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Severe immune-related adverse events (irAEs) occur in up to 60% of patients with melanoma treated with immune checkpoint inhibitors (ICIs). However, it is unknown whether a common baseline immunological state precedes irAE development. Here we applied mass cytometry by time of flight, single-cell RNA sequencing, single-cell V(D)J sequencing, bulk RNA sequencing and bulk T cell receptor (TCR) sequencing to study peripheral blood samples from patients with melanoma treated with anti-PD-1 monotherapy or anti-PD-1 and anti-CTLA-4 combination ICIs. By analyzing 93 pre- and early on-ICI blood samples and 3 patient cohorts (n= 27, 26 and 18), we found that 2 pretreatment factors in circulation—activated CD4 memory T cell abundance and TCR diversity—are associated with severe irAE development regardless of organ system involvement. We also explored on-treatment changes in TCR clonality among patients receiving combination therapy and linked our findings to the severity and timing of irAE onset. These results demonstrate circulating T cell characteristics associated with ICI-induced toxicity, with implications for improved diagnostics and clinical management.

The conserved WD40-repeat protein WDR5 interacts with multiple proteins both inside and outside the nucleus. However, it is currently unclear whether and how the distribution of WDR5 between complexes is regulated. Here, we show that an unannotated microprotein EMBOW (endogenous microprotein binder of WDR5) dually encoded in the human SCRIBgene interacts with WDR5 and regulates its binding to multiple interaction partners, including KMT2A and KIF2A. EMBOW is cell cycle regulated, with two expression maxima at late G1 phase and G2/M phase. Loss of EMBOW decreases WDR5 interaction with KIF2A, aberrantly shortens mitotic spindle length, prolongs G2/M phase, and delays cell proliferation. In contrast, loss of EMBOW increases WDR5 interaction with KMT2A, leading to WDR5 binding to off-target genes, erroneously increasing H3K4me3 levels, and activating transcription of these genes. Together, these results implicate EMBOW as a regulator of WDR5 that regulates its interactions and prevents its off-target binding in multiple contexts.

We performed exome sequencing of 77 melanocytic specimens composed of Spitz nevi (n=29), Spitzoid melanomas (n=27), and benign melanocytic nevi (n=21), and compared the results with published melanoma sequencing data. Our study highlights the prominent similarity between Spitzoid and conventional melanomas with similar copy number changes and high and equal numbers of ultraviolet-induced coding mutations affecting similar driver genes. Mutations in MEN1, PRKAR1A, and DNMT3Ain Spitzoid melanomas may indicate involvement of the protein kinase A pathway, or a role of DNA methylation in the disease. Other than activating HRASvariants, there were few additional mutations in Spitz nevi, and few copy number changes other than 11p amplification and chromosome 9 deletions. Similarly, there were no large-scale copy number alterations and few somatic alterations other than activating BRAFor NRASmutations in conventional nevi. A presumed melanoma driver mutation (IDH1Arg132Cys) was revealed in one of the benign nevi. In conclusion, our exome data show significantly lower somatic mutation burden in both Spitz and conventional nevi compared with their malignant counterparts, and high genetic similarity between Spitzoid and conventional melanoma.

We performed exome sequencing of 77 melanocytic specimens composed of Spitz nevi (n=29), Spitzoid melanomas (n=27), and benign melanocytic nevi (n=21), and compared the results with published melanoma sequencing data. Our study highlights the prominent similarity between Spitzoid and conventional melanomas with similar copy number changes and high and equal numbers of ultraviolet-induced coding mutations affecting similar driver genes. Mutations in MEN1, PRKAR1A, and DNMT3A in Spitzoid melanomas may indicate involvement of the protein kinase A pathway, or a role of DNA methylation in the disease. Other than activating HRAS variants, there were few additional mutations in Spitz nevi, and few copy number changes other than 11p amplification and chromosome 9 deletions. Similarly, there were no large-scale copy number alterations and few somatic alterations other than activating BRAF or NRAS mutations in conventional nevi. A presumed melanoma driver mutation (IDH1Arg132Cys) was revealed in one of the benign nevi. In conclusion, our exome data show significantly lower somatic mutation burden in both Spitz and conventional nevi compared with their malignant counterparts, and high genetic similarity between Spitzoid and conventional melanoma.

Next-generation sequencing of melanomas has unraveled critical driver genes and genomic abnormalities, mostly defined as occurring at high frequency. In addition, less abundant mutations are present that link melanoma to a set of disorders, commonly called RASopathies. These disorders, which include neurofibromatosis and Noonan and Legius syndromes, harbor germline mutations in various RAS/mitogen-activated protein kinase signaling pathway genes. We highlight shared amino acid substitutions between this set of RASopathy mutations and those observed in large-scale melanoma sequencing data, uncovering a significant overlap. We review the evidence that these mutations activate the RAS/mitogen-activated protein kinase pathway in melanoma and are involved in melanomagenesis. Furthermore, we discuss the observations that two or more RASopathy mutations often co-occur in melanoma and may act synergistically on activating the pathway.

Despite the remarkable clinical response of melanoma harboring BRAF mutations to BRAF inhibitors (BRAFi), most tumors become resistant. Here, we identified the downregulation of the ubiquitin ligase RNF125 in BRAFi-resistant melanomas and demonstrated its role in intrinsic and adaptive resistance to BRAFi in cultures as well as its association with resistance in tumor specimens. Sox10/MITF expression correlated with and contributed to RNF125 transcription. Reduced RNF125 was associated with elevated expression of receptor tyrosine kinases (RTKs), including EGFR. Notably, RNF125 altered RTK expression through JAK1, which we identified as an RNF125 substrate. RNF125 bound to and ubiquitinated JAK1, prompting its degradation and suppressing RTK expression. Inhibition of JAK1 and EGFR signaling overcame BRAFi resistance in melanoma with reduced RNF125 expression, as shown in culture and in in vivo xenografts. Our findings suggest that combination therapies targeting both JAK1 and EGFR could be effective against BRAFi-resistant tumors with de novo low RNF125 expression.

Melanoma is an aggressive cancer that is highly resistance to therapies once metastasized. We studied microRNA (miRNA) expression in clinical melanoma subtypes and evaluated different miRNA signatures in the background of gain of function somatic and inherited mutations associated with melanoma. Total RNA from 42 patient derived primary melanoma cell lines and three independent normal primary melanocyte cell cultures was evaluated by miRNA array. MiRNA expression was then analyzed comparing subtypes and additional clinicopathologic criteria including somatic mutations. The prevalence and association of an inherited variant in a miRNA binding site in the 3’UTR of the KRASoncogene, referred to as the KRAS-variant, was also evaluated. We show that seven miRNAs, miR-142-3p, miR-486, miR-214, miR-218, miR-362, miR-650 and miR-31, were significantly correlated with acral as compared to non-acral melanomas (p < 0.04). In addition, we discovered that the KRAS-variant was enriched in non-acral melanoma (25%), and that miR-137 under expression was significantly associated with melanomas with the KRAS-variant. Our findings indicate that miRNAs are differentially expressed in melanoma subtypes and that their misregulation can be impacted by inherited gene variants, supporting the hypothesis that miRNA misregulation reflects biological differences in melanoma.

BRAFV600EKis a frequent mutationally active tumor-specific kinase in melanomas that is currently targeted for therapy by the specific inhibitor PLX4032. Our studies with melanoma tumor cells that are BRAFV600EKand BRAFWTshowed that, paradoxically, while PLX4032 inhibited ERK12 in the highly sensitive BRAFV600EK, it activated the pathway in the resistant BRAFWTcells, via RAF1 activation, regardless of the status of mutations in NRAS or PTEN. The persistently active ERK12 triggered downstream effectors in BRAFWTmelanoma cells and induced changes in the expression of a wide-spectrum of genes associated with cell cycle control. Furthermore, PLX4032 increased the rate of proliferation of growth factor-dependent NRAS Q61L mutant primary melanoma cells, reduced cell adherence and increased mobility of cells from advanced lesions. The results suggest that the drug can confer an advantage to BRAFWTprimary and metastatic tumor cells in vivo and provide markers for monitoring clinical responses.

HMB45 is a mouse monoclonal antibody raised against Pmel17/gp100, a melanoma-specific marker, which is routinely used in the diagnosis of primary cutaneous malignant melanoma. The standard expression pattern for a positive HMB45 staining result on immunohistochemistry is based upon the results of chromogenic-based methods. We re-evaluated the patterns of HMB45 staining across the 480-core ‘SPORE melanoma progression array’ containing lesions representing the spectrum of melanocytic lesions ranging from thin nevus to visceral metastasis using the fluorescence-based staining technique and automated quantitative analysis (AQUA) of the obtained digital images. The methods validated the expected cytoplasmic HMB45 staining pattern in 70/108 malignant lesions and in the epithelial components of nevus specimens. However, the fluorescence-based approach revealed a nuclear gp100 localization present in the dermal component of all nevi that was not seen before. This nuclear localization could not be observed on routine chromogenic stains, because the standard hematoxylin nuclear counterstain overwhelms the weak nuclear HMB45 stain. The thin (0.450±0.253) and thick (0.513±0.227) nevi had strongly positive mean ln(nuclear/non-nuclear AQUA score ratios), which are significantly higher than those from the group of malignant lesions (P<0.0001). This finding was reproduced on a smaller but independent progression array composed of nevi and melanomas from the Yale Pathology archives (P<0.01). The odds ratio associated with a sample being a nevus was 2.24 (95% CI: 1.87–2.69, P<0.0001) for each 0.1 unit increase of the ln(nuclear/non-nuclear AQUA score ratio) to yield an ROC curve with 0.93 units of area and a simultaneously maximized sensitivity of 0.92 and specificity of 0.80 for distinguishing benign nevi from malignant melanomas. On the basis of this preliminary study, we propose that the ratio of nuclear to non-nuclear HMB45 staining may be useful for diagnostic challenges in melanocytic lesions.

Investigators representing all major melanoma research areas present an overview of the most important challenges for the field. Four major research areas are covered plus the training of new investigators. For each area we first describe the present status, its strengths and weaknesses, and then outline specific recommendations. In basic research of melanoma, we outline the pertinent issues for melanoma classification, understanding melanocyte development and transformation, melanoma resistance, tumor microenvironment, metastasis, animal models, immune response, and blood and tissue diagnostics. In clinical research we provide an overview of the current challenges and the strategies for characterization, monitoring, and therapy. It will be important to develop strong research and clinical infrastructures by establishing tumor banks, identifying and validating biomarkers, developing new imaging techniques, and increasing multidisciplinary collaboration and communication. To strengthen the field we need to recruit both young and established investigators and foster career development plans that cover all disciplines. Recent research advances provide significant opportunities to have a major impact on this devastating disease. This group provides recommendations for both short- and long-term strategies that build on research strengths and opportunities established by the many members of the research community.

Abstract Rb is a tumor suppressor that represses the expression of E2F regulated genes required for cell cycle progression. It is inactivated in melanomas and other cancer cells by phosphorylation catalyzed by persistent cyclin dependent kinase (CDK) activity. CDK activity is sustained in melanoma cells mostly by the elimination of the CDK inhibitor p16INK4A and by high levels of cyclins whose expression is maintained by stimuli emanating from activated cell surface receptors and/or mutated intracellular intermediates, such as N-Ras and B-Raf. However, Rb also suppresses the expression of apoptosis genes, and its presence protects normal melanocytes from cell death. Its high expression in human melanoma cells and tumors suggests a similar role in malignant cells as well. The differential release and suppression of E2F transcriptional activity is likely to depend on promoter-specific E2F/Rb interaction. Phosphorylated Rb is displaced from cell cycle genes but not from others. In addition, Rb gene repression is dependent on the nature of Rb-E2F interaction and the activity of the Rb-bound proteins recruited to the promoter. Deciphering the differences in Rb/E2F complex formation in normal and malignant melanocytes is likely to shed light on the mechanism by which Rb can exert tumor suppressing and promoting activities in this cellular system. The Rb/E2F pathway provides opportunities for efficient therapy at multiple levels. Novel drugs can reactivate Rb potential to suppress growth cycle promoting genes. In addition, the high E2F transcriptional activity in melanoma cells can be exploited to deliver cytotoxic molecules specifically to tumors, sparing the normal tissues.

In tyrosinase-positive amelanotic melanoma cells, inactive tyrosinase accumulates in the endoplasmic reticulum. Based on studies described here, we propose that aberrant vacuolar proton ATPase (V-ATPase)-mediated proton transport in melanoma cells disrupts tyrosinase trafficking through the secretory pathway. Amelanotic but not melanotic melanoma cells or normal melanocytes display elevated proton export as observed by the acidification of the extracellular medium and their ability to maintain neutral intracellular pH. Tyrosinase activity and transit through the Golgi were restored by either maintaining the melanoma cells in alkaline medium (pH 7.4–7.7) or by restricting glucose uptake. The translocation of tyrosinase out of the endoplasmic reticulum and the induction of cell pigmentation in the presence of the ionophore monensin or the specific V-ATPase inhibitors concanamycin A and bafilomycin A1 supported a role for V-ATPases in this process. Because it was previously shown that V-ATPase activity is increased in solid tumors in response to an acidified environment, the appearance of hypopigmented cells in tyrosinase-positive melanoma tumors may indicate the onset of enhanced glycolysis and extracellular acidification, conditions known to favor metastatic spread and resistance to weak base chemotherapeutic drugs.

Tyrosinase is essential for pigmentation and is a source of tumor-derived antigenic peptides and cellular immune response. Wild type tyrosinase in melanoma cells and certain albino mutants in untransformed melanocytes are targeted to proteolytic degradation by the 26 S proteasome due to retention of the misfolded protein in the endoplasmic reticulum and its subsequent retranslocation to the cytosol. Here, we demonstrate that the substrates DOPA and tyrosine induced in melanoma cells a transition of misfolded wild type tyrosinase to the native form that is resistant to proteolysis, competent to exit the endoplasmic reticulum, and able to produce melanin. Because the enzymatic activity of tyrosinase is induced by DOPA, we propose that proper folding of the wild type protein, just like mutant forms, is tightly linked to its catalytic state. Loss of pigmentation, therefore, in tyrosinase-positive melanoma cells is a consequence of tumor-induced metabolic changes that suppress tyrosinase activity and DOPA production within these cells.

Tyrosinase is a type I membrane glycoprotein essential for melanin synthesis. Mutations in tyrosinase lead to albinism due, at least in part, to aberrant retention of the protein in the endoplasmic reticulum and subsequent degradation by the cytosolic ubiquitin-proteasomal pathway. A similar premature degradative fate for wild type tyrosinase also occurs in amelanotic melanoma cells. To understand critical cotranslational events, the glycosylation and rate of translation of tyrosinase was studied in normal melanocytes, melanoma cells, an in vitrocell-free system, and semi-permeabilized cells. Site-directed mutagenesis revealed that all seven N-linked consensus sites are utilized in human tyrosinase. However, glycosylation at Asn-290 (Asn-Gly-Thr-Pro) was suppressed, particularly when translation proceeded rapidly, producing a protein doublet with six or sevenN-linked core glycans. The inefficient glycosylation of Asn-290, due to the presence of a proximal Pro, was enhanced in melanoma cells possessing 2–3-fold faster (7.7–10.0 amino acids/s) protein translation rates compared with normal melanocytes (3.5 amino acids/s). Slowing the translation rate with the protein synthesis inhibitor cycloheximide increased the glycosylation efficiency in live cells and in the cell-free system. Therefore, the rate of protein translation can regulate the level of tyrosinaseN-linked glycosylation, as well as other potential cotranslational maturation events.

Inactivation of the retinoblastoma tumor suppressor protein (pRb) has been implicated in melanoma cells, but the molecular basis for this phenotype has not yet been elucidated, and the status of additional family members (p107 and p130, together termed pocket proteins) or the consequences on downstream targets such as E2F transcription factors are not known. Because cell cycle progression is dependent on the transcriptional activity of E2F family members (E2F1–E2F6), most of them regulated by suppressive association with pocket proteins, we characterized E2F–pocket protein DNA binding activity in normal versus malignant human melanocytes. By gel shift analysis, we show that in mitogen-dependent normal melanocytes, external growth factors tightly controlled the levels of growth-promoting free E2F DNA binding activity, composed largely of E2F2 and E2F4, and the growth-suppressive E2F4–p130 complexes. In contrast, in melanoma cells, free E2F DNA binding activity (E2F2 and E2F4, to a lesser extent E2F1, E2F3, and occasionally E2F5), was constitutively maintained at high levels independently of external melanocyte mitogens. E2F1 was the only family member more abundant in the melanoma cells compared with normal melanocytes, and the approximately fivefold increase in DNA binding activity could be accounted for mostly by a similar increase in the levels of the dimerization partner DP1. The continuous high expression of cyclin D1, A2, and E, the persistent cyclin-dependent kinase 4 (CDK4) and CDK2 activities, and the presence of hyperphosphorylated forms of pRb, p107, and p130, suggest that melanoma cells acquired the capacity for autonomous growth through inactivation of all three pocket proteins and release of E2F activity, otherwise tightly regulated in normal melanocytes by external growth factors.

Cell lineage analysis aims to uncover the developmental history of an organism back to its cell of origin. Recently, novel in vivomethods utilizing genome editing enabled important insights into the cell lineages of animals. In contrast, human cell lineage remains restricted to retrospective approaches, which still lack resolution and cost-efficient solutions. Here, we demonstrate a scalable platform based on short tandem repeats targeted by duplex molecular inversion probes. With this human cell lineage tracing method, we accurately reproduced a known lineage of DU145 cells and reconstructed lineages of healthy and metastatic single cells from a melanoma patient who matched the anatomical reference while adding further refinements. This platform allowed us to faithfully recapitulate lineages of developmental tissue formation in healthy cells. In summary, our lineage discovery platform can profile informative somatic mutations efficiently and provides solid lineage reconstructions even in challenging low-mutation-rate healthy single cells.

Transformation of normal melanocytes to metastatic melanoma cells is characterized by loss of dependency on external growth factors required for the viability and proliferation of normal melanocytes. The molecular events that lead to melanoma cell autonomous growth are not well defined, but are likely to include sustained activity of cyclin-dependent kinases (CDK2, CDK4 and CDK6) as a result of loss of CDK inhibitors (such as p16INK4aand possibly p27KIP1), and persistent upregulation of several cyclins (cyclin D1, cyclin A and cyclin E), the positive regulators of CDKs. CDKs phosphorylate, and consequently, inactivate the retinoblastoma family of tumor suppressor proteins (pRb, p107 and p130), termed pocket proteins. The inactivation of pocket proteins liberates E2F transcription factors from suppressive complexes ('free' E2F) that, in turn, induces the continuous expression of target genes whose products promote cell cycle progression. In normal melanocytes, external growth factors suppress the activity of all three pocket proteins, allowing E2F activity to accumulate and sustain transcription of target genes required for cell proliferation. In contrast, in melanoma cells from advanced lesions, all three pocket proteins are highly phosphorylated and inactive, even in the absence of environmental mitogens, and free E2F activity is constitutively high. Manipulations of normal mouse melanocytes in vitro, and in vivoin transgenic mouse expressing ectopic genes, further support the notion that growth rate, and release from dependency on external mitogens, positively correlate with inactivation of pocket proteins. The latter has been accomplished by sustained cell surface receptor stimulation, such as constitutive high expression of a growth factor, or by sequestration with dominantly acting viral proteins. Taken together, chronic hyperphosphorlyation/inactivation of pRb, p107 and p130 is probably one of the key events in converting growth-factor dependent normal melanocytes, to autonomously growing melanoma cells. Since all pocket proteins are regulated by CDKs activity, it is likely that agents that inhibit this class of enzymes will be effective in treating melanoma patients.

Melanomas are highly variable with respect to aberrant gene expression and chromosomal lesions but share a common characteristic of an acquired independence from environmental growth factors that are needed for proliferation of normal melanocytes. Receptors with tyrosine kinase activity play a critical role in normal melanocyte proliferation and in the uncontrolled growth of melanomas. Normal human melanocytes depend on exogenous peptide growth factors such as basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), or mast cell growth factor (MGF), all of which stimulate receptors with tyrosine kinase activity. In contrast, human melanoma cells from primary nodular and metastatic lesions grow autonomously partially because of inappropriate production of bFGF and continuous activation of the bFGF-receptor kinase. Animal models also provide evidence for the importance of receptor-tyrosine kinases in normal melanocyte proliferation and in malignant transformation. In the mouse, genes residing in three loci in which inactivation mutations lead to piebaldism, the dominant spotting (W), patch (Ph), and Sl encode, respectively, the receptor-kinases c-kit and platelet derived growth factor receptor, and the ligand for c-kit: MGF. In vivo transformation of mouse melanocytes to melanoma, due to constitutive expression of a transmembrane tyrosine kinase, the oncogene ret, was recently demonstrated in transgenic mice. Studies on a fish model, Xiphophorus, in which melanoma is inherited, showed that the dominant tumor inducing gene, Tu, encodes an EGF-receptor related tyrosine kinase which is expressed only in melanomas and not in normal tissues. Taken together, the results suggest that the uncontrolled growth of melanomas is due, in large part, to constitutive activation of receptors with tyrosine kinase activity.

Summary: A major obstacle to applying the techniques of molecular biology to the genetics and cell biology of pigmentation has been our inability to grow normal murine melanocytes in culture. We report here the establishment and characterization of continuously proliferating cultures of cutaneous pigment cells from seven strains of mice. Melanocytes were grown from the dermis of newborn mice in medium containing 12-0-tetradecanoyl-13-phorbol-acetate; a substance, such as melanotropin, that raises intracellular levels of cyclic AMP; and an extract made from human placenta.

Summary: Normal human melanocytes, unlike pigment cells from metastatic melanomas, do not survive in culture in routine, serum-supplemented media. The search for natural growth factors for melanocytes has shown that mitogenic activity is ubiquitous in several tissues and in melanomas. Of several known growth factors tested, basic fibroblast growth factor (bFGF) was the only one mitogenic for melanocytes but only in the presence of cyclic-adenosine-monophosphate (cAMP) stimulators. The mitogenic activity toward melanocytes in tissues and melanoma cell extracts had high affinity for heparin and antibodies to bFGF synthetic peptides. These results suggest that one of the growth factors for melanocytes might be bFGF or a bFGF-like polypeptide and that autocrine production of bFGF-like molecules by melanoma cells may contribute to the malignant phenotype of melanocytes. Because acidic FGF (aFGF) did not stimulate growth, the receptors for bFGF on melanocytes might be significantly different from those for a FGF.

Summary: The main obstacle to establishing pure normal human melanocytes in vitro is contamination of the cultures by fibroblasts. The obstacle can be overcome by selective destruction of fibroblasts with geneticin (G418 sulfate). Treatment of mixed cultures with this drug at a concentration of 100 μg/ml for two days results in pure cultures of normal human melanocytes.

Karyotype analysis of first passage cells from a melanoma, occurring in a patient with a giant congenital nevus, revealed monosomy of chromosomes #6 and #11, and trisomy of chromosomes #8 and #22. Five marker chromosomes were present, including two ring chromosomes. The origin of three of the marker chromosomes was determined, and all were found to contain the region 6p21.1–6p23. The ring chromosomes were also thought to contain this region, to which the major histocompatibility locus has been mapped. The significance of these findings is discussed in the light of recent reports of chromosome studies of melanoma cells.

Four vascular endothelial growth factor (VEGF) splice variants containing 121, 165, 189, and 206 amino acids are produced from a single human gene as a result of alternative splicing. VEGF121is not a heparin-binding protein, while the other VEGF species possess heparin binding ability. YU-ZAZ6 human melanoma cells expressed the mRNA encoding the VEGF receptor flt-1,but not the mRNA encoding the VEGF receptor KDR/flk-1.Both VEGF121and VEGF165bound to the VEGF receptors of these cells. Unexpectedly, heparin inhibited the binding of VEGF121as well as the binding of VEGF165to the VEGF receptors of the melanoma cells. Digestion of the cells with heparinase also inhibited the binding of both VEGF variants. The VEGF165binding ability of heparinase-digested cells could be partially restored by the addition of exogenous heparin to the binding reaction. In contrast, the addition of heparin to heparinase-digested cells did not restore VEGF121binding. These results suggest that cell-surface heparan sulfates may regulate the binding ability of the VEGF receptors of the melanoma cells. They also indicate that heparin is not able to fully substitute for cell surface-associated heparan sulfates since VEGF121binding to the VEGF receptors of heparinase-treated cells is not restored by heparin. These data suggest that changes in the composition of cell-surface heparin-like molecules may differentially affect the interaction of various VEGF isoforms with VEGF receptors.

La plupart des mélanomes présentent une hyperactivation de la voie des MAPK secondaire à une mutation activatrice de BRAF (50 %) ou NRAS (20 %). La voie des MAPK est soumise à plusieurs formes de régulation. Un exemple est celui de l’AMP cyclique (AMPc) qui par la phosphorylation de la protéine kinase A (PKA) inhibe CRAF. L’AMPc est produit par 2 types d’enzymes : l’adénylate cyclase transmembranaire et une adénylate cyclase soluble. L’expression de sAC est dérégulée dans de nombreux cancers dont le mélanome. De plus, sAC est mutée dans 10 % des mélanomes et est localisée différemment dans les mélanocytes tumoraux suggérant qu’elle pourrait jouer un rôle dans la carcinogenèse cutanée.

THE cyclic forms of AMP, GMP and CMP have been implicated as agents which either stimulate or inhibit cell division. Pastan et al. reviewed this topic1and others have dealt with it2–6. Our studies of the growth of cultured melanoma cells have focused on the inhibitory effects of cyclic AMP7. We analysed variant cells that can grow in the presence of agents, such as melanocyte stimulating hormone (MSH), which increase the intracellular levels of cyclic AMP and inhibit the growth of wild-type cells. We describe here a class of variants which are not only resistant to the inhibition, but require cyclic AMP to grow at the rate normally observed in wild-type cells. In one such variant, division was markedly stimulated when either MSH, theophylline, isobutyl methyl xanthine, cholera toxin, prostaglandin E1(PGE1) or dibutyryl cyclic AMP was added to the medium. Each agent inhibited the growth of wild-type cells. The cells were temperature-sensitive in that they expressed the variant phenotype at 37 °C, but behaved as the wild type at 40 °C. Studies of dose responses to dibutyryl cyclic AMP revealed that growth was also enhanced in the wild type by lower concentrations of the nucleotide (10−5M), a fact that had gone unnoticed in melanoma cells but has been reported for other cell types1. These findings suggest a stimulatory as well as inhibitory role for cyclic AMP in control of growth of melanoma cells.

This laboratory has established in previous studies that Pmel 17, a gene expressed specifically in melanocytes, maps near the silver coat color locus (si/si) on mouse chromosome 10. In the current study, we have focused on determining whether or not the si allele carries a mutation in Pmel 17. Pmel 17 cDNA clones, isolated from wild-type and si/si murine melanocyte cDNA libraries, were sequenced and compared. A single nucleotide (A) insertion was found in the putative cytoplasmic tail of the sUsi Pmel 17 cDNA clone. This insertion is predicted to alter the last 24 amino acids at the C-terminus. Also predicted is the extension of the Pmel 17 protein by 12 residues because a new termination signal created downstream from the wild-type reading frame. The mutation was confirmed by the sequence of the PCR-amplified genomic region flanking and including the mutation site. The fact that si/si Pmel 17 was not recognized by antibodies directed toward the C-terminal 15 amino acids of wild-type Pmel 17, indicated a defect in this region. We conclude from these results that silverpmel 17 protein has a major defect at the carboxyl terminus. The chromosomal location and the identification of a potentially pathologic mutation in si-Pmel 17 support our conclusion that Pmel 17 is encoded at the silver locus.

Doxorubicin (DXR) conjugated to a monoclonal antibody (MAb), 9.2.27, which recognizes a human melanoma-associated proteoglycan, effectively suppresses the growth of human melanoma xenografts and prolongs the life span of tumor-bearing athymk nude (nulnu) mice. We have investigated further the mechanism(s) of this in viva antitu-mor activity. Our results indicate that following iv injection, the DXR-MAb 9.2.27 conjugate is cleared from the circulation with typical Diphasic kinetics, similar to the clearance process of the unconjugated MAb 9.2.27. In contrast, less than 10% of injected dose per milliliter of blood is found in the circulation at any given time after ip injection. Toxicity studies further indicate that DXR-MAb 9.2.27 conjugate is less toxic in vivo than the freely administered DXR, which is known to cause considerable cardiotoxic effects. Direct autoradiography demonstrates that the DXR-MAb 9.2.27 conjugate binds specifically to the tissue sections derived from a human melanoma xenograft of a nude mouse. A critical evaluation is given of the relevance of these findings and their impact on the design of future strategies for the immunochemotherapy of malignant melanoma. [J Natl Cancer Inst 1988;80:l154–1159]