Uveal melanoma (UM) is the second most common form of melanoma and the most common primary cancer of the eye, resulting not only in vision loss, but in metastatic death in up to half of patients. There are no effective treatment options once the tumor metastasizes and patients usually die within a few months of diagnosis despite systemic therapy. UMs harbor activating oncogenic mutations in GNAQ, GNA11 or CYSLTR2 that occur early in tumor formation and that are unrelated to metastatic risk. We showed that over 80% of class 2 UMs harbor loss of function mutations of BAP1 and that recurrent mutations altering R625 of SF3B1 are found in tumors with slower rates of metastasis. Mutations in EIF1AX are associated with tumors with minimal chance of metastasis. The presence of BAP1, SF3B1 and EIF1AX mutations, are nearly always mutually exclusive. However, overlaid on this are genomic rearrangements that include gain of chromosomes 8q and 6p, and loss of chromosomes 1p, 6q and 8p. Our studies of these karyotypic/copy number alterations in UM have identified a critical chromatin modifier mapping to chromosome 6q (PHF10) and candidate genes in other regions of recurrent chromosomal alteration. The consequences of these changes in the development of UM are poorly understood. To extend and refine our copy number studies we will now will perform long-read single molecule sequencing of DNA and RNA isolated from primary UMs with different driver mutations and CNAs.
In Aims 1 and 2 we will perform long-read single molecule sequencing (SMRT) to develop detailed maps of the structural variations (SVs) of the genomes of these tumors, identifying and characterizing critical breakpoints, gene fusions and tumor specific isoforms. Besides obtaining insights into the generation of chromosomal alterations we will validate and refine focal deletions and identify critical isoforms in such regions. Functional analyses in cell lines will examine the consequences of gene fusions and isoforms highly correlated with copy number changes. In parallel and in Aim 3 we will use the power of fly genetics to investigate the consequence of major alterations in UM in D. Melanogaster. We will first generate and characterize flies with loss of function mutations in the BAP1 ortholog calypso, and generate activating mutations of the GNAQ ortholog G?Q so that the combined effect of loss of calypso and activation of G?Q can be investigated. We will focus on larval eye discs and wing discs that represented well-ordered epithelial monolayers and examine the the phenotypic, signaling and epigenomic changes that arise from these mutations. The fly model is capable of addressing the functional relevance of the genomic insights from all three Aims by performing rapid genetic interactions. In the future, candidate UM drivers identified from Aims 1-2 will be tested for the ability to modify the cancer-like phenotypes we see in our two hit G?Q and calypso fly model. Genetic suppressors of overgrowth, tissue transformation, and metastasis phenotypes would represent exciting therapeutic candidates to pursue in future studies.
Uveal melanoma is a devastating eye cancer that is untreatable once it metastasizes. We have identified major genetic alterations and will now use long read single molecule sequencing to expand our findings. These changes will be modeled in the fly model system to identify novel therapeutic candidates.
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