Molecular Heterogeneity and Function of EWS-WT1 Fusion Transcripts in Desmoplastic Small Round Cell Tumors

Desmoplastic small round cell tumor (DSRCT) is a primitive sarcoma with a consistent cytogenetic abnormality, t(11;22)(p13;q12). This chromosomal translocation generates a chimeric transcript that is formed by fusion of the 5′ region of the Ewing’s sarcoma gene, EWS , with the 3′ DNA-binding segment of WT1 , the Wilms’ tumor suppressor gene. We collected 14 DSRCT tumor samples and examined the hybrid transcripts. We identified: ( a ) combinatorial heterogeneity of EWS exons fused to WT1 including use of EWS exons 7, 8, and 9; ( b ) subpopulations of variant transcripts in 6 of 14 tumors characterized by aberrant splicing resulting in loss of EWS exon 6 or WT1 exon 9; ( c ) multiple cDNA products with large internal deletions; and ( d ) insertion of small stretches of heterologous DNA at the fusion site or exon splice region in transcripts from two tumors. Most of the splice variants were in-frame, and in vitro translated fusion proteins with intact DNA-binding motifs formed complexes with a WT 1 response element in gel mobility assays. Each of the chimeric proteins retains the ability to bind to the GC and TC elements of the early transcription factor EGR-1 as well as WT1 consensus sequences. We present evidence that various EWS-WT1 proteins up-regulated EGR-1 promoter activity and that this up-regulation is specifically dependent upon the absence of the exon 9 KTS domain of WT1 . The molecular diversity and functionality exhibited by these fusion transcripts may have significant biological implications for their transactivating and tumorigenic potential.