Molecular analysis of the chromosomal translocation t(11;22)(p13;q12) in intra-abdominal desmoplastic small round cell tumors: Fusion of the EWS1, Ewing\u27s sarcoma gene, to WT1 creates a novel oncogenic transcription factor

The Intraabdominal Desmoplastic Small Round Cell Tumor (IADSRCT) is a pediatric/adolescent tumor believed to arise from a primitive cell in the peritoneal mesothelium. My characterization of the t(11;22)(p13;q12) in IADSRCT has demonstrated the consistent association of this tumor with a fusion transcript containing the $5\sp\prime$ end of the Ewing\u27s sarcoma gene (EWS1), and the $3\sp\prime$ end from the Wilms\u27 tumor (WT1) gene. My survey of frozen and formalin-fixed specimens demonstrated this fusion gene in 11/12 IADSRCT specimens examined. In each case the RNA fusion junction was the same, and a predicted protein was formed containing the amino-terminal domain (NTD) from EWS1 and the last three zinc fingers of WT1. These analyses have also demonstrated the maintenance of alternative splicing in the zinc-finger region derived from WT1, suggesting the presence of two protein isoforms in the tumor. Analysis of DNA-binding using zinc-finger recombinant proteins illustrated that the splice-generated isoform EWS-WTZF (+KTS) had reduced binding abilities to EGR and WTE-based consensus target sequences compared to EWS-WTZF (-KTS). Interestingly, EWS-WTZF (-KTS) and WTZF bound similar DNA targets, but EWS-WTZF did so with increased affinity and decreased sensitivity to mutations. The similarity between the DNA binding domains of EGR-1 and EWS-WT1 (-KTS) suggested that t(11;22)(p13;q12) had created an EGR-1-like transcriptional activator. However, a comparison of EWS-WT1 and EGR-1 revealed differences in their binding preferences. We have shown that the EWS-WT1 fusion protein has the ability to activate transcription from target promoters containing EGR-consensus-like sequences in NIH 3T3 cells and human mesothelial cells. Finally, I demonstrated the oncogenic potential of EWS-WT1 using retroviral infection of NIH 3T3 cells followed by growth in soft agar