Mucosal and acral melanomas are rare subtypes of melanoma with unique clinical and biological features. A dearth of well-established and characterized melanoma cell lines that reflect rare subtypes impedes research on these lethal diseases. We present a protocol developed by exploring various combinations of established techniques that we used to generate mucosal and acral cell lines from patient tissues and patient derived xenografts.
Skin pigmentation relies on the coordinated regulation of melanin production and dendritic morphology to ensure effective pigment distribution. While staurosporine is widely used as a pro-apoptotic agent in malignant cells, its effects on normal human melanocytes remain poorly understood. Here, we investigated the effects of staurosporine on melanocyte biology and identified it as a potent inducer of non-canonical melanocyte maturation at sub-cytotoxic concentrations without stimulating melanocyte proliferation. In primary human melanocytes, staurosporine enhanced melanogenesis and promoted dendrite formation, elongation, and branching, resulting in acquisition of a mature melanocyte phenotype distinct from its apoptotic effects in melanoma cells. These phenotypic changes were accompanied by activation of β-catenin signaling together with coordinated remodeling of Rho family protein expression and the actin cytoskeleton. Topical application of staurosporine increased physiological pigmentation in guinea pig skin without detectable inflammation or melanocyte proliferation. Furthermore, staurosporine accelerated repigmentation in a rhododendrol-induced leukoderma model by promoting functional maturation of epidermal melanocytes and enhancing nuclear localization of β-catenin. Collectively, our findings identify staurosporine as a non-canonical regulator of melanocyte maturation and demonstrate that functional maturation, rather than proliferative expansion, is sufficient to enhance pigmentation under both physiological and depigmented conditions.
In-transit melanoma (ITM) is a unique presentation of metastatic cutaneous melanoma associated with therapeutic challenges. Despite its clinical importance, the molecular drivers of ITM remain poorly defined. We aimed to characterize the mutational landscape of ITM and identify genetic alterations distinguishing it from other melanoma metastases. Tumor samples from 528 patients in the MSK-IMPACT dataset, comprised of over 300 cancer-related genes, were analyzed. Samples were classified as primary, in-transit, regional lymph node, or distant metastases. Driver mutation frequencies were compared across groups, and mutual information (MI) and principal component analysis (PCA) were used to identify ITM-associated genes and mutation patterns. NRAS Q61 mutations were enriched in ITM compared with other melanoma sites, while NF1 mutations were less common. Exploratory MI, PCA, and pairwise analyses identified recurrent NRASMUT/wild-type gene patterns that may distinguish ITM from other melanoma samples. Genes retained in the wild-type state alongside NRASMUT were associated with PI3K/AKT/mTOR, TGF-β, and E2F-related pathways. Collectively, these findings suggest that ITM is enriched for NRAS Q61 mutations and may have a comparatively lower co-mutation burden, providing a basis for future studies of ITM biology and clinical behavior.
Germline genetic variation can influence the number of nevi on the skin. However, nevi can be classified in many ways (e.g., by shape, size or evolution); little is known of how genetics influences the density of different nevus morphologies on the body. Here, we explore how germline genetics influences the density of flat, raised and atypical nevi, and identify which classification correlates most with genetic risk of melanoma. We estimate heritability and perform genome-wide association analysis of flat, raised, and atypical nevus count within the Brisbane Twin Nevus Study (N = 3862). We compare the effect estimates of genetic loci associated with each morphology. We also assess how well each morphology correlates with the genetic risk of melanoma using a polygenic risk score of melanoma. Heritability estimates revealed both unique and shared genetic effects between morphologies. We identified two loci near IRF4 and MC1R showing opposing effects on flat and raised nevus counts. Variation in raised nevus count showed the strongest correlation with melanoma risk using polygenic risk scores. Although some genetic effects are shared between nevus morphologies, they appear to be genetically distinct phenotypes. Given the opposing effects of IRF4 and MC1R on flat and raised nevus counts, future studies of nevus count should consider, where possible, analysing each nevus morphology separately as well as combined.
The tripartite motif (TRIM) family of E3 ubiquitin ligases is known to play a crucial role in the initiation, growth, and metastasis of various tumors. However, little is known about the biological features and relevant molecular mechanism of Tripartite motif‐containing 63 (TRIM 63) in melanoma. The expression levels of TRIM63 and purinergic receptor P2Y1 (P2RY1) in melanoma were examined by online database. Cell counting kit‐8 (CCK‐8) and colony formation assay were carried out to explore the effects of TRIM63 on melanoma cells proliferation. Transwell assay was used to explore the influence of TRIM63 on melanoma cells invasion and migration. Bioinformatics, co‐immunoprecipitation (co‐IP) assay, ubiquitination assay, and protein stability assay were used to detect the regulatory mechanism of TRIM63 on P2RY1. TRIM63 was upregulated in melanoma samples, and a higher expression level of TRIM63 indicated a shorter overall survival of melanoma patients. Knocked down of TRIM63 obviously suppressed the proliferation, invasion, and migration abilities of melanoma cells. Mechanistically, TRIM63 was regarded as a posttranslational mediator of P2RY1, and TRIM63 was co‐immunoprecipitated with P2RY1 and degraded its protein level. Notably, silencing P2RY1 alleviated melanoma cells progression by TRIM63 depletion. Collectively, these data suggested that TRIM63 contributed to melanoma cells growth and mobility by ubiquitination of P2RY1 and may be a promising candidate as a potential diagnostic and therapeutic marker for patients with melanoma.
Vitiligo is a common autoimmune disorder characterized by melanocyte loss, yet the molecular mechanisms linking genetic susceptibility to melanocyte dysfunction remain incompletely understood. In this study, we integrated multi-tissue transcriptome-wide association studies (TWAS), summary-data-based Mendelian randomization (SMR), and colocalization analyses to identify causal genes within vitiligo susceptibility loci. CTSS was prioritized as a candidate susceptibility gene at the 1q21 locus. Single-cell and bulk RNA sequencing analyses revealed that CTSS was significantly upregulated in lesional melanocytes and associated with an MHC class II-related transcriptional program. Mechanistically, CTSS expression was induced downstream of IFN-γ/STAT1 signaling and positively associated with oxidative phosphorylation, suggesting potential metabolic regulation. Functional experiments demonstrated that CTSS overexpression was associated with increased HLA-DRA expression, enhanced apoptosis, and reduced proliferation in melanocytes. Furthermore, the selective CTSS inhibitor RO5459072 attenuated HLA-DRA induction in vitro. Clinically, serum CTSS levels were elevated in patients with active vitiligo, showed moderate diagnostic performance for disease activity (AUC = 0.787), and correlated with disease severity. Collectively, these findings suggest that CTSS may serve as a key mediator linking genetic susceptibility, inflammatory signaling, and metabolic status in vitiligo. Rather than establishing melanocytes as professional antigen-presenting cells, our results indicate that melanocytes may acquire partial immunogenic features under inflammatory stress conditions. This study provides new insights into melanocyte-intrinsic mechanisms underlying vitiligo pathogenesis and highlights CTSS as a potential therapeutic target for future investigation.
The 2025 Cure Ocular Melanoma (CURE OM) Global Science Meeting took place in Amsterdam, The Netherlands on October 25th, 2025. Several promising drug candidates are in development; however, there is still an urgent need for better prevention, detection, and treatment of uveal melanoma. The purpose of this meeting was to promote international collaboration and idea exchange between scientists, industry, and patient advocates.
Uveal melanoma is a hard-to-treat arginine-dependent cancer secondary to argininosuccinate synthetase 1 (ASS1) loss with half of patients succumbing to liver-dominant metastases. Arginine deprivation with pegargiminase is a novel antimetabolite strategy for patients with uveal melanoma. We investigated the preclinical rationale for combining pegargiminase with melphalan, an alkylating agent approved recently for the treatment of hepatic-centric disease. Drug sensitivity of ASS1-deficient uveal melanoma cell lines was performed in 2D culture using proliferation and cytotoxicity assays, with analysis of cell death, cell cycle, DNA double-strand breaks, and interrogation of the molecular mechanism of action by RNA-seq. ADI-PEG20 and melphalan suppressed uveal melanoma cell line proliferation and triggered cytotoxicity, effects which were enhanced with the drug combination. ADI-PEG20 downregulated multiple genes of the Fanconi anemia pathway and synergized with melphalan to increase DNA double-strand breaks. Melphalan and pegargiminase is a rational new drug combination that warrants clinical testing in uveal melanoma.
We present MelOD (Melanoma Omics Dashboard), a free, web-based interactive platform integrating preprocessed data from 16 melanoma studies, including eight bulk transcriptomics, six single-cell RNA-seq, and two proteomics datasets. MelOD provides user-friendly visualization and analysis tools, differential expression, dimensionality reduction, clustering, correlation, and survival analysis without requiring local computational resources. Several datasets include annotations for immunotherapy response, facilitating exploration of resistance and response signatures. Built on RShiny with optimized handling of large datasets, MelOD supports real-time hypothesis generation, cross-study validation, and community dataset contributions. Freely accessible online, MelOD lowers barriers to multi-omics research in melanoma and related fields.
While the molecular characteristics of cutaneous melanomas (CMs) in individuals with light skin have been extensively studied, less is known about acral melanoma (AM), a non-ultraviolet radiation-induced melanoma more frequently found in individuals with darker skin. Exome and whole genome sequencing of 25 AMs from South African patients with a range of ancestral backgrounds were performed. While close to 50% in CMs, only one AM in a patient of European ancestry harbored an alteration at p.V600 in BRAF. 76% of tumors were triple wild-type. There were hotspot mutations in KIT, mutations in genes encoding components of the MAPK pathway (K57N in MAP2K1 and Y71H in HRAS), and novel candidate driver mutations in genes including PROS1, RGPD3, and SF3A3. Copy number gains included regions with TERT, CDK4, PAK1/GAB2, and DAD1 (chr14q11.2). Gain of chr17q25.1 harboring CDK3 was seen in samples with African ancestry. Eleven significantly deleted regions included chr1q43 (MAP1LC3C) and homozygous deletions of HLA-DRB1, PHF10 (chr6q27), and ATMIN (chr16q23). Mutational signatures included chromosomal instability and chromothripsis. There was a robust ancestry-related difference in tumor evolutionary dynamics where African ancestry was correlated with more genome doubling and clonality with less evolutionary branching.
Acral melanoma (AM) is a rare subtype of melanoma with high malignancy. Although the transcriptional landscape of the tumor microenvironment (TME) in AM has been characterized, the heterogeneity of malignant cells and underlying molecular features remain to be fully delineated. We analyzed 23 AM samples across three single cell RNA sequencing (scRNA-seq) datasets and quantified the intra-tumoral melanoma cell heterogeneity (ITMH) based on the degree of deviation for individual cells in principal component space. We compared the molecular characteristics of melanoma cells and TME between high-intra-tumoral-heterogeneity (ITMHhi) and low-intra-tumoral-heterogeneity (ITMHlo) groups. Differentially expressed genes (DEGs) in melanoma cells were extracted and a single sample Gene Set Enrichment Analysis (ssGSEA)-based differential scoring method was applied to stratify patients into ITMHhi and ITMHlo subtypes in bulk RNA-seq cohorts. Weighted Gene Co-expression Network Analysis (WGCNA) was employed to explore the gene modules related to tumor heterogeneity. ScRNA-seq analysis revealed diverse transcriptional heterogeneity among melanoma cells within and between patients. ITMHhi and ITMHlo samples distributed separately in the transcriptional space. We observed that the heterogeneity of melanoma cells was correlated with genomic, immunological, and clinical characteristics. The ITMHhi group exhibited distinct molecular programs and high genome instability in tumor cells, as well as high progenitor exhausted and inflammatory microenvironment. We also defined ITMH-related AM subtypes with different gene expression patterns and clinical outcomes in bulk RNA-seq data. Hub genes associated with high heterogeneity were identified by DEG and WGCNA analysis, and a prognostic risk model was developed to predict patients' survival based on the hub genes. Our study provided insight into understanding the intra-tumoral heterogeneity of AM and its potential impact on patient stratification, TME remodeling and prognosis prediction.
Mucosal melanoma (MM) is a rare and lethal subtype of melanoma, disproportionately affecting Asian populations and exhibiting distinct clinicopathological and genetic features compared to cutaneous melanoma (CM). Often diagnosed at advanced stages, MM shows poor responses to conventional therapies, and no standardized treatment regimen currently exists. Progress in preclinical modeling, including cell lines, patient-derived xenografts (PDXs), organoids (PDOs), and comparative animal models-has provided valuable tools for studying MM pathogenesis and therapeutic resistance. Yet these models remain limited in number, heterogeneity, and standardization, restricting their ability to capture MM's molecular diversity and immunosuppressive microenvironment. Beyond physical platforms, emerging virtual strategies-including computational simulations, artificial intelligence-driven multi-omics integration, and in silico clinical trials-offer scalable, cost-effective complements. By simulating tumor-immune-drug interactions using minimal biospecimens, these models offer a unique platform for hypothesis testing and patient stratification in this rare cancer type. This review summarizes MM's clinicopathological features and therapeutic challenges, evaluates current preclinical models, and highlights the synergistic integration of biological and virtual approaches. Future efforts should prioritize MM-specific repositories, multi-model integration, and incorporation of in silico pipelines to accelerate translational research and improve outcomes in this highly lethal malignancy.
While prior studies have described the development of vitiligo and alopecia among patients with metastatic melanoma undergoing cancer immunotherapy, there are scant reports of secondary poliosis. Here we detail a case series of patients with melanoma who developed poliosis secondary to cancer immunotherapy. A clinical database of melanoma patients treated at the University of Southern California, Norris Comprehensive Cancer Center, from 2018 to 2023 was reviewed to identify patients who developed poliosis during cancer immunotherapy treatment. Patients with both cutaneous and non-cutaneous melanoma (acral and uveal) were included. Cancer immunotherapies included checkpoint inhibitors, bispecifics, and tumor infiltrating lymphocyte (TIL) therapy. Patient characteristics, tumor features (histology, stage), treatment variables, and clinical outcomes were collected for all patients. Immunotherapy-related side effects, including poliosis, were recorded. A total of 10 melanoma patients with poliosis were identified, including 5 women; 8 had stage IV disease. The median duration of immunotherapy treatment was 20 months (range 10-46). Median onset of poliosis was 4 months (range 2-12). Amongst the 10 patients, 9 (90%) remain in continued response to treatment without disease progression at this time. This case series describes 10 melanoma patients who developed poliosis while undergoing immunotherapy, all of whom developed clinical benefit from treatment. This cohort included patients with rare and challenging melanoma subtypes, e.g., acral lentiginous and uveal melanoma. The development of poliosis may portend a positive clinical benefit from these immunotherapy treatments. Further research is necessary to elucidate the underlying mechanisms of immune-mediated melanocyte destruction and depigmentation in melanoma patients receiving immunotherapy.
NRAS mutations occur in 10%-30% of cutaneous melanomas and are associated with high tumor mutational burden. Mutant NRAS signaling drives aberrant cell growth and proliferation, in part, through activation of the RAF-MEK-ERK1/2 kinase pathway; however, targeted therapies to this pathway have limited effectiveness in patients with NRAS mutant melanoma. The role of other targetable signaling pathways in NRAS mutant melanoma is poorly characterized. Here, we demonstrated that one isoform of diacylglycerol kinase, diacylglycerol kinase eta (DGKη), a lipid signaling regulator, was highly expressed in NRAS mutant melanoma patient samples. Knockdown of DGKH in NRAS mutant melanoma cell lines resulted in significant growth inhibition in vitro. Transcriptomic data indicated downregulation of the estrogen response late signature, including decreased CCND1 (cyclin D1) expression following DGKH knockdown. Cell growth inhibition and decreased cyclin D1 expression correlated to an inhibition of cell cycle after DGKH knockdown. These data suggest that DGKη mediates cell cycle progression in NRAS mutant melanoma cells and represents a potential therapeutic target for these patients.
Amelanotic melanoma lacks melanin and is more challenging to diagnose than pigmented melanoma, often leading to delayed detection and worse outcomes. We conducted a retrospective cohort study of 322 patients (28 with amelanotic melanoma and 294 with melanotic melanoma) treated at Sheba Medical Center between 2017 and 2023. Clinical features, treatment modalities, and 1-year outcomes were analyzed. Tumor stage at diagnosis was the strongest predictor of remission, disease progression, and mortality in both groups. Pigmentation was associated with a higher likelihood of treatment modification (p = 0.02) and showed a borderline association with progression (p = 0.08). Subgroup analysis by stage revealed no significant outcome differences except for increased therapy change in stage 2 melanotic melanoma. The findings reinforce the importance of early detection, especially in amelanotic melanoma.
Non-V600E/K BRAF mutations have been reported in melanoma, but data on their clinical relevance are conflicting. This study investigated the distribution, prognostic role, and functional impact of rare BRAF mutations in melanoma. We retrospectively assessed frequency, response to therapy and outcome of rare BRAF mutations compared to V600E/K in cases from 19 Italian Melanoma group (IMI) centers. 258/14,081 samples (1.8%) harbored rare BRAF mutations, 40% encompassing codon 600. Overall and progression-free survival (OS, PFS) following target therapy with BRAF/MEK inhibitors were comparable to V600E/K mutant melanoma (HR = 0.85 and 0.89, p > 0.1). Response to target therapy was lower, albeit not significantly, in rare BRAF mutant melanomas compared to V600E/K (48% vs. 66%, p > 0.05). OS, PFS, and objective response in cases treated with immunotherapy were unaffected by BRAF status. Molecular dynamics simulation assessing whether selected BRAF variants affected BRAF structure similarly to V600E showed variable degrees of destabilization towards constitutive protein activation, particularly for mutations encompassing codons 599-601. These results indicate that rare BRAF mutations can modify BRAF kinase activity including a subset of mutations outside but close to codon 600. Molecular approaches able to detect rare BRAF mutations could identify additional melanoma cases eligible for therapies with BRAF/MEK inhibitors.
OCA2, a melanosome transmembrane spanning protein, functions to regulate melanosomal pH, optimizing production of melanin pigment. OCA2 is one of eight non-syndromic autosomal recessive oculocutaneous albinism (OCA) loci and is the second most common cause of OCA worldwide. Genome wide association studies (GWAS) have identified OCA2 coding and regulatory variants linked to common skin and eye color pigment variation, skin cancer susceptibility, and retinal pigment epithelium tissue metrics. Within a cohort of 106 OCA2 probands with two biallelic OCA2 variants, a total of 74 distinct OCA2 rare variants were identified (11 large structural, 17 small indel/frameshift, 12 splice site, and 34 missense coding variants). Phase-validated haplotypes, comprised of both OCA2 common pigmentation trait GWAS alleles and rare variants, were obtained for 95/106 probands. In total, 41 distinct multi-allele OCA2 haplotypes were identified with 27 haplotypes containing either rs1800404-A and/or rs12913832-G alleles, each of which is known to reduce correct isoform splicing or gene expression by ~20%. These results find that common GWAS alleles with known OCA2 functional impact are present on haplotypes with variants of unknown significance in OCA2 probands and highlight the need for haplotype-based analysis at the OCA2 locus in addition to individual variant pathogenic assessment.
At the 2025 ESPCR (European Society for Pigment Cell Research) meeting in Erlangen, a workshop on "Pigment Cell Models: Sensitivity, Innovation, and the Challenges of Cell Culture" brought together researchers to discuss technical, methodological, and reproducibility issues in culturing melanocytes, keratinocytes, fibroblasts, and melanoma cells. The discussion between experts in the field highlighted key and recurrent pitfalls affecting experimental outcomes, including low-density seeding, temperature fluctuations, over-passaging, and mycoplasma contamination, as well as sources of variability arising from media composition, batch effects, and environmental conditions. Importantly, the workshop distinguished between practices supported by evidence and consensus-based guidance derived from collective expert experience. Species- and donor-specific differences, especially between human, mouse, and zebrafish melanocyte models, were identified as additional major determinants of experimental variability. Emerging systems, including human and mouse pluripotent stem cell (PSC)-derived melanocytes, as well as avian and zebrafish melanoma lines, were discussed for their complementary mechanistic and translational value. Overall, the workshop concluded that transparent documentation, explicit reporting standards, and shared best practices are essential to improve reproducibility and further advance pigment cell research.
