Zebrafish models in melanoma research: analysis of coding and non-coding BRAFV600E

Malignant melanoma is one of the most aggressive types of cancer. While early-stage melanoma can be cured by surgical excision, late-stage melanoma remains a highly lethal disease. Current therapeutic strategies, including single agents or combined therapies, are hampered by low response rates and by diverse resistance mechanisms. The most frequent mutation in malignant melanoma is the V600E substitution in the BRAF oncogene. This mutation constitutively activates the MAPK pathway, promoting cell survival, proliferation, and motility. Among the impacting therapies, BRAFV600E inhibitors (BRAFi) are initially very effective, but, due to quick development of acquired resistance, they can be used for short periods of time (4-6 months). The development of current drug combinations just postpones the acquired resistance. With the final aim to identify new molecular factors involved in BRAFV600E-driven malignant transformation, hence, to improve the response to BRAFi, we are developing and characterizing new melanoma models in zebrafish. In this 3-year PhD project, using the Tol2 system, I generated melanoma-prone transgenic lines in which tumors are driven by BRAFV600E in its reference and X1 isoforms (BRAFV600E-ref and BRAFV600E-X1). While BRAFV600E-ref is the isoform commonly used for similar models, BRAFV600E-X1 is a poorly characterized isoform that, as we discovered in our laboratory, always coexists with the ref. The novelty of this project also lies in the study of the 3’UTR (three prime untranslated region) regulatory regions. These lines express either BRAFV600E ref/X1 coding sequence only or BRAFV600E ref/X1 coding sequence plus their respective 3’UTR. Our data in a mosaic condition show alterations in the pigmentation patterns and in the development of nevi, from which tumors originate, as well as a higher melanoma incidence in presence of BRAFV600E-ref compared to BRAFV600E-X1. Moreover, tumor development resulted to be faster in fish expressing each coding sequence with respect to the coding sequence + 3'UTR. Likewise, we are also generating stable lines. The determination of the location of the transgene is done through an innovative genotyping technique that combines CRISPR/Cas9-mediated DNA editing and Oxford Nanopore Technology (ONT) based sequencing. Stable lines will be studied for BRAFV600E variant-specific coding-(in)dependent activities and drug sensitivity. With respect to drug screening, we will exploit the neural crest signature, which is present in progenitor cells during the early stages of embryonic development and is aberrantly reactivated in melanoma cells. We will use crestin, a common marker in zebrafish, to generate a dual reporter zebrafish line expressing mCherry and Luciferase reporter genes under the control of the crestin promoter. Crossed with the BRAFV600E variant-specific melanoma prone lines, this crestin line can be used as a tool for high-throughput quantitative screening of novel BRAFi-focused drug combinations in zebrafish embryos. Preliminarily, our data confirm a higher expression of crestin in the embryos of the BRAFV600E transgenic line compared to the wild type line. Furthermore, we observed reduced expression of this marker when treating BRAFV600E embryos with anticancer drugs, including BRAFi