Abstract 5342: Assessing EWS/FLI1 activity as a read-out for small-molecule drug screening in Ewing's sarcoma
Authors: Rechfeld Florian , Boro Aleksandar , Niggli Felix K. , Schäfer Beat W. 
Published: June 2012

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expression of three well-described EWS/FLI1 target genes: NR0B1, NKX2.2 and CAV1 and one new target gene, PHLDA1 (unpublished data) as well as the expression of the fusion protein itself.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