SUMMARYEwing’s sarcoma, a malignant bone tumor of children and young adults, is a member of the small-round-blue-cell tumor family. Ewing’s sarcoma family tumors (ESFTs), which include peripheral primitive neuroectodermal tumors (PNETs), are characterized by chromosomal translocations that generate fusions between the EWS gene and ETS-family transcription factors, most commonly FLI1. The EWS-FLI1 fusion oncoprotein represents an attractive therapeutic target for treatment of Ewing’s sarcoma. The cell of origin of ESFT and the molecular mechanisms by which EWS-FLI1 mediates tumorigenesis remain unknown, and few animal models of Ewing’s sarcoma exist. Here, we report the use of zebrafish as a vertebrate model of EWS-FLI1 function and tumorigenesis. Mosaic expression of the human EWS-FLI1 fusion protein in zebrafish caused the development of tumors with histology strongly resembling that of human Ewing’s sarcoma. The incidence of tumors increased in a p53 mutant background, suggesting that the p53 pathway suppresses EWS-FLI1-driven tumorigenesis. Gene expression profiling of the zebrafish tumors defined a set of genes that might be regulated by EWS-FLI1, including the zebrafish ortholog of a crucial EWS-FLI1 target gene in humans. Stable zebrafish transgenic lines expressing EWS-FLI1 under the control of the heat-shock promoter exhibit altered embryonic development and defective convergence and extension, suggesting that EWS-FLI1 interacts with conserved developmental pathways. These results indicate that functional targets of EWS-FLI1 that mediate tumorigenesis are conserved from zebrafish to human and provide a novel context in which to study the function of this fusion oncogene.

Summary. Two pregnant cats showed unilateral ovarian dysgenesis at surgery. Cytogenetic examination revealed a 37,X/39,XXX karyotype in Cat 1, a 37,X/38,XX karyotype in Cat 2 and a marked difference in frequency of mosaicism between fibroblasts and lymphocytes in both cats. Histologically the ovarian morphology ranged from normal to complete dysgenesis in both cats. Three fetuses examined showed a normal feline karyotype.

Ewing's sarcoma, a member of the small round blue cell family of tumors, is an aggressive and malignant bone tumor of children and young adults. The cells are generally poorly differentiated, although can also have characteristics of neuroectodermal cells. Ewing's tumors are characterized by a t(11:22) that generates a fusion of the EWS protein and the FLI1 transcription factor. This fusion oncoprotein, EWS-FLI1, represents a potential therapeutic target for treatment of Ewing's sarcoma. However, the molecular mechanisms by which EWS-FLI1 mediates tumorigenesis remain unknown. Few animal models exist in which to study the function of EWS-FLI1. Here, we report the use of zebrafish as a new vertebrate model of EWS-FLI1 function and tumorigenesis. Using the zebrafish Tol2 transposon system, we have developed several transgenic models to study the function of EWS-FLI1 in a vertebrate model organism. First, we have injected zebrafish with transposons containing human EWS-FLI1 driven by either the heat shock or the β-actin promoter. A subset of the injected fish developed tumors when raised to adulthood. These tumors strongly resemble Ewing's sarcoma. This finding demonstrates that the functional targets of EWS-FLI1 that mediate tumor development are conserved from zebrafish to human. We observe an increased incidence of tumors in a p53 mutant background, suggesting that the p53 pathway may suppress EWS-FLI1 driven tumorigenesis. We are performing microarray analysis to identify candidate downstream effector genes and to compare to previously identified EWS-FLI1 target genes. In addition to these tumors, we identified transgenic lines that transmit the EWS-FLI1 transgene to their progeny. The embryos that express EWS-FLI1 exhibit abnormal embryonic development. This result suggests that EWS-FLI1 may regulate conserved developmental pathways. These transgenic lines represent a novel physiological context in which to study the cellular function of EWS-FLI1. From these two models of EWS-FLI1 expression in the zebrafish, we conclude that human EWS-FLI1 functions in zebrafish and that these models provide a novel context in which to study the function of this fusion oncogene. Additionally, the rapid development and small size of the zebrafish will allow us to utilize these models for genetic and chemical screens for specific EWS-FLI1 inhibitors for the treatment of Ewing's sarcoma. Citation Information: Cancer Res 2009;69(23 Suppl):B36.