Abstract Embryonal rhabdomyosarcoma (ERMS) is a common pediatric malignancy of muscle with relapse being the major clinical challenge. Self-renewing tumor-propagating cells (TPCs) drive cancer relapse and are confined to a molecularly definable subset of ERMS cells. To identify drugs that suppress TPC function, a large-scale chemical screen comprising of ∼40,000 compounds including 47% FDA-approved drugs was completed, identifying GSK3-inhibitors as potent suppressors of ERMS growth through inducing terminal differentiation of TPCs into myosin-expressing cells. In support of GSK3 inhibitors functioning through activation of the canonical WNT pathway, recombinant WNT3A and a stabilized β-catenin enhanced differentiation and reduced self-renewal in human ERMS cell lines. Moreover, treatment of tumor-bearing zebrafish with a GSK3 inhibitor activated the WNT/β-catenin pathway, resulting in suppressed ERMS growth, depleted TPCs, and diminished self-renewal capacity in vivo. As canonical WNT signaling is essential for transition from muscle stem cell proliferation to myogenic differentiation during regeneration, our findings suggest that the same developmental pathways that regulate muscle stem cell self-renewal also contribute to tumorigenesis in ERMS. GSK3 inhibitors are being assessed for their efficacy in inhibiting growth as well as inducing differentiation of human ERMS in xenograft mouse models. Our work has identified the vital role of canonical WNT pathway in regulating differentiation and self-renewal of ERMS and demonstrated the effective application of small molecule inhibitors to target differentiation of TPCs in ERMS. Citation Format: Eleanor Chen, Michael DeRan, Katherine Brooke Grandinetti, Myron Ignatius, Ryan Clagg, Karin McCarthy, Riadh Lobbardi, Xu Wu, David Langenau. Canonical WNT/β;-catenin pathway activation suppresses embryonal rhabdomyosarcoma growth and self-renewal. [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 A67.

Significance Embryonal rhabdomyosarcoma (ERMS) is a cancer of skeletal muscle and is one of the most common pediatric cancers of soft tissue. There is no effective treatment for patients with relapsed ERMS, with less than 50% surviving the disease. The self-renewing and molecularly defined tumor propagating cells (TPCs) drive continued tumor growth and relapse. Yet to date, drugs targeting ERMS self-renewal and differentiation of TPCs have not been identified. Our study describes a large-scale chemical screen to identify targetable pathways essential for modulating self-renewal and differentiation of ERMS and demonstrates the feasibility of inducing differentiation of TPCs in ERMS by small molecules.

Abstract Human cancers are often associated with large chromosomal amplifications and deletions comprising megabases of sequence - making it challenging to identify drivers of cancer growth and progression. To bypass this obstacle, we have applied array comparative genomic hybridization (aCGH) to zebrafish embryonal rhabdomyosaroma (ERMS) and utilized cross-species comparison to identify novel collaborating oncogenes important to human disease. Because zebrafish ERMS predominantly contain small, focal amplifications that span only 50-100kb of sequence, candidate oncogenes were easily identified by cross-species comparisons with human ERMS. Remarkably, 16 of 19 chromosomal gains identified in zebrafish ERMS also exhibited focal, copy gains in human disease. Candidate oncogenes found in these regions were also over-expressed in a majority of human ERMS as assessed by microarray gene expression analysis and immunohistochemical stain of primary human ERMS and normal fetal muscle. siRNA and shRNA knockdown studies identified important roles for candidate ocogenes in regulating human ERMS growth. For example, knockdown of Cyclin D2, Homeobox Protein C6, and Plexin A1 (PLXNA1) resulted in reduced proliferation in both human RD and SMS-CTR cell lines. PLXNA1 knockdown also resulted in impaired migratory potential in scratch and transwell migration assays. Moreover, PLXNA1 loss resulted in increased numbers of terminally-differentiated myosin-expressing myocytes, suggesting that dysregulated expression of PLXNA1 plays a key role in the differentiation arrest of human ERMS. Finally, vascular endothelial growth factor (VEGF) was also amplified and over-expressed in a subset of human and zebrafish ERMS. Chemical inhibition of VEGF in ERMS-bearing zebrafish led to significantly reduced tumor growth which was associated with decreased tumor neovascularization. Analysis of human microarray gene expression data revealed that increased VEGFA expression correlated with poor clinical outcome in patients with ERMS independent of tumor grade and stage, indicating high VEGFA expression as an independent prognostic indicator and implicating inhibitors of the VEGF pathway as a promising therapy for improving patient survival. In total, our results demonstrate the utility of aCGH and cross-species comparisons to rapidly identify candidate genes essential for the pathogenesis of human cancer. Citation Format: Eleanor Chen, Kimberly P. Dobrinski, Kim H. Brown, Ryan Clagg, Elena Edelman, Myron Ignatius, Jillian Brockmann, G. Petur Nielsen, Sridhar Ramaswamy, Charles Keller, Charles Lee, David M. Langenau. Cross-species array comparative genomic hybridization identifies novel driver genes in embryonal rhabdomyosarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1412. doi:10.1158/1538-7445.AM2013-1412

Human cancer genomes are highly complex, making it challenging to identify specific drivers of cancer growth, progression, and tumor maintenance. To bypass this obstacle, we have applied array comparative genomic hybridization (array CGH) to zebrafish embryonal rhabdomyosaroma (ERMS) and utilized cross-species comparison to rapidly identify genomic copy number aberrations and novel candidate oncogenes in human disease. Zebrafish ERMS contain small, focal regions of low-copy amplification. These same regions were commonly amplified in human disease. For example, 16 of 19 chromosomal gains identified in zebrafish ERMS also exhibited focal, low-copy gains in human disease. Genes found in amplified genomic regions were assessed for functional roles in promoting continued tumor growth in human and zebrafish ERMS--identifying critical genes associated with tumor maintenance. Knockdown studies identified important roles for Cyclin D2 (CCND2), Homeobox Protein C6 (HOXC6) and PlexinA1 (PLXNA1) in human ERMS cell proliferation. PLXNA1 knockdown also enhanced differentiation, reduced migration, and altered anchorage-independent growth. By contrast, chemical inhibition of vascular endothelial growth factor (VEGF) signaling reduced angiogenesis and tumor size in ERMS-bearing zebrafish. Importantly, VEGFA expression correlated with poor clinical outcome in patients with ERMS, implicating inhibitors of the VEGF pathway as a promising therapy for improving patient survival. Our results demonstrate the utility of array CGH and cross-species comparisons to identify candidate oncogenes essential for the pathogenesis of human cancer.

Embryonal rhabdomyosarcoma (ERMS) is an aggressive pediatric sarcoma of muscle. Here, we show that ERMS-propagating potential is confined to myf5+ cells and can be visualized in live, fluorescent transgenic zebrafish. During early tumor growth, myf5+ ERMS cells reside adjacent normal muscle fibers. By late-stage ERMS, myf5+ cells are reorganized into distinct regions separated from differentiated tumor cells. Time-lapse imaging of late-stage ERMS revealed that myf5+ cells populate newly formed tumor only after seeding by highly migratory myogenin+ ERMS cells. Moreover, myogenin+ ERMS cells can enter the vasculature, whereas myf5+ ERMS-propagating cells do not. Our data suggest that non-tumor-propagating cells likely have important supportive roles in cancer progression and facilitate metastasis.