Establishing an in-vivo Sleeping-Beauty Screen to Identify and Characterize Novel Drivers of Melanomagenesis in a Nevi-Prone Zebrafish Model.

University Of Iowa, Iowa City IA

Investigators

Abstract

Abstract Melanoma, a deadly cutaneous malignancy, is increasing globally despite treatment advances. Melanocytic nevi ("moles") are often studied for early diagnosis, but their risk of progressing to melanoma is low. While BRAFV600E or NRASQ61R mutations are common in both melanomas and nevi, additional mutations in tumor-suppressor genes are needed for melanoma development. Similar to human nevi, BRAFV600E and NRASQ61R nevi in zebrafish remain benign. Even in tp53-deficient BRAFV600E and NRASQ61R fish, only a small percentage of nevi advance to melanoma. These findings emphasize the robust constraints on nevus growth and the need for cooperative transcriptional changes for melanocyte transformation. A knowledge gap exists in our understanding of the transcriptional mechanisms outside of somatic mutations that are necessary for transformation. The Sleeping Beauty (SB) transposon mutagenesis system has been utilized to identify novel genes associated with melanoma recurrence. It is unique in comparison to other genetic screens (i.e. CRISPR gRNA) in that transposon integration allows for gene silencing, activation or expression of truncated transcripts through an internal promoter within the transposon. We have established two transgenic nevi-prone zebrafish line, that harbors a germline T2/OncZ transposon, Tg(mitfa-BRAFv600e: T2/OncZ): mitfa-/- and Tg(mitfa-NRASQ61R: T2/OncZ): mitfa-/-. Introducing plasmids expressing mitfa into melanocyte stem cells in our transgenic mitfa-/- fish rescues melanocytes, achieved through injection and electroporation of plasmids carrying the mitfa-promoter driving expression of mitfa, thus inducing nevi formation. While rescued melanocytes propagate into nevi within 4 weeks, these zebrafish remain melanoma-free for up to ~1 year. Injecting mitfa-SB100 transposase plasmids into formed nevi at 6 weeks induces SB mutagenesis and results in transposition of the OncZ transposon, and thus anticipated to accelerate melanocyte transformation. Sequencing melanomas and control nevi will identify significantly enriched transposon integration sites and a short list of candidate genes will be identified. The function of such genes, predicted to be accelerators of melanomagenesis and in the context of both BRAF and NRAS- induced nevi, will be validated by in vivo and in vitro assays utilizing zebrafish and human melanocyte cell lines. Our proposal presents a novel in vivo application of the SB system to uncover unique accelerators of melanomagenesis. This approach combines established zebrafish lines with transgene electroporation techniques, allowing for precise spatial and temporal control over tumor initiation. We aim to identify new transcriptional mechanisms underlying nevi transformation, offering critical insights into melanoma biology and advancing genetic methodologies in cancer research.