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Melanoma shows frequent Ras-Raf-MAPK pathway activation via frequent mutations in NRAS and BRAF. Novel mutations in other components of this pathway such as MEK1, MEK2, MAP3K5, and MAP3K9 have been reported recently by high-throughput sequencing efforts. In addition, NF1 (neurofibromatosis type 1), one of the Ras GTPase activating proteins (RasGAPs), has been shown to be mutated or suppressed in melanoma. However, importance of other RasGAPs has not been addressed in melanoma. To obtain a comprehensive view of melanoma genomes, we conducted whole genome sequencing (WGS) of 15 metastatic melanomas and matched normal PBMC genomes from 13 melanoma patients. All melanoma genomes from these 13 patients contained at least one mutation in genes of Ras-Raf-MAPK pathway (MAPK1, MAP3K1, MAP4K2, MAP3K14, NRAS, and BRAF). Our whole genome analyses also identified two novel somatic missense mutations (p.Tyr472His and p.Leu481Phe) in or around the PH domain in RASA1 gene. RASA1 (RAS p21 protein activator (GTPase activating protein) 1, also called as p120RasGAP) is a GTPase activation protein that negatively regulates Ras by catalyzing the hydrolysis of active Ras-GTP to inactive Ras-GDP. In this study, we addressed possible roles of RASA1 in melanoma tumorigenesis. To determine RASA1 level in melanocytic lesions, immunohistochemical analysis of RASA1 on human melanoma tissue microarray (TMA) containing nevi (n=34), primary melanomas (n=63), lymph node metastasis (n=35), and distal metastasis (n=29) was performed. This analysis has shown the inverse correlation of RASA1 expression to melanoma progression. Functionally, ectopic expression of RASA1 mutant increased soft-agar colony formation and tumor growth while that of wild-type RASA1 decreased. shRNA-mediated knock down of RASA1 promoted soft-agar colony formation and invasion of melanocytes and melanoma cells with BRAF mutation as a consequence of the elevated Ras activity. Here, we established the functional significance of novel somatic missense mutations in RASA1 identified in our WGS study. Collectively, these data implicated that RASA1 inactivation by loss of expression and/or mutation may promote melanoma tumorigenesis. Citation Format: Hyeran Sung, Li Ding, Krishna L. Kanchi, Jane L. Messina, Vernon K. Sondak, Mulé J. James, Richard K. Wilson, Jeffrey S. Weber, Minjung Kim. RASA1 alteration promotes melanoma tumorigenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 442. doi:10.1158/1538-7445.AM2014-442