The “cancerized field” concept posits that cells in a given tissue share an oncogenic mutation or insult and are thus cancer-prone, yet only discreet clones within the field initiate tumors. Nearly all benign nevi carry oncogenic BRAFV600E mutations, but they only rarely become melanoma. The zebrafish crestin gene is expressed embryonically in neural crest progenitors (NCP’s) and is specifically re-expressed in melanoma. We show by live imaging of transgenic zebrafish crestin reporters that, within a cancerized field (BRAFV600E-mutant; p53-deficient), a single melanocyte reactivates the NCP state, and this establishes that a fate change occurs at melanoma initiation in this model. We show the crestin element is regulated by NCP transcription factors, including sox10. Forced sox10 overexpression in melanocytes accelerated melanoma formation, consistent with activation of a NCP gene signature and super-enhancers leading to melanoma. Our work highlights the importance of NCP state reemergence as a key event in melanoma initiation.

SUMMARY In mammalian embryonic stem cells, the acquisition of pluripotency is dependent upon Nanog, but the in vivo analysis of Nanog has been hampered by its requirement for early mouse development. In an effort to examine the role of Nanog in vivo, we identified a zebrafish Nanog ortholog, and found that its knockdown impaired endoderm formation. Genome-wide transcription analysis revealed that nanog-like morphants fail to develop the extra-embryonic yolk syncytial layer (YSL), which produces Nodal required for endoderm induction. We examined the genes that were regulated by Nanog-like, and identified the homeobox gene mxtx2, which is both necessary and sufficient for YSL induction. Chromatin immunoprecipitation assays and genetic studies indicated that Nanog-like directly activates mxtx2, which in turn specifies the YSL lineage by directly activating YSL genes. Our study identifies a Nanog-like-Mxtx2-Nodal pathway and establishes a role for Nanog-like in regulating the formation of the extra-embryonic tissue required for endoderm induction.

Summary Studying cancer metabolism gives insight into tumorigenic survival mechanisms and susceptibilities. In melanoma, we identify HEXIM1, a transcription elongation regulator, as a melanoma tumor suppressor that responds to nucleotide stress. HEXIM1 expression is low in melanoma. Its overexpression in a zebrafish melanoma model suppresses cancer formation while its inactivation accelerates tumor onset in vivo. Knockdown of HEXIM1 rescues zebrafish neural crest defects and human melanoma proliferation defects that arise from nucleotide depletion. Under nucleotide stress, HEXIM1 is induced to form an inhibitory complex with P-TEFb, the kinase that initiates transcription elongation, to inhibit elongation at tumorigenic genes. The resulting alteration in gene expression also causes anti-tumorigenic RNAs to bind to and be stabilized by HEXIM1. HEXIM1 plays an important role in inhibiting cancer cell-specific gene transcription while also facilitating anti-cancer gene expression. Our study reveals an important role for HEXIM1 in coupling nucleotide metabolism with transcriptional regulation in melanoma.

Chemical genetics is the use of small molecules to perturb biological pathways. This technique is a powerful tool for implicating genes and pathways in developmental programs and disease, and simultaneously provides a platform for the discovery of novel therapeutics. The zebrafish is an advantageous model for in vivo high-throughput small molecule screening due to translational appeal, high fecundity, and a unique set of developmental characteristics that support genetic manipulation, chemical treatment, and phenotype detection. Chemical genetic screens in zebrafish can identify hit compounds that target oncogenic processes– including cancer initiation and maintenance, metastasis, and angiogenesis–and may serve as cancer therapies. Notably, by combining drug discovery and animal testing, in vivo screening of small molecules in zebrafish has enabled rapid translation of hit anti-cancer compounds to the clinic, especially through the repurposing of FDA-approved drugs. Future technological advancements in automation and high-powered imaging, as well as the development and characterization of new mutant and transgenic lines, will expand the scope of chemical genetics in zebrafish.

Within a group of cancer-prone cells that harbor a shared oncogenic mutation, only rare clones transition to a malignant state. These rare and transient events occurring during cancer initiation remain incompletely understood, and we thus sought to visualize and molecularly characterize this crucial early step in oncogenesis of melanoma. We previously found that the zebrafish crestin gene, which specifically marks embryonic neural crest and normally turns off at three days of life, is specifically re-expressed in BRAFV600E/p53 mutant melanoma tumors in adult zebrafish. We cloned the crestin promoter/enhancer and developed an EGFP reporter that recapitulates the embryonic expression pattern of crestin mRNA and, crucially, marks de novo melanomas in living BRAFV600E/p53 mutant zebrafish. Remarkably, we also found that crestin:EGFP becomes active when lesions are only a few cells in number, potentially in the first cell of the melanoma. These crestin:EGFP positive patches are transplantable, and these precursor lesions are enriched for expression of neural crest progenitor (NCP) genes including the transcription factors sox10 and dlx2a, among other melanoma- and NCP-associated genes. In order to favor the readoption of an NCP-state, we forced misexpression of sox10 in melanocytes of BRAFV600E/p53 melanoma-prone zebrafish, and this led to enhanced melanoma formation. We then analyzed the chromatin landscape of both human and zebrafish melanoma cells using ChIP-Seq and ATAC-Seq and identified super-enhancers at crestin (in zebrafish), sox10, dlx2a, and other NCP/melanoma loci, which together described an overall chromatin signature consistent with features of the NCP state. These data support a model in which 1) an NCP program stochastically reactivates, as read out by crestin expression, in rare BRAFV600E/p53 mutated melanocytes at the initiation of melanoma formation and 2) reemergence of this NCP state is an important and potentially rate-limiting event in melanoma initiation. We anticipate that progenitor identity reemergence will prove to be a general feature of cancer initiation. This abstract is also presented as Poster B19. Citation Format: Charles K. Kaufman, Christian Mosimann, Andrew Thomas, Zi Peng Fan, Song Yang, Justin Tan, Rachel D. Fogley, Ellen van Rooijen, Elliott Hagedorn, Christie Ciarlo, Richard White, Dominick Matos, Ann-Christin Puller, Cristina Santoriello, Eric Liao, Richard A. Young, Leonard I. Zon. The reemergence of neural crest progenitor identity is a key event in the initiation of melanoma from a field of cancer-prone melanocytes. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr PR06.

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