Zebrafish Melanoma Research Articles

Abstract 4180: Rewiring the cytokine network in melanoma

Abstract Malignant melanoma is one of the most aggressive types of malignancies in humans resulting in 50,000 deaths worldwide annually. Fortuitously, malignant melanoma is highly immunogenic and it is now well established that the host immune system can detect and kill melanoma. T helper type-1 lymphocytes (TH1) play a pivotal role in inducing and maintaining anti-tumour immune responses. In melanoma patients, it has been observed that higher number of TH1 cells in the tumour micro-environment lead to better prognosis. In this study, using an autochthonous zebrafish melanoma model, when we forced the expression of interferon gamma (IFNg) locally in the tumour microenvironment, thereby potentially enhancing TH1 differentiation, we observed an inflammatory response against melanoma that eventually lead to complete tumour regression. Using transposon mediated BAC transgenesis, we have generated a Tg(cd4:mcherry) reporter line labelling CD4+ cells in zebrafish. By driving Human NRASQ61L in zebrafish melanocytes we have modelled melanoma in zebrafish and later when these fish developed tumours we forced the expression of IFNg in melanoma cells using a tamoxifen inducible LSL/CreERT2 system driven by mitfa minimal promoter. Experiments were performed using animals with and without CreERT2. Following tamoxifen administration, we observed a loss of pigmentation in all the IFNg induced tumours (n = 12) by 2 weeks post induction which gradually increased over the study period. Whereas no loss of pigmentation was observed in control tumours (n = 12) throughout the study period. At 9 weeks post induction, the study was terminated and tumours were analysed using histology. In tumours were IFNg was induced, we observed that the tumour tissue was now replaced with fibrotic tissue accompanied by a marked lymphocyte infiltration and patches of necrosis. However, new tumour nodules were also observed to develop in the vicinity of regressing nodules which are unpigmented and potentially hypo-immunogenic. We observed no signs of tumour regression in control tumours. Although preliminary, our data is very exciting and promising as it will open new avenues for developing combinatorial therapies in melanoma. Using a high throughput in-vivo model system such as zebrafish, it is an exciting proposition to test the impact of combining anti-PD1/PDL1 inhibitors with various cytokines/chemokines in inducing effective and sustained tumour regression in melanoma. Citation Format: Raghavendar Nagaraju, Christopher Dee, Helen Young, Jorge Barriuso, Christopher Secombes, Adam Hurlstone. Rewiring the cytokine network in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4180.

Reprofiling using a zebrafish melanoma model reveals drugs cooperating with targeted therapeutics.

Phenotype-guided re-profiling of approved drug molecules presents an accelerated route to developing anticancer therapeutics by bypassing the target-identification bottleneck of target-based approaches and by sampling drugs already in the clinic. Further, combinations incorporating targeted therapies can be screened for both efficacy and toxicity. Previously we have developed an oncogenic-RAS-driven zebrafish melanoma model that we now describe display melanocyte hyperplasia while still embryos. Having devised a rapid method for quantifying melanocyte burden, we show that this phenotype can be chemically suppressed by incubating V12RAS transgenic embryos with potent and selective small molecule inhibitors of either MEK or PI3K/mTOR. Moreover, we demonstrate that combining MEK inhibitors (MEKi) with dual PI3K/mTOR inhibitors (PI3K/mTORi) resulted in a super-additive suppression of melanocyte hyperplasia. The robustness and simplicity of our novel screening assay inspired us to perform a modest screen of FDA approved compounds for their ability to potentiate MEKi PD184352 or PI3K/mTORi NVPBEZ235 suppression of V12RAS-driven melanocyte hyperplasia. Through this route, we confirmed Rapamycin as a compound that could synergize with MEKi and even more so with PI3K/mTORi to suppress melanoma development, including suppressing the growth of cultured human melanoma cells. Further, we discovered two additional compounds—Disulfiram and Tanshinone—that also co-operate with MEKi to suppress the growth of transformed zebrafish melanocytes and showed activity toward cultured human melanoma cells. In conclusion, we provide proof-of-concept that our phenotype-guided screen could be used to identify compounds that affect melanoma development and prompt further evaluation of Disulfiram and Tanshinone as possible partners for combination therapy.

628 CD271 downregulation promotes melanoma invasion in multicellular tumor spheroids and in a zebrafish melanoma model

628 CD271 downregulation promotes melanoma invasion in multicellular tumor spheroids and in a zebrafish melanoma model

Abstract PR06: The reemergence of neural crest progenitor identity is a key event in the initiation of melanoma from a field of cancer-prone melanocytes

Abstract 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.

Stress from Nucleotide Depletion Activates the Transcriptional Regulator HEXIM1 to Suppress Melanoma

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.

A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation.

The "cancerized field" concept posits that cancer-prone cells in a given tissue share an oncogenic mutation, but only discreet clones within the field initiate tumors. Most benign nevi carry oncogenic BRAF(V600E) mutations but rarely become melanoma. The zebrafish crestin gene is expressed embryonically in neural crest progenitors (NCPs) and specifically reexpressed in melanoma. Live imaging of transgenic zebrafish crestin reporters shows that within a cancerized field (BRAF(V600E)-mutant; p53-deficient), a single melanocyte reactivates the NCP state, revealing a fate change at melanoma initiation in this model. NCP transcription factors, including sox10, regulate crestin expression. Forced sox10 overexpression in melanocytes accelerated melanoma formation, which is consistent with activation of NCP genes and super-enhancers leading to melanoma. Our work highlights NCP state reemergence as a key event in melanoma initiation.

Visualizing the beginnings of melanoma

Melanoma Initiation In cancer biology, a tumor begins from a single cell within a group of precancerous cells that share genetic mutations. Kaufman et al. used a zebrafish melanoma model to visualize cancer initiation (see the Perspective by Boumahdi and Blanpain). They used a fluorescent reporter

Long-term drug administration in the adult zebrafish using oral gavage for cancer preclinical studies.

ABSTRACTZebrafish are a major model for chemical genetics, and most studies use embryos when investigating small molecules that cause interesting phenotypes or that can rescue disease models. Limited studies have dosed adults with small molecules by means of water-borne exposure or injection techniques. Challenges in the form of drug delivery-related trauma and anesthesia-related toxicity have excluded the adult zebrafish from long-term drug efficacy studies. Here, we introduce a novel anesthetic combination of MS-222 and isoflurane to an oral gavage technique for a non-toxic, non-invasive and long-term drug administration platform. As a proof of principle, we established drug efficacy of the FDA-approved BRAFV600E inhibitor, Vemurafenib, in adult zebrafish harboring BRAFV600E melanoma tumors. In the model, adult casper zebrafish intraperitoneally transplanted with a zebrafish melanoma cell line (ZMEL1) and exposed to daily sub-lethal dosing at 100 mg/kg of Vemurafenib for 2 weeks via oral gavage resulted in an average 65% decrease in tumor burden and a 15% mortality rate. In contrast, Vemurafenib-resistant ZMEL1 cell lines, generated in culture from low-dose drug exposure for 4 months, did not respond to the oral gavage treatment regimen. Similarly, this drug treatment regimen can be applied for treatment of primary melanoma tumors in the zebrafish. Taken together, we developed an effective long-term drug treatment system that will allow the adult zebrafish to be used to identify more effective anti-melanoma combination therapies and opens up possibilities for treating adult models of other diseases.

Generation and analysis of zebrafish melanoma models.

The rapid emergence of the zebrafish as a cancer model has been aided by advances in genetic, chemical, and imaging technologies. Melanoma in particular highlights both the power and challenges associated with cancer modeling inzebrafish. This chapterfocuses on the lessons that have emerged from the melanoma models as paradigmatic of what will apply to nearly all cancer models in the zebrafish system. Wespecifically focus on methodologies related to germline and mosaic transgenic melanoma generation, and how these can be used to deeply interrogate additional cooperating oncogenes or tumor suppressors. These transgenic tumors can in turn be used to generate zebrafish-specific, stable melanoma cell lines which can be fluorescently labeled, modified by cDNA/CRISPR techniques, and used for detailed in vivo imaging of cancer progression in real time. These zebrafish melanoma models are beginning to elucidate both cell intrinsic and microenvironmental factors in melanoma that have broader implications for human disease. We envision that nearly all of the techniques described here can be applied to other zebrafish cancer models, and likely expanded beyond what we describe here.

Neutrophils fan cancer's flames.

A new study published in this issue of The EMBO Journal looks into the crosstalk between inflammation and cancer (Antonio et al , [2015][1]). By using a zebrafish melanoma model, the authors reveal that neutrophils recruited at the wound site directly interact with cells undergoing oncogenic transformation and provide them with a paracrine proliferative support. Importantly, the authors demonstrate the clinical relevance of this association, showing that neutrophil infiltration has an independent prognostic value for human melanoma. This study reinforces the notion that inflammation flames carcinogenesis, which might have important implications for the improvement of antitumour therapies. [1]: #ref-1

Abstract PR10: From melanocyte to melanoma: Identifying key molecular events at the onset of cancer

Abstract A fundamental challenge in cancer biology is understanding the earliest events that switch a premalignant or premetastatic cell into a fully competent cancer cell. Driven by this question, we focused on the zebrafish gene crestin that marks the embryonic neural crest from the onset of somitogenesis through 72 hpf when it normally turns off; surprisingly, our lab previously discovered that crestin mRNA is reexpressed in melanoma tumors in adult zebrafish. We have developed a fluorescent reporter gene using the crestin promoter that recapitulates the embryonic expression pattern of crestin, and when bred into our BRAFV600E/p53 mutant melanoma zebrafish model, marks grossly visible melanoma tumors. Remarkably, we found that crestin:EGFP becomes active when lesions are only a few cells in number, potentially in the first cell of the melanoma. We are able to isolate crestin (+) patches at the few to single cell level. Using a crestin:CreERt2 inducible recombinase together with a lox-dependent fluorescent lineage tracer line ubi:switch, multiple neural crest descendant lineages including melanocytes can be traced. Further by expressing the melanocyte lineage master transcription factor mitf under the control of the crestin promoter, we can rescue melanocyte formation in mitf null embryos, and in the Na/BRAFV600E/p53 melanoma-prone mutant background, some adult fish develop darkly pigmented patches and melanoma tumors. These data support a model in which BRAFV600E/p53 mutated melanocytes stochastically reactivate an early melanocyte or neural crest progenitor program as read out by crestin reactivation at the onset of melanoma formation. Using our novel reporter, we are able to analyze in vivo the earliest steps of cancer formation as well as the neural crest lineage program. This abstract is also presented as Poster A47. Citation Format: Charles K. Kaufman, Christian Mosimann, Richard White, Dominic Matos, Leonard I. Zon. From melanocyte to melanoma: Identifying key molecular events at the onset of cancer. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr PR10.

Abstract 2045: Identifying key molecular events at the onset of melanoma using a neural crest stem cell marker

Abstract An ongoing challenge in cancer biology is understanding the earliest, initiating events that switch a premalignant cell into a fully competent cancer cell. Driven by this challenge, we focused on the zebrafish gene crestin that marks the embryonic neural crest from the onset of somitogenesis through 72 hours post fertilization when it normally turns off. Surprisingly, our lab previously showed that crestin mRNA is reexpressed in melanoma tumors in adult zebrafish, reading out a neural crest progenitor state. We have developed a fluorescent reporter using the crestin promoter/enhancer that recapitulates the embryonic expression pattern of crestin, and when bred into our BRAFV600E/p53 mutant melanoma zebrafish model, marks grossly visible melanoma tumors. Remarkably, we found that crestin:EGFP becomes active when lesions are only a few cells in number, potentially in the first cell of the melanoma. Using a crestin:CreERt2 inducible recombinase together with a lox-dependent fluorescent lineage tracer line ubi:switch, multiple neural crest descendant lineages including melanocytes can be traced. Further by expressing the melanocyte lineage master transcription factor mitf under the control of the crestin promoter, we can rescue melanocyte formation in mitf null embryos, and in the Na/BRAFV600E/p53 melanoma-prone mutant background, some adult fish develop darkly pigmented patches and melanoma tumors. Finally, we isolated small patches of crestin:EGFP positive cells and show that these precursor lesions are enriched for expression of the neural crest progenitor transcription factor sox10. These data support a model in which BRAFV600E/p53 mutated melanocytes stochastically reactivate an early melanocyte or neural crest progenitor program as read out by crestin reactivation at the initiation of melanoma formation. Using our novel reporter, we are able to analyze in vivo the earliest steps of cancer formation as well as the neural crest lineage program. Citation Format: Charles K. Kaufman, Christian Mosimann, Richard M. White, Dominic Matos, Leonard I. Zon. Identifying key molecular events at the onset of melanoma using a neural crest stem cell marker. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2045. doi:10.1158/1538-7445.AM2014-2045

898: Impact of inflammation in a spontaneous model of melanoma in zebrafish

898: Impact of inflammation in a spontaneous model of melanoma in zebrafish

Immunohistochemistry for Histone H3 Lysine 9 Methyltransferase and Demethylase Proteins in Human Melanomas

Methylation and demethylation of histone H3 lysine 9 (H3K9) play a role in the transcriptional regulation of several cancer-related genes and are closely associated with malignant tumor behavior. A novel study has recently demonstrated that SETDB1, a member of the H3K9 methyltransferases, accelerates tumor formation significantly in a zebrafish melanoma model. However, the expression of H3K9 methyltransferases including SETDB1 and demethylases has not been systematically examined in samples of human melanoma. Here, we used immunohistochemistry to examine the expression of the H3K9 methyltransferases, EHMT2 and SETDB1, and a H3K9 demethylase, LSD1, in 67 patients with melanoma. Overexpression of EHMT2, SETDB1, and LSD1 was observed in 14 (21%), 38 (57%), and 53 (79%) of the 67 patients, respectively. A significant relationship was observed between overexpression of EHMT2 or SETDB1 and aggressive tumor behavior such as lymph node metastasis and/or distant metastasis (P < 0.05), whereas no significant relationship was evident for LSD1 immunoreactivity. Univariate log-rank tests demonstrated that patients with melanoma overexpressing EHMT2 had a poorer outcome (P < 0.001), whereas overexpression of SETDB1 or LSD1 had no prognostic impact. These results suggest that overexpression of EHMT2 might be a prognostic marker in patients with melanoma.

Abstract CN03-03: Use of the zebrafish for studying cancer genetics.

Abstract Melanoma is a devastating disease and there are few therapies that lead to substantial long-term remission for metastatic disease. We have created a zebrafish model of melanoma by overexpressing BRAF-V600E in the combination of P53 mutation. This fish develops melanoma at a characteristic rate and the tumors greatly resemble those of human melanomas. Examination of gene expression in the melanomas demonstrated neural crest transcription factor expression in the zebrafish melanomas. A neural crest marker called Crestin was substantially upregulated in the tumors compared to adjacent skin. A chemical screen was undertaken to find small molecules that prevent neural crest development during embryogenesis in the zebrafish, hoping to find a small molecule that would inhibit the gene expression signature in the tumor. A drug called leflunomide was found to inhibit transcription elongation in the crest and also in human melanomas. Combination of a BRAF inhibitor plus leflunomide led to a substantial decrease in human tumors when placed into nude mice. A Phase II clinical trial has been undertaken using the combination of BRAF inhibitor and leflunomide. Leflunomide has an important effect on transcription elongation that is dependent on a new tumor suppressor complex called the HEXIM complex. Inactivation of the HEXIM complex interferes with the effect of the drug. HEXIM has also been shown to be a tumor suppressor in our zebrafish model system. In an effort to understand the effects of leflunomide, we undertook a screen for a chemical suppressor of leflunomide. This demonstrated pathways that affected drug metabolism as well as transcription elongation. Our studies demonstrate a novel pathway for interrupting cancer development by blocking transcription elongation in combination with BRAF inhibitors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):CN03-03. Citation Format: Leonard I. Zon. Use of the zebrafish for studying cancer genetics. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr CN03-03.

Abstract 1273: Adaptive immunity in a zebrafish model of melanoma.

Abstract The recent success of the anti-CLTA-4 antibody, ipilimumab, for late stage metastatic melanoma, provides proof of principle that stimulating the immune system can have profound effects on patient outcome. However, a detailed understanding of the role of immune-modulating cells within the tumor microenvironment has been confounded by the lack of a suitable model system that would allow for direct visualization of cells within the intact tumor microenvironment. Previous work has demonstrated that the zebrafish, Danio Rerio, can be genetically manipulated to produce melanomas that recapitulate BRAFV600E;P53-/- human tumors. Due to its translucency, ease of genetic manipulation and high fecundity, the zebrafish is an attractive model system that allows for in vivo characterization of cells within intact tissue. Using this model system, we have begun investigating the adaptive immune response to melanoma in zebrafish. We have focused our initial investigations on determining whether transcript expression of key adaptive immune-modulatory proteins are present in the zebrafish and to what extent they are expressed intratumorally . Our studies have found that orthologs of CLTA-4, FOXP3, and CD8 are all expressed in wild-type zebrafish, but not in rag-/- zebrafish. Furthermore, transcript levels of CTLA-4 were greatest in the melanoma tumor relative to other regions of the zebrafish, which is consistent with the constitutive expression of CTLA-4 in human melanomas. Additionally, we have cloned the promoters for zebrafish FOXP3 and CD8 and are developing fluorescent reporter lines that will enable us to visualize and ablate subpopulations of T cells within the tumor. These lines, along with previously described reporter lines for MHC II and lck, will enable us to determine the role of adaptive immune cells during melanoma onset and progression in the zebrafish. Our research supports the notion that there is conservation between the human and zebrafish adaptive immune systems, and that similar immune evasion mechanisms may be contributing to melanoma growth in zebrafish. These data position the zebrafsh as a powerful model system for immunological investigations that focus on the adaptive immune response to melanoma. Citation Format: Corrie A. Painter, Craig Ceol. Adaptive immunity in a zebrafish model of melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1273. doi:10.1158/1538-7445.AM2013-1273

Abstract 3234: Comparative oncogenomics: identifying melanoma drivers from zebrafish to humans.

Abstract The elucidation of core networks driving tumorigenesis is greatly enhanced by studying tumors from multiple species. A comparison of tumors between evolutionarily distant organisms such as humans and fish offers a particularly powerful method to identify such core functionalities. In the past several years, the zebrafish has emerged as an important vertebrate model of cancer because it develops tumors that are histologically and functionally similar to the human disease. Overexpression of the BRAFV600E oncogene under the melanocyte-specific mitf promoter leads to a 100% penetrant melanoma in the context of p53 loss of function. We have previously shown that these zebrafish tumors exhibit characteristics of neural crest progenitors, rather than differentiated melanocytes, similar to many human melanomas which also overexpress the neural crest markers sox10 and ednrb. To identify the genomic lesions that are common to both species, we are performing RNA and DNA-sequencing of the zebrafish tumors and comparing them to human melanoma resequencing samples to identify common driver events. To facilitate this, we have developed technologies for both short and long term culture of zebrafish melanoma cell lines, along with normal tissue from each individual animal. One such melanoma line, ZMEL1, has been comprehensively characterized. RNA-seq demonstrated upregulation of neural crest genes such as ednrb, and pathway analysis indicated enrichment for ERK/MAP kinase and PI3K/AKT signaling pathways, and these tumors are sensitive to inhibition by the BRAF inhibitor PLX4720. Whole-genome sequencing (WGS) was performed on tumor cell line and matched normal tissue to a depth of 39X coverage on the Illumina platform. A total of 5193 single basepair mutations were identified, of which 2% were found in the coding region, 41% in intronic regions and 56% in the intergenic DNA. Within the coding region, 25% were G&amp;gt;C and 18% T&amp;gt;C substitutions, with approximately equal number of non-synonymous to silent mutations. Coding mutations in several genes such as rps6kb1b and plb1 are of particular interest because of their reported mutation in human cancer as well. 35 regions of copy number changes were found, comprising over 600 genes, and these include amplification of known oncogenes such as SETDB1. Functional analysis of the genomic changes common to both zebrafish and human tumors is now being undertaken using in vitro and in vivo melanoma models. In summary, comparative oncogenomics between zebrafish and human melanoma provides a powerful method for filtering the vast number of changes down to a list of manageable, testable hypotheses. This method can be broadly applied to other tumors types. Citation Format: Richard M. White, Erin Langdon, Charles Kaufman, Ellen van Rooijen, Jennifer Yen, Peter Van Loo, Leonard Zon. Comparative oncogenomics: identifying melanoma drivers from zebrafish to humans. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3234. doi:10.1158/1538-7445.AM2013-3234 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

The genetic heterogeneity and mutational burden of engineered melanomas in zebrafish models

BackgroundMelanoma is the most deadly form of skin cancer. Expression of oncogenic BRAF or NRAS, which are frequently mutated in human melanomas, promote the formation of nevi but are not sufficient for tumorigenesis. Even with germline mutated p53, these engineered melanomas present with variable onset and pathology, implicating additional somatic mutations in a multi-hit tumorigenic process.ResultsTo decipher the genetics of these melanomas, we sequence the protein coding exons of 53 primary melanomas generated from several BRAFV600E or NRASQ61K driven transgenic zebrafish lines. We find that engineered zebrafish melanomas show an overall low mutation burden, which has a strong, inverse association with the number of initiating germline drivers. Although tumors reveal distinct mutation spectrums, they show mostly C > T transitions without UV light exposure, and enrichment of mutations in melanogenesis, p53 and MAPK signaling. Importantly, a recurrent amplification occurring with pre-configured drivers BRAFV600E and p53-/- suggests a novel path of BRAF cooperativity through the protein kinase A pathway.ConclusionThis is the first analysis of a melanoma mutational landscape in the absence of UV light, where tumors manifest with remarkably low mutation burden and high heterogeneity. Genotype specific amplification of protein kinase A in cooperation with BRAF and p53 mutation suggests the involvement of melanogenesis in these tumors. This work is important for defining the spectrum of events in BRAF or NRAS driven melanoma in the absence of UV light, and for informed exploitation of models such as transgenic zebrafish to better understand mechanisms leading to human melanoma formation.

Conserved Expression Signatures between Medaka and Human Pigment Cell Tumors

Aberrations in gene expression are a hallmark of cancer cells. Differential tumor-specific transcript levels of single genes or whole sets of genes may be critical for the neoplastic phenotype and important for therapeutic considerations or useful as biomarkers. As an approach to filter out such relevant expression differences from the plethora of changes noted in global expression profiling studies, we searched for changes of gene expression levels that are conserved. Transcriptomes from massive parallel sequencing of different types of melanoma from medaka were generated and compared to microarray datasets from zebrafish and human melanoma. This revealed molecular conservation at various levels between fish models and human tumors providing a useful strategy for identifying expression signatures strongly associated with disease phenotypes and uncovering new melanoma molecules.

The histone methyltransferase SETDB1 is recurrently amplified in melanoma and accelerates its onset

The most common mutation in melanoma, BRAF(V600E), activates the BRAF serine/threonine kinase and causes excessive MAPK pathway activity1,2. BRAF(V600E)mutations are also present in benign melanocytic nevi3, highlighting the importance of additional genetic alterations in the genesis of malignant tumors. Such changes include recurrent copy number variations that result in the amplification of oncogenes4,5. For certain amplifications, the large number of genes in the interval has precluded an understanding of cooperating oncogenic events. Here, we have used a zebrafish melanoma model to test genes in a recurrently amplified region on chromosome 1 for the ability to cooperate with BRAF(V600E) and accelerate melanoma. SETDB1, an enzyme that methylates histone H3 on lysine 9 (H3K9), was found to significantly accelerate melanoma formation in the zebrafish. Chromatin immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-Seq) and gene expression analyses revealed target genes, including Hox genes, that are transcriptionally dysregulated in response to elevated SETDB1. Our studies establish SETDB1 as an oncogene in melanoma and underscore the role of chromatin factors in regulating tumorigenesis.