Project Summary Ewing sarcoma is an aggressive bone tumor that has a peak incidence in children and adolescents. Systemic chemotherapy and local control measures have improved survival rates for patients with localized tumors however, survival rates for patients with metastatic disease are dismal. Ewing sarcoma is defined by tumor- initiating EWS-ETS fusion proteins, most commonly EWS-FLI1, that have remained undruggable, highlighting a need for new strategies and therapeutic opportunities to improve patient survival. EWS-ETS fusions induce malignant transformation by hijacking gene regulatory networks via enhancer reprogramming. The putative cell of origin is a mesenchymal stem/progenitor cell derived from the neural crest or mesoderm lineage. Aside from EWS-FLI1 fusion proteins, Ewing sarcoma tumors rarely present with other recurrent mutations, however present with clinical heterogeneity. Tumor heterogeneity has become an important concept in cancer research for studying metastasis and treatment response. Studies of other pediatric tumors have demonstrated that the epigenetic and transcriptomic state of tumor cells and their inherent plasticity reflects differences in biologic phenotypes, of which are required for metastatic progression. Abnormal expression and regulation of developmental programs is evident and may in part explain the undifferentiated features of Ewing tumors. Homeobox (HOX) genes are evolutionarily conserved transcription factors that play essential roles in body patterning and embryogenesis. We have shown that posterior HOXD gene expression is widely deregulated in Ewing sarcoma. In particular, our findings revealed HOXD13 to be overexpressed in tumor samples relative to normal and other malignant tissues and critical for Ewing sarcoma tumorigenicity. My preliminary data reveal HOXD13 expression is maintained through EWS-FLI1-dependent enhancer reprogramming and modulation of HOXD13 leads to opposing shifts in neural and mesenchymal lineage programs, supporting the role of HOXD13 as a determinant of cell state. The working hypothesis is that HOXD13 regulates epigenomic and transcriptomic transitions in Ewing sarcoma cells and that these molecular transitions drive phenotypic transitions between more proliferative and metastatic cell states. This hypothesis will be tested in two aims. In Aim 1, I will determine if and how HOXD13 regulates Ewing sarcoma cell state. Aim 2 will define the contribution of Ewing tumor cell state to the metastatic phenotype. Functional characterization of cell state transitions, and the mechanisms underlying these transitions, will generate new insights into the biology of Ewing sarcoma metastasis and provide novel opportunities for the development of therapies.