Purpose: The EWS gene is implicated in the chromosomal translocation t(11;22)(q24;q12) that leads to the EWS/FLI-1 gene fusion, in 85% of Ewing sarcomas. EWS is a member of the FET family of proteins, which includes two other members named FUS and TAF15. FUS and TAF15 are also involved in oncogenic gene fusions. All three native proteins possess an RNA-binding domain as well as an N-terminal domain that acts as a powerful transactivation domain within the fusion protein. Mutations in the FUS gene have been associated with certain types of amyotrophic lateral sclerosis, a fatal neuro-degenerative disease. The literature indicates a role for these proteins in the modulation of gene expression. This project aims to improve understanding of the normal functions of the FET protein family, and of the impact that the EWS-FLI-1 oncogenic fusion protein may have on them, in the context of Ewing sarcoma.

Methods: To elucidate their function, we have developed an efficient knock-down of the three protein-coding genes by RNA interference. We then chose to study classical processes of tumoral development and progression, as well as gene expression regulation mechanisms.

Results: In Ewing and non-Ewing cell lines, we observe a decrease in live cell count after efficient simultaneous knock-down of the three proteins, as well as after knockdown of EWS or TAF15 alone. Preliminary data suggest that this phenotype might be connected to an increased cell death rather than to a cell cycle arrest.

RNA-sequencing of RNA extracted in different knockdown conditions - in Ewing and non-Ewing cell lines - shows a large number of modulated transcripts both in terms of quantitative and qualitative (alternative splicing of known transcripts or novel transcripts) expression. We find a highly significant overlap between groups of transcripts that are differentially expressed after EWS, FUS or TAF15 knock-down within a given cell line, as well as between differentially expressed transcripts after same FET knockdown in two different cell backgrounds. Ingenuity-IPA analysis of transcripts at the intersection of the three FET knockdown conditions yields a significant over-representation of functions compatible with the observed phenotype such as “cell growth and proliferation”, “cell death and survival” and “cell cycle”. By crossing RNA-seq data from our Ewing cell line either after EWS or EWS-FLI-1 knockdown, we came across a small subset of 19 transcripts that were conversely regulated by EWS and EWS-FLI. This overlap allows us to hypothesize in a robust manner that EWS-FLI-1 may exert a dominant negative effect on EWS, at least in terms of regulation of gene expression. Among this subset, one target of interest is under further validation and functional assessment. Besides, we are currently validating the impact of EWS knockdown on alternative splicing of specific targets, both in Ewing and non-Ewing cells. More global aspects concerning the role of the different FET proteins in splicing and expression of alternative transcripts are currently being established.

Conclusions: By deciphering the roles of the FET family of proteins in cell proliferation and regulation of gene expression, as well as how EWS-FLI-1 may disrupt them, we may identify new downstream transcripts relevant for Ewing's sarcoma biology as well as possible therapeutic targets.

Citation Format: Sarah Cohen-Gogo, Franck Tirode, Olivier Delattre. Deciphering the role(s) of the FET family of proteins in the Ewing sarcoma model: Is EWS-FLI-1 crashing the party? [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A59.