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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. In this report, we addressed this challenge by developing a two-armed screen for druggable upstream regulatory kinases in the PAX3/7-FOXO1 pathway. Screening libraries of kinome siRNA and small molecules, we defined PLK1 as an upstream-acting regulator. Mechanistically, PLK1 interacted with and phosphorylated PAX3-FOXO1 at the novel site S503, leading to protein stabilization. Notably, PLK1 inhibition led to elevated ubiquitination and rapid proteasomal degradation of the PAX3-FOXO1 chimeric oncoprotein. On this basis, we embarked on a preclinical validation of PLK1 as a target in a xenograft mouse model of aRMS, where the PLK1 inhibitor BI 2536 reduced PAX3-FOXO1–mediated gene expression and elicited tumor regression. Clinically, analysis of human aRMS tumor biopsies documented high PLK1 expression to offer prognostic significance for both event-free survival and overall survival. Taken together, these preclinical studies validate the PLK1–PAX3-FOXO1 axis as a rational target to treat aRMS. Cancer Res; 75(1); 98–110. ©2014 AACR.

Ewing sarcoma belongs to the family of pediatric tumors which arise most commonly in bone. The majority of Ewing sarcoma is characterized by a balanced translocation between chromosomes 11 and 22 which encodes for the uniquely expressed fusion protein EWS/FLI1. Tumor cells are crucially dependent on expression of the fusion protein. Protein degradation is an important and highly regulated process in all cells and novel insights are beginning to be applied for cancer therapy. We aim to investigate the mechanism of turnover with the goal to diminish EWS/FLI1 protein and thereby identify novel targets for Ewing sarcoma treatment. Here, we show first that EWS/FLI1 protein stabilizes upon proteasome inhibition. Co-immunoprecipitation of EWS/FLI1 with ubiquitin shows a characteristic ubiquitination smear indicating that EWS/FLI1 is ubiquitinated and a substrate of the ubiquitin-proteasome system. We characterized the turnover itself as rather slow. Interestingly, the half-life time of the fusion protein is increased by four-fold compared to the full length protein FLI1. This increased stability possibly contributes to the oncogenic and resistant features of the fusion protein. To identify a deubiquitinating enzyme capable of regulating EWS/FLI1 turnover, we selected 20 deubiquitinating enzymes based on their expression in Ewing cell lines and tumors in-silico. Then, we established a screening approach directly measuring EWS/FLI1 protein levels as a read-out by an ELISA-like assay, allowing us to monitor the amount of 3x flag-tagged EWS/FLI1 protein. After transient knocked down of the 20 candidates, the screening revealed ubiquitin specific protease 19 (USP19) as a promising candidate. Depletion of USP19 via siRNA or shRNA was capable to diminish endogenous EWS/FLI1 protein level which led to modulation of its target gene levels. Vice versa, USP19 overexpression stabilized EWS/FLI1 protein level by twofold. Most importantly, stable knockdown of USP19 in Ewing cell lines led to a 40% reduction of cell growth and proliferation. Currently, we further characterize the influence of USP19 on EWS/FLI1 turnover and Ewing cell viability in vivo to identify novel possibilities for therapeutic interventions. Citation Format: Maria E. Gierisch, Laura A. Lopez-Garcia, Franziska Pfistner, Felix K. Niggli, Beat W. Schaefer. Identification of deubiquitinating enzyme USP19 as a regulator of EWS/FLI1 protein turnover in Ewing sarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4966. doi:10.1158/1538-7445.AM2015-4966

Ewing sarcoma (ES) is the second most frequent bone cancer in childhood and it is characterized by the presence of the balanced t(11;22)(q24;q12) translocation in more than 85% of cases, generating a dysregulated transcription factor EWS/FLI1. ES belongs to small-round-blue-cell tumors and it is a very aggressive osteolytic cancer with early tendency for development of metastasis. Mostly it affects bones such as pelvis, femour and ribs but can also arise in soft tissues, mainly in adults. EWS/FLI1 is an essential oncogenic component of ES development which is necessary for tumor cell maintenance, through inappropriate regulation of target genes that are crucial for the fully malignant phenotype. Therefore, EWS/FLI1 represents an attractive therapeutic target. Screening of a small library of 153 targeted compounds has identified inhibitors of the PI3K pathway as main modulators of EWS/FLI1 activity and surprisingly this was due to an effect of the compound on EWS/FLI1 transcription. Indeed, treatment of four different ES cell lines with BEZ235 (PI3K-mTOR inhibitor) resulted in downregulation of EWS/FLI1 by 50% expression 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, named Del2 and Del23 region, as being important for EWS/FLI1 transcription. Based on in silico prediction and on in vivo data, we were able to predict 4 transcription factor candidates to bind this region and being responsive to PI3K regulation. Among these, SP1 was identified as modulator of EWS/FLI1 gene expression by siRNA. Various approches such as qRT-PCR, Western Blot and Immunofluorescence confirmed a reduction of EWS/FLI1 in Ewing cells when PI3Kαγδ and SP1 were depleted; Electrophoretic Mobility Shift Assay (EMSA) and Chromatine Immuno Precipitation (ChIP) both confirmed that the transcription factor SP1 is indeed binding to Del23 region of the EWS/FLI1 promoter. In summary, our results provide the very first insights on the transcriptional regulation of EWS/FLI1, an area that has not been investigated so far, and offer a molecular explanation for the known sensitivity of ES cell lines to PI3K inhibition. Citation Format: Chiara Giorgi, Alexander Boro, Laura A. Lopez-Garcia, Beat W. Schaefer, Felix K. Niggli. EWS/FLI1 transcription is modulated by the PI3K pathway via SP1 in Ewing sarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 478. doi:10.1158/1538-7445.AM2015-478

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.

Oncogenic addiction provides an opportunity to develop new treatment options, especially for childhood cancers. Pediatric tumors contain a lower number of oncogenic mutations compared to most adult cancers, suggesting stronger dependency on individual oncogenes, such as chimeric transcription factors that have the ability to control multiple oncogenic pathways. Taking advantage of this addiction, targeting of oncogenic transcription factors becomes a new powerful strategy for therapy of translocation positive pediatric tumors like alveolar rhabdomyosarcoma (aRMS), which is characterized by a very dismal prognosis. As transcription factors are considered to be difficult to target, we developed an approach to screen for druggable upstream regulators like kinases. In two parallel activity reporter assays, using a kinome siRNA library and a small molecule library, we identified PLK1 to contribute to the activity of PAX3-FOXO1, the oncogenic transcription factor specific to aRMS. Mechanistically, we were able to demonstrate a direct interaction of the two proteins by co-immunoprecipitation. PLK1 silencing or inhibition increased degradation of PAX3-FOXO1, which suggests a stabilization of PAX3-FOXO1 due to phosphorylation by PLK1. Candidate phosphorylation sites were identified by in vitro kinase assays and mass spectrometric analysis and site-specific mutagenesis is currently utilized to validate the impact of these sites on fusion protein turnover. To highlight PLK1 as therapeutic target in aRMS, in vivo treatment studies using the PLK1 inhibitor BI 2536 resulted in complete tumor regression in two aRMS xenograft mouse models. Finally, tissue microarray analysis of human aRMS tumor biopsies showed PLK1 to be overexpressed and predictive of overall survival. Hence, our preclinical studies validate PLK1 as highly relevant drug target in alveolar rhabdomyosarcoma, similar to recent findings in other pediatric tumors. Our data suggests that PLK1 inhibitors warrant further investigations to evaluate the potential to be translated into a clinical setting. Citation Format: Verena Thalhammer, David Herrero-Martin, Regina Hecker, Dominik Laubscher, Laura Lopez-Garcia, Marco Wachtel, Peter Bode, Beat Schäfer. PLK1 regulates PAX3-FOXO1 stability and its inhibition mediates regression of alveolar rhabdomyosarcoma xenograft tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3966. doi:10.1158/1538-7445.AM2014-3966