Abstract LB-240: Modulating transcription factor abnormalities in pediatric cancer
Authors: Stegmaier Kimberly , Crompton Brian , Jessneck Jonathan , Ross Kenneth , Gupta Supriya , Plank Lynn Ver , Winckler Wendy , Tolliday Nicola 
Published: June 2012

Abstract
Transcription factor abnormalities are commonly involved in the pathogenesis of malignancy. In principle, these cancer-promoting proteins make for potential therapeutic targets. In practice, this class of proteins has been considered largely intractable to traditional pharmacological approaches. Emerging chemical genomic approaches offer new opportunity in targeting transcription factor abnormalities. We have focused our efforts on the pediatric solid tumor, Ewing sarcoma, associated in the majority of cases with the tumor dependent, ETS transcription factor rearrangement EWS/FLI.
Today, treatment of Ewing sarcoma relies on cytotoxic chemotherapy, with outcome for patients with metastatic or recurrent disease dismal, and targeted treatments only beginning to emerge. In order to tackle the challenge of modulating EWS/FLI in Ewing sarcoma, we built upon an innovation developed by our group using gene expression signatures as surrogates for different biological states in a small molecule library screen: Gene Expression-based High-throughput Screening (GE-HTS). GE-HTS employs ligation-mediated amplification and a fluorescent bead-based detection to measure the expression of up to 500 genes in 384-well format. Using genome-wide expression data sets profiling EWS/FLI-directed shRNA, induced expression of EWS/FLI, and primary human tumors, we identified a 123 gene signature for EWS/FLI on versus off. We next adapted this signature to our GE-HTS assay and tested it across 6 Ewing sarcoma cell lines and multiple shRNA constructs. We confirmed a robust assay signal window with Z-scores above 0.7. In a pilot screen of 1600 bioactive compounds, our assay identified among other novel hits, rapamycin, a drug known to decrease EWS/FLI protein levels. We have completed a screen of over 10,000 small molecules, including bioactives (FDA-approved drugs), natural products, and diversity oriented synthesis (DOS) molecules. DOS compounds offer a novel chemistry with the structural complexity and chirality of natural products, and yet the chemical tractability (e.g. rapid optimization and chemical modification for target identification) of more planar, simple compounds. 150 top scoring molecules are now under evaluation in secondary screening assays.
We have also explored the concept of in silico “screens-within-a-screen,” including the measurement of a 10 gene “death” sub-signature of the original 123 gene EWS/FLI signature. Importantly, chemotherapeutic agents, including two currently used to treat patients with Ewing sarcoma (vincristine and etoposide), were identified to induce the “death” sub-signature. We also developed and validated with shRNA, a 23 gene sub-signature for NR0B1, a validated downstream target of EWS/FLI. Candidate NR0B1 modulators are also under evaluation. It is our expectation that the tiling of sub-signature hits in combination will reveal molecules with additive or synergistic activity.
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 LB-240. doi:10.1158/1538-7445.AM2011-LB-240