Ewing's sarcoma family of tumors (EFT) is characterized by the presence of chromosomal translocations leading to the expression of oncogenic transcription factors such as, in the majority of cases, EWS/FLI1. Because of its key role in Ewing's sarcoma development and maintenance, EWS/FLI1 represents an attractive therapeutic target. Here, we characterize PHLDA1 as a novel direct target gene whose expression is repressed by EWS/FLI1. Using this gene and additional specific well‐characterized target genes such as NROB1, NKX2.2 and CAV1, all activated by EWS/FLI1, as a read‐out system, we screened a small‐molecule compound library enriched for FDA‐approved drugs that modulated the expression of EWS/FLI1 target genes. Among a hit‐list of nine well‐known drugs such as camptothecin, fenretinide, etoposide and doxorubicin, we also identified the kinase inhibitor midostaurin (PKC412). Subsequent experiments demonstrated that midostaurin is able to induce apoptosis in a panel of six Ewing's sarcoma cell lines in vitro and can significantly suppress xenograft tumor growth in vivo. These results suggest that midostaurin might be a novel drug that is active against Ewing's cells, which might act by modulating the expression of EWS/FLI1 target genes.

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.

Background: Ewing's sarcoma (ES) is the second most common bone malignancy in children and adolescents and accounts for approximately three percent of pediatric cancers. The hallmark of Ewing Sarcoma Family of Tumors (ESFT) is a chromosomal translocation involving the EWS gene on chromosome 22q12 with a member of the ETS transcription factor family. The derived EWS/FLI1 chimeric fusion protein, found in 85% of tumors, is a potent transcription factor, regulating malignant transformation and maintenance of the oncogenic phenotype by aberrant expression of its target genes. Hence, EWS/FLI1 represents an attractive therapeutic target whose transcriptional activity might be modulated by already existing small molecule inhibitors. Material and Methods: We screened the LOPAC1280 library (Sigma-Aldrich) consisting of 1280 different drug-like and well annotated compounds covering all major drug types. In addition we screened a smaller library composed of the latest targeted agents in the field of oncology, including most characterized kinase inhibitors. The effect of the compounds on EWS/FLI1 transcriptional activity was measured in three ES cell lines, using quantitative RT-PCR. Our read out was relative expression of three well-described EWS/FLI1 target genes: NR0B1, NKX2.2 and CAV1 and one new target gene, PHLDA1 (unpublished data) as well as the expression of the fusion protein itself. In parallel we measured proliferation using WST-1 assays. Results: We identified several well known chemotherapeutics such as camptothecin, doxorubicin, vincristine and etoposide as modulators of EWS/FLI1 target gene expression. The current usage of some of these agents in the treatment of Ewing's sarcoma validated our screening approach in an ideal and unbiased way. In addition we identified inhibitors targeting several signalling pathways, both known and unknown to play a role in sarcomas. The most prominent among them is the phosphoinositide-3-kinase (PI3K) pathway, playing a prominent role in many cancer types. Three inhibitors targeting this pathway revealed a significant modulation of EWS/FLI1 target genes and a strong inhibition of cell proliferation in all three ES cell lines. Conclusion: Screening of small molecule inhibitors was used to identify inhibitors of potential molecular pathways regulating the transcriptional activity of EWS/FLI1. Work continues to investigate these pathways in more detail on the molecular level. Genetic-loss of function experiments will be carried out to exclude unspecific off-target effects. Our findings will help to prioritize specific pathway inhibitors for more detailed investigations to accelerate the development of improved and novel therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5342. doi:10.1158/1538-7445.AM2011-5342