Project Summary The goal of this project is to understand how brachyury expression is maintained in chordoma and to identify approaches to attack brachyury function as a therapeutic strategy. Chordoma is a rare primary tumor that develops in the skull base and spine and currently has no targeted therapies. Strong evidence supports the hypothesis that chordoma is driven by expression of the T gene, which encodes the protein brachyury. Brachyury is a transcription factor (TF) that is responsible for controlling the genes required for mesoderm and notochord formation. Brachyury expression is normally turned off early in development, however, chordoma cells fail to turn off the T gene, resulting in sustained brachyury expression. Within our previous work, we have mapped a cluster super-enhancers adjacent to the T gene that we believe are necessary to drive this aberrant brachyury expression. Genetic knockout of brachyury arrests chordoma cells, which suggests that inhibiting brachyury function presents a promising therapeutic strategy. However, historically transcription factors have been difficult to directly target because, unlike other proteins that contain active sites, transcription factors lack obvious pockets for small molecule ligand binding. The overarching goal of this project is to gain insight into how brachyury expression is propagated and to identify other effective approaches for targeting brachyury function in conjunction with its direct inhibition. We hypothesize that we can inhibit brachyury expression upstream of its transcriptional activation as a therapeutic strategy. To test this hypothesis, we will engineer a model system where complete brachyury degradation can be achieved to model the consequences of its perturbation. Examining the consequences of brachyury degradation in comparison to inhibiting its activation upstream will elucidate the efficacy between direct and indirect brachyury inhibition. Finally, functionally dissecting the brachyury super-enhancers to identify the critical regions for trans-factor binding may elucidate novel chordoma therapeutic targets. Together, these proposed studies will lend insight into the role of brachyury in chordoma and will help to achieve the common goal of developing new approaches to therapeutically target brachyury.