (HDAC) activity. Furthermore, in proliferation assays ES were more susceptible to treatment with HDAC inhibitors than other small round blue cell tumors such as neuroblastoma or pediatric cALL cells. Treatment of enzymatic inhibitors blocking EZH2 activity could not mimic the results observed after EZH2 RNA interference indicating that EZH2-containing PRC2 complexes may serve as a building block of class I HDAC activity in ES. Conclusion: Class I HDACs seem to be important mediators of the pathognomonic EWS-ETS-mediated transcription program in ES and thus interesting new treatment opportunities for this malignant disease. Citation Format: Kristina von Heyking, Tim Hensel, Julia Singer, Oxana Schmidt, Stefan Burdach, Günther H. Richter. Investigating the role of class I HDACs in Ewing sarcoma pathology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3866. doi:10.1158/1538-7445.AM2017-3866

A phase I, first-in-human, dose escalation study of intravenous TK216 in patients with relapsed or refractory Ewing sarcoma.

TPS11626 Background: Ewing sarcoma (ES) is a rare cancer that affects children and young adults. Patients with recurrent/refractory ES have a poor prognosis (5-year survival 10-15%) with no improvement despite advances in cytotoxic and targeted therapies. Genomic rearrangements resulting in fusion proteins and over-expression of ets family transcription factors occur in 95% of ES. In particular, the EWS-FLI1 oncogenic fusion creates a constitutively active transcription factor that drives the malignant ES phenotype. Strategies to target the EWS-FLI1 fusion protein have been limited by lack of specificity. A promising approach is to target the interaction of the ets transcription factor with its critical protein partner, RNA helicase A (RHA). TK216 is a novel small-molecule that directly binds to EWS-FLI1 and inhibits its function by blocking binding to RHA. TK216 demonstrates potent anti-proliferative effects on ES cell lines and xenografts. Methods: We initiated a Phase 1, first-in-human, open-label, multi-center, dose-escalation/dose-expansion trial of TK216 in patients with recurrent/refractory ES who are ≥12 years of age (ClinicalTrials.gov: NCT02657005). TK216 is dosed based on body surface area and administered as a continuous intravenous infusion for 7 days followed by 14 days rest every 21 days. Treatment may continue in the absence of disease progression. One intrapatient dose escalation is allowed. Enrollment of 6 to 8 cohorts using a 3+3 dose-escalation design is anticipated. During dose expansion, a total of 18 patients with ES will be accrued at the recommended Phase 2 dose (RP2D). The primary objective of the study is to determine the maximum tolerated dose and RP2D of TK216. Secondary objectives are to assess the safety profile, pharmacokinetics, pharmacodynamics, and antitumor activity of TK216. Molecular assays will be performed to characterize EWS-FLI or EWS-ets abnormalities in archival tumor tissue. The overall response rate, duration of response, progression-free survival, and overall survival will be determined in the expansion cohort. Nine patients have been enrolled since June 2016. Accrual to cohorts 1, 2, and 3 completed and cohort 4 opened in January 2017. Clinical trial information: NCT02657005.

Abstract A81: Adoptively transferred Chondromodulin 1/HLA-A:0201 allo-restricted T cell receptor transgenic CD8+ T cells are well tolerated and cause partial regression in a patient with disseminated Ewing Sarcoma

Abstract Background: Patients with Ewing sarcoma (ES) metastatic to bone and bone marrow (BM) do not survive irrespective of therapy. We assessed toxicity, viability, BM homing and anti-tumor activity of adoptively transferred HLA-A*0201/chondromodulin 1 (CHM1) allo-restricted T cell receptor (TCR) transgenic CD8+ T cells in an ES patient with refractory disease including marrow. Patient and Methods: An twelve years old HLA-A2+ boy was diagnosed with ES of the right femur. After treatment according to the Euro-Ewing 2008 protocol he suffered early relapse 11 months after diagnosis. For relapse treatment he received MetaEICESS 2007 including consolidation with haploidentical stem cell transplantation (allo-SCT) from his HLA-A2- mother. Before and shortly after transplant the patient relapsed twice. Thereafter he suffered from progressive disease under combined rescue radiochemotherapy. Within one week after the last progression, he received 1 x 106/kg (1st transfer, d0) and 8.2 x 106/kg CHM1319-T cell receptor (TCR) transgenic donor CD8+ T cells (2nd transfer, d32; in combination with ipilimumab and nivolumab), respectively. A patient derived ES cell line was isolated. Results: CHM1319-TCR transgenic donor T cells showed specific in vitro lysis of the patient's ES cell line. One week after 1st transfer, CHM1319-TCR transgenic T cells stained CHM1319 multimer positive in BM but negative in peripheral blood (PB). After 2nd transfer, BM of posterior iliac spines as well as PB stained CHM1319 multimer positive at a high rate and RT-PCR and flow-cytometric CD99+/CD45- ES phenotype as well as partial PET-signal reduction were noted. Five further metastatic bone/ marrow compartments showed significant PET signal regression. The patient never developed GvHD symptoms and died of disease 10 weeks after 1st transfer due to resistant lung metastasis. Conclusions: CHM1319-TCR transgenic T cells home to affected BM and cause partial disease regression. These cells may proliferate in vivo for at least four weeks without causing GvHD. Citation Format: Uwe Thiel, Andreas Kirschner, Ingo Einspieler, Stefan Burdach. Adoptively transferred Chondromodulin 1/HLA-A:0201 allo-restricted T cell receptor transgenic CD8+ T cells are well tolerated and cause partial regression in a patient with disseminated Ewing Sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A81.

Systemic levels of neuropeptide Y and dipeptidyl peptidase activity in patients with Ewing sarcoma--associations with tumor phenotype and survival.

Ewing sarcoma (ES) is driven by fusion of the Ewing sarcoma breakpoint region 1 gene (EWSR1) with an E26 transformation-specific (ETS) transcription factor (EWS-ETS), most often the Friend leukemia integration 1 transcription factor (FLI1). Neuropeptide Y (NPY) is an EWS-FLI1 transcriptional target; it is highly expressed in ES and exerts opposing effects, ranging from ES cell death to angiogenesis and cancer stem cell propagation. The functions of NPY are regulated by dipeptidyl peptidase IV (DPPIV), a hypoxia-inducible enzyme that cleaves the peptide and activates its growth-promoting actions. The objective of this study was to determine the clinically relevant functions of NPY by identifying the associations between patients' ES phenotype and their NPY concentrations and DPP activity. NPY concentrations and DPP activity were measured in serum samples from 223 patients with localized ES and 9 patients with metastatic ES provided by the Children's Oncology Group. Serum NPY levels were elevated in ES patients compared with the levels in a healthy control group and an osteosarcoma patient population, and the elevated levels were independent of EWS-ETS translocation type. Significantly higher NPY concentrations were detected in patients with ES who had tumors of pelvic and bone origin. A similar trend was observed in patients with metastatic ES. There was no effect of NPY on survival in patients with localized ES. DPP activity in sera from patients with ES did not differ significantly from that in healthy controls and patients with osteosarcoma. However, high DPP levels were associated with improved survival. Systemic NPY levels are elevated in patients with ES, and these high levels are associated with unfavorable disease features. DPPIV in serum samples from patients with ES is derived from nontumor sources, and its high activity is correlated with improved survival.

Abstract 3973: BRICHOS genes CHM1 and ITM2A maintain an undifferentiated, invasive phenotype in Ewing sarcoma

Abstract Ewing sarcomas (ES) are highly malignant, osteolytic bone or soft tissue tumors, which are characterized by ews/ets translocations and early metastasis into lung and bone. In this study, we analyzed the role of the BRICHOS domain-containing genes CHM1 and ITM2A in ES pathogenesis, especially in bone-associated tumor growth and metastasis. Both genes are overexpressed in ES and among others appear to be important for chondrogenic differentiation. CHM1 in addition seems directly up-regulated by EWS/FLI1. Using RNA interference, we demonstrate that CHM1 and ITM2A slightly reduced bone invasiveness and osteolysis in vivo that is likely mediated by the suppression of the osteolytic genes HIF1α, IL6, JAG1 and VEGF. Furthermore, CHM1 and ITM2A suppressed the chondrogenic and to a lesser extend the osteogenic differentiation status of ES cells indicating that osteomimicry might play a role in homing, colonizing and invasion into bone tissues. Additionally, we show that both BRICHOS domain-containing genes significantly increased the expression of the stem cell genes, ABCG2 and PROM1. Together with the observed undifferentiated phenotype of ES cells, we demonstrate that CHM1 and ITM2A clearly enhanced lung metastasis in a xenograft mouse model in vivo. Our results suggest that CHM1 and ITM2A are essential players with respect to the undifferentiated phenotype and the metastatic behavior of ES cells. Citation Format: Kristina von Heyking, Annette Fasan, Stefan Burdach, Günther H. Richter. BRICHOS genes CHM1 and ITM2A maintain an undifferentiated, invasive phenotype in Ewing sarcoma. [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 3973. doi:10.1158/1538-7445.AM2014-3973

ERBB4 confers metastatic capacity in Ewing sarcoma

Metastatic spread is the single-most powerful predictor of poor outcome in Ewing sarcoma (ES). Therefore targeting pathways that drive metastasis has tremendous potential to reduce the burden of disease in ES. We previously showed that activation of the ERBB4 tyrosine kinase suppresses anoikis, or detachment-induced cell death, and induces chemoresistance in ES cell lines in vitro. We now show that ERBB4 is transcriptionally overexpressed in ES cell lines derived from chemoresistant or metastatic ES tumours. ERBB4 activates the PI3K-Akt cascade and focal adhesion kinase (FAK), and both pathways contribute to ERBB4-mediated activation of the Rac1 GTPase in vitro and in vivo. ERBB4 augments tumour invasion and metastasis in vivo, and these effects are blocked by ERBB4 knockdown. ERBB4 expression correlates significantly with reduced disease-free survival, and increased expression is observed in metastatic compared to primary patient-matched ES biopsies. Our findings identify a novel ERBB4-PI3K-Akt-FAK-Rac1 pathway associated with aggressive disease in ES. These results predict that therapeutic targeting of ERBB4, alone or in combination with cytotoxic agents, may suppress the metastatic phenotype in ES.

Abstract 1958: Disregulation of TARBP-2 dependent miRNA maturation : A potential new therapeutic target in Ewing sarcoma..

Abstract We have recently shown that microRNAs (miRNAs) play essential roles is the generation of the cancer stem cell (CSC) phenotype in Ewing sarcoma familly tumors (ESFT). Here we compared the miRNA expression profile of ESFT CSCs to that of embryonic stem cells and CSCs from different tumor, which seem to be shared by these different cell types. Our data suggests that the mechanisms implicated in the miRNAs CSC signature rely on a reversible TARBP-2 dependent miRNA maturation defect. Restoration of TARBP2 activity either through chemical compounds or systemic delivery of synthetic forms of two of its targets, miRNA-143 or miRNA-145, inhibited ESFT CSC growth in vitro and in vivo. Taken together our observations provide new therapeutic strategies through restoration of normal miRNA expression profile, inhibiting CSC self-renewal and tumor maintenance. Citation Format: Claudio De Vito, Nicolo Riggi, Sandrine Cornaz, Ivan Stamenkovic. Disregulation of TARBP-2 dependent miRNA maturation : A potential new therapeutic target in Ewing sarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1958. doi:10.1158/1538-7445.AM2013-1958

BCL11B is up-regulated by EWS/FLI and contributes to the transformed phenotype in Ewing sarcoma.

The EWS/FLI translocation product is the causative oncogene in Ewing sarcoma and acts as an aberrant transcription factor. EWS/FLI dysregulates gene expression during tumorigenesis by abnormally activating or repressing genes. The expression levels of thousands of genes are affected in Ewing sarcoma, however, it is unknown which of these genes contribute to the transformed phenotype. Here we characterize BCL11B as an up-regulated EWS/FLI target that is necessary for the maintenance of transformation in patient derived Ewing sarcoma cells lines. BCL11B, a zinc finger transcription factor, acts as a transcriptional repressor in Ewing’s sarcoma and contributes to the EWS/FLI repressed gene signature. BCL11B repressive activity is mediated by the NuRD co-repressor complex. We further demonstrate that re-expression of SPRY1, a repressed target of BCL11B, limits the transformation capacity of Ewing sarcoma cells. These data define a new pathway downstream of EWS/FLI required for oncogenic maintenance in Ewing sarcoma.

EWS/Fli-1 Fusion Protein Is Involved in Neural Phenotype in Ewing^|^apos;s Sarcoma Cells

Ewing's sarcoma (ES) and other peripheral primitive neuroectodermal tumor (PNET) show limited neural differentiation and are thought to have a neural crest origin. The fusion gene, EWS/Fli-1 found in ES and PNET are formed by a tumor specific 11; 22 chromosomal translocations. They have been shown to encode a transcriptional activator and promote cellular transformation. However, the precise biological functions of the fusion gene products remain to be elucidated. Especially it is controversial whether the gene products may inhibit neural differentiation or they may keep neural phenotype in ES. In the present study, we first investigated EWS/Fli-1 expression after induction of neural differentiation by addition of nerve growth factor (NGF) or c-AMP and demonstrated that EWS/Fli-1 protein expression did not change in response to those treatments. Moreover, overexpression of EWS/Fli-1 did not affect differentiation process of C6 glial cells. On the contrary, morphological differentiation and expression of NSE (neuron specific enolase) and trkA, one of the high-affinity NGF receptors, were inhibited in ES cells, in which the endogeneous EWS/Fli-1 protein was not detected. In conclusion, our data are compatible with a view that EWS/Fli-1 fusion gene may not inhibit neural differentiation process, but may play an important role in preserving the potentiality of neural phenotype in ES cells.

Inhibition of EWS-FLI-1 fusion protein with antisense oligodeoxynucleotides.

Ewing's sarcoma family of tumors (EFT) contain reciprocal translocations, of which approximately 90% occur between the long arm of chromosomes 11 and 22,t(11;22)(q24;q12) resulting in the formation of chimeric proteins generated by a fusion of the EWS and FLI-1 genes. To determine if EWS-FLI-1 protein is responsible for the Ewing sarcoma phenotype we have used sequence-specific antisense oligodeoxynucleotides (ODN) to block its expression. We have evaluated a series of antisense ODN directed toward the breakpoint region in an effort to prevent translation of the fusion messenger RNA. ODN were first evaluated in a cell-free in vitro translation system. Exogenously added RNase H was found to be required for translation inhibition. ODN that showed complete inhibition of translation were electroporated into TC-32 cells, a EFT cell line. Fusion protein and EWS protein levels were evaluated by Western blot analysis. A 40-60% decrease in the fusion protein was observed in TC-32 cells with antisense ODN directed toward the breakpoint region. Cell viability was reduced with antisense sequences in TC-32 cells but not in a prostate cancer cell line. Since inhibition of t(11:22) gene product is correlated to effects on cell viability reduction of the fusion protein may thus offer insight into the biology of EFT.