Abstract LB-233: Modeling Ewing sarcoma initiation in human neural crest stem cells

Abstract

Abstract Ewing sarcoma family tumors (ESFT) are aggressive bone and soft tissue tumors of putative neural crest (NCSC) and/or mesenchymal (MSC) stem cell origin. It is our hypothesis that the initiation and progression of ESFT depends on aberrant silencing of genes that normally instruct terminal differentiation of NCSC and their progeny. Further, we hypothesize that this is a downstream consequence of EWS-FLI1-mediated activation of polycomb proteins. In the current study we are using human NCSC to define the consequences of EWS-FLI1 oncogene expression on DNA methylation in developing neural crest cells. NCSC were isolated from in vitro differentiating embryonic stem cells by FACS-based sorting. NCSC were expanded in culture in self-renewal media or induced to differentiate either as unmanipulated cells or following lentiviral transduction with EWS-FLI1-EGFP or EGFP control virus. Cells were studied for differentiation capacity and for gene expression by RT-PCR and Affymetrix Human Exon array profiling. Genomic DNA was isolated at various timepoints for evaluation of genome wide DNA methylation using Illumina 450K BeadChips. Our studies thus far show that clonal populations of NCSC can be induced to generate neural, glial and myofibroblastic progeny. In addition, NCSC rapidly adopt an MSC-like phenotype in adherent culture and these cells (NC-MSC) retain the ability to differentiate into peripherin positive peripheral nerves as well as adipogenic and osteogenic progeny. Both NCSC and NC-MSC tolerate EWS-FLI1 and expression of EWS-FLI1 in NC-MSC partially reactivates the more primitive NCSC genetic program. EWS-FLI1-expressing cells up-regulate expression of the polycomb proteins BMI-1 and EZH2 and avoid cellular senescence. Kinetic transcriptional evaluation of transduced cells revealed that changes in gene expression downstream of EWS-FLI1 are highly dynamic. In particular, with increasing passage, EWS-FLI1+ cells progressively down-regulate transcripts involved in neural differentiation. Preliminary Illumina GoldenGate assays revealed that the promoters of many polycomb target genes involved in neural differentiation are relatively hypermethylated in ESFT compared to normal adult tissues and stem cells. We are now performing Illumina 450K assays of differentiating NCSC in the presence or absence of EWS-FLI1 to define the consequences of oncogene activation on the normal evolution of DNA methylation over time. Updated data from these studies will be presented at the meeting. In summary, we have developed a human developmental model in which to study the the molecular mechanisms of EWS-FLI1-induced transformation. Our studies thus far suggest that ESFT is a stem cell tumor in which a single genetic mutation induces profound epigenetic changes that ultimately cooperate to induce tumor formation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-233. doi:10.1158/1538-7445.AM2011-LB-233