Proteasomal Degradation of the EWS-FLI1 Fusion Protein Is Regulated by a Single Lysine Residue.

E-26 transformation-specific (ETS) proteins are transcription factors directing gene expression through their conserved DNA binding domain. They are implicated as truncated forms or interchromosomal rearrangements in a variety of tumors including Ewing sarcoma, a pediatric tumor of the bone. Tumor cells express the chimeric oncoprotein EWS-FLI1 from a specific t(22;11)(q24;12) translocation. EWS-FLI1 harbors a strong transactivation domain from EWSR1 and the DNA-binding ETS domain of FLI1 in the C-terminal part of the protein. Although Ewing cells are crucially dependent on continuous expression of EWS-FLI1, its regulation of turnover has not been characterized in detail. Here, we identify the EWS-FLI1 protein as a substrate of the ubiquitin-proteasome system with a characteristic polyubiquitination pattern. Using a global protein stability approach, we determined the half-life of EWS-FLI1 to lie between 2 and 4 h, whereas full-length EWSR1 and FLI1 were more stable. By mass spectrometry, we identified two ubiquitin acceptor lysine residues of which only mutation of Lys-380 in the ETS domain of the FLI1 part abolished EWS-FLI1 ubiquitination and stabilized the protein posttranslationally. Expression of this highly stable mutant protein in Ewing cells while simultaneously depleting the endogenous wild type protein differentially modulates two subgroups of target genes to be either EWS-FLI1 protein-dependent or turnover-dependent. The majority of target genes are in an unaltered state and cannot be further activated. Our study provides novel insights into EWS-FLI1 turnover, a critical pathway in Ewing sarcoma pathogenesis, and lays new ground to develop novel therapeutic strategies in Ewing sarcoma.

Gierisch ME ，Pfistner F ，Lopez-Garcia LA ，Harder L ，Schäfer BW ，Niggli FK ... -  《-》

High-throughput RNAi screen in Ewing sarcoma cells identifies leucine rich repeats and WD repeat domain containing 1 (LRWD1) as a regulator of EWS-FLI1 driven cell viability.

A translocation leading to the formation of an oncogenic EWS-ETS fusion protein defines Ewing sarcoma. The most frequent gene fusion, present in 85 percent of Ewing sarcomas, is EWS-FLI1. Here, a high-throughput RNA interference screen was performed to identify genes whose function is critical for EWS-FLI1 driven cell viability. In total, 6781 genes were targeted by siRNA molecules and the screen was performed both in presence and absence of doxycycline-inducible expression of the EWS-FLI1 shRNA in A673/TR/shEF Ewing sarcoma cells. The Leucine rich repeats and WD repeat Domain containing 1 (LRWD1) targeting siRNA pool was the strongest hit reducing cell viability only in EWS-FLI1 expressing Ewing sarcoma cells. LRWD1 had been previously described as a testis specific gene with only limited information on its function. Analysis of LRWD1 mRNA levels in patient samples indicated that high expression associated with poor overall survival in Ewing sarcoma. Gene ontology analysis of LRWD1 co-expressed genes in Ewing tumors revealed association with DNA replication and analysis of differentially expressed genes in LRWD1 depleted Ewing sarcoma cells indicated a role in connective tissue development and cellular morphogenesis. Moreover, EWS-FLI1 repressed genes with repressive H3K27me3 chromatin marks were highly enriched among LRWD1 target genes in A673/TR/shEF Ewing sarcoma cells, suggesting that LRWD1 contributes to EWS-FLI1 driven transcriptional regulation. Taken together, we have identified LRWD1 as a novel regulator of EWS-FLI1 driven cell viability in A673/TR/shEF Ewing sarcoma cells, shown association between high LRWD1 mRNA expression and aggressive disease and identified processes by which LRWD1 may promote oncogenesis in Ewing sarcoma.

He T ，Surdez D ，Rantala JK ，Haapa-Paananen S ，Ban J ，Kauer M ，Tomazou E ，Fey V ，Alonso J ，Kovar H ，Delattre O ，Iljin K ... -  《-》

USP19 deubiquitinates EWS-FLI1 to regulate Ewing sarcoma growth.

Gierisch ME ，Pedot G ，Walser F ，Lopez-Garcia LA ，Jaaks P ，Niggli FK ，Schäfer BW ... -  《-》

EWS-FLI1 and Menin Converge to Regulate ATF4 Activity in Ewing Sarcoma.

Ewing sarcomas are driven by EWS-ETS fusions, most commonly EWS-FLI1, which promotes widespread metabolic reprogramming, including activation of serine biosynthesis. We previously reported that serine biosynthesis is also activated in Ewing sarcoma by the scaffolding protein menin through as yet undefined mechanisms. Here, we investigated whether EWS-FLI1 and/or menin orchestrate serine biosynthesis via modulation of ATF4, a stress-response gene that acts as a master transcriptional regulator of serine biosynthesis in other tumors. Our results show that in Ewing sarcoma, ATF4 levels are high and that ATF4 modulates transcription of core serine synthesis pathway (SSP) genes. Inhibition of either EWS-FLI1 or menin leads to loss of ATF4, and this is associated with diminished expression of SSP transcripts and proteins. We identified and validated an EWS-FLI1 binding site at the ATF4 promoter, indicating that the fusion can directly activate ATF4 transcription. In contrast, our results suggest that menin-dependent regulation of ATF4 is mediated by transcriptional and post-transcriptional mechanisms. Importantly, our data also reveal that the downregulation of SSP genes that occurs in the context of EWS-FLI1 or menin loss is indicative of broader inhibition of ATF4-dependent transcription. Moreover, we find that menin inhibition similarly leads to loss of ATF4 and the ATF4-dependent transcriptional signature in MLL-rearranged B-cell acute lymphoblastic leukemia, extending our findings to another cancer in which menin serves an oncogenic role. IMPLICATIONS: These studies provide new insights into metabolic reprogramming in Ewing sarcoma and also uncover a previously undescribed role for menin in the regulation of ATF4.

Jiménez JA ，Apfelbaum AA ，Hawkins AG ，Svoboda LK ，Kumar A ，Ruiz RO ，Garcia AX ，Haarer E ，Nwosu ZC ，Bradin J ，Purohit T ，Chen D ，Cierpicki T ，Grembecka J ，Lyssiotis CA ，Lawlor ER ... -  《-》

Impairment of p53 acetylation by EWS-Fli1 chimeric protein in Ewing family tumors.

The chromosomal translocation t(11;22)(q24;q12) yields the EWS-Fli1 fusion gene, which contributes to the development of Ewing Family Tumors (EFTs). Previous studies have shown the ability of EWS-Fli1 chimeric protein to silence p53 activity. Here we demonstrate that the introduction of EWS-Fli1 significantly inhibited p300-mediated acetylation of p53 at Lys-382 and depletion of EWS-Fli1 protein by small interfering RNAs (siRNA) in EFTs cells facilitated it in response to DNA damage. Furthermore, the deacetylation of p53 by EWS-Fli1 suppressed its transcriptional activity and enhanced mdm2-mediated p53 degradation. On the other hand, immunoprecipitation study shows that N-terminal region of EWS-Fli1 associated with histone deacetylase 1 (HDAC1) to forms a complex with p53. Knockdown of HDAC1, but not HDAC2 or HDAC3 protein restored the expression of p53 Lys-382 in EFTs cells. Overexpression of HDAC1 also significantly inhibited p53 transcriptional activity. Pharmacologic inhibitor of HDAC, trichostatin A (TSA) promoted p53-p300 interaction and recruitment of p53 Lys-382 to promoter regions of its target genes p21 and Puma, consequently inducing apoptosis and stabilizing the acetylation of p53 at Lys-382 together with the upregulation of p21 and Puma, which were impaired in EFTs cells after the knockdown of p53 expression. Our data indicate EWS-Fli1 might deacetylate p53 to inhibit its transcriptional function and protein stability via the recruitment of HDAC1. These results might elucidate a novel molecular mechanism about the abrogation of p53 pathway by EWS-Fli1 in EFTs pathogenesis.

Li Y ，Li X ，Fan G ，Fukushi J ，Matsumoto Y ，Iwamoto Y ，Zhu Y ... -  《-》