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Purpose: Propagation of Ewing sarcoma requires precise regulation of EWS::FLI1 transcriptional activity. Determining the mechanisms of fusion regulation will advance our understanding of tumor progression. Here we investigated whether HOXD13, a developmental transcription factor that promotes Ewing sarcoma metastatic phenotypes, influences EWS::FLI1 transcriptional activity. Experimental Design: Existing tumor and cell line datasets were used to define EWS::FLI1 binding sites and transcriptional targets. Chromatin immunoprecipitation and CRISPR interference were employed to identify enhancers. CUT&RUN and RNA sequencing defined binding sites and transcriptional targets of HOXD13. Transcriptional states were investigated using bulk and single-cell transcriptomic data from cell lines, patient-derived xenografts, and patient tumors. Mesenchymal phenotypes were assessed by gene set enrichment, flow cytometry, and migration assays. Results: We found that EWS::FLI1 creates a de novo GGAA microsatellite enhancer in a developmentally conserved regulatory region of the HOXD locus. Knockdown of HOXD13 led to widespread changes in expression of developmental gene programs and EWS::FLI1 targets. HOXD13 binding was enriched at established EWS::FLI1 binding sites where it influenced expression of EWS::FLI1-activated genes. More strikingly, HOXD13 bound and activated EWS::FLI1-repressed genes, leading to adoption of mesenchymal and migratory cell states that are normally suppressed by the fusion. Single-cell analysis confirmed that direct transcriptional antagonism between HOXD13-mediated gene activation and EWS::FLI1-dependent gene repression defines the state of Ewing sarcoma cells along a mesenchymal axis. Conclusions: Ewing sarcoma tumors are comprised of tumor cells that exist along a mesenchymal transcriptional continuum. The identity of cells along this continuum is, in large part, determined by the competing activities of EWS::FLI1 and HOXD13. See related commentary by Weiss and Bailey, p. 4360

Primary bone sarcomas, including osteosarcoma (OS) and Ewing sarcoma (ES), are aggressive tumors with peak incidence in childhood and adolescence. The intense standard treatment for these patients consists of combined surgery and/or radiation and maximal doses of chemotherapy; a regimen that has not seen improvement in decades. Like other tumor types, ES and OS are characterized by dysregulated cellular metabolism and a rewiring of metabolic pathways to support the biosynthetic demands of malignant growth. Not only are cancer cells characterized by Warburg metabolism, or aerobic glycolysis, but emerging work has revealed a dependence on amino acid metabolism. Aside from incorporation into proteins, amino acids serve critical functions in redox balance, energy homeostasis, and epigenetic maintenance. In this review, we summarize current studies describing the amino acid metabolic requirements of primary bone sarcomas, focusing on OS and ES, and compare these dependencies in the normal bone and malignant tumor contexts. We also examine insights that can be gleaned from other cancers to better understand differential metabolic susceptibilities between primary and metastatic tumor microenvironments. Lastly, we discuss potential metabolic vulnerabilities that may be exploited therapeutically and provide better-targeted treatments to improve the current standard of care.

Glioblastoma (GBM) is an aggressive primary brain cancer; with a 5-year survival of ~5%. Challenges that hamper GBM therapeutic efficacy include: (i) tumor heterogeneity, (ii) treatment resistance, (iii) immunosuppressive tumor microenvironment (TME) and (iv) the blood-brain barrier (BBB). The C-X-C Motif Chemokine Ligand-12/ C-X-C Motif Chemokine Receptor-4 (CXCL12/CXCR4) signaling pathway is activated in GBM and is associated with tumor progression. Although the CXCR4 antagonist (AMD3100) has been proposed as an attractive anti-GBM therapeutic target, it’s poor pharmacokinetic properties, and unfavorable bioavailability have hampered its clinical implementation. Thus, we developed synthetic protein nanoparticles (SPNPs) coated with the transcytotic peptide iRGD (AMD3100-SPNPs) to target the CXCL2/CXCR4 pathway in GBM via systemic delivery. We showed that AMD3100-SPNPs block CXCL12/CXCR4 signaling in three mouse and human GBM cell cultures in vitro and in a GBM mouse model in vivo. This results in (i) inhibition of GBM proliferation, (ii) reduced infiltration of CXCR4(+) monocytic myeloid derived suppressor cells (M-MDSCs) into the TME, (iii) restoration of BBB integrity, and (iv) induction of immunogenic cell death (ICD), sensitizing the tumor to radiotherapy, and leading to anti-GBM immunity. Additionally, we showed that combining AMD3100-SPNPs with radiation led to long term survival; with ~60% of GBM tumor bearing mice remaining tumor free, after rechallenging with a second GBM in the contralateral hemisphere. This was due to a sustained anti-GBM immunological memory response that prevented tumor recurrence without additional treatment. In view of the potent ICD induction and reprogrammed tumor microenvironment, this SPNP-mediated strategy has a significant clinical translation applicability.

Ewing sarcomas are driven by EWS–ETS fusions, most commonly EWS-FLI1, which promotes widespread metabolic reprogramming, including activation of serine biosynthesis. We previously reported that serine biosynthesis is also activated in Ewing sarcoma by the scaffolding protein menin through as yet undefined mechanisms. Here, we investigated whether EWS-FLI1 and/or menin orchestrate serine biosynthesis via modulation of ATF4, a stress-response gene that acts as a master transcriptional regulator of serine biosynthesis in other tumors. Our results show that in Ewing sarcoma, ATF4 levels are high and that ATF4 modulates transcription of core serine synthesis pathway (SSP) genes. Inhibition of either EWS-FLI1 or menin leads to loss of ATF4, and this is associated with diminished expression of SSP transcripts and proteins. We identified and validated an EWS–FLI1 binding site at the ATF4 promoter, indicating that the fusion can directly activate ATF4 transcription. In contrast, our results suggest that menin-dependent regulation of ATF4 is mediated by transcriptional and post-transcriptional mechanisms. Importantly, our data also reveal that the downregulation of SSP genes that occurs in the context of EWS-FLI1 or menin loss is indicative of broader inhibition of ATF4-dependent transcription. Moreover, we find that menin inhibition similarly leads to loss of ATF4 and the ATF4-dependent transcriptional signature in MLL-rearranged B-cell acute lymphoblastic leukemia, extending our findings to another cancer in which menin serves an oncogenic role. Implications: These studies provide new insights into metabolic reprogramming in Ewing sarcoma and also uncover a previously undescribed role for menin in the regulation of ATF4.

Wnt/β-catenin signaling is active in small subpopulations of Ewing sarcoma cells, and these cells display a more metastatic phenotype, in part due to antagonism of EWS-FLI1-dependent transcriptional activity. Importantly, these β-catenin-activated Ewing sarcoma cells also alter secretion of extracellular matrix (ECM) proteins. We thus hypothesized that, in addition to cell-autonomous mechanisms, Wnt/β-catenin-active tumor cells might contribute to disease progression by altering the tumor microenvironment (TME). Analysis of transcriptomic data from primary patient biopsies and from β-catenin-active versus -nonactive tumor cells identified angiogenic switch genes as being highly and reproducibly upregulated in the context of β-catenin activation. In addition, in silico and in vitro analyses, along with chorioallantoic membrane assays, demonstrated that β-catenin-activated Ewing cells secreted factors that promote angiogenesis. In particular, activation of canonical Wnt signaling leads Ewing sarcoma cells to upregulate expression and secretion of proangiogenic ECM proteins, collectively termed the angiomatrix. Significantly, our data show that induction of the angiomatrix by Wnt-responsive tumor cells is indirect and is mediated by TGF-β. Mechanistically, Wnt/β-catenin signaling antagonizes EWS-FLI1-dependent repression of TGF-β receptor type 2, thereby sensitizing tumor cells to TGF-β ligands. Together, these findings suggest that Wnt/β-catenin-active tumor cells can contribute to Ewing sarcoma progression by promoting angiogenesis in the local TME.

// Laurie K. Svoboda 1 , Natashay Bailey 1 , Raelene A. Van Noord 2 , Melanie A. Krook 1 , Ashley Harris 1 , Cassondra Cramer 1 , Brooke Jasman 1 , Rajiv M. Patel 3, 4 , Dafydd Thomas 3 , Dmitry Borkin 3 , Tomasz Cierpicki 3 , Jolanta Grembecka 3 , Elizabeth R. Lawlor 1, 3 1 Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, MI 48109, USA 2 Department of Surgery, University of Michigan Medical School, Ann Arbor, MI 48109, USA 3 Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109, USA 4 Department of Dermatology, University of Michigan Medical School, Ann Arbor, MI 48109, USA Correspondence to: Elizabeth R. Lawlor, email: elawlor@med.umich.edu Keywords: Ewing sarcoma, trithorax, MLL, menin, HOX Received: August 15, 2016 Accepted: November 14, 2016 Published: November 18, 2016 ABSTRACT Developmental transcription programs are epigenetically regulated by the competing actions of polycomb and trithorax (TrxG) protein complexes, which repress and activate genes, respectively. Ewing sarcoma is a developmental tumor that is associated with widespread de-regulation of developmental transcription programs, including HOX programs. Posterior HOXD genes are abnormally over-expressed by Ewing sarcoma and HOXD13 , in particular, contributes to the tumorigenic phenotype. In MLL1 fusion-driven leukemia, aberrant activation of HOXA genes is epigenetically mediated by the TrxG complex and HOXA gene expression and leukemogenesis are critically dependent on the protein-protein interaction between the TrxG proteins MLL1 and menin. Based on these data, we investigated whether posterior HOXD gene activation and Ewing sarcoma tumorigenicity are similarly mediated by and dependent on MLL1 and/or menin. Our findings demonstrate that Ewing sarcomas express high levels of both MLL1 and menin and that continued expression of both proteins is required for maintenance of tumorigenicity. In addition, exposure of Ewing sarcoma cells to MI-503, an inhibitor of the MLL1-menin protein-protein interaction developed for MLL1-fusion driven leukemia, leads to loss of tumorigenicity and down-regulated expression of the posterior HOXD gene cluster. Together these data demonstrate an essential role for MLL1 and menin in mediating tumor maintenance and posterior HOXD gene activation in Ewing sarcoma. A critical dependency of these tumors on the MLL1-menin interaction presents a potentially novel therapeutic target.

Background: Previous studies have shown that aberrant activation of the Wnt/β-catenin pathway is associated with many malignant neoplasms. This includes some soft-tissue sarcoma phenotypes, most notably synovial sarcoma, implicating potential targets for novel molecular therapies. Objective: We investigate the level of Wnt/β-catenin pathway activation present in leiomyosarcomas relative to synovial sarcomas, using expression of LEF1 and β-catenin as surrogates. Methods: Cancer outlier profile analysis was performed on messenger RNA expression datasets in Oncomine (70 synovial sarcomas, 178 leiomyosarcomas). Results for LEF1 and β-catenin messenger RNA expression were reported in terms of median-centered intensity. Separate immunohistochemical studies were performed on tissue microarrays created from 77 synovial sarcomas and 89 leiomyosarcomas using antibodies to LEF1 and β-catenin. Tumors with unequivocal strong nuclear staining involving ⩾5% of cells were interpreted as positive. Results: Cancer outlier profile analysis demonstrated a higher level of LEF1 messenger RNA expression in synovial sarcomas than in leiomyosarcomas ( p Conclusion: These results provide further evidence that aberrant activation of the Wnt/β-catenin pathway is present in most synovial sarcomas, but not in most leiomyosarcomas. While targeting the constituents of this pathway might be effective in the treatment of synovial sarcomas, it is not likely to be an effective strategy in the treatment of leiomyosarcomas.

Metastatic Ewing sarcoma has a very poor prognosis and therefore new investigations into the biologic drivers of metastatic progression are key to finding new therapeutic approaches. The tumor microenvironment is highly dynamic, leading to exposure of different regions of a growing solid tumor to changes in oxygen and nutrient availability. Tumor cells must adapt to such stress in order to survive and propagate. In the current study, we investigate how Ewing sarcoma cells respond to the stress of growth factor deprivation and hypoxia. Our findings reveal that serum deprivation leads to a reversible change in Ewing cell cytoskeletal phenotypes. Using an array of migration and invasion techniques, including gelatin matrix degradation invadopodia assays, we show that exposure of Ewing sarcoma cells to serum deprivation and hypoxia triggers enhanced migration, invadopodia formation, matrix degradation and invasion. Further, these functional changes are accompanied by and dependent on activation of Src kinase. Activation of Src, and the associated invasive cell phenotype, were blocked by exposing hypoxia and serum-deprived cells to the Src inhibitor dasatinib. These results indicate that Ewing sarcoma cells demonstrate significant plasticity in response to rapidly changing micro-environmental stresses that can result from rapid tumor growth and from necrosis-causing therapies. In response to these stresses, Ewing cells transition to a more migratory and invasive state and our data show that Src is an important mediator of this stress response. Our data support exploration of clinically available Src inhibitors as adjuvant agents for metastasis prevention in Ewing sarcoma.

Multi-agent chemotherapeutic regimes remain the cornerstone treatment for Ewing sarcoma, the second most common bone malignancy diagnosed in pediatric and young adolescent populations. We have reached a therapeutic ceiling with conventional cytotoxic agents, highlighting the need to adopt novel approaches that specifically target the drivers of Ewing sarcoma oncogenesis. As KDM1A/lysine-specific demethylase 1 (LSD1) is highly expressed in Ewing sarcoma cell lines and tumors, with elevated expression levels associated with worse overall survival (P = 0.033), this study has examined biomarkers of sensitivity and mechanisms of cytotoxicity to targeted KDM1A inhibition using SP-2509 (reversible KDM1A inhibitor). We report, that innate resistance to SP-2509 was not observed in our Ewing sarcoma cell line cohort (n = 17; IC50 range, 81 –1,593 nmol/L), in contrast resistance to the next-generation KDM1A irreversible inhibitor GSK-LSD1 was observed across multiple cell lines (IC50 > 300 μmol/L). Although TP53/STAG2/CDKN2A status and basal KDM1A mRNA and protein levels did not correlate with SP-2509 response, induction of KDM1B following SP-2509 treatment was strongly associated with SP-2509 hypersensitivity. We show that the transcriptional profile driven by SP-2509 strongly mirrors KDM1A genetic depletion. Mechanistically, RNA-seq analysis revealed that SP-2509 imparts robust apoptosis through engagement of the endoplasmic reticulum stress pathway. In addition, ETS1/HIST1H2BM were specifically induced/repressed, respectively following SP-2509 treatment only in our hypersensitive cell lines. Together, our findings provide key insights into the mechanisms of SP-2509 cytotoxicity as well as biomarkers that can be used to predict KDM1A inhibitor sensitivity in Ewing sarcoma. Mol Cancer Ther; 17(9); 1902–16. ©2018 AACR.

Relapse after clinical remission remains a leading cause of cancer-associated death. Although the mechanisms of tumor relapse are complex, the ability of cancer cells to survive physiological stress is a prerequisite for recurrence. Ewing sarcoma (ES) and neuroblastoma (NB) are aggressive cancers that frequently relapse after initial remission. In addition, both tumors overexpress the polycomb group (PcG) proteins BMI-1 and EZH2, which contribute to tumorigenicity. We have discovered that ES and NB resist hypoxic stress-induced death and that survival depends on PcG function. Epigenetic repression of developmental programs is the most well-established cancer-associated function of PcG proteins. However, we noted that voltage-gated potassium (Kv) channel genes are also targets of PcG regulation in stem cells. Given the role of potassium in regulating apoptosis, we reasoned that repression of Kv channel genes might have a role in cancer cell survival. Here we describe our novel finding that PcG-dependent repression of the Kv1.5 channel gene KCNA5 contributes to cancer cell survival under conditions of stress. We show that survival of cancer cells in stress is dependent upon suppression of Kv1.5 channel function. The KCNA5 promoter is marked in cancer cells with PcG-dependent chromatin repressive modifications that increase in hypoxia. Genetic and pharmacological inhibition of BMI-1 and EZH2, respectively, restore KCNA5 expression, which sensitizes cells to stress-induced death. In addition, ectopic expression of the Kv1.5 channel induces apoptotic cell death under conditions of hypoxia. These findings identify a novel role for PcG proteins in promoting cancer cell survival via repression of KCNA5.

Background Advances in cancer therapeutics depend on reliable in vivo model systems. To develop biologically relevant xenografts, ultrasound was utilized for tissue-directed implantation of neuroblastoma (NB) cell line and patient-derived tumors in the adrenal gland, and for renal subcapsular engraftment of Ewing's sarcoma (ES). Materials and methods NB xenografts were established by direct adrenal injection of luciferase-transfected NB cell lines (IMR32, SH-SY5Y, SK-N-BE2) or NB patient-derived tumor cells (UMNBL001, UMNBL002). ES xenografts were established by renal subcapsular injection of TC32, A673, CHLA-25, or A4573 cells. Progression was monitored by in vivo imaging. Results Tumors progressed to local disease with metastasis evident by 5 weeks. Metastatic sites included cortical bone, lung, liver, and lymph nodes. Xenografted tumors retained immunochemical features of the original cancer. Conclusion Human NB adrenal xenografts, including two patient-derived orthotopic, and ES renal subcapsular xenografts were established by ultrasound without open surgery. Tissue-directed implantation is an effective technique for developing metastatic preclinical models.

Metastasis is the major cause of cancer deaths and control of gene transcription has emerged as a critical contributing factor. RhoA- and RhoC-induced gene transcription via the actin-regulated transcriptional co-activator megakaryocytic leukemia (MKL) and serum response factor (SRF) drive metastasis in breast cancer and melanoma. We recently identified a compound, CCG-1423, which blocks Rho/MKL/SRF-mediated transcription and inhibits PC-3 prostate cancer cell invasion. Here, we undertook a genome-wide expression study in PC-3 cells to explore the mechanism and function of this compound. There was significant overlap in the genes modulated by CCG-1423 and Latrunculin B (Lat B), which blocks the Rho/MKL/SRF pathway by preventing actin polymerization. In contrast, the general transcription inhibitor 5,6-dichloro-1-β-d-ribofuranosyl-1H-benzimidazole (DRB) showed a markedly different pattern. Effects of CCG-1423 and Lat B on gene expression correlated with literature studies of MKL knock-down. Gene sets involved in DNA synthesis and repair, G1/S transition, and apoptosis were modulated by CCG-1423. It also upregulated genes involved in endoplasmic reticulum stress. Targets of the known Rho target transcription factor family E2F and genes related to melanoma progression and metastasis were strongly suppressed by CCG-1423. These results confirm the ability of our compound to inhibit expression of numerous Rho/MKL-dependent genes and show effects on stress pathways as well. This suggests a novel approach to targeting aggressive cancers and metastasis.

// Kristina von Heyking 1 , Laura Roth 1, * , Miriam Ertl 1, * , Oxana Schmidt 1 , Julia Calzada-Wack 2 , Frauke Neff 2 , Elizabeth R. Lawlor 3 , Stefan Burdach 1 , Gunther H.S. Richter 1 1 Laboratory for Functional Genomics and Transplantation Biology, Children’s Cancer Research Center and Department of Pediatrics, Klinikum rechts der Isar, Technische Universitat Munchen, Munich Comprehensive Cancer Center (CCCM), and German Translational Cancer Research Consortium (DKTK), Munich, Germany 2 Institute of Pathology, Helmholtz Zentrum Munchen - German Research Center for Environmental Health (GmbH), Neuherberg, Germany 3 Departments of Pediatrics and Pathology, University of Michigan, Ann Arbor, Michigan, United States of America * These authors contributed equally to this work Correspondence to: Gunther H.S. Richter, email: guenther.richter@tum.de Keywords: HOXD, metastasis, Ewing sarcoma, WNT signaling, endochondral development Received: December 02, 2015 Accepted: May 13, 2016 Published: May 30, 2016 ABSTRACT Microarray analysis revealed genes of the posterior HOXD locus normally involved in bone formation to be over-expressed in primary Ewing sarcoma (ES). The expression of posterior HOXD genes was not influenced via ES pathognomonic EWS/ETS translocations. However, knock down of the dickkopf WNT signaling pathway inhibitor 2 (DKK2) resulted in a significant suppression of HOXD10, HOXD11 and HOXD13 while over-expression of DKK2 and stimulation with factors of the WNT signaling pathway such as WNT3a, WNT5a or WNT11 increased their expression. RNA interference demonstrated that individual HOXD genes promoted chondrogenic differentiation potential, and enhanced expression of the bone-associated gene RUNX2 . Furthermore, HOXD genes increased the level of the osteoblast- and osteoclast-specific genes, osteocalcin ( BGLAP ) and platelet-derived growth factor beta polypeptide ( PDGFB ), and may further regulate endochondral bone development via induction of parathyroid hormone-like hormone ( PTHLH ). Additionally, HOXD11 and HOXD13 promoted contact independent growth of ES, while in vitro invasiveness of ES lines was enhanced by all 3 HOXD genes investigated and seemed mediated via matrix metallopeptidase 1 (MMP1). Consequently, knock down of HOXD11 or HOXD13 significantly suppressed lung metastasis in a xeno-transplant model in immune deficient mice, providing overall evidence that posterior HOXD genes promote clonogenicity and metastatic potential of ES.

Despite multimodal treatment, long term outcome for patients with Ewing sarcoma is still poor. The second >European interdisciplinary Ewing sarcoma research summit> assembled a large group of scientific experts in the field to discuss their latest unpublished findings on the way to the identification of novel therapeutic targets and strategies. Ewing sarcoma is characterized by a quiet genome with presence of an EWSR1-ETS gene rearrangement as the only and defining genetic aberration. RNAsequencing of recently described Ewing-like sarcomas with variant translocations identified them as biologically distinct diseases. Various presentations adressed mechanisms of EWS-ETS fusion protein activities with a focus on EWS-FLI1. Data were presented shedding light on the molecular underpinnings of genetic permissiveness to this disease uncovering interaction of EWS-FLI1 with recently discovered susceptibility loci. Epigenetic context as a consequence of the interaction between the oncoprotein, cell type, developmental stage, and tissue microenvironment emerged as dominant theme in the discussion of the molecular pathogenesis and inter- and intratumor heterogeneity of Ewing sarcoma, and the difficulty to generate animal models faithfully recapitulating the human disease. The problem of preclinical development of biologically targeted therapeutics was discussed and promising perspectives were offered from the study of novel in vitro models. Finally, it was concluded that in order to facilitate rapid pre-clinical and clinical development of novel therapies in Ewing sarcoma, the community needs a platform to maintain knowledge of unpublished results, systems and models used in drug testing and to continue the open dialogue initiated at the first two Ewing sarcoma summits., The conference was supported by the European Union’s Seventh Framework Programme grants 261743(ENCCA) and 259348 (ASSET).

The synthesis and processing of mRNA, from transcription to translation initiation, often requires splicing of intragenic material. The final mRNA composition varies based on proteins that modulate splice site selection. EWS-FLI1 is an Ewing sarcoma (ES) oncoprotein with an interactome that we demonstrate to have multiple partners in spliceosomal complexes. We evaluate the effect of EWS-FLI1 on posttranscriptional gene regulation using both exon array and RNA-seq. Genes that potentially regulate oncogenesis, including CLK1, CASP3, PPFIBP1, and TERT, validate as alternatively spliced by EWS-FLI1. In a CLIP-seq experiment, we find that EWS-FLI1 RNA-binding motifs most frequently occur adjacent to intron–exon boundaries. EWS-FLI1 also alters splicing by directly binding to known splicing factors including DDX5, hnRNP K, and PRPF6. Reduction of EWS-FLI1 produces an isoform of γ-TERT that has increased telomerase activity compared with wild-type (WT) TERT. The small molecule YK-4–279 is an inhibitor of EWS-FLI1 oncogenic function that disrupts specific protein interactions, including helicases DDX5 and RNA helicase A (RHA) that alters RNA-splicing ratios. As such, YK-4–279 validates the splicing mechanism of EWS-FLI1, showing alternatively spliced gene patterns that significantly overlap with EWS-FLI1 reduction and WT human mesenchymal stem cells (hMSC). Exon array analysis of 75 ES patient samples shows similar isoform expression patterns to cell line models expressing EWS-FLI1, supporting the clinical relevance of our findings. These experiments establish systemic alternative splicing as an oncogenic process modulated by EWS-FLI1. EWS-FLI1 modulation of mRNA splicing may provide insight into the contribution of splicing toward oncogenesis, and, reciprocally, EWS-FLI1 interactions with splicing proteins may inform the splicing code.

Motivated by the recent implication of cysteine protease cathepsin L as a potential target for anti-cancer drug development, we used a conditional MycER(TAM); Bcl-x(L) model of pancreatic neuroendocrine tumorigenesis (PNET) to assess the role of cathepsin L in Myc-induced tumor progression. By employing a cysteine cathepsin activity probe in vivo and in vitro, we first established that cathepsin activity increases during the initial stages of MycER(TAM); Bcl-x(L) tumor development. Among the cathepsin family members investigated, only cathepsin L was predominately produced by beta-tumor cells in neoplastic pancreata and, consistent with this, cathepsin L mRNA expression was rapidly upregulated following Myc activation in the beta cell compartment. By contrast, cathepsins B, S and C were highly enriched in tumor-infiltrating leukocytes. Genetic deletion of cathepsin L had no discernible effect on the initiation of neoplastic growth or concordant angiogenesis. However, the tumors that developed in the cathepsin L-deficient background were markedly reduced in size relative to their typical wild-type counterparts, indicative of a role for cathepsin L in enabling expansive tumor growth. Thus, genetic blockade of cathepsin L activity is inferred to retard Myc-driven tumor growth, encouraging the potential utility of pharmacological inhibitors of cysteine cathepsins in treating late stage tumors.

A goal of the COG Ewing Sarcoma Biology Committee is enabling identification of reliable biomarkers that can predict treatment response and outcome through the use of prospectively collected tissues and correlative studies in concert with COG therapeutic studies. In this report, we aim to provide a concise review of the most well-characterized prognostic biomarkers in Ewing Sarcoma (ES), and to provide recommendations concerning design and implementation of future biomarker studies. Of particular interest and potentially high clinical relevance are studies of cell cycle proteins, sub-clinical disease, and copy number alterations. We discuss findings of particular interest from recent biomarker studies and examine factors important to the success of identifying and validating clinically relevant biomarkers in ES. A number of promising biomarkers have demonstrated prognostic significance in numerous retrospective studies and now need to be validated prospectively in larger cohorts of equivalently treated patients. The eventual goal of refining the discovery and use of clinically relevant biomarkers is the development of patient specific ES therapeutic modalities.

Despite intensive treatment with chemotherapy, radiotherapy and surgery, over 70% of patients with metastatic Ewing's Sarcoma Family of Tumors (EFT) will die of their disease. We hypothesize that properly characterized laboratory models reflecting the drug resistance of clinical tumors will facilitate the application of new therapeutic agents to EFT. To determine resistance patterns, we studied newly established EFT cell lines derived from different points in therapy: two established at diagnosis (CHLA-9, CHLA-32), two after chemotherapy and progressive disease (CHLA-10, CHLA-25), and two at relapse after myeloablative therapy and autologous bone marrow transplantation (post-ABMT) (CHLA-258, COG-E-352). The new lines were compared to widely studied EFT lines TC-71, TC-32, SK-N-MC, and A-673. These lines were extensively characterized with regard to identity (short tandem repeat (STR) analysis), p53, p16/14 status, and EWS/ETS breakpoint and target gene expression profile. The DIMSCAN cytotoxicity assay was used to assess in vitro drug sensitivity to standard chemotherapy agents. No association was found between drug resistance and the expression of EWS/ETS regulated genes in the EFT cell lines. No consistent association was observed between drug sensitivity and p53 functionality or between drug sensitivity and p16/14 functionality across the cell lines. Exposure to chemotherapy prior to cell line initiation correlated with drug resistance of EFT cell lines in 5/8 tested agents at clinically achievable concentrations (CAC) or the lower tested concentration (LTC): (cyclophosphamide (as 4-HC) and doxorubicin at CAC, etoposide, irinotecan (as SN-38) and melphalan at LTC; P

The role of aberrant DNA methylation in Ewing sarcoma is not completely understood. The methylation status of 503 genes in 52 formalin-fixed paraffin-embedded EWS tumors and 3 EWS cell lines was compared to human mesenchymal stem cell primary cultures (hMSCs) using bead chip methylation analysis. Relative expression of methylated genes was assessed in 5-Aza-2-deoxycytidine-(5-AZA)-treated EWS cell lines and in a cohort of primary EWS samples and hMSCs by gene expression and quantitative RT-PCR. 129 genes demonstrated statistically significant hypermethylation in EWS tumors compared to hMSCs. Thirty-six genes were profoundly methylated in EWS and unmethylated in hMSCs. 5-AZA treatment of EWS cell lines resulted in upregulation of expression of hundreds of genes including 162 that were increased by at least 2-fold. The expression of 19 of 36 candidate hypermethylated genes was increased following 5-AZA. Analysis of gene expression from an independent cohort of tumors confirmed decreased expression of six of nineteen hypermethylated genes (AXL, COL1A1, CYP1B1, LYN, SERPINE1,) andVCAN. Comparing gene expression and DNA methylation analyses proved to be an effective way to identify genes epigenetically regulated in EWS. Further investigation is ongoing to elucidate the role of these epigenetic alterations in EWS pathogenesis.

The Ewing Sarcoma Family Tumors (ESFT) consist of the classical pathologic entities of Ewing Sarcoma and peripheral Primitive Neuroectodermal Tumor. Occurring largely in the childhood through young adult years, these tumors have an unsurpassed propensity for metastasis and have no defined cell of origin. The biology of these aggressive malignancies centers around EWS/FLI1 and related EWS/ETS chimeric transcription factors, which are largely limited to this tumor class. Much progress has been made in the identification of a network of loci whose expression is modulated by EWS/FLI1 and its congeners. To date, little progress has been made in reconstructing the sequence of direct and indirect events that produce this network of modulated loci. The recent identification of GLI1 as an upregulated target of EWS/ETS transcription factors suggests a target which may be a more central mediator in the ESFT signaling network. In this paper, we further define the relationship of EWS/FLI1 expression and GLI1 upregulation in ESFT. This relationship is supported with data from primary tumor specimens. It is consistently observed across multiple ESFT cell lines and with multiple means of EWS/FLI1 inhibition. GLI1 inhibition affects tumor cell line phenotype whether shRNA or endogenous or pharmacologic inhibitors are employed. As is seen in model transformation systems, GLI1 upregulation by EWS/FLI1 appears to be independent of Hedgehog stimulation. Consistent with a more central role in ESFT pathogenesis, several known EWS/FLI1 targets appear to be targeted through GLI1. These findings further establish a central role for GLI1 in the pathogenesis of Ewing Tumors.