Abstract 3897: EWS-FLI1 is regulated by acetylation

Abstract

Abstract Introduction: EWS-FLI1 (EF) is the oncogenic transcription factor that is pathognomonic for the Ewing's Sarcoma Family of Tumors (ESFT). Lysine acetylation is an important mechanism in transcription factor regulation including protein stability, protein-protein interaction, DNA-binding, and transcription activation. We hypothesized that EF is acetylated and that acetylation modulates its function. Methods: We used in vitro acetylation assays, mass spectrometry, mutagenesis, EMSA, reporter luciferase assays, western blot and cellular cytotoxicity studies to test this hypothesis. Results: We found that ESFT cells express all three major eukaryotic histone acetyl transferases (HAT): PCAF, CBP and p300. The EF amino acid sequence possesses a number of lysine residues that might constitute potential acetylation sites almost all reside in the C-terminal FLI1 domain (CTD), which contains the DNA binding ets-domain. We show that the CTD is acetylated by CBP, PCAF and P300 and identified four lysines as major sites for acetylation (K240, K252, K380 and K397). Full length EF directly binds to both PCAF and P300, and can be acetylated in vitro. Expression of several HAT(s) stimulates transcription of a typical EF target promoter (NR0B1), placed upstream of a luciferase reporter. Furthermore, histone deacetylase inhibitor (HDACi) treated Cos7 cells that artificially express EF, show increased activity of the NR0B1 reporter compared to non-treated cells. We provide evidence that this increased reporter activity relies upon the ability of acetylation to increase EF DNA binding activity and thus, transcription activation. HDACi (SAHA and TSA) demonstrated potent inhibition of ESFT cell lines with each of the different fusion types including TC71 (type 1, IC50 SAHA/TSA 1068/24 nM), SKES-1 (type 2, IC50 SAHA/TSA 1068/24 nM), and A4573 (type 3, IC50 SAHA/TSA 739/16 nM). Discussion: In conclusion, we show for the first time that EF is acetylated at functionally important residues, and provide evidence that acetylation may play a positive role in the oncogenic activity of EF fusion proteins. At a molecular level, at least one key function of acetylation might consist of the enhancement of EF DNA binding- and transcriptional activity. On the other side, our data pose an apparent contradiction, because ESFT expressing cells undergo cell death when treated with HDAC inhibitors. We believe that one likely explanation for this phenomenon is that the anti-tumor effects of HDACi are rather pleiotropic, and may converge on pro-apoptotic molecules not necessarily related to EF function. Since the expression of EF is crucial for the survival of ESFT cells and maintenance of the tumor the therapy must not support the oncogene's function. Current studies are investigating whether the hyper-sensitivity of ESFT cells to HDACi treatment is sustained by broader effects of protein acetylation, and to determine the specific contribution of EF acetylation in regulation of proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3897.