Project Abstract Infantile hemangioma (IH) is a common vascular tumor with a unique lifecycle of rapid blood vessel formation over the first 6-9 months of infancy, followed by a slow spontaneous involution of blood vessels over several years. For most children, IH does not pose a serious threat and therapy is unnecessary; however, in about 10% of cases, IH can enlarge dramatically, threaten organs and cause permanent disfigurement. Over the last 10 years, propranolol, a well-known non-selective β-adrenergic receptor antagonist, has emerged as first-line therapy for endangering IH, yet how and why it works so well in reducing the vascular overgrowth in IH has remained a mystery. There is a significant need to improve propranolol therapy because up to 18% of IHs fail to respond, up to 25% resume growth when the drug is stopped, and 37% of propranolol-treated infants require surgery at 5-6 years of age to minimize deformity caused by remaining fibrofatty residua. To improve on propranolol, it is essential to elucidate it’s mechanism of action against vascular overgrowth, which will then provide a path forward to advance IH medical therapy, and potentially other neovascular diseases as well. In previous funding cycles, we identified a hemangioma stem cell (HemSC) from human IH surgical specimens that can differentiate into endothelial cells, pericytes and adipocytes and form hemangioma-like vessels within 7 days when implanted into immune-deficient mice. Subsequent studies from our lab and others validate HemSCs as the IH-initiating cell. Our recent results show that a small molecule inhibitor of the transcription factor SOX18 and propranolol both effectively block HemSC-to-endothelial differentiation. Furthermore, the R(+) enantiomer of propranolol, which lacks β-adrenergic receptor antagonistic activity, is equally effective. This novel discovery identifies a β-adrenergic receptor-independent, SOX18-dependent mechanism by which propranolol reduces vascular overgrowth in IH. To investigate deeply, we propose three specific aims. Aim 1 will directly and rigorously test the requirement for SOX18 in IH vessel formation using our in vivo model in which IH-derived HemSC form IH-like blood vessels in nude mice. Aim 2 will investigate dimerization status of SOX18 in IH (sub-aim 2a), how propranolol and the R(+) enantiomer disrupt SOX18 dimerization and sub- cellular localization (sub-aim 2b), and how this alters transcription to prevent HemSC-blood vessel formation (sub-aim 2c). Aim 3, conducted in parallel, will analyze our existing next generation sequencing data using new bioinformatic tools to identify potential chromosomal translocations or small copy number variants that could produce fusion transcripts with new activities (sub-aim 3a) and will perform deep coverage RNA-Seq on IH tissue and freshly isolated IH cells as an alternative method to identify fusion transcripts (sub-aim 3b); once identified, the connection to SOX18 and IH vessel formation w...