Project Summary/Abstract: Ewing sarcoma (ES) is a malignant bone and soft-tissue tumor with extremely poor prognosis for patients with metastatic or relapsed disease. In the absence of representative genetic models, the developmental cell of origin as well as the mechanisms of tumor initiation remain poorly understood. To address those questions, I have developed an innovative zebrafish model of ES by introducing the human EWSR1-FLI1 oncofusion to the zebrafish genome. This model allows studying the behavior of GFP-labeled cancer cells during tumor initiation and progression in a complex developmental context which is not currently possible in mammals. The fish develop tumors positive for known ES markers, recapitulating the main aspects of human disease. Preliminary data reveal dysregulation of heparan sulfate proteoglycan (HSPG) metabolism and associated activation of ERK signaling in ES cells. Targeting HSPGs with the specific antagonist surfen reduces ERK1/2 signaling and decreases tumorigenicity in vitro and in vivo. My preliminary data suggest that dysregulated HSPG turnover can affect normal cell differentiation leading to cell transformation. I hypothesize that such HSPG- mediated dysregulation of signaling between cancer and normal cells can facilitate ES progression. The aims outlined in this proposal will take advantage of the genetic model of ES in combination with innovative single-cell transcriptomic, genetic, and high-resolution imaging approaches to characterize the type of cells giving rise to ES (Aim 1), identify key effectors in cancer and cancer niche cells promoting the cancer progression (Aim 2), and to target HSPG mediated signaling in genetic zebrafish and xenograft mouse models for ES treatment (Aim 3). Aim1 will be completed in the K99 phase and will be co-mentored by Dr. Crump. Aims 2 and 3 will be initiated during the K99 phase under the mentorship of Dr. Amatruda and my advisory committee and then completed during the R00 phase. Completion of these aims will inform what type of cells contribute to ES development. These findings will determine the role of HSPG in tumor initiation and progression building the foundation for a new strategy of targeting the enzymes involved in HSPG metabolism for ES treatment. The proposed project will lay the groundwork for my career goal of obtaining a position as a tenure-track Assistant Professor at a top-tier academic research institution. During the K99 phase, I will receive mentorship in zebrafish and cancer biology from Dr. Amatruda and training in developmental biology from my co-mentor Dr. Crump. Regular interactions with my other advisory committee will allow me to acquire new expertise in single- cell genomics and tumor microenvironment (Dr. Asgharzadeh), ES pathology (Dr. Triche), and data analysis (Dr. Gai). Both CHLA and USC will provide extensive resources for my career and professional development. As CHLA hosts one of the most experienced communities of pediatric cance...