Specific oncogene activation of the cell of origin in mucosal melanoma

Abstract

Mucosal melanoma (MM) is a deadly cancer derived from mucosal melanocytes. To test the consequences of MM genetics, we developed a zebrafish model in which all melanocytes experienced CCND1 expression and loss of PTEN and TP53. Surprisingly, melanoma only developed from melanocytes lining internal organs, analogous to the location of patient MM. We found that zebrafish MMs had a unique chromatin landscape from cutaneous melanoma. Internal melanocytes could be labeled using a MM-specific transcriptional enhancer. Normal zebrafish internal melanocytes shared a gene expression signature with MMs. Patient and zebrafish MMs have increased migratory neural crest gene and decreased antigen presentation gene expression, consistent with the increased metastatic behavior and decreased immunotherapy sensitivity of MM. Our work suggests the cell state of the originating melanocyte influences the behavior of derived melanomas. Our animal model phenotypically and transcriptionally mimics patient tumors, allowing this model to be used for MM therapeutic discovery.

Figure 1:. Zebrafish model recapitulates localization of human MM.

A) Light micrographs of zebrafish melanoma generated from melanocyte-specific expression or CRISPR-deletion in transparent “Casper” zebrafish. Cutaneous model (upper). Mucosal model (lower). B) MM free survival of zebrafish with melanocyte-specific expression of human CCND1 and melanocyte-specific CRISPR of pten a and b and tp53. C-F) Zebrafish melanoma histology. C-D) H&E stain of MM (C) and CM (D). E-F) phospho ERK (pERK) immunohistochemistry (IHC) of MM (E) and CM (F). Scale bar = 1mm; inset scale bar = 100um. Black arrow = tumor, red arrow = brain. G) % zebrafish melanomas pERK positive. H) % zebrafish MM penetrance.

Figure 2:. Zebrafish MM has a distinct cellular state from CM.

A-B) Volcano plots of A) bulk RNA-seq and B) ATAC-seq from zebrafish MM vs. CM. C) IGV plot of ATAC-seq showing chromatin accessibility at tfap2a and tfap2b in zebrafish MM vs. CM. Boxes = loci used for tfap2b reporter. D-E) tfap2b enhancer-driving GFP preferentially labels internal mitfa-mCherry labeled melanocytes in 6-day old Casper zebrafish. D) Fluorescent images. Scale bar = 500um. E) Reporter expression quantification. p-value = 0.0003, 2-way ANOVA with multiple comparisons.

Figure 3:. Zebrafish internal melanocytes have properties consistent with MM initiating cells.

A) Light micrographs demonstrating internal GFP-expressing melanocytes from adult Casper zebrafish. Scale bar = 500um. B) Schematic for single-cell RNA-seq (scRNA-seq) of cutaneous and internal adult zebrafish melanocytes. C) UMAP-plot of scRNA-seq from GFP-sorted external and internal melanocytes. D) mitfa-expressing cells from C). E) UMAP of re-clustered mitfa+ melanocytes. Blue = internal. Red = external. Labels = cluster names. F) tfap2a and tfap2b expression in “internal”, “external”, and “both” melanocytes. G) Dot plot showing gene expression of top differentially expressed genes from zebrafish MMs for “internal”, “external”, and “both” melanocytes from E).

Figure 4:. MM cell state is conserved in patients.

A) UMAP-plot of single-cell RNA-seq (scRNA-seq) from patient tumors, CM patients (n=23) and MM patients (n=10). B-C) Mean normalized gene expression in CM vs. MM cells from scRNA-seq for B) MAPK target genes, p-value = 0.0036 two-sided t-test and C) HLA Class I antigen presentation genes. p-value = 0.017 two-sided t-test. Dots = patients. D) Average gene expression of expressed zebrafish mhc class I/II genes in a zebrafish CM vs. MM. Dots = genes. Lines indicate the same gene in two conditions. p-value = 0.0017, two-tailed Wilcoxon matched-pairs rank test. E) Mean normalized gene expression in MM vs. CM cells from scRNA-seq for migratory neural crest genes. p-value = 0.036 two-sided t-test. F) Average gene expression (FPKM) of migratory neural crest genes in zebrafish CMs compared to the MMs. Dots = genes. Lines indicate the same gene in two conditions. p-value= 0.031, two-tailed Wilcoxon matched-pairs rank test. G-H) Melanocyte-specific expression of hPAX3 vs. control in zebrafish with labeled internal melanocytes (double positive mitfamCherry and tfap2b-GFP) and pigment removal at 5dpf. G) Representative immunofluorescent images. Scale bar = 500μm. H) Quantification of double positive melanocytes inside and outside zebrafish embryos. p-value = 0.0014, ordinary one-way ANOVA with multiple comparisons.