Abstract NG03: Zebrafish models of PAX3-FOXO1 pathogenesis reveal novel targets in human rhabdomyosarcoma
Authors: Kendall Genevieve , Watson Sarah , Xu Lin , Tirode Franck , Delattre Olivier , Amatruda James 
Published: July 2016

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children, and is associated with a misregulation of skeletal muscle developmental pathways. Of the two histological subtypes, embryonal (ERMS) and alveolar (ARMS), ARMS is more aggressive and prone to metastases. The majority of ARMS cases have a defining t(1;13) or t(2;13) chromosomal translocation and a fusion of either PAX3 or PAX7 with FOXO1, creating a transcriptionally active chimeric protein in which the PAX DNA binding domain is juxtaposed with the transactivation domain of FOXO1. PAX3-FOXO1 is the most prevalent ARMS chromosomal translocation, and is associated with reduced overall survival. However, the biological impact of this PAX3-FOXO1 fusion oncogene and its gain of function activities remains to be fully understood, and the fusion has been intractable to target therapeutically. Remaining questions include understanding how PAX3-FOXO1 initiates and drives ARMS tumorigenesis in the context of a vertebrate developmental system, including susceptible cell types and targeted signaling pathways, with the aim of guiding therapeutic strategies.

We are implementing human PAX3-FOXO1 transgenic zebrafish models to address these remaining questions. Previously, we demonstrated that the introduction of human fusion oncogenes, such as EWS-FLI1, in developing zebrafish initiates tumor formation that faithfully recapitulates the human disease (Leacock et al, 2012). Here, we use a transposon-based system to express the human PAX3-FOXO1 fusion during embryonic development, and find that PAX3-FOXO1 is active in a zebrafish system, functions to induce early embryonic defects, such as cyclopia, and is tumorigenic. These developmental phenotypes and tumor formation are unique to PAX3-FOXO1, as over-expression of the normal PAX3 gene in this context does not have the same effect. To identify the developmental signaling pathways PAX3-FOXO1 is leveraging to induce these distinct phenotypes, we FACS sorted cells expressing GFP tagged PAX3 or PAX3-FOXO1 from embryos, and performed a microarray gene expression analysis. Contrasting PAX3 and PAX3-FOXO1 revealed distinct gene expression signatures and aberrant developmental signaling pathways likely responsible for PAX3-FOXO1 driven cell transformation. Of the identified up-regulated genes, the most up-regulated PAX3-FOXO1 target is hairy-related 3 (her3), which we validate as being both unique to and dependent on PAX3-FOXO1 expression.

The human homolog of her3 is HES3, which is a basic helix-loop-helix transcription factor that is activated by Notch signaling. To determine its relevance in the human disease, we compared HES3 expression from RNAseq data derived from PAX3/7-FOXO1 fusion positive RMS patient tumors, and found that HES3 is over-expressed in fusion-positive RMS as compared to the fusion-negative RMS subtype. Furthermore, the expression status of HES3 is predictive of overall survival. Over-expression of HES3 predicts reduced overall survival only in the context of the PAX3/7-FOXO1 fusion, and not for embryonal or fusion negative RMS, suggesting this gene and related signaling pathways are down-stream targets of PAX3-FOXO1 that likely contribute to its pathogenesis.

These data highlight the utility of zebrafish developmental assays to identify new oncogenic targets in a whole animal context that are relevant in the human disease. Our findings identify a new PAX3-FOXO1 target gene, and implicate Notch signaling as a contributor to PAX3-FOXO1 pathogenesis. We are currently applying these transgenic zebrafish models, in parallel to human cell models and primary patient genomic data, to determine the mechanisms underlying her3/HES3 over-expression and its potential for therapeutic targeting. Overall, an ARMS transgenic zebrafish model with embryonic phenotypes that are informative and predictive of tumorigenesis has relevance ranging from studying ARMS biology to translational applications, including in vivo drug discovery.

Citation Format: Genevieve Kendall, Sarah Watson, Lin Xu, Franck Tirode, Olivier Delattre, James Amatruda. Zebrafish models of PAX3-FOXO1 pathogenesis reveal novel targets in human rhabdomyosarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr NG03.