Several aggressive pediatric sarcomas contain translocations resulting in the formation of fusion genes encoding chimeric transcription factors (CTF). Most prominent among these are EWS-FLI1 in Ewing's sar- coma (ES), PAX3-FKHR in alveolar rhabdomyosarcoma (ARMS), and EWS-WT1 in the desmoplastic small round cel tumor (DSRCT). A comprehensive analysis of the transcriptional targets of these CTFs is needed to understand their pathogenesis and to identify key genes and pathways as candidate therapeutic targets. We will use a novel combination of approaches to identify and validate direct transcriptional targets of these 3 CTFs in the most genuine cellular context by combining RNA interference (RNAi), expression profiling, and chromatin immunoprecipitation (CHIP) in cell lines of these three major pediatric sarcomas, as well as bioin- formatic mining of ranked lists of genes highly associated with these specific CTFs in our existin,q dataset of Affymetrix U133A expression profiles obtained from over 150 pediatric translocation sarcomas. The biologi- cally validated target genes thus identified will then be prioritized as potential therapeutic targets (either functional or immunologic). Selected potential functional targets will be further evaluated for their role in cancer cell growth properties using RNAi in the same and additional cell lines. A.,,.!m1: Analysis of CTF- regulated gene expression in pediatric sarcoma cell lines. We will use microarray-based expression profiling to screen for genes downregulated or upregulated upon knockdown of the endogenous CTF tran- script in ES, ARMS, and DSRCT cell lines by RNAi using stably transfected tetracycline-inducible constructs encoding short hairpin RNAs (processed to short interfering RNAs).
Aim 2 : Identification of direct tran- scriptional tarqets of CTFs. To select putative direct CTF targets for biological validation, we will use bioin- formatic approaches to identify promoters with predicted CTF binding sites within two sets of ranked gene lists: 1. CTF-regulated genes derived from Aim 1;and 2. genes highly associated with specific CTFs in expression profiles from primary tumors. Poorly defined promoters will be verified by experimental delinea- tion of transcription start sites and bioinformatic approaches. Biological CTF target gene validation ap- proaches will consist of ChIP assays to confirm in vivo promoter binding in the same cell lines, mapping of promoter binding (electrophoretic mobility shift assays), and analysis of functional effects (transactivation assays).
Aim 3 : Evaluation of direct targets of CTF transactivation as therapeutic tar clets by RNAi. In order to identify pathways and gene products critical to the growth and survival of these 3 pediatric sarcomas and therefore representing potentially tractable therapeutic targets, we will examine the effects of RNAi- mediated knockdown of selected direct targets of transactivation by these 3 CTFs in the corresponding cell lines.
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