Summary A single driver mutation, BRAFV600E, drives half of all melanomas. However, in the majority of cases, acquisition of BRAFV600E instead drives benign tumors, such as melanocytic nevi (the common mole). We seek to decipher the intracellular mechanisms that prevent full transformation, and harness this knowledge to develop candidate early diagnosis and chemoprevention strategies. We have discovered a signature of microRNAs (miRNA) as the most differentially expressed transcripts distinguishing nevi from either normal melanocytes or melanomas. We have reported that expression of these miRNAs classifies biopsied pigmented neoplasms with high diagnostic accuracy. To determine whether knowledge of these miRNAs could aid in the prevention of melanoma: First, we conducted a comprehensive identification and functional screen of the targets of the most predictive miRNA, MIR211-5p. Using freshly isolated and CRISPR engineered human normal, nevus and melanoma melanocytes, we identified inhibition of AURKB expression as a critical mechanism driving both BRAFV600E and MIR211-5p associated growth arrest in vitro. Therefore, in Aim 1, our objective is to assess the roll of the MIR211-5p/AURKB axis in nevus formation and transformation in vivo, and the efficacy of disrupting this axis in melanoma chemoprevention. Second, we generated a non-invasive assay for miRNA screening of pigmented lesions prior to biopsy. In a small pilot study, we found the high accuracy of classification of melanocytic neoplasia was retained. In Aim 2, our objective is to validate the utility of using non-invasive profiling of the miRNA signature to screen pigmented skin lesions. Three advances distinguish our proposal. The first is the reproducibility of our miRNA classifier, currently validated on six independent datasets. Second, our model systems for this disease—engineered primary human melanocytes, nevi, and melanomas, combined with an in vivo system that recapitulates both the genetics and progression of melanoma—puts us in a unique position to control for context-specific effects when studying these critical events. Third, is our development of a non-invasive miRNA profiling assay, a molecular profiling technique that is both non-invasive and lesion-specific. Our team, consisting of experts in both the basic biology of miRNA and melanoma, in vivo models of melanoma, topical drug delivery, and the daily practice of melanoma surveillance, allows us to comprehensively tackle this project. This project has both basic and clinical potential significance. Our studies explore novel explanations for nevus initiation, driven by observations made from clinical lesions. We expect these studies to directly result in an increase in the early detection of melanomas and preclinical validation of a strategy for topical chemoprevention for particularly high-risk individuals and/oror anatomic areas.