Functional analysis of recurrent CDC20 promoter variants in human melanoma.

Small nucleotide variants in non-coding regions of the genome can alter transcriptional regulation, leading to changes in gene expression which can activate oncogenic gene regulatory networks. Melanoma is heavily burdened by non-coding variants, representing over 99% of total genetic variation, including the well-characterized TERT promoter mutation. However, the compendium of regulatory non-coding variants is likely still functionally under-characterized. We developed a pipeline to identify hotspots, i.e. recurrently mutated regions, in melanoma containing putatively functional non-coding somatic variants that are located within predicted melanoma-specific regulatory regions. We identified hundreds of statistically significant hotspots, including the hotspot containing the TERT promoter variants, and focused on a hotspot in the promoter of CDC20. We found that variants in the promoter of CDC20, which putatively disrupt an ETS motif, lead to lower transcriptional activity in reporter assays. Using CRISPR/Cas9, we generated an indel in the CDC20 promoter in human A375 melanoma cell lines and observed decreased expression of CDC20, changes in migration capabilities, increased growth of xenografts, and an altered transcriptional state previously associated with a more proliferative and less migratory state. Overall, our analysis prioritized several recurrent functional non-coding variants that, through downregulation of CDC20, led to perturbation of key melanoma phenotypes.

Transcriptional profile and chromatin accessibility in zebrafish melanocytes and melanoma tumors.

Transcriptional and epigenetic characterization of melanocytes and melanoma cells isolated from their in vivo context promises to unveil key differences between these developmentally related normal and cancer cell populations. We therefore engineered an enhanced Danio rerio (zebrafish) melanoma model with fluorescently labeled melanocytes to allow for isolation of normal (wild type) and premalignant (BRAFV600E-mutant) populations for comparison to fully transformed BRAFV600E-mutant, p53 loss-of-function melanoma cells. Using fluorescence-activated cell sorting to isolate these populations, we performed high-quality RNA- and ATAC-seq on sorted zebrafish melanocytes vs. melanoma cells, which we provide as a resource here. Melanocytes had consistent transcriptional and accessibility profiles, as did melanoma cells. Comparing melanocytes and melanoma, we note 4128 differentially expressed genes and 56,936 differentially accessible regions with overall gene expression profiles analogous to human melanocytes and the pigmentation melanoma subtype. Combining the RNA- and ATAC-seq data surprisingly revealed that increased chromatin accessibility did not always correspond with increased gene expression, suggesting that though there is widespread dysregulation in chromatin accessibility in melanoma, there is a potentially more refined gene expression program driving cancerous melanoma. These data serve as a resource to identify candidate regulators of the normal vs. diseased states in a genetically controlled in vivo context.

Synergistic melanoma cell death mediated by inhibition of both MCL1 and BCL2 in high-risk tumors driven by NF1/PTEN loss.

Melanomas driven by loss of the NF1 tumor suppressor have a high risk of treatment failure and effective therapies have not been developed. Here we show that loss-of-function mutations of nf1 and pten result in aggressive melanomas in zebrafish, representing the first animal model of NF1-mutant melanomas harboring PTEN loss. MEK or PI3K inhibitors show little activity when given alone due to cross-talk between the pathways, and high toxicity when given together. The mTOR inhibitors, sirolimus, everolimus, and temsirolimus, were the most active single agents tested, potently induced tumor-suppressive autophagy, but not apoptosis. Because addition of the BCL2 inhibitor venetoclax resulted in compensatory upregulation of MCL1, we established a three-drug combination composed of sirolimus, venetoclax, and the MCL1 inhibitor S63845. This well-tolerated drug combination potently and synergistically induces apoptosis in both zebrafish and human NF1/PTEN-deficient melanoma cells, providing preclinical evidence justifying an early-stage clinical trial in patients with NF1/PTEN-deficient melanoma.

Functional in vivo characterization of sox10 enhancers in neural crest and melanoma development.

The role of a neural crest developmental transcriptional program, which critically involves Sox10 upregulation, is a key conserved aspect of melanoma initiation in both humans and zebrafish, yet transcriptional regulation of sox10 expression is incompletely understood. Here we used ATAC-Seq analysis of multiple zebrafish melanoma tumors to identify recurrently open chromatin domains as putative melanoma-specific sox10 enhancers. Screening in vivo with EGFP reporter constructs revealed 9 of 11 putative sox10 enhancers with embryonic activity in zebrafish. Focusing on the most active enhancer region in melanoma, we identified a region 23 kilobases upstream of sox10, termed peak5, that drives EGFP reporter expression in a subset of neural crest cells, Kolmer-Agduhr neurons, and early melanoma patches and tumors with high specificity. A ~200 base pair region, conserved in Cyprinidae, within peak5 is required for transgenic reporter activity in neural crest and melanoma. This region contains dimeric SoxE/Sox10 dimeric binding sites essential for peak5 neural crest and melanoma activity. We show that deletion of the endogenous peak5 conserved genomic locus decreases embryonic sox10 expression and disrupts adult stripe patterning in our melanoma model background. Our work demonstrates the power of linking developmental and cancer models to better understand neural crest identity in melanoma.

Isotope tracing in adult zebrafish reveals alanine cycling between melanoma and liver.

The cell-intrinsic nature of tumor metabolism has become increasingly well characterized. The impact that tumors have on systemic metabolism, however, has received less attention. Here, we used adult zebrafish harboring BRAFV600E-driven melanoma to study the effect of cancer on distant tissues. By applying metabolomics and isotope tracing, we found that melanoma consume ~15 times more glucose than other tissues measured. Despite this burden, circulating glucose levels were maintained in disease animals by a tumor-liver alanine cycle. Excretion of glucose-derived alanine from tumors provided a source of carbon for hepatic gluconeogenesis and allowed tumors to remove excess nitrogen from branched-chain amino acid catabolism, which we found to be activated in zebrafish and human melanoma. Pharmacological inhibition of the tumor-liver alanine cycle in zebrafish reduced tumor burden. Our findings underscore the significance of metabolic crosstalk between tumors and distant tissues and establish the adult zebrafish as an attractive model to study such processes.

Melanoma models for the next generation of therapies.

There is a lack of appropriate melanoma models that can be used to evaluate the efficacy of novel therapeutic modalities. Here, we discuss the current state of the art of melanoma models including genetically engineered mouse, patient-derived xenograft, zebrafish, and ex vivo and in vitro models. We also identify five major challenges that can be addressed using such models, including metastasis and tumor dormancy, drug resistance, the melanoma immune response, and the impact of aging and environmental exposures on melanoma progression and drug resistance. Additionally, we discuss the opportunity for building models for rare subtypes of melanomas, which represent an unmet critical need. Finally, we identify key recommendations for melanoma models that may improve accuracy of preclinical testing and predict efficacy in clinical trials, to help usher in the next generation of melanoma therapies.

SATB2 induction of a neural crest mesenchyme-like program drives melanoma invasion and drug resistance.

Recent genomic and scRNA-seq analyses of melanoma demonstrated a lack of recurrent genetic drivers of metastasis, while identifying common transcriptional states correlating with invasion or drug resistance. To test whether transcriptional adaptation can drive melanoma progression, we made use of a zebrafish mitfa:BRAFV600E;tp53-/- model, in which malignant progression is characterized by minimal genetic evolution. We undertook an overexpression-screen of 80 epigenetic/transcriptional regulators and found neural crest-mesenchyme developmental regulator SATB2 to accelerate aggressive melanoma development. Its overexpression induces invadopodia formation and invasion in zebrafish tumors and human melanoma cell lines. SATB2 binds and activates neural crest-regulators, including pdgfab and snai2. The transcriptional program induced by SATB2 overlaps with known MITFlowAXLhigh and AQP1+NGFR1high drug-resistant states and functionally drives enhanced tumor propagation and resistance to Vemurafenib in vivo. In summary, we show that melanoma transcriptional rewiring by SATB2 to a neural crest mesenchyme-like program can drive invasion and drug resistance in autochthonous tumors.

Difference in biophysical properties of cancer-initiating cells in melanoma mutated zebrafish.

Despite sharing oncogenetic mutations, only a small number of cells within a given tissue will undergo malignant transformation. Biochemical and physical factors responsible for this cancer-initiation process are not well understood. Here we study biophysical differences of pre-melanoma and melanoma cells in a BRAFV600E/P53 zebrafish model. The AFM indentation technique was used to study the cancer-initiating cells while the surrounding melanocytes were the control. We observed a statistically significant decrease in the modulus of elasticity (the effective Young's modulus) of cancer-initiating cells compared to the surrounding melanocytes. No significant differences in the pericellular coat surrounding cells were observed. These results contribute to a better understanding of the factors responsible for the initiation of cancer.

Feeding amount significantly alters overt tumor onset rate in a zebrafish melanoma model.

The manner in which zebrafish are fed may have important impacts on the behavior of disease models. We examined the effect of different feeding regimens on the rate of overt melanoma tumor onset in a p53/BRAF-dependent model, a commonly used read-out in this and many other cancer models. We demonstrate that increased feeding leads to more rapid melanoma onset. The ability to modulate overt tumor onset rates with this regimen indicates additional flexibility to 'tune' the system to more quickly generate tumors for study and to begin to address questions related to cancer metabolism using the zebrafish model.

A chemical screen in zebrafish embryonic cells establishes that Akt activation is required for neural crest development.

The neural crest is a dynamic progenitor cell population that arises at the border of neural and non-neural ectoderm. The inductive roles of FGF, Wnt, and BMP at the neural plate border are well established, but the signals required for subsequent neural crest development remain poorly characterized. Here, we conducted a screen in primary zebrafish embryo cultures for chemicals that disrupt neural crest development, as read out by crestin:EGFP expression. We found that the natural product caffeic acid phenethyl ester (CAPE) disrupts neural crest gene expression, migration, and melanocytic differentiation by reducing Sox10 activity. CAPE inhibits FGF-stimulated PI3K/Akt signaling, and neural crest defects in CAPE-treated embryos are suppressed by constitutively active Akt1. Inhibition of Akt activity by constitutively active PTEN similarly decreases crestin expression and Sox10 activity. Our study has identified Akt as a novel intracellular pathway required for neural crest differentiation.

Tumor diversity and evolution revealed through RADseq.

Cancer is an evolutionary disease, and there is increasing interest in applying tools from evolutionary biology to understand cancer progression. Restriction-site associated DNA sequencing (RADseq) was developed for the field of evolutionary genetics to study adaptation and identify evolutionary relationships among populations. Here we apply RADseq to study tumor evolution, which allows for unbiased sampling of any desired frequency of the genome, overcoming the selection bias and cost limitations inherent to exome or whole-genome sequencing. We apply RADseq to both human pancreatic cancer and zebrafish melanoma samples. Using either a low-frequency (SbfI, 0.4% of the genome) or high-frequency (NsiI, 6-9% of the genome) cutter, we successfully identify single nucleotide substitutions and copy number alterations in tumors, which can be augmented by performing RADseq on sublineages within the tumor. We are able to infer phylogenetic relationships between primary tumors and metastases. These same methods can be used to identify somatic mosaicism in seemingly normal, non-cancerous tissues. Evolutionary studies of cancer that focus on rates of tumor evolution and evolutionary relationships among tumor lineages will benefit from the flexibility and efficiency of restriction-site associated DNA sequencing.

Efficient Transduction of Zebrafish Melanoma Cell Lines and Embryos Using Lentiviral Vectors.

The establishment of in vitro cultures of zebrafish cancer cells has expanded the potential of zebrafish as a disease model. However, the lack of effective methods for gene delivery and genetic manipulation has limited the experimental applications of these cultures. To overcome this barrier, we tested and optimized vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral and retroviral vector transduction protocols. We show that lentivirus successfully and efficiently transduced zebrafish melanoma cell lines in vitro, allowing antibiotic selection, fluorescence-based sorting, and in vivo allotransplantation. In addition, injection of concentrated lentiviral particles into embryos and tumors established the feasibility of in vivo gene delivery.

Zebrafish Melanoma.

Melanoma skin cancer is a potentially deadly disease in humans and has remained extremely difficult to treat once it has metastasized. In just the last 10 years, a number of models of melanoma have been developed in the zebrafish that are biologically faithful to the human disease and have already yielded important insights into the fundamental biology of melanoma and offered new potential avenues for treatment. With the diversity and breadth of the molecular genetic tools available in the zebrafish, these melanoma models will continue to be refined and expanded upon to keep pace with the rapidly evolving field of melanoma biology.

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.

Ubiquitous transgene expression and Cre-based recombination driven by the ubiquitin promoter in zebrafish.

Molecular genetics approaches in zebrafish research are hampered by the lack of a ubiquitous transgene driver element that is active at all developmental stages. Here, we report the isolation and characterization of the zebrafish ubiquitin (ubi) promoter, which drives constitutive transgene expression during all developmental stages and analyzed adult organs. Notably, ubi expresses in all blood cell lineages, and we demonstrate the application of ubi-driven fluorophore transgenics in hematopoietic transplantation experiments to assess true multilineage potential of engrafted cells. We further generated transgenic zebrafish that express ubiquitous 4-hydroxytamoxifen-controlled Cre recombinase activity from a ubi:cre(ERt2) transgene, as well as ubi:loxP-EGFP-loxP-mCherry (ubi:Switch) transgenics and show their use as a constitutive fluorescent lineage tracing reagent. The ubi promoter and the transgenic lines presented here thus provide a broad resource and important advancement for transgenic applications in zebrafish.

GATA-3: an unexpected regulator of cell lineage determination in skin.

Multipotent skin stem cells give rise to epidermis and its appendages, including the hair follicle. The Lef-1/Tcf family of Wnt-regulated transcription factors plays a major role in specification of the hair shaft, but little is known about how the equally important hair channel, the inner root sheath (IRS), develops in concert to shape and guide the hair. In a microarray screen to search for transcriptional regulators of hair follicle morphogenesis, we identified GATA-3, a key regulator of T-cell lineage determination. Surprisingly, this transcription factor is essential for stem cell lineage determination in skin, where it is expressed at the onset of epidermal stratification and IRS specification in follicles. GATA-3-null/lacZ knock-in embryos can survive up to embryonic day 18.5 (E18.5), when they fail to form the IRS. Skin grafting unveiled additional defects in GATA-3-null hairs and follicles. IRS progenitors failed to differentiate, whereas cortical progenitors differentiated, but produced an aberrant hair structure. Curiously, some GATA-3-null progenitor cells expressed mixed IRS and hair shaft markers. Taken together, these findings place GATA-3 with Lef-1/Wnts at the crossroads of the IRS versus hair shaft cell fate decision in hair follicle morphogenesis. This newfound function for GATA-3 in skin development strengthens the parallels between the differentiation programs governing hair follicle and lymphocyte differentiation.