Abstract IA32: Integrating genomic approaches to discover new therapeutic strategies for Ewing sarcoma

Abstract

Abstract While next-generation sequencing studies are unveiling cancer genomes at an unprecedented pace, for most pediatric cancers, only a minority of the tumor samples have been shown to possess lesions that are immediately targetable by our current drug armamentarium. Quite commonly, the signature cancer-promoting events in childhood malignancies disrupt chromatin architecture and/or transcription (e.g., SMARB1 deletion/mutation in malignant rhabdoid tumor, MLL rearrangements in acute leukemia, and PAX3-FKHR in alveolar rhabdomyosarcoma). In Ewing sarcoma, the second most common type of primary bone cancer in children, the defining translocation fuses the EWSR1 gene to an ETS family member gene, most commonly FLI1, resulting in the oncoprotein EWS-FLI and an aberrant gene expression profile. While this translocation event was discovered approximately 20 years ago, current therapies do not directly target the fusion product. With the exception of nuclear hormone receptors, transcription factors have proven largely refractory to conventional drug discovery approaches. Our laboratory is taking a multi-pronged, integrative approach to discovering new therapeutic strategies for Ewing sarcoma, leveraging next-generation sequencing, functional genomic and proteomic studies, and chemical biology. We have co-led a collaborative effort to characterize the genomic landscape of childhood Ewing sarcoma tumors using next-generation sequencing approaches. One immediate and striking observation from this study is that Ewing sarcoma genomes are among the most quiet sequenced to date, with strikingly few samples demonstrating immediately actionable lesions. These results speak to the critical importance of understanding, and either directly targeting the expressed EWS-ETS fusion, or identifying synthetic lethal vulnerabilities in the presence of these fusion proteins. Toward this end, we have focused on the development and implementation of a panel of screening assays to identify new small-molecule modulators of EWS-FLI. We have completed a primary screen of over 350,000 compounds for those inhibiting the activity of an EWS-FLI reporter and a secondary counter-screen for selective killing of Ewing sarcoma cell lines. We are now focusing our attention on 10 promising hits to be studied in a panel of EWS-FLI-relevant assays. In parallel, we have conducted a near genome-wide shRNA screen in a panel of Ewing sarcoma cell lines to identify Ewing sarcoma cell vulnerabilities and are now integrating this data with our next-generation sequencing study and small-molecule screen. Finally, we took a proteomic approach to identify more easily targetable tyrosine kinase vulnerabilities in Ewing sarcoma tumors. Utilizing a Luminex fluorescent, bead-based technology we profiled activated tyrosine kinases using phosphorylation status as a surrogate for kinase activation. We identified focal adhesion kinase (FAK), a tyrosine kinase critical for cellular adhesion, growth, and survival, as a candidate target in Ewing sarcoma. In this study, we demonstrated that FAK is highly phosphorylated in primary Ewing sarcoma tumor samples and that downregulation of FAK by shRNA and treatment with a FAK-selective kinase inhibitor, PF-562271, impaired growth and colony formation in Ewing sarcoma cell lines. Moreover, shRNA-directed against FAK and small-molecule inhibition of FAK attenuated Ewing sarcoma tumor growth in vivo in two xenograft models of Ewing sarcoma. In collaboration with the NIH, we are now screening a library of clinically relevant compounds to identify highly active compound combinations with FAK inhibitors in Ewing sarcoma. Citation Format: Kimberly Stegmaier. Integrating genomic approaches to discover new therapeutic strategies for Ewing sarcoma. [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 IA32.