Genome-edited uveal melanoma cell lines for investigating constitutively active GNAQ and GNA11

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 DESCRIPTION (provided by applicant): Heterotrimeric G proteins (αβγ) are well known for their function in linking G protein- coupled receptors (GPCRs) to intracellular responses, and thereby playing essential roles in transmitting a wide variety of extracellular signals into regulation of countless physiological process. G proteins function as molecular switches whereby a GPCR promotes GDP release from the α subunit (Gα), followed by subsequent binding of GTP by Gα and dissociation of Gα and Gβγ. Gα-GTP and Gβγ can then regulate a variety of signaling proteins until the GTP hydrolysis activity of Gα turns off signaling by generating the inactive Gα-GDP, which then re- associates with Gβγ to complete the cycle of activation and inactivation. However, in uveal melanoma this tightly controlled G protein cycle is corrupted by a mutation in the closely related Gα, αq or α1. Such mutations, occurring in over 90% of uveal melanoma, generate a constitutively active αq or α11 in which GTP hydrolysis activity is abrogated, thereby locking the Gα in an active GTP bound state and turning αq or α11 into an oncogenic driver of this cancer. Uveal melanoma, the most common cancer of the eye in adults, metastasizes in up to 50% of uveal melanoma patients, and, once metastasis occurs, it is invariably fatal with an average survival of less than six months. There are currently no effective therapies for metastatic uveal melanoma, and thus there exists an urgent need to better understand the molecular mechanisms that promote the development of uveal melanoma. Based on numerous basic research studies on G protein function, it is clear that mutationally activated Gα exhibit key cell biology properties, such as changes in subcellular localization, trafficking, lipid modification, degradation and activation of unique signaling pathways, that set them apart from the wild type Gα counterpart. In uveal melanoma, it has been difficult to compare mutationally activated αq or α11 with wild type αq or α11 due to a lack of tools to differentiate between and effectively study the endogenous mutant versus wild type protein. Therefore, the objective of this project is to develop such a tool by using gene-editing approaches with uveal melanoma cell lines to generate cells in which the endogenous αq or α11, either the wild type or mutationally activated form, is fused in-frame to GFP. These genome-edited uveal melanoma cell lines will be a novel and much-needed tool to better understand how mutationally activated αq or α11 functions in uveal melanoma and to identify key aspects of regulation of mutationally activated αq or α11 that differ from that of wld type αq or α11.