Evaluating Pathways Involved in Melanoma Initiation - ABSTRACT Melanoma is a devastating type of cancer, and despite recent advances in targeted or immunologic therapies, many patients relapse. We study melanoma using a zebrafish model with melanocyte-specific expression of human BRAFV600E or other oncogenes in the background of a variety of tumor suppressor mutations. With zebrafish genetic and chemical biology tools, we hope to reveal critical pathways that participate in the initiation and formation of melanoma. We have observed the onset of melanoma using a neural crest specific transgenic reporter for crestin, a neural crest progenitor gene that is expressed early in neural crest development and shuts off by day 5 of zebrafish development. When a melanoma arises, crestin expression reactivates in single cells transforming into melanoma. We recently found that the initiating cell is derived from a zone of hundreds of cells that express higher levels of the master melanocyte regulator MITF and display aberrant morphology. We called this region a cancer precursor zone (CPZ) and found analogous regions that express MITF and another conserved gene, ID1, in early human melanocyte lesions and melanoma in situ. Using novel cellular barcoding technologies such as CRISPR-based GESTALT, we have shown the CPZ is oligoclonal from which a single malignant clone expands to form melanoma. We also showed that BMP, which induces ID1 clonally expands CPZs. Using the color-based Palmbow, we will study gene expression in clones of CPZ and early tumors and use imaging to study the clones. Clonal mapping using mitochondrial sequencing of human melanomas will confirm our observations in zebrafish. We plan to use proteomics and zebrafish genetics to examine the pathways involved in neural crest reactivation from the CPZ. Using proteomics, we have found growth factors and receptors that are expressed in crestin+ patches and not CPZs, such as midkine b that is known to regulate neural crest development. Overexpression and knockout experiments will be used to test the sufficiency and requirement of these proteins for causing tumor initiation. The CPZ is also associated with increased oxidative stress, and we found the antioxidant gene ApoD is expressed at higher levels in early crestin+ patches and drives the transition to overt tumors. One function of ApoD is to convey resistance to ferroptotic death and to enable tumor initiating cells to handle more reactive oxygen species (ROS). Overexpression of ApoD leads to an increase in both tumor incidence and abundance in zebrafish. We plan to use genetics and cell biology, including leveraging MAZERATI, a tissue-specific CRISPR technology, to understand the mechanism by which ApoD controls ferroptosis and how the protection from ROS and ferroptosis plays a role in melanoma initiation. We also plan to use our color based and CRISPR cellular barcoding technologies to investigate if ROS resistance and suppression of ferroptosis by ApoD leads to clonal selection and tumor initiation. Our studies will improve the understanding of cancer initiation and progression. This may lead to the identification of new biomarkers or drug targets, yielding early diagnosis and new preventative therapies for melanoma.
