Molecular heterogeneity and function of EWS-WT1 fusion transcripts in desmoplastic small round cell tumors.

ABSTRACT

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 WT1 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.