Abstract A68: Posttranslational modifications of the oncogenic fusion protein EWS/FLI1
Authors: Garcia Laura A. Lopez , Gierisch Maria E. , Schäfer Beat W. 
Published: May 2015

Abstract
Ewing sarcoma is a pediatric tumor that affects bone and soft tissues. It is characterized in the majority of cases by the presence of a chromosomal translocation that leads to the expression of the oncogenic transcription factor, EWS/FLI1. Continuing expression of EWS/FLI1 is essential for tumor growth, cellular transformation and tumor cell survival, suggesting it being an important target for therapy. However, very little is known about the regulation of its oncogenic activity that might be modulated by posttranslational modifications (PTMs).
PTMs can modulate protein function in several different ways such as by altering its conformation, stability, activity, protein-protein and protein-DNA interactions, cellular localization etc. Here, we aimed to identify and characterize PTMs of EWS/FLI1 to understand their relevance for its oncogenic potential in order to identify novel therapeutic approaches.
We purified flag-EWS/FLI1 from A673 cells (Ewing cell line) and analyzed it by mass spectrometry to detect PTMs. For the first time, we were able to identify novel Ser/Thr residues which are phosphorylated in EWS/FLI1 apart from the phospho-Thr79 residue which was previously described. Except Thr79, all phosphorylation sites are present in the FLI1 part of the fusion protein. Furthermore, EWS/FLI1 was found to be methylated at a single arginine residue. Non evidence of acetylation of EWS/FLI1 was detected. Individual mutations of the phospho-Ser/Thr to alanine or the methylated arginine to lysine did not affect the cellular localization of EWS/FLI1. In order to assess whether phosphorylation of EWS/FLI1 has an impact on its transcriptional activity, different cellular systems were used to overexpress EWS/FLI1 wild type (wt) and mutants and to measure EWS/FLI1 target genes level by qRT-PCR. Transient overexpression of EWS/FLI1 wt and mutants in Ewing cell lines (A673 and RD ES) did not lead to a sufficient upregulation of EWS/FLI1 target genes. However, overexpression of EWS/FLI1 by retroviral transduction of RD (embryonal rhabdomyosarcoma cell line) and hMSC (human messenchymal stem cells) induced significant upregulation of EWS/FLI1 target genes when comparing EWS/FLI1 wt with EWS/FLI1 R386N, a DNA binding site mutant completely lacking transcriptional activity. With these systems, we are now in the process to elucidate the importance of individual PTMs of EWS/FLI1. Preliminary results indicate that phosphorylation of the fusion protein may be necessary for full transcriptional activity by increasing its stability.
Characterization of PTMs in EWS/FLI1 is therefore expected to lead to the discovery of new target options for the treatment of Ewing sarcoma.
Citation Format: Laura A. Lopez Garcia, Maria E. Gierisch, Beat W. Schäfer. Posttranslational modifications of the oncogenic fusion protein EWS/FLI1. [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 A68.