Synthetic Lethal Screen to Identify Molecular Mechanisms that Drive Uveal Melanoma

Abstract

Melanoma that strikes the uveal cells of the eye represents about 5% of all melanoma cases. Uveal melanoma is a highly aggressive form of cancer that exhibits a propensity to metastasize to the liver. At this time there are no effective treatments for metastatic uveal melanoma and new therapeutic strategies are urgently needed. The majority of uveal melanomas are derived from early activating mutations in the G-proteins GNA11 or GNAQ. In order to further explore and identify molecular mechanisms that uveal melanoma cell lines rely upon for proliferation and survival, we will perform CRISPR/Cas9 mediated synthetic lethality screening. To accomplish this, we will generate a uveal melanoma cell line 92.1 that expresses the endonuclease Cas9 and introduce a guide RNA library into it that targets the functional domains of epigenetic regulator proteins. Thus, we will be able to determine which epigenetic regulator genes are required for the survival and proliferation of these cells by measuring which guide RNAs "drop out" over time. The guide RNAs that drop out likely disrupt essential genes that the cell relies on for growth.  Cells carrying these gRNAs are consequently depleted from the culture. This will indicate that Cas9 mediated disruption of the gene targeted by that guide RNA causes cell death and will therefore identify the key dependencies of the uveal melanoma cells. We expect that the results of these experiments will reveal novel genetic mechanisms characteristic of GNAQ mutant uveal melanoma cells which could be therapeutically targeted in the future.