Project Summary/Abstract Within a group of cancer-prone cells that share an oncogenic mutation, sometimes described as a “cancerized field”, only select cells transition to a malignant state. Due to the rarity and transient nature of these events, cancer initiation has been difficult to study and remains incompletely understood. Identifying cells at the earliest stages of transformation and understanding the underlying mechanisms leading to their malignant conversion would allow for earlier detection and treatment of cancer, which should lead to better patient outcomes. Such is the case in the skin, where non-cancerous overgrowths of pigmented melanocytes, called nevi or moles, harbor a potentially cancer-causing mutation, termed BRAFV600E, which is also found in over half of melanoma cancers. Yet nevi rarely progress to invasive melanoma. To enable studies of melanoma initiation and the mechanisms driving this conversion to cancer, we developed a novel reporter of melanoma initiation in a zebrafish melanoma model (that relies on this same BRAFV600E mutation) that expresses a fluorescent protein (EGFP) in the first cell of melanoma and can be visualized in a live animal. Using this model, we found that embryonic neural crest identity reemerges during melanoma initiation, and that regulation of sox10 neural crest transcription factor levels in melanocytes is one key control point in this process. In this proposal, we will test the hypothesis that a specific subset of normally embryonic transcriptional inputs modulates sox10 activity during melanoma initiation and that upregulation of neural crest transcription factors -- in addition to and potentially upstream of sox10 -- directs this transition to malignancy. By defining the role of key transcriptional inputs in modulating sox10 in its native context, we will understand the molecular mechanisms modulating the melanocyte to melanoma transition. We further identify additional neural crest transcription factors that may modulate this process and determine their role and regulation in melanoma initiation. Finally, we use human melanoma patient-derived xenografts (PDX) to further clarify if ongoing expression of these factors is needed for melanoma viability or if their role is primarily during tumor initiation. Overall, this proposal describes innovative approaches to determine the precise regulation of altered transcriptional and epigenetic programs driving melanoma cancer initiation, which may define new and earlier- acting therapeutic targets for treating melanoma.