Uveal melanoma is the most common intraocular malignancy in the United States and has a 10-year disease specific survival rate of 50%. No effective treatment options exist. While activation of the mitogen-activated protein (MAP) kinase pathway is a common feature in uveal melanoma, mutations in known melanoma oncogenes such as BRAF, NRAS, or KIT are absent. We recently identified somatic mutations in the heterotrimeric G protein alpha q subunit, GNAQ, in 46% of uveal melanomas. Mutations of GNAQ were found in primary and metastatic lesions as well as over 80% of blue nevi and 2% of cutaneous melanomas. GNAQ mutations are found in a mutually exclusive pattern with BRAF, NRAS, or KIT and turn GNAQ into a dominant acting oncogene. siRNA-mediated knockdown in GNAQ -mutant melanoma cell lines leads to marked apoptosis. We identified MAP-kinase activation as one contributing factor to GNAQ-mediated oncogenesis. However, the factors involved in this activation and what other pathways are involved in oncogenesis are currently unknown. In this study we will fill this gap, evaluate GNAQ as a therapeutic target in a preclinical setting, and search for additional oncogenes that are functionally related. In Aim 1 we will determine how GNAQ mutation contributes to melanoma formation using candidate and unbiased approaches to identify the downstream effectors of mutant GNAQ in melanoma. We will carry out gain and loss of function studies in primary melanocytes and uveal melanoma cells, respectively and determine the protein expression and phosphorylation kinetics of canonical downstream signaling targets. These analyses will be complemented by data from already ongoing genome wide expression analyses aimed at identifying the signaling networks downstream of GNAQ as well as proteomic analysis to identify GNAQ's immediate effectors in melanoma. In aim 2 we will validate mutant GNAQ as a therapeutic target in melanoma by carrying out a pre-clinical trial in murine xenograft models of liver metastasis, which is the predominant metastatic site in humans. This aim will be carried out in collaboration with Alnylam, who will provide us with anti- GNAQ siRNA formulated for in vivo use. In aim 3 we will identify possible additional oncogenes using an integrated genomics approach. We will perform detailed comparisons of the clinicopathological features and the activation status of the downstream pathways (aim 1) between uveal melanomas with and without GNAQ mutations to determine whether uveal melanomas without mutations harbor alterations in functionally related genes. We will attempt to identify these genes by determining genomic regions of divergent copy number between the two groups, using high-resolution array-based comparative genomic hybridization and re-sequence the implicated candidate genes. PUBLIC HEALTH RELEVANCE: Uveal melanoma is a highly aggressive form of cancer with no effective treatment, in part because the underlying mechanistic alterations were previously unknown. We have recently discovered mutations in the gene GNAQ, which drive aberrant cell growth in 50% of this melanoma type. The goal of this project is to study its function and develop rationally-based treatment strategies and to discover the equivalent genetic alterations in the remaining 50% of uveal melanomas.