Differential histone acetylation and super-enhancer regulation underlie melanoma cell dedifferentiation.

Dedifferentiation or phenotype switching refers to the transition from a proliferative to an invasive cellular state. We previously identified a 122-gene epigenetic gene signature that classifies primary melanomas as low versus high risk (denoted as Epgn1 or Epgn3). We found that the transcriptomes of the Epgn1 low-risk and Epgn3 high-risk cells are similar to the proliferative and invasive cellular states, respectively. These signatures were further validated in melanoma tumor samples. Examination of the chromatin landscape revealed differential H3K27 acetylation in the Epgn1 low-risk versus Epgn3 high-risk cell lines that corroborated with a differential super-enhancer and enhancer landscape. Melanocytic lineage genes (MITF, its targets and regulators) were associated with super-enhancers in the Epgn1 low-risk state, whereas invasiveness genes were linked with Epgn3 high-risk status. We identified the ITGA3 gene as marked by a super-enhancer element in the Epgn3 invasive cells. Silencing of ITGA3 enhanced invasiveness in both in vitro and in vivo systems, suggesting it as a negative regulator of invasion. In conclusion, we define chromatin landscape changes associated with Epgn1/Epgn3 and phenotype switching during early steps of melanoma progression that regulate transcriptional reprogramming. This super-enhancer and enhancer-driven epigenetic regulatory mechanism resulting in major changes in the transcriptome could be important in future therapeutic targeting efforts.

B7-H3 drives immunosuppression and Co-targeting with CD47 is a new therapeutic strategy in ß-catenin activated melanomas.

In melanoma, immune cell infiltration into the tumor is associated with better patient outcomes and response to immunotherapy. T-cell non-inflamed tumors (cold tumors) are associated with tumor cell-intrinsic Wnt/ß-catenin activation, and are typically resistant to anti-PD-1 alone or in combination with anti-CTLA-4 therapy. Reversal of the 'cold tumor' phenotype and identifying new effective immunotherapies are challenges. We sought to investigate the role of a newer immunotherapy agent, B7-H3, in this setting. RNA sequencing was used to identify co-targeting strategies upon B7-H3 inhibition in a well-defined preclinical melanoma model driven by ß-catenin. We found that immune checkpoint molecule B7-H3 confers a suppressive tumor microenvironment by modulating antiviral signals and innate immunity. B7-H3 inhibition led to an inflamed microenvironment, up-regulation of CD47/SIRPa signaling, and together with blockade of the macrophage checkpoint CD47 resulted in additive antitumor responses. We found that the antitumor effects of the B7-H3/CD47 antibody combination were dependent on cytokine signaling pathways (CCR5/CCL5 and IL4).

Gab2-mediated signaling promotes melanoma metastasis.

Metastatic melanoma is a disease with a poor prognosis that currently lacks effective treatments. Critical biological features of metastasis include acquisition of migratory competence, growth factor independence, and invasive potential. In an attempt to identify genes that contribute to melanoma pathogenesis, a genome-wide search using bacterial artificial chromosome array comparative genomic hybridization and single nucleotide polymorphism arrays in a series of 64 metastatic melanoma samples and 20 melanoma cell lines identified increased copy numbers of Gab2 located on 11q14.1. Gab2 is an adaptor protein that potentiates the activation of the Ras-Erk and PI3K-Akt pathways and has recently been implicated in human cancer; however, its role in melanoma has not been explored. In this study, we found that Gab2 was either amplified (approximately 11%) and/or overexpressed (approximately 50%) in melanoma. Gab2 protein expression correlated with clinical melanoma progression, and higher levels of expression were seen in metastatic melanomas compared with primary melanoma and melanocytic nevi. We found that overexpression of Gab2 potentiates, whereas silencing of Gab2 reduces, migration and invasion of melanoma cells. Gab2 mediated the hyperactivation of Akt signaling in the absence of growth factors, whereas inhibition of the PI3K-Akt pathway decreased Gab2-mediated tumor cell migration and invasive potential. Gab2 overexpression resulted in enhanced tumor growth and metastatic potential in vivo. These studies demonstrate a previously undefined role for Gab2 in melanoma tumor progression and metastasis.

Association of MDM2 SNP309, age of onset, and gender in cutaneous melanoma.

PURPOSE: In certain cancers, MDM2 SNP309 has been associated with early tumor onset in women. In melanoma, incidence rates are higher in women than in men among individuals less than 40 years of age, but among those older than 50 years of age, melanoma is more frequent in men than in women. To investigate this difference, we examined the association among MDM2 SNP309, age at diagnosis, and gender among melanoma patients. EXPERIMENTAL DESIGN: Prospectively enrolled melanoma patients (N = 227) were evaluated for MDM2 SNP309 and the related polymorphism, p53 Arg72Pro. DNA was isolated from patient blood samples, and genotypes were analyzed by PCR-restriction fragment length polymorphism. Associations among MDM2 SNP309, p53 Arg72Pro, age at diagnosis, and clinicopathologic features of melanoma were analyzed. RESULTS: The median age at diagnosis was 13 years earlier among women with a SNP309 GG genotype (46 years) compared with women with TG+TT genotypes (59 years; P = 0.19). Analyses using age dichotomized at each decade indicated that women with a GG genotype had significantly higher risks of being diagnosed with melanoma at ages <50 years compared with women >or=50 years, but not when the comparison was made between women <60 and >or=60 years. At ages <50 years, women with a GG genotype had a 3.89 times greater chance of being diagnosed compared with women with TG+TT genotypes (P = 0.01). Similar observations were not seen among men. CONCLUSIONS: Our data suggest that MDM2 may play an important role in the development of melanoma in women. The MDM2 SNP309 genotype may help identify women at risk of developing melanoma at a young age.

An integrative approach identifies dysregulated long non-coding RNAs as microRNA decoys during nevus to melanoma transformation.

Mounting evidence supports a role for dysregulated long non-coding RNAs (lncRNA) in the development of many cancers. A recently discovered function of lncRNAs is to act as microRNA (miR) decoys or competing endogenous RNAs, which sequester specific miRs and relieve negative regulation of mRNA expression by miRs. Although a large number of non-coding RNAs are thought to function as competing endogenous RNAs, miR-sequestering lncRNAs involved in nevus to melanoma transformation remain largely unknown. In this study, we applied a bioinformatics approach to a unique dataset of benign melanocytic nevi and primary melanomas of the skin in order to fill this research gap. We modified a previously published miR target prediction algorithm, RNAhybrid, and improved its search efficiency. We reported the presence of many lncRNAs and miRs deregulated when transitioning from a senescence-like state of nevi to melanoma. We provided evidence of a relatively new and understudied mechanism of gene regulation during this process and identified for the first time lncRNAs (n = 122) that may potentially function as miR decoys as well as their target miRs during nevus to melanoma transformation. The knowledge presented here can be employed for developing biomarkers for diagnostic and risk stratification purposes.

Dual suppression of inner and outer mitochondrial membrane functions augments apoptotic responses to oncogenic MAPK inhibition.

Mitogen-activated protein kinase (MAPK) pathway inhibitors show promise in treating melanoma, but are unsuccessful in achieving long-term remission. Concordant with clinical data, BRAFV600E melanoma cells eliminate glycolysis upon inhibition of BRAFV600E or MEK with the targeted therapies Vemurafenib or Trametinib, respectively. Consequently, exposure to these therapies reprograms cellular metabolism to increase mitochondrial respiration and restrain cell death commitment. As the inner mitochondrial membrane (IMM) is sub-organellar site of oxidative phosphorylation (OXPHOS), and the outer mitochondrial membrane (OMM) is the major site of anti-apoptotic BCL-2 protein function, we hypothesized that suppressing these critical mitochondrial membrane functions would be a rational approach to maximize the pro-apoptotic effect of MAPK inhibition. Here, we demonstrate that disruption of OXPHOS with the mitochondria-specific protonophore BAM15 promotes the mitochondrial pathway of apoptosis only when oncogenic MAPK signaling is inhibited. Based on RNA-sequencing analyses of nevi and primary melanoma samples, increased pro-apoptotic BCL-2 family expression positively correlates with high-risk disease suggesting a highly active anti-apoptotic BCL-2 protein repertoire likely contributes to worse outcome. Indeed, combined inhibition of the anti-apoptotic BCL-2 repertoire with BH3-mimetics, OXPHOS, and oncogenic MAPK signaling induces fulminant apoptosis and eliminates clonogenic survival. Altogether, these data suggest that dual suppression of IMM and OMM functions may unleash the normally inadequate pro-apoptotic effects of oncogenic MAPK inhibition to eradicate cancer cells, thus preventing the development of resistant disease, and ultimately, supporting long-term remission.

Genomic Characterization of Dysplastic Nevi Unveils Implications for Diagnosis of Melanoma.

A well-defined risk factor and precursor for cutaneous melanoma is the dysplastic nevus. These benign tumors represent clonal hyperproliferation of melanocytes that are in a senescent-like state, but with occasional malignant transformation events. To portray the mutational repertoire of dysplastic nevi in patients with the dysplastic nevus syndrome and to determine the discriminatory profiles of melanocytic nevi (including dysplastic nevi) from melanoma, we sequenced exomes of melanocytic nevi including dysplastic nevi (n = 19), followed by a targeted gene panel (785 genes) characterization of melanocytic nevi (n = 46) and primary melanomas (n = 42). Exome sequencing revealed that dysplastic nevi harbored a substantially lower mutational load than melanomas (21 protein-changing mutations versus >100). Known "driver" mutations in genes for melanoma, including CDKN2A, TP53, NF1, RAC1, and PTEN, were not found among any melanocytic nevi sequenced. Additionally, melanocytic nevi including dysplastic nevi showed a significantly lower frequency and a different UV-associated mutational signature. These results show that although melanocytic nevi and dysplastic nevi harbor stable genomes with relatively few alterations, progression into melanomas requires additional mutational processes affecting key tumor suppressors. This study identifies molecular parameters that could be useful for diagnostic platforms.

Transcriptional dissection of melanoma identifies a high-risk subtype underlying TP53 family genes and epigenome deregulation.

BACKGROUND: Melanoma is a heterogeneous malignancy. We set out to identify the molecular underpinnings of high-risk melanomas, those that are likely to progress rapidly, metastasize, and result in poor outcomes. METHODS: We examined transcriptome changes from benign states to early-, intermediate-, and late-stage tumors using a set of 78 treatment-naive melanocytic tumors consisting of primary melanomas of the skin and benign melanocytic lesions. We utilized a next-generation sequencing platform that enabled a comprehensive analysis of protein-coding and -noncoding RNA transcripts. RESULTS: Gene expression changes unequivocally discriminated between benign and malignant states, and a dual epigenetic and immune signature emerged defining this transition. To our knowledge, we discovered previously unrecognized melanoma subtypes. A high-risk primary melanoma subset was distinguished by a 122-epigenetic gene signature ("epigenetic" cluster) and TP53 family gene deregulation (TP53, TP63, and TP73). This subtype associated with poor overall survival and showed enrichment of cell cycle genes. Noncoding repetitive element transcripts (LINEs, SINEs, and ERVs) that can result in immunostimulatory signals recapitulating a state of "viral mimicry" were significantly repressed. The high-risk subtype and its poor predictive characteristics were validated in several independent cohorts. Additionally, primary melanomas distinguished by specific immune signatures ("immune" clusters) were identified. CONCLUSION: The TP53 family of genes and genes regulating the epigenetic machinery demonstrate strong prognostic and biological relevance during progression of early disease. Gene expression profiling of protein-coding and -noncoding RNA transcripts may be a better predictor for disease course in melanoma. This study outlines the transcriptional interplay of the cancer cell's epigenome with the immune milieu with potential for future therapeutic targeting. FUNDING: National Institutes of Health (CA154683, CA158557, CA177940, CA087497-13), Tisch Cancer Institute, Melanoma Research Foundation, the Dow Family Charitable Foundation, and the Icahn School of Medicine at Mount Sinai.

Mutations in the CYLD gene in Brooke-Spiegler syndrome, familial cylindromatosis, and multiple familial trichoepithelioma: lack of genotype-phenotype correlation.

Brooke-Spiegler syndrome (BSS), familial cylindromatosis (FC), and multiple familial trichoepithelioma (MFT), originally described as distinct entities, share overlapping clinical findings. Patients with BSS are predisposed to multiple skin appendage tumors such as cylindroma, trichoepithelioma, and spiradenoma. FC, however, is characterized by cylindromas and MFT by trichoepitheliomas as the only tumor type. These disorders have recently been associated with mutations in the CYLD gene. In this report, we describe three families with BSS, one with FC, and two with MFT phenotypes associated with novel and recurrent mutations in CYLD. We provide evidence that these disorders represent phenotypic variation of a single entity and lack genotype-phenotype correlation.

B-RAF and melanocytic neoplasia.

High frequency of B-RAF gene mutations has recently been identified in benign melanocytic nevi and melanoma. This review focuses on clinical studies that evaluate the role of B-RAF in melanocytic neoplasia.

Identification of recurrent mutations in the ARS (component B) gene encoding SLURP-1 in two families with mal de Meleda.

Mal de Meleda is a rare, autosomal recessive form of palmoplantar keratoderma. The disease has been mapped to chromosome 8qter, and in a recent study mutations in the ARS gene have been identified in families with this disorder. Here, we report two unrelated families with mal de Meleda, in which two different homozygous mutations in the ARS gene were identified. These findings support the notion that mutations in the ARS gene are pathogenic in mal de Meleda.

A novel missense mutation in CYLD in a family with Brooke-Spiegler syndrome.

Brooke-Spiegler syndrome (BSS, familial cylindromatosis or turban tumor syndrome) is an inherited disease characterized by neoplasms of the skin appendages such as cylindroma, trichoepithelioma, and spiradenoma. The disease has been mapped to 16q12-13, and mutations in the CYLD gene have been identified in families with this disorder. Of interest, multiple familial trichoepithelioma (MFT) has been described as a distinct disorder characterized by the familial occurrence of trichoepitheliomas. MFT has been mapped to 9p21; however, to date a candidate gene has not been identified. In this report, we describe a four-generation family with BSS presenting predominantly with trichoepitheliomas (resembling MFT phenotype). We identified a novel missense mutation in the CYLD gene, designated E474G, in the affected individuals of this family. Our findings exemplify clinical heterogeneity within BSS and extend the body of evidence that mutations in CYLD are implicated in this disease. Although not conclusive, these findings suggest that BSS and MFT may represent a single entity.

A recurrent mutation in the ARS (component B) gene encoding SLURP-1 in Turkish families with mal de Meleda: evidence of a founder effect.

Mal de Meleda is a rare form of palmoplantar keratoderma, and recently mutations in the ARS (component) B gene have been identified in families with this disease. We identified a recurrent nonsense mutation, R96X, in four families of Turkish descent. In this report, we demonstrate that these families share a common ancestral haplotype at the mal de Meleda locus, suggesting a founder effect.

FBXW7 mutations in melanoma and a new therapeutic paradigm.

BACKGROUND: Melanoma is a heterogeneous tumor with subgroups requiring distinct therapeutic strategies. Genetic dissection of melanoma subgroups and identification of therapeutic agents are of great interest in the field. These efforts will ultimately lead to treatment strategies, likely combinatorial, based on genetic information. METHODS: To identify "driver" genes that can be targeted therapeutically, we screened metastatic melanomas for somatic mutations by exome sequencing followed by selecting those with available targeted therapies directed to the gene product or its functional partner. The FBXW7 gene and its substrate NOTCH1 were identified and further examined. Mutation profiling of FBXW7, biological relevance of these mutations and its inactivation, and pharmacological inhibition of NOTCH1 were examined using in vitro and in vivo assays. RESULTS: We found FBXW7 to be mutated in eight (8.1%) melanoma patients in our cohort (n = 103). Protein expression analysis in human tissue samples (n = 96) and melanoma cell lines (n = 20) showed FBXW7 inactivation as a common event in melanoma (40.0% of cell lines). As a result of FBXW7 loss, we observed an accumulation of its substrates, such as NOTCH1. Ectopic expression of mutant forms of FBXW7 (by 2.4-fold), as well as silencing of FBXW7 in immortalized melanocytes, accelerated tumor formation in vivo (by 3.9-fold). Its inactivation led to NOTCH1 activation, upregulation of NOTCH1 target genes (by 2.6-fold), and promotion of tumor angiogenesis and resulted in tumor shrinkage upon NOTCH1 inhibition (by fivefold). CONCLUSIONS: Our data provides evidence on FBXW7 as a critical tumor suppressor mutated and inactivated in melanoma that results in sustained NOTCH1 activation and renders NOTCH signaling inhibition as a promising therapeutic strategy in this setting.

GAB2 amplifications refine molecular classification of melanoma.

PURPOSE: Gain-of-function mutations in BRAF, NRAS, or KIT are associated with distinct melanoma subtypes with KIT mutations and/or copy number changes frequently observed among melanomas arising from sun-protected sites, such as acral skin (palms, soles, and nail bed) and mucous membranes. GAB2 has recently been implicated in melanoma pathogenesis, and increased copy numbers are found in a subset of melanomas. We sought to determine the association of increased copy numbers of GAB2 among melanoma subtypes in the context of genetic alterations in BRAF, NRAS, and KIT. EXPERIMENTAL DESIGN: A total of 85 melanomas arising from sun-protected (n = 23) and sun-exposed sites (n = 62) were analyzed for copy number changes using array-based comparative genomic hybridization and for gain-of-function mutations in BRAF, NRAS, and KIT. RESULTS: GAB2 amplifications were found in 9% of the cases and were associated with melanomas arising from acral and mucosal sites (P = 0.005). Increased copy numbers of the KIT locus were observed in 6% of the cases. The overall mutation frequencies for BRAF and NRAS were 43.5% and 14%, respectively, and were mutually exclusive. Among the acral and mucosal melanomas studied, the genetic alteration frequency was 26% for GAB2, 13% for KIT, 30% for BRAF, and 4% for NRAS. Importantly, the majority of GAB2 amplifications occurred independent from genetic events in BRAF, NRAS, and KIT. CONCLUSIONS: GAB2 amplification is critical for melanomas arising from sun-protected sites. Genetic alterations in GAB2 will help refine the molecular classification of melanomas.

Influence of age on survival in childhood spitzoid melanomas.

BACKGROUND: Melanoma occurring during childhood and adolescence is rare. Although a few limited studies suggest that the prognosis of childhood melanomas is similar to those in adults, and is dependent on the initial stage of the tumor, there is controversy with respect to the biologic behavior of childhood melanomas. Spitzoid melanoma is a subtype of melanoma with distinct clinical and histopathologic features. The prognosis of spitzoid melanoma in children, despite metastasis, has been suggested to be better than that observed in adults; however, this assertion remains controversial. Whereas a number of spitzoid melanomas with regional lymph node metastasis with no further progression have been reported, cases leading to widespread metastasis and fatal outcomes are also well documented. METHODS: A retrospective review of the literature was conducted between 1949 and 2006. A total of 82 cases of spitzoid melanoma with regional and/or widespread metastasis that occurred in children, 17 years of age and under, were selected for the analysis. RESULTS: The 5-year survival rate in children diagnosed with metastatic spitzoid melanomas between 0 and 10 years of age was 88% compared with 49% in those between 11 and 17 years of age. CONCLUSIONS: The findings support the notion that younger age (< or =10) may be associated with longer survival in children with metastatic spitzoid melanomas.

Are all melanomas the same? Spitzoid melanoma is a distinct subtype of melanoma.

BACKGROUND: Although the majority of melanomas demonstrate high rates of mutations in B-RAF or N-RAS that result in constitutive activation of the mitogen-activated protein kinase-signaling pathway, emerging data suggest molecular differences among melanoma subtypes. In this study, the authors evaluated the contribution of B-RAF and N-RAS mutations to the pathogenesis of Spitzoid melanomas. METHODS: In total, 33 Spitzoid melanomas were analyzed for clinical and pathologic characteristics as well as for hot-spot mutations in the B-RAF and N-RAS genes. In the majority of patients (28 of 33 melanomas), the tumors were confined to the skin with no evidence of metastasis (average follow-up, 32.5 mos). There were five metastasizing melanomas (5 of 33 tumors) with regional or systemic spread. RESULTS: Of 33 Spitzoid melanomas, only 1 showed the V600E mutation in the B-RAF gene (1 of 33 tumors; 3%). It was noteworthy that none of the metastatic Spitzoid melanomas (0 of 5 tumors; 0%), of which 2 resulted in fatal outcomes, demonstrated mutations in B-RAF or N-RAS. CONCLUSIONS: In contrast to the majority of cutaneous melanomas, activating hot-spot mutations in B-RAF or N-RAS were not involved in the pathogenesis of Spitzoid melanoma. These data suggested that Spitzoid melanoma is a distinct form of melanoma with unknown genes and/or signaling pathways involved in its development.