Melanomas arise from pigmented cells called melanocytes, typically in the skin. Melanomas can spread to distal parts of the body, through a process called metastasis. Metastasis is the primary cause of mortality in all cancers; melanomas have the highest propensity to metastasize to the brain, contributing to the poor prognosis. Although significant progress has been made in melanocyte biology, the factors influencing metastasis still remain largely unknown. The major cause of melanoma is due to ultra-violet radiation exposure, which causes a large number of mutations in melanocytes. Large-scale sequencing efforts have identified frequently occurring DNA mutations in melanoma, but this approach fails to distinguish between passenger mutations and mutations that actually drive the disease. The role of neighboring cells such as adipocytes or immune cells adds further complexity to understanding factors driving metastasis. For example, the White lab recently uncovered a novel role for adipocytes in driving melanoma. Thus, animal models with an intact immune system that faithfully recapitulate human melanoma are crucial to identify mechanisms regulating metastasis.

Here, we propose to utilize the zebrafish to study melanoma. Zebrafish are easy to breed in large numbers and form melanomas histologically and genetically similar to human melanomas. The optically transparent zebrafish casper, developed in the White lab, allows for easy visualization of melanoma by fluorescence imaging. For example, we can image cancer cells at the single cell level and track its fate within the animal. We collaborated with the Adams lab at the Sanger Center to obtain data from the AVAST-M clinical trial, the largest adjuvant study in high-risk primary melanoma. In this study, 466 patients underwent sequencing of the primary tumor to generate high quality sequencing data. Extensive clinical details were collected such as pathological details on the primary tumors and importantly, whether the patient did or did not ultimately metastasize. We used this powerful dataset to ask whether the expression of certain genes might predict metastasis in general, or more specifically to the brain, which has led us to a list of 60 exciting candidate genes. Additionally, the White lab has developed a method called Transgene Electroporation in Adult Zebrafish (TEAZ) to model melanoma. In TEAZ, mutations are induced in melanocytes, similar to what occurs in human melanomas, using powerful CRISPR-Cas9 based genome editing, which results in aggressive melanoma.

In this study, we propose to use TEAZ and CRISPR-Cas9 to delete candidate genes in zebrafish and monitor metastasis by fluorescence imaging. We will also assess if there are specific factors driving metastasis to the brain, which remains the most aggressive subtype of melanoma. We anticipate that this study will help identify new regulators of metastasis and pave the way for novel therapeutic targets in melanoma.