Mutations in BRAF and NRAS that lead to constitutive activation of MAPK signalling have been found at high frequencies in many melanomas, both benign and malignant. However, they have not been found in uveal melanomas (arising from in the cells that give colour to the eye) or in blue naevi melanomas (a type of benign blue–black mole). Now a genetic screen of biopsy samples shows that these melanoma subtypes instead show frequent activating mutations in the G protein α-subunit GNAQ, also leading to the activation of the MAPK pathway. This identifies signalling components downstream of GNAQ as potential therapeutic targets. Mutations in BRAF and NRAS that lead to constitutive activation of MAP kinase signalling have been found at high frequencies in many melanomas. However, they have not been found in the uveal melanomas and blue nevi subtypes of melanoma. This paper shows that these subtypes instead show frequent activating mutations in the G protein α-subunit GNAQ, also leading to the activation of the MAP kinase pathway. BRAF and NRAS are common targets for somatic mutations in benign and malignant neoplasms that arise from melanocytes situated in epithelial structures, and lead to constitutive activation of the mitogen-activated protein (MAP) kinase pathway1,2. However, BRAF and NRAS mutations are absent in a number of other melanocytic neoplasms in which the equivalent oncogenic events are currently unknown3. Here we report frequent somatic mutations in the heterotrimeric G protein α-subunit, GNAQ, in blue naevi (83%) and ocular melanoma of the uvea (46%). The mutations occur exclusively in codon 209 in the Ras-like domain and result in constitutive activation, turning GNAQ into a dominant acting oncogene. Our results demonstrate an alternative route to MAP kinase activation in melanocytic neoplasia, providing new opportunities for therapeutic intervention.

Uveal melanoma is the most common intraocular cancer. There are no effective therapies for metastatic disease. Mutations in GNAQ, the gene encoding an alpha subunit of heterotrimeric G proteins, are found in 40% of uveal melanomas. We sequenced exon 5 of GNAQ and GNA11, a paralogue of GNAQ, in 713 melanocytic neoplasms of different types (186 uveal melanomas, 139 blue nevi, 106 other nevi, and 282 other melanomas). We sequenced exon 4 of GNAQ and GNA11 in 453 of these samples and in all coding exons of GNAQ and GNA11 in 97 uveal melanomas and 45 blue nevi. We found somatic mutations in exon 5 (affecting Q209) and in exon 4 (affecting R183) in both GNA11 and GNAQ, in a mutually exclusive pattern. Mutations affecting Q209 in GNA11 were present in 7% of blue nevi, 32% of primary uveal melanomas, and 57% of uveal melanoma metastases. In contrast, we observed Q209 mutations in GNAQ in 55% of blue nevi, 45% of uveal melanomas, and 22% of uveal melanoma metastases. Mutations affecting R183 in either GNAQ or GNA11 were less prevalent (2% of blue nevi and 6% of uveal melanomas) than the Q209 mutations. Mutations in GNA11 induced spontaneously metastasizing tumors in a mouse model and activated the mitogen-activated protein kinase pathway. Of the uveal melanomas we analyzed, 83% had somatic mutations in GNAQ or GNA11. Constitutive activation of the pathway involving these two genes appears to be a major contributor to the development of uveal melanoma. (Funded by the National Institutes of Health and others.)

Chemical mutagenesis in the mouse is a powerful approach for phenotype-driven genetics, but questions remain about the efficiency with which new mutations ascertained by their phenotype can be localized and identified, and that knowledge applied to a specific biological problem. During a global screen for dominant phenotypes in about 30,000 animals, a novel class of pigmentation mutants were identified by dark skin (Dsk). We determined the genetic map location, homozygous phenotype, and histology of 10 newDskand 2 new dark coat (Dcc) mutations, and identified mutations inAgouti(Met1Leu,Dcc4),Sox18(Leu220ter,Dcc1),Keratin 2e(Thr500Pro,Dsk2), andEgfr(Leu863Gln,Dsk5). Cutaneous effects of mostDskmutations are limited to melanocytes, except for theKeratin 2eandEgfrmutations, in which hyperkeratosis and epidermal thickening precede epidermal melanocytosis by 3–6 wk. TheDsk2mutation is likely to impair intermediate filament assembly, leading to cytolysis of suprabasal keratinocytes and secondary hyperkeratosis and melanocytosis. TheDsk5mutation causes increased tyrosine kinase activity and a decrease in steady-state receptor levels in vivo. TheDskmutations represent genes or map locations not implicated previously in pigmentation, and delineate a developmental pathway in which mutations can be classified on the basis of body region, microscopic site, and timing of pigment accumulation.

Many members of the animal kingdom display coat or skin color differences along their dorsoventral axis. To determine the mechanisms that control regional differences in pigmentation, we have studied how a classical mouse mutation, droopy ear (de^H), affects dorsoventral skin characteristics, especially those under control of the Agouti gene. Mice carrying the Agouti allele black-and-tan (a^t) normally have a sharp boundary between dorsal black hair and yellow ventral hair; the de^H mutation raises the pigmentation boundary, producing an apparent dorsal-to-ventral transformation. We identify a 216 kb deletion in de^H that removes all but the first exon of the Tbx15 gene, whose embryonic expression in developing mesenchyme correlates with pigmentary and skeletal malformations observed in de^H/de^H animals. Construction of a targeted allele of Tbx15 confirmed that the de^H phenotype was caused by Tbx15 loss of function. Early embryonic expression of Tbx15 in dorsal mesenchyme is complementary to Agouti expression in ventral mesenchyme; in the absence of Tbx15, expression of Agouti in both embryos and postnatal animals is displaced dorsally. Transplantation experiments demonstrate that positional identity of the skin with regard to dorsoventral pigmentation differences is acquired by E12.5, which is shortly after early embryonic expression of Tbx15. Fate-mapping studies show that the dorsoventral pigmentation boundary is not in register with a previously identified dermal cell lineage boundary, but rather with the limb dorsoventral boundary. Embryonic expression of Tbx15 in dorsolateral mesenchyme provides an instructional cue required to establish the future positional identity of dorsal dermis. These findings represent a novel role for T-box gene action in embryonic development, identify a previously unappreciated aspect of dorsoventral patterning that is widely represented in furred mammals, and provide insight into the mechanisms that underlie region-specific differences in body morphology.

GNAQ and GNA11 are heterotrimeric G protein alpha subunits, which are mutated in a mutually exclusive pattern in most cases of uveal melanoma, one of the most aggressive cancers. Here we introduce the first transgenic mouse model of uveal melanoma, which develops cancers induced by expression of oncogenic GNAQ^Q209L under control of the Rosa26 promoter. Disease penetrance is 100% by 3 months of age, with 94% of mice also developing lung tumors. In this model, the Yap protein of the Hippo pathway is activated in the eyes, and blood vessels near the lesions in the head and lungs exhibit melanocytic invasion. While full transcription levels are not necessary for GNAQ^Q209L to transform mouse melanocytes, we obtained suggestive evidence of a selective advantage for increased GNAQ^Q209L expression in human tumors. Intriguingly, enforced expression of GNAQ^Q209L progressively eliminated melanocytes from the interfollicular epidermis in adults, possibly explaining the near absence of GNAQ^Q209 mutations in human epithelial melanomas. The mouse model also exhibited dermal nevi and melanocytic neoplasms of the central nervous system, accompanied by impaired hearing and balance, identifying a novel role for GNAQ in melanocyte-like cells of the inner ear. Overall, this model offers a new tool to dissect signaling by oncogenic GNAQ and to test potential therapeutics in an in vivo setting where GNAQ^Q209L mutations contribute to both the initiation and metastatic progression of uveal melanoma. Cancer Res; 75(16); 3384–97. ©2015 AACR.