Abstract IA04: EWS-FLI1, polycomb proteins and stem cells: Clues to the developmental origins of Ewing sarcoma
Authors: Elizabeth R. Lawlor, Russell G. Adderley
Journal: Cancer Research (2014)

Abstract Ewing sarcomas (ES) are aggressive bone and soft tissue tumors that primarily affect children and young adults. Despite intensive therapy, relapse is common and for patients who relapse or present with overt metastatic disease survival rates are at best 20%. Intensification of current treatment regimens is impossible due to dose-limiting toxicities. Therefore, novel, less toxic approaches to therapy are desperately needed. For ES as well as other pediatric tumors, these novel therapies will ideally target tumor cells whilst sparing normal developing tissues. Attainment of this important goal demands that we better understand the molecular underpinnings of ES and how its development deviates from normal. Many pediatric malignancies arise as a result of developmental errors in which genetic mutations in fetal or immature cells initiate the complex process of malignant transformation, leading to eventual formation of a tumor. ES can occur in any anatomic location and their precise cellular origin remains elusive. Genetically they are characterized by expression of chimeric fusion oncogenes, most commonly EWS-FLI1. Although it functions as an oncogene in ES, EWS-FLI1 is toxic to most normal cells. In contrast, studies from our lab and others have shown that stem cells, in particular bone marrow-derived mesenchymal (BM-MSC) and neural crest-derived (NCSC) stem cells are tolerant of the fusion protein. Moreover, the genetic profile of ES, combined with its ability to differentiate in vivo and in vitro into both neural and mesenchymal lineages implicates multipotent neuro-mesenchymal stem cells as putative cells of origin. Significantly, studies in model organisms have demonstrated that rare populations of NCSC persist in adult tissues and that some MSC are actually of neural crest rather than mesodermal origin. Based on these data we are investigating the molecular consequences of EWS-FLI1 on neural crest (NC) development using both human embryonic stem cell-derived and primary mouse NCSC. Our studies have shown that both human and murine NCSC cells tolerate EWS-FLI1. This tolerance is dependent on cooperation with polycomb proteins as demonstrated by dose-dependent tolerance of the oncoprotein in NCSC derived from Bmi-1 wild-type, heterozygous, and knockout mice and by induction of cellular senescence following BMI-1 knockdown in EWS-FLI1+ human NC cells. Ectopic expression of EWS-FLI1 in human NC cells leads to upregulation of BMI-1 and EZH2, maintenance of stemness, and initiation of an ES-like transcriptional program. Given the essential role that polycomb proteins play in regulation of transcriptional programs during embryonic development we evaluated gene expression profiles of control vector- and EWS-FLI1-transduced NC cells that had been exposed to differentiation media. After six weeks in differentiation media the transcriptional profiles of both cells evolved significantly and changes in expression were detected in hundreds of genes. Interestingly, however, the changes in gene expression were largely divergent. In particular, while exposure of control NC cells to differentiation media activated a mesenchymal transcriptional program, in EWS-FLI1+ cells a neuronal transcriptional program was instead dominant. Significantly, expression of numerous polycomb-regulated target genes was disrupted in EWS-FLI1+ cells, including expression of multiple HOX genes. Gene expression profiling of primary tumors confirmed that the polycomb-target and HOX gene profiles of ES differs markedly from normal stem cells and terminally differentiated adult tissues as well as other pediatric solid tumors and other cancer types. In particular, posterior HOXD genes, normally involved in controlling distal limb development, are abnormally upregulated in tumors and EWS-FLI1+ cells. Thus, expression of the fusion oncogene alters the transcriptional response of stem cells to differentiation signals, thus disrupting normal development. Polycomb target genes that are normally reversibly silenced by histone modifications in normal stem cells are often aberrantly and heritably silenced by DNA hypermethylation in cancer. Given our discovery that EWS-FLI1 profoundly deregulates polycomb proteins and expression of polycomb-regulated transcriptional programs, we next investigated whether these changes are associated with changes in DNA methylation. Illumina 450K and GoldenGate BeadArrays were used to evaluate CpG methylation in ES tumors, cell lines, normal stem cells (MSC, NCSC), and EWS-FLI1+ cells. These studies confirmed that ES tumors and cell lines demonstrate tissue specific patterns of DNA methylation that differ from both adult tissues as well as normal stem cells. In addition, unlike the very stable patterns of methylation displayed by normal tissues and stem cells, ES displayed substantial inter-tumor heterogeneity. Hyper-methylation of CpG islands was prevalent in ES, and was enriched in the promoter regions of polycomb target genes, in particular the promoters of bivalent loci. However, abnormal methylation was not limited to gene promoters but was evident at CpG islands across the genome. To determine if changes in DNA methylation are a consequence of EWS-FLI1 we evaluated EWS-FLI1+ NC cells. EWS-FLI1+ NC cells progressively altered their methylation state when exposed to differentiation media and evolved over time to acquire promoter and CpG island methylation profiles that resembled those of established ES cells. Thus, the DNA methylation profile of ES is abnormal and a downstream consequence of EWS–FLI1. Thus, our studies together support a model of ES tumorigenesis in which epigenomic deregulation drives tumor initiation downstream of the initiating oncogenic mutation. Specifically, we propose that ES arises from BMI-1+ stem cells that are inherently tolerant of EWS-FLI1. Expression of the fusion then leads to upregulation of polycomb proteins, deregulation of polycomb target gene expression in response to differentiation signals, and global disruption of DNA methylation, in particular at CpG islands. The progressive acquisition of and selection for growth-promoting epigenetic changes ultimately combine to induce full malignant transformation. These findings, along with the recent findings of other groups, demonstrate the central role of epigenetic deregulation in ES tumor initiation and progression and suggest that epigenetic modifiers should be aggressively investigated as novel agents for the treatment of these devastating tumors. Citation Format: Elizabeth R. Lawlor, Russell G. Adderley. EWS-FLI1, polycomb proteins and stem cells: Clues to the developmental origins of 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 IA04.