Pediatric tumors harbor very low numbers of somatic mutations and therefore offer few targets to improve therapeutic management with targeted drugs. In particular, outcomes remain dismal for patients with metastatic alveolar rhabdomyosarcoma (aRMS), where the chimeric transcription factor PAX3/7-FOXO1 has been implicated but problematic to target.

Ewing sarcoma is the second most common pediatric bone and soft tissue tumor presenting with an aggressive behavior and prevalence to metastasize. The diagnostic translocation t(22;11)(q24;12) leads to expression of the chimeric oncoprotein EWS-FLI1 which is uniquely expressed in all tumor cells and maintains their survival. Constant EWS-FLI1 protein turnover is regulated by the ubiquitin proteasome system. Here, we now identified ubiquitin specific protease 19 (USP19) as a regulator of EWS-FLI1 stability using an siRNA based screening approach. Depletion of USP19 resulted in diminished EWS-FLI1 protein levels and, vice versa, upregulation of active USP19 stabilized the fusion protein. Importantly, stabilization appears to be specific for the fusion protein as it could not be observed neither for EWSR1 nor for FLI1 wild type proteins even though USP19 binds to the N-terminal EWS region to regulate deubiquitination of both EWS-FLI1 and EWSR1. Further, stable shUSP19 depletion resulted in decreased cell growth and diminished colony forming capacity in vitro, and significantly delayed tumor growth in vivo. Our findings not only provide novel insights into the importance of the N-terminal EWSR1 domain for regulation of fusion protein stability, but also indicate that inhibition of deubiquitinating enzyme(s) might constitute a novel therapeutic strategy in treatment of Ewing sarcoma.

Cullin-RING ubiquitin (Ub) ligases (CRLs) are responsible for ubiquitinylation of approximately 20% of all proteins degraded by the Ub proteasome system (UPS). CRLs are regulated by the COP9 signalosome (CSN) and by Cullin-associated Nedd8-dissociated protein 1 (CAND1). The CSN is responsible for removal of Nedd8 from cullins inactivating CRLs. CAND1 modulates the assembly of F-box proteins into cullin 1-RING Ub ligases (CRL1s). We show that CAND1 preferentially blocks the integration of Skp2 into CRL1s. Suppression of CAND1 expression in HeLa cells leads to an increase of the Skp2 assembly into CRL1s and to significant reduction of the cyclin-dependent kinase (CDK) inhibitor p27. In contrary, CAND1 overexpression causes elevation of p27. The observed CAND1-dependent effects and CAND1 expression are independent of the CSN as demonstrated in CSN1 knockdown cells. Increase of p27 is a hallmark of preadipocyte differentiation to adipocytes (adipogenesis). We demonstrate that the accumulation of p27 is associated with an increase of CAND1 and a decrease of Skp2 during adipogenesis of human LiSa-2 preadipocytes. CAND1 knockdown reduces p27 and blocks adipogenesis. Due to the impact of CAND1 on Skp2 control, CAND1 could represent an important effector molecule in adipogenesis, but also in cancer development.

Edited by George DeMartinoEHere, 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. E-26 transformation-specific (ETS)2 family members are strong activators or repressors of transcription with a highly conserved ETS domain (1-3). ETS transcription factors (TFs) bind most commonly in complexes to a GGA core region to mediate gene expression (4, 5). Their main biological functions include regulation of differentiation, lineage determination of the hematopoietic system, and control of angiogenesis (6, 7). Most of the ETS family members have oncogenic potential because truncated or overexpressed ETS proteins have been linked to several cancer entities (8 -11). ERG and ETV1 are frequently fused to the TMPRSS2 promoter in prostate cancer, whereas ETV1 and ETV6 are implicated in leukemia (12, 13). Like other aberrant fusion proteins, they act as drivers of uncontrolled cell growth and survival (14, 15). However, most TFs do not harbor an enzymatic pocket and are therefore difficult to target directly. Novel strategies that uncover vulnerable sites in TFs are urgently needed to develop novel targeted therapies (16).Ewing sarcoma is a rare pediatric bone and soft tissue tumor with an aggressive behavior and prevalence to metastasize (17,18). Its main genetic abnormalities are EWS-ETS rearrangements, among them most commonly the EWS gene on chromosome 22 fused to FLI1 on chromosome 11, which results in expression of the chimeric transcription factor . Continuous expression of the fusion protein is crucial for tumor formation, progression, and maintenance (22, 23), and its down-regulation inhibits proliferation and reduces tumor cell growth (24 -26). EWS-FLI1 is thought to function mainly as a modulator to activate and repress a wide range of target genes but also as a regulator of splicing processes or as a component of large interaction networks (27-31). However, inhibition of a single downstream target gene has not been proven effective yet for Ewing sarcoma therapy.The turnover of most intracellular proteins is mediated via the ubiquitin-proteasome ...

Ewing sarcoma (ES) is the second most frequent bone cancer in childhood and is characterized by the presence of the balanced translocation t(11;22)(q24;q12) in more than 85% of cases, generating a dysregulated transcription factor EWS/FLI1. This fusion protein is an essential oncogenic component of ES development which is necessary for tumor cell maintenance and represents an attractive therapeutic target. To search for modulators of EWS/FLI1 activity we screened a library of 153 targeted compounds and identified inhibitors of the PI3K pathway to directly modulate EWS/FLI1 transcription. Surprisingly, treatment of four different ES cell lines with BEZ235 resulted in down regulation of EWS/FLI1 mRNA and protein by ∼50% with subsequent modulation of target gene expression. Analysis of the EWS/FLI1 promoter region (−2239/+67) using various deletion constructs identified two 14bp minimal elements as being important for EWS/FLI1 transcription. We identified SP1 as modulator of EWS/FLI1 gene expression and demonstrated direct binding to one of these regions in the EWS/FLI1 promoter by EMSA and ChIP experiments. These results provide the first insights on the transcriptional regulation of EWS/FLI1, an area that has not been investigated so far, and offer an additional molecular explanation for the known sensitivity of ES cell lines to PI3K inhibition.