Solid tumors, especially those with aberrant MYCN activation, often harbor an immunosuppressive microenvironment to fuel malignant growth and trigger treatment resistance. Despite this knowledge, there are no effective strategies to tackle this problem. We found that chemokine-like factor ( CKLF ) is highly expressed by various solid tumor cells and transcriptionally up-regulated by MYCN. Using the MYCN-driven high-risk neuroblastoma as a model system, we demonstrated that as early as the premalignant stage, tumor cells secrete CKLF to attract CCR4-expressing CD4 + cells, inducing immunosuppression and tumor aggression. Genetic depletion of CD4 + T regulatory cells abolishes the immunorestrictive and protumorigenic effects of CKLF. Our work supports that disrupting CKLF-mediated cross-talk between tumor and CD4 + suppressor cells represents a promising immunotherapeutic approach to battling MYCN-driven tumors.

Abstract Neuroblastoma, especially those with aberrant MYCN activation, often harbor an immunosuppressive microenvironment to fuel malignant growth and trigger treatment resistance. Despite this knowledge, there are no effective strategies to tackle this problem. Here we combined analyses of human neuroblastoma with live tracking and functional studies of the tumor microenvironment (TME) in zebrafish. We identified chemokine-like factor (CKLF) as a key driver of MYCN-mediated immunosuppression and neuroblastoma aggression. We showed that human MYCN-activated neuroblastoma upregulates and secretes CKLF, which is a reliable predictor of poor patient survival. Analysis of primary patient samples demonstrates a strong positive association between MYCN and CKLF expression in neuroblastoma cells together with the enrichment of FOXP3+ T cells in the TME. Taking advantage of in vivo zebrafish models of MYCN-driven neuroblastoma that resemble human high-risk disease, we demonstrated that cklf overexpression in zebrafish neural crest cells induces an immunosuppressive TME while promoting rapid tumor onset and progression. We also demonstrated that as early as the premalignant stage, tumor cells secrete CKLF to attract CCR4-expressing Cd4+ cells to induce immunosuppression and tumor aggression. Conversely, genetic depletion of cklf in tumor cells reduces the recruitment of Cd4+ cells to the TME while increasing cytotoxic Cd8+ and natural killer cells infiltration, inhibiting neuroblastoma development in zebrafish. Our work provides the first example that MYCN can activate CKLF to allure CD4+ immune cells to the TME and incite immunosuppression, positioning CKLF as a potential novel immunotherapeutic target for the treatment of MYCN-driven high-risk neuroblastoma. Citation Format: Xiaodan Qin, Hui Feng, Andrew Lam, Xu Zhang, Satyaki Sengupta, Bryan Iorgulescu, Sanjukta Das, Zhenwei Zhou, Tao Zuo, Grace Meara, Madison Rager, Alexander Floru, Hongru Ni, Chinyere Kemet, Divya Veerapaneni, Daniel Kashy, Liang Lin, Kenneth Lloyd, Lauren Kwok, Kaylee Smith, Raghavendar Nagaraju, Rob Meijers, Craig Ceol, Ching-Ti Liu, Sanda Alexandrescu, Catherine J. Wu, Derin Keskin, Rani George. CKLF attracts CCR4-expressing CD4+ cells to foster immune repression and tumor aggressiveness in MYCN-driven neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C002.

Polycystic kidney disease (PKD) is an important cause of kidney failure, but treatment options are limited. While later stages of the disease have been extensively studied, mechanisms driving the initial conversion of kidney tubules into cysts are not understood. To identify genes with the potential to promote cyst initiation, we deleted polycystin-2 (Pkd2) in mice and surveyed transcriptional changes before and immediately after cysts developed. We identified 74 genes which we term cyst initiation candidates (CICs). To identify conserved changes with relevance to human disease we compared these murine CICs to single cell transcriptomic data derived from patients with PKD and from healthy controls. Tumor-associated calcium signal transducer 2 (Tacstd2) stood out as an epithelial-expressed gene with elevated levels early in cystic transformation that further increased with disease progression. Human tissue biopsies and organoids show that TACSTD2 protein is low in normal kidney cells but is elevated in cyst lining cells, making it an excellent candidate for mechanistic exploration of its role in cyst initiation. While TACSTD2 has not been studied in PKD, it has been studied in cancer where it is highly expressed in solid tumors while showing minimal expression in normal tissue. This property is being exploited by antibody drug conjugates that target TACSTD2 for the delivery of cytotoxic drugs. Our finding that Tacstd2/TACSTD2 is prevalent in cysts, but not normal tissue, suggests that it should be explored as a candidate for drug development in PKD. More immediately, our work suggests that PKD patients undergoing TACSTD2-directed treatment for breast and urothelial cancer should be monitored for kidney effects.

<p>This file contains Supplementary Figures S1-S9 with accompanying legends. Supplementary Fig. S1 examines the relationship between KIT expression and BRAFV600E mutations in TCGA human melanoma samples. Supplementary Fig. S2 shows the rescue of melanocytes in kit(lf)-mutant zebrafish by BRAFV600E. Supplementary Fig. S3 shows tumor onset for an additional kit loss-of-function allele as well as tumor invasion images representative of a mosaic analysis. Supplementary Fig. S4 portrays the levels of Mitfa and pAkt in zebrafish melanomas. Supplementary Fig. S5 shows RAF dimerization and signaling experiments performed with V-BRAFV600E stimulated by NRASQ61K. Supplementary Fig. S6 shows RAF dimerization and signaling experiments performed with V-BRAFWT stimulated by KIT and SCF. Supplemental Fig. S7 shows results of experiments overexpressing KIT in A375 and UACC257 human melanoma cells with accompanying growth and signaling changes. Supplementary Fig. S8 shows survival curves for TCGA patients with melanomas expressing high or low KIT. Lastly, Supplementary Fig. S9 displays a measurement of BRAFV600E levels in zebrafish melanomas overexpressing BRAFWT.</p>

<p>Supplementary Table S1 and S2 show the BRET50 data for Supplementary Fig. S5A and S6B, respectively. Supplementary Table S3 and S4 show further analyses of the relationship between KIT expression and several clinical parameters of melanoma progression or tumor suppressor gene alteration.</p>

Abstract Neuroblastoma, especially those with aberrant MYCN activation, often harbor an immunosuppressive microenvironment to fuel malignant growth and trigger treatment resistance. Despite this knowledge, there are no effective strategies to tackle this problem. Here we combined analyses of human neuroblastoma with live tracking and functional studies of the tumor microenvironment (TME) in zebrafish. We identified a tumor-associated chemokine (TAC) as a key driver of MYCN-mediated immunosuppression and neuroblastoma aggression. Taking advantage of in vivo zebrafish models of MYCN-driven neuroblastoma that resemble human high-risk disease, we demonstrated that TAC overexpression in zebrafish neural crest cells induces an immunosuppressive TME while promoting rapid tumor onset and progression. We also demonstrated that as early as the premalignant stage, tumor cells secrete TAC to attract CCR4-expressing Cd4+ cells to induce immunosuppression and tumor aggression. In addition, genetically depleting Cd4+ T regulatory cells abolishes the immunorestrictive and pro-tumorigenic effects of TAC. We showed that human MYCN-activated neuroblastoma upregulates and secretes TAC, which is a reliable predictor of poor patient survival. Analysis of primary patient samples demonstrates a strong positive association between MYCN and TAC expression in neuroblastoma cells together with the enrichment of FOXP3+ T cells in the TME. Our work provides the first example that MYCN can activate TAC to allure CD4+ immune cells to the TME and incite immunosuppression, positioning TAC as a potential novel immunotherapeutic target for the treatment of MYCN-driven high-risk neuroblastoma. Citation Format: Xiaodan Qin, Andrew Lam, Xu Zhang, Satyaki Sengupta, J. Bryan Iorgulescu, Sanjukta Das, Zhenwei Zhou, Tao Zuo, Grace K. Meara, Madison Rager, Alexander E. Floru, Hongru Ni, Chinyere Kemet, Divya Veerapaneni, Daniel Kashy, Liang Lin, Kenneth Lloyd, Lauren Kwok, Kaylee S. Smith, Raghavendar T. Nagaraju, Rob Meijers, Craig Ceol, Ching-Ti Liu, Sanda Alexandrescu, Catherine J. Wu, Derin B. Keskin, Rani E. George, Hui Feng. A tumor-associated chemokine attracts CCR4-expressing CD4+ cells to foster immune repression and tumor aggressiveness in MYCN-driven neuroblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C20.

Summary: GABA signaling by melanoma cells was found by Tagore and colleagues to trigger keratinocyte-driven growth of melanomas. This study reveals new roles for nonneuronal signaling by a neurotransmitter in regulating tumor initiation and outgrowth. See related article by Tagore et al., p. 2270 (4).

Tissue-resident stem and progenitor cells are present in many adult organs, where they are important for organ homeostasis and repair in response to injury. However, the signals that activate these cells and the mechanisms governing how these cells renew or differentiate are highly context-dependent and incompletely understood, particularly in non-hematopoietic tissues. In the skin, melanocyte stem and progenitor cells are responsible for replenishing mature pigmented melanocytes. In mammals, these cells reside in the hair follicle bulge and bulb niches where they are activated during homeostatic hair follicle turnover and following melanocyte destruction, as occurs in vitiligo and other skin hypopigmentation disorders. Recently, we identified melanocyte progenitors in adult zebrafish skin. To elucidate mechanisms governing melanocyte progenitor renewal and differentiation we analyzed individual transcriptomes from thousands of melanocyte lineage cells during the regeneration process. We identified transcriptional signatures for progenitors, deciphered transcriptional changes and intermediate cell states during regeneration, and analyzed cell–cell signaling changes to discover mechanisms governing melanocyte regeneration. We identified KIT signaling via the RAS/MAPK pathway as a regulator of melanocyte progenitor direct differentiation and asymmetric division. Our findings show how activation of different subpopulations of mitfa -positive cells underlies cellular transitions required to properly reconstitute the melanocyte pigmentary system following injury.

<p>This file contains Supplementary Figures S1-S9 with accompanying legends. Supplementary Fig. S1 examines the relationship between KIT expression and BRAFV600E mutations in TCGA human melanoma samples. Supplementary Fig. S2 shows the rescue of melanocytes in kit(lf)-mutant zebrafish by BRAFV600E. Supplementary Fig. S3 shows tumor onset for an additional kit loss-of-function allele as well as tumor invasion images representative of a mosaic analysis. Supplementary Fig. S4 portrays the levels of Mitfa and pAkt in zebrafish melanomas. Supplementary Fig. S5 shows RAF dimerization and signaling experiments performed with V-BRAFV600E stimulated by NRASQ61K. Supplementary Fig. S6 shows RAF dimerization and signaling experiments performed with V-BRAFWT stimulated by KIT and SCF. Supplemental Fig. S7 shows results of experiments overexpressing KIT in A375 and UACC257 human melanoma cells with accompanying growth and signaling changes. Supplementary Fig. S8 shows survival curves for TCGA patients with melanomas expressing high or low KIT. Lastly, Supplementary Fig. S9 displays a measurement of BRAFV600E levels in zebrafish melanomas overexpressing BRAFWT.</p>

This file contains Supplementary Figures S1-S9 with accompanying legends. Supplementary Fig. S1 examines the relationship between KIT expression and BRAFV600E mutations in TCGA human melanoma samples. Supplementary Fig. S2 shows the rescue of melanocytes in kit(lf)-mutant zebrafish by BRAFV600E. Supplementary Fig. S3 shows tumor onset for an additional kit loss-of-function allele as well as tumor invasion images representative of a mosaic analysis. Supplementary Fig. S4 portrays the levels of Mitfa and pAkt in zebrafish melanomas. Supplementary Fig. S5 shows RAF dimerization and signaling experiments performed with V-BRAFV600E stimulated by NRASQ61K. Supplementary Fig. S6 shows RAF dimerization and signaling experiments performed with V-BRAFWT stimulated by KIT and SCF. Supplemental Fig. S7 shows results of experiments overexpressing KIT in A375 and UACC257 human melanoma cells with accompanying growth and signaling changes. Supplementary Fig. S8 shows survival curves for TCGA patients with melanomas expressing high or low KIT. Lastly, Supplementary Fig. S9 displays a measurement of BRAFV600E levels in zebrafish melanomas overexpressing BRAFWT.

Dysregulated cellular metabolism is a cancer hallmark for which few druggable oncoprotein targets have been identified. Increased fatty acid (FA) acquisition allows cancer cells to meet their heightened membrane biogenesis, bioenergy, and signaling needs. Excess FAs are toxic to non-transformed cells but surprisingly not to cancer cells. Molecules underlying this cancer adaptation may provide alternative drug targets. Here, we demonstrate that diacylglycerol O-acyltransferase 1 (DGAT1), an enzyme integral to triacylglyceride synthesis and lipid droplet formation, is frequently up-regulated in melanoma, allowing melanoma cells to tolerate excess FA. DGAT1 over-expression alone transforms p53-mutant zebrafish melanocytes and co-operates with oncogenic BRAF or NRAS for more rapid melanoma formation. Antagonism of DGAT1 induces oxidative stress in melanoma cells, which adapt by up-regulating cellular reactive oxygen species defenses. We show that inhibiting both DGAT1 and superoxide dismutase 1 profoundly suppress tumor growth through eliciting intolerable oxidative stress.

Melanoma is commonly driven by activating mutations in the MAP kinase BRAF; however, oncogenic BRAF alone is insufficient to promote melanomagenesis. Instead, its expression induces a transient proliferative burst that ultimately ceases with the development of benign nevi comprised of growth-arrested melanocytes. The tumor suppressive mechanisms that restrain nevus melanocyte proliferation remain poorly understood. Here we utilize cell and murine models to demonstrate that oncogenic BRAF leads to activation of the Hippo tumor suppressor pathway, both in melanocytes in vitro and nevus melanocytes in vivo. Mechanistically, we show that oncogenic BRAF promotes both ERK-dependent alterations in the actin cytoskeleton and whole-genome doubling events, which independently reduce RhoA activity to promote Hippo activation. We also demonstrate that functional impairment of the Hippo pathway enables oncogenic BRAF -expressing melanocytes to bypass nevus formation and rapidly form melanomas. Our data reveal that the Hippo pathway enforces the stable arrest of nevus melanocytes and represents a critical barrier to melanoma development.

Melanomas and other solid tumors commonly have increased ploidy, with near-tetraploid karyotypes being most frequently observed. Such karyotypes have been shown to arise through whole-genome doubling events that occur during early stages of tumor progression. The generation of tetraploid cells via whole-genome doubling is proposed to allow nascent tumor cells the ability to sample various pro-tumorigenic genomic configurations while avoiding the negative consequences that chromosomal gains or losses have in diploid cells. Whereas a high prevalence of whole-genome doubling events has been established, the means by which whole-genome doubling arises is unclear. Here, we find that BRAF V600E , the most common mutation in melanomas, can induce whole-genome doubling via cytokinesis failure in vitro and in a zebrafish melanoma model. Mechanistically, BRAF V600E causes decreased activation and localization of RhoA, a critical cytokinesis regulator. BRAF V600E activity during G1/S phases of the cell cycle is required to suppress cytokinesis. During G1/S, BRAF V600E activity causes inappropriate centriole amplification, which is linked in part to inhibition of RhoA and suppression of cytokinesis. Together these data suggest that common abnormalities of melanomas linked to tumorigenesis – amplified centrosomes and whole-genome doubling events – can be induced by oncogenic BRAF and other mutations that increase RAS/MAPK pathway activity.

Solid tumors, especially those with aberrant MYCN activation, harbor an immunosuppressive microenvironment to fuel malignant growth and trigger treatment resistance1,2, yet the underlying mechanisms are elusive and effective strategies to tackle this challenge are lacking. Here we demonstrated the crucial role of T regulatory (Treg) cells in MYCN-mediated immune repression and tumor aggression using high-risk neuroblastoma (NB) as a model system. Human MYCN-activated NB attracts CD4+ Treg cells, which are also found enriched in MYCN-high primary patient samples. Zebrafish MYCN-overexpressing neural crests recruit Cd4+ cells before tumor formation and induce an immunosuppressive microenvironment, thereby promoting tumor onset and progression. Strikingly, disruption of Treg cells through depletion of forkhead box protein 3a restores anti-tumor immunity and impairs NB development. Together, our studies establish Treg cells as the key driver of MYCN-mediated immunosuppression and tumor aggressiveness, providing mechanistic insights and therapeutic implications.

Skin diseases affect nearly one third of the world's population. Disease types range from oncologic to inflammatory, and outcomes can be as severe as death and disfigurement. Although many skin diseases have been modeled in murine models, the advantages of zebrafish models have led to recent increasing use in modeling human disease. Their rapid development, comparable skin architecture, tractable genetics, unparalleled optical properties, and straightforward drug screens make them an excellent model to study skin disease. In this review, we discuss the attributes of the zebrafish model system as well as current zebrafish models for dermatologic diseases, including melanoma, squamous cell carcinoma, vitiligo, epidermal bullosa, psoriasis, and wounding.

Differentiation status has been shown to be important at various stages in melanoma development. Acquisition of a less differentiated or neural crest–like state in melanomas is associated with tumor initiation (Kaufman et al., 2016Kaufman C.K. Mosimann C. Fan Z.P. Yang S. Thomas A.J. Ablain J. et al.A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation.Science. 2016; 351: aad2197Crossref PubMed Scopus (220) Google Scholar), progression and metastasis (Carreira et al., 2006Carreira S. Goodall J. Denat L. Rodriguez M. Nuciforo P. Hoek K.S. et al.Mitf regulation of Dia1 controls melanoma proliferation and invasiveness.Genes Dev. 2006; 20: 3426-3439Crossref PubMed Scopus (424) Google Scholar; Gupta et al., 2005Gupta P.B. Kuperwasser C. Brunet J.P. Ramaswamy S. Kuo W.L. Gray J.W. et al.The melanocyte differentiation program predisposes to metastasis after neoplastic transformation.Nat Genet. 2005; 37: 1047-1054Crossref PubMed Scopus (362) Google Scholar), and adaptive resistance to standard-of-care therapies (Fallahi-Sichani et al., 2017Fallahi-Sichani M. Becker V. Izar B. Baker G.J. Lin J.R. Boswell S.A. et al.Adaptive resistance of melanoma cells to RAF inhibition via reversible induction of a slowly dividing de-differentiated state.Mol Syst Biol. 2017; 13: 905Crossref PubMed Scopus (123) Google Scholar; Mehta et al., 2018Mehta A. Kim Y.J. Robert L. Tsoi J. Comin-Anduix B. Berent-Maoz B. et al.Immunotherapy resistance by inflammation-induced dedifferentiation.Cancer Discov. 2018; 8: 935-943Crossref PubMed Scopus (78) Google Scholar; Sun et al., 2014Sun C. Wang L. Huang S. Heynen G.J. Prahallad A. Robert C. et al.Reversible and adaptive resistance to BRAF(V600E) inhibition in melanoma.Nature. 2014; 508: 118-122Crossref PubMed Scopus (541) Google Scholar). Currently, little is known about specific factors and pathways that act to initially promote neural crest identity during tumor initiation. Recently, GDF6-activated BMP signaling was shown to regulate differentiation and survival both in melanomas and during melanocyte development (Gramann et al., 2019Gramann A.K. Venkatesan A.M. Guerin M. Ceol C.J. Regulation of zebrafish melanocyte development by ligand-dependent BMP signaling.Elife. 2019; 8: e50047Crossref PubMed Scopus (10) Google Scholar; Venkatesan et al., 2018Venkatesan A.M. Vyas R. Gramann A.K. Dresser K. Gujja S. Bhatnagar S. et al.Ligand-activated BMP signaling inhibits cell differentiation and death to promote melanoma.J Clin Invest. 2018; 128: 294-308Crossref PubMed Scopus (27) Google Scholar). Together, these results have implicated BMP signaling as a key regulator of neural crest and melanocyte programs in normal and pathologic states of the melanocyte lineage. However, it is unknown whether BMP signaling plays a role early in melanoma initiation and progression. It was reported previously that adult differentiated melanocytes do not have active BMP signaling, whereas 80% of melanomas have active BMP signaling (Gramann et al., 2019Gramann A.K. Venkatesan A.M. Guerin M. Ceol C.J. Regulation of zebrafish melanocyte development by ligand-dependent BMP signaling.Elife. 2019; 8: e50047Crossref PubMed Scopus (10) Google Scholar; Venkatesan et al., 2018Venkatesan A.M. Vyas R. Gramann A.K. Dresser K. Gujja S. Bhatnagar S. et al.Ligand-activated BMP signaling inhibits cell differentiation and death to promote melanoma.J Clin Invest. 2018; 128: 294-308Crossref PubMed Scopus (27) Google Scholar). Here, we analyzed only primary melanomas for the presence of phosphorylated SMAD (pSMAD)-1/5/8, a marker of canonical BMP activity. Clinical data associated with these samples is included in Supplementary Table S1. We observed that 65.4% of primary melanoma samples stained positive for pSMAD (Figure 1a). This indicates that BMP signaling, despite being inactive in adult differentiated melanocytes, is active in a large proportion of primary melanomas. To further evaluate the potential for a functional role of BMP signaling during melanoma initiation, we employed two complementary approaches using zebrafish: the MiniCoopR system (Ceol et al., 2011Ceol C.J. Houvras Y. Jane-Valbuena J. Bilodeau S. Orlando D.A. Battisti V. et al.The histone methyltransferase SETDB1 is recurrently amplified in melanoma and accelerates its onset.Nature. 2011; 471: 513-517Crossref PubMed Scopus (332) Google Scholar), which allows mosaic expression of a target gene of interest specifically within melanocytes in the tumorigenic strain Tg(mitfa:BRAF-V600E);p53(lf);mitfa(lf), and the Tg(crestin:eGFP) neural crest reporter system. crestin:eGFP expression was previously shown to be absent in wild-type adult zebrafish but expressed in single cells in the skin of Tg(mitfa:BRAFV600E);p53(lf) adults, some of which were likely melanoma-initiating cells (Kaufman et al., 2016Kaufman C.K. Mosimann C. Fan Z.P. Yang S. Thomas A.J. Ablain J. et al.A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation.Science. 2016; 351: aad2197Crossref PubMed Scopus (220) Google Scholar). We constructed a melanocyte reporter MiniCoopR vector, mitfa:nls-mCherry, to identify melanocyte-lineage cells. We bred Tg(mitfa:BRAF-V600E);p53(lf);mitfa(lf);Tg(crestin:eGFP) zebrafish; injected individual MiniCoopR vectors into embryos at the single-cell stage; and monitored for development of crestin:eGFP-positive cells and lesions, which were previously shown to give rise to melanomas (Figure 1b) (Kaufman et al., 2016Kaufman C.K. Mosimann C. Fan Z.P. Yang S. Thomas A.J. Ablain J. et al.A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation.Science. 2016; 351: aad2197Crossref PubMed Scopus (220) Google Scholar). In initial experiments, we observed eGFP signal only in nls-mCherry–positive cells, indicating specificity of crestin:eGFP for the melanocyte lineage. Just 8.2% of mitfa:nls-mCherry–positive cells were also crestin:eGFP-positive (Figure 1c), indicating that a small subset of melanocyte-lineage cells were competent to form tumors. Of these crestin:eGFP-positive cells, a majority were pigmented (Supplementary Figure S1). Because nascent melanomas in the zebrafish model are generally unpigmented, a pigmented cell of origin would presumably undergo dedifferentiation in its progression to a nascent tumor. Alternatively, it is possible that unpigmented crestin-positive cells are cells of origin and possess a less differentiated state even before their progression. To determine whether BMP signaling was active in crestin:eGFP-positive single cells and nascent lesions that were in the earliest stages of expansion into tumors (∼10–20 cells per lesion), we plucked scales from MiniCoopR-injected animals to isolate eGFP-positive lesions for immunofluorescence and stained with the pSMAD antibody to detect BMP signaling activity. We found that 4 of 4 single crestin:eGFP-positive cells were pSMAD-positive and 6 of 6 crestin:eGFP-positive lesions also contained pSMAD-positive cells. Of crestin:eGFP-positive cells within lesions, 90.9% were also pSMAD-positive (Figure 1d), indicating that BMP signaling was active in early melanoma lesions in zebrafish, similar to our findings within human primary melanomas. pSMAD staining was not observed in crestin:eGFP-negative cells. Together, these data show a strong association between BMP activity and neural crest identity, as read out by crestin:eGFP expression. Because the presence of BMP activity does not necessarily indicate a functional role within eGFP-positive cells, we sought to modulate BMP signaling and assess any changes in the development of eGFP-positive lesions. Using MiniCoopR, we expressed a dominant-negative BMP receptor (dnBMPR) that was previously shown to suppress BMP signaling in zebrafish (Pyati et al., 2005Pyati U.J. Webb A.E. Kimelman D. Transgenic zebrafish reveal stage-specific roles for Bmp signaling in ventral and posterior mesoderm development.Development. 2005; 132: 2333-2343Crossref PubMed Scopus (116) Google Scholar) and a phosphomimetic of SMAD1 (SMAD1-DVD) that provides constitutive intracellular BMP signaling activity (Nojima et al., 2010Nojima J. Kanomata K. Takada Y. Fukuda T. Kokabu S. Ohte S. et al.Dual roles of smad proteins in the conversion from myoblasts to osteoblastic cells by bone morphogenetic proteins.J Biol Chem. 2010; 285: 15577-15586Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) to modulate BMP activity (Supplementary Figure S2). In zebrafish expressing dnBMPR, we observed fewer crestin:eGFP-positive lesions than in controls, whereas in zebrafish expressing SMAD1-DVD, we observed a greater number of crestin:eGFP-positive lesions (Figure 2a and b). These results indicate that BMP signaling is capable of altering development of early melanoma lesions, which suggests that the BMP pathway impacts acquisition of neural crest identity during melanoma initiation. Given these results, we hypothesized that BMP signaling may generally have a role in disease onset. We monitored our zebrafish expressing dnBMPR and SMAD1-DVD for development of bona fide melanomas. We observed a delay in onset in the dnBMPR group, with median tumor-free survival of approximately 26 weeks, compared with controls, with median tumor-free survival of 17 weeks. In the SMAD1-DVD group, we observed an acceleration in tumor onset, with median tumor-free survival of 12 weeks (Figure 2c). Thus, the ability of BMP signaling to promote development of early melanoma lesions is likely reflected by the accelerated onset of macroscopically visible tumors. Our studies have demonstrated the presence of active BMP signaling in human primary melanomas and identified a role for BMP signaling during melanoma initiation in zebrafish. Our previous studies identified a role for GDF6-activated BMP signaling during melanocyte development and within established melanoma cells (Venkatesan et al., 2018Venkatesan A.M. Vyas R. Gramann A.K. Dresser K. Gujja S. Bhatnagar S. et al.Ligand-activated BMP signaling inhibits cell differentiation and death to promote melanoma.J Clin Invest. 2018; 128: 294-308Crossref PubMed Scopus (27) Google Scholar). In both situations, BMP signaling functions to promote expression of neural crest genes and suppress expression of melanocyte differentiation genes. Inhibition of BMP signaling causes decreased expression of specific neural crest genes and increased expression of melanocyte differentiation factors, which, during development, leads to an increase in the number of embryonic melanocytes and, in tumors, leads to differentiation of melanoma cells (Gramann et al., 2019Gramann A.K. Venkatesan A.M. Guerin M. Ceol C.J. Regulation of zebrafish melanocyte development by ligand-dependent BMP signaling.Elife. 2019; 8: e50047Crossref PubMed Scopus (10) Google Scholar; Venkatesan et al., 2018Venkatesan A.M. Vyas R. Gramann A.K. Dresser K. Gujja S. Bhatnagar S. et al.Ligand-activated BMP signaling inhibits cell differentiation and death to promote melanoma.J Clin Invest. 2018; 128: 294-308Crossref PubMed Scopus (27) Google Scholar). Results from this study suggest an analogous mechanism whereby BMP signaling acts to put melanocyte-lineage cells in a more neural crest–like state to facilitate melanoma initiation. The question of differentiation state and cell of origin during melanoma initiation is still debated within the field, with data supporting multiple mechanisms through different model systems (Köhler et al., 2017Köhler C. Nittner D. Rambow F. Radaelli E. Stanchi F. Vandamme N. et al.Mouse cutaneous melanoma induced by mutant BRaf arises from expansion and dedifferentiation of mature pigmented melanocytes.Cell Stem Cell. 2017; 21: 679-693.e6Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar; Moon et al., 2017Moon H. Donahue L.R. Choi E. Scumpia P.O. Lowry W.E. Grenier J.K. et al.Melanocyte stem cell activation and translocation initiate cutaneous melanoma in response to UV exposure.Cell Stem Cell. 2017; 21: 665-678.e6Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Here, we show in our crestin:eGFP reporter system that melanocyte-lineage cells in adult zebrafish that re-express crestin:eGFP are more likely to be pigmented than unpigmented. We suspect that this may be due to temporal overlap between initial crestin:eGFP expression and degradation of pigment following downregulation of pigmentation genes during dedifferentiation. This would support the hypothesis that melanomas arise from differentiated melanocytes that must undergo dedifferentiation to a more neural crest–like state before becoming a melanoma. However, it is possible that although pigmented cells more frequently re-express crestin:eGFP, it is the unpigmented crestin:eGFP-positive cells that develop into melanomas because they are already in a less differentiated state. Further investigation is needed to characterize the nature of the cell of origin of melanomas. The presence and functional role of BMP signaling in promoting early melanoma-initiating lesions indicates potential value in diagnosis, prognosis, and therapy for patients with melanoma. All zebrafish study protocols were approved by the Institutional Animal Care and Use Committee at University of Massachusetts Medical School (Worcester, MA). Approval for the tissue microarray study was granted by the Brigham and Women’s Partners Human Research Committee. Informed consent was not necessary, as all tissue samples were discardable and deidentified. The data supporting the findings of this study are included in this published article. Alec K. Gramann: http://orcid.org/0000-0001-7527-5533 William Tyler Frantz: http://orcid.org/0000-0003-1207-9652 Karen Dresser: http://orcid.org/0000-0002-8599-4352 Camilla Borges Ferreira Gomes: http://orcid.org/0000-0003-0967-7600 Christine G. Lian: http://orcid.org/0000-0003-4626-1612 April Deng: http://orcid.org/0000-0002-1373-7613 Craig J. Ceol: http://orcid.org/0000-0002-7188-7580 The authors state no conflicts of interest. AKG is supported by the Melanoma Research Foundation Medical Student Award, a University of Massachusetts Medical School (Worcester, MA) Center for Clinical and Translational Science TL1 Fellowship (UL1-TR001453), and the National Cancer Institute National Research Service Award (1F31CA239478-01). WTF is supported by National Institutes of Health T32 (CA130807). CJC is supported by the Kimmel Scholar Award (SKF-13-123); Department of Defense Peer Reviewed Cancer Research Program (W8IXWH-13-0107); and the National Institutes of Health, National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01AR063850). The content is solely the responsibility of the authors and does not necessarily represent the official views of the Department of Defense or National Institutes of Health. Conceptualization: AKG, CJC; Data Curation: AKG, WTF, KD, CBFG, CGL, AD; Formal Analysis: AKG, CJC, CGL; Funding Acquisition: AKG, CJC; Investigation: AKG, WTF, CGL, CJC; Methodology: AKG, CJC; Resources: CGL, CJC; Writing - Original Draft Preparation: AKG, CJC, WTF, AD; Writing - Review and Editing: AKG, CJC Download .xlsx (.02 MB) Help with xlsx files Supplemental Table 1 For patient samples stained for phosphorylated SMAD, a modified visual semiquantitative method was used. Sections, as described previously (Lian et al., 2012Lian C.G. Xu L. Ceol C. Wu F. Larson A. Dresser K. et al.Loss of 5-hydroxymethylcytosine is an epigenetic hallmark of melanoma.Cell. 2012; 150: 1135-1146Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar), were scored for immunointensity (0–4) and immunopositivity (0–3), which were then multiplied. Scoring was conducted independently by CGL and CBFG, and the scores were averaged. Sections with scores <2 were binned into the negative group, and sections with scores ≥2 were binned into the positive staining group. MiniCoopR constructs Pmitfa:dnBMPR:pA, Pmitfa:SMAD1-DVD:pA, and Pmitfa:nls-mCherry:pA (control) were used in the MiniCoopR assay as previously described (Ceol et al., 2011Ceol C.J. Houvras Y. Jane-Valbuena J. Bilodeau S. Orlando D.A. Battisti V. et al.The histone methyltransferase SETDB1 is recurrently amplified in melanoma and accelerates its onset.Nature. 2011; 471: 513-517Crossref PubMed Scopus (370) Google Scholar). In brief, Tg(Pmitfa:BRAFV600E); p53(lf); mitfa(lf); Tg(Pcrestin:eGFP) animals were bred and embryos were harvested. Single-cell stage embryos were then injected with 25 pg of a single construct and 25 pg of Tol2 transposase RNA to integrate within the genome. On successful integration of the MiniCoopR constructs, the mitfa minigene in a construct rescued the mitfa(lf) mutation and allowed development of melanocytes. Embryos were screened for incorporation of the transgene by rescue of melanocytes at 5 days after fertilization. Animals were then grown and monitored for the onset of crestin:eGFP-positive cells and lesions and for the development of macroscopic melanomas.Supplementary Figure S2Validation of dnBMPR and SMAD1-DVD modulation of BMP signaling. RNA was harvested from zebrafish expressing dnBMPR or SMAD1-DVD and qPCR was performed for id3 expression. n = 3. Error bars represent mean ± SEM. P-values by one-way ANOVA with Dunnet’s multiple comparison correction. ∗∗∗P < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

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.

In this issue of Developmental Cell, Campbell et al. (2021) show that melanoma cells with distinct invasive or proliferative gene signatures can form heterotypic clusters that extravasate collectively and readily seed the growth of metastatic lesions. These findings highlight interactions between heterogenous tumor cells as being critical for metastasis.