Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma in the United States. Fusion- negative (FN) RMS are the most common subtype and are driven by RAS-pathway activation. Despite intensive treatment with radiation, chemotherapy, and surgery, a large fraction of patients develop refractory, metastatic, and relapsed RMS that has survival rates of less than 20%. A major hurdle to the design of new and effective treatments for aggressive RMS can be attributed to our limited understanding of the drivers of cancer stem cell (CSC) self-renewal and metastasis. The long-term goal and overall objective of our studies is to identify CSCs and metastatic cells in FN-RMS and then define molecular pathways that can differentiate these cells into non- proliferative, non-migratory cell types or kill them completely. Our central hypothesis is that FN-RMS CSCs drive tumor growth, therapy-resistance and metastasis. We also hypothesize that the genes and pathways promoting the transition of RMS cells into differentiated non-proliferative, non-metastatic cell types can be therapeutically targeted. The rationale and feasibility of our approach comes from our recent discovery of a novel, molecularly- defined FN-RMS CSC that expresses mesenchymal pathway-enriched genes and shares remarkable similarity to a recently discovered bi-potent, muscle mesenchyme progenitor that can make both muscle and osteogenic cells between 9-14 weeks of human development. This FN-RMS CSC is molecularly-distinct from CSCs reported by others in the field, can be isolated using CD44/CD90 antibodies and FACs, and expresses genes associated with epithelial-to-mesenchymal transition (EMT), which is a major driver of metastasis in other cancers. Aim 1 will identify FN-RMS cell heterogeneity and cell types that drive tumor growth and metastasis using single cell sequencing, lineage and cell fate barcode tracing, and mouse xenograft studies. This work will test the hypothesis that CD44+/CD90+ FN-RMS cells define the CSCs and that these cells are largely quiescent under steady state growth conditions, and yet undergo self-renewal divisions following chemo- and radiation-therapy to drive tumor regrowth and metastasis. Aim 2 will quantify human FN-RMS CSC self-renewal and cell state transitions in vivo at single cell resolution using fluorescent cell state reporters, photoconvertible cell lineage tracing tools, and engraftment into optically-clear immune deficient zebrafish. This aim will test the hypothesis that CSCs undergo asymmetric/symmetric self-renewal divisions following therapy to re-create all the functionally diverse cell types in RMS. Aim 3 will identify the molecular mechanisms driving FN-RMS cell states, testing the hypothesis that DNA binding proteins and transcription factors, including NOTCH3 and MEF2C, are dominant oncogenic drivers of RMS cell fate and independently regulate gene networks that promote CSC and/or differentiated muscle cell states. This work will have...