Topical therapy for regression and melanoma prevention of congenital giant nevi.

Giant congenital melanocytic nevi are NRAS-driven proliferations that may cover up to 80% of the body surface. Their most dangerous consequence is progression to melanoma. This risk often triggers preemptive extensive surgical excisions in childhood, producing severe lifelong challenges. We have presented preclinical models, including multiple genetically engineered mice and xenografted human lesions, which enabled testing locally applied pharmacologic agents to avoid surgery. The murine models permitted the identification of proliferative versus senescent nevus phases and treatments targeting both. These nevi recapitulated the histologic and molecular features of human giant congenital nevi, including the risk of melanoma transformation. Cutaneously delivered MEK, PI3K, and c-KIT inhibitors or proinflammatory squaric acid dibutylester (SADBE) achieved major regressions. SADBE triggered innate immunity that ablated detectable nevocytes, fully prevented melanoma, and regressed human giant nevus xenografts. These findings reveal nevus mechanistic vulnerabilities and suggest opportunities for topical interventions that may alter the therapeutic options for children with congenital giant nevi.

Skin ß-endorphin mediates addiction to UV light.

UV light is an established carcinogen, yet evidence suggests that UV-seeking behavior has addictive features. Following UV exposure, epidermal keratinocytes synthesize proopiomelanocortin (POMC) that is processed to melanocyte-stimulating hormone, inducing tanning. We show that, in rodents, another POMC-derived peptide, ß-endorphin, is coordinately synthesized in skin, elevating plasma levels after low-dose UV. Increases in pain-related thresholds are observed and reversed by pharmacologic opioid antagonism. Opioid blockade also elicits withdrawal signs after chronic UV exposure. This effect was sufficient to guide operant behavioral choices to avoidance of opioid withdrawal (conditioned place aversion). These UV-induced nociceptive and behavioral effects were absent in ß-endorphin knockout mice and in mice lacking p53-mediated POMC induction in epidermal keratinocytes. Although primordial UV addiction, mediated by the hedonic action of ß-endorphin and anhedonic effects of withdrawal, may theoretically have enhanced evolutionary vitamin D biosynthesis, it now may contribute to the relentless rise in skin cancer incidence in humans.

B-cell-specific checkpoint molecules that regulate anti-tumour immunity.

The role of B cells in anti-tumour immunity is still debated and, accordingly, immunotherapies have focused on targeting T and natural killer cells to inhibit tumour growth1,2. Here, using high-throughput flow cytometry as well as bulk and single-cell RNA-sequencing and B-cell-receptor-sequencing analysis of B cells temporally during B16F10 melanoma growth, we identified a subset of B cells that expands specifically in the draining lymph node over time in tumour-bearing mice. The expanding B cell subset expresses the cell surface molecule T cell immunoglobulin and mucin domain 1 (TIM-1, encoded by Havcr1) and a unique transcriptional signature, including multiple co-inhibitory molecules such as PD-1, TIM-3, TIGIT and LAG-3. Although conditional deletion of these co-inhibitory molecules on B cells had little or no effect on tumour burden, selective deletion of Havcr1 in B cells both substantially inhibited tumour growth and enhanced effector T cell responses. Loss of TIM-1 enhanced the type 1 interferon response in B cells, which augmented B cell activation and increased antigen presentation and co-stimulation, resulting in increased expansion of tumour-specific effector T cells. Our results demonstrate that manipulation of TIM-1-expressing B cells enables engagement of the second arm of adaptive immunity to promote anti-tumour immunity and inhibit tumour growth.

Intratumoral Activity of the CXCR3 Chemokine System Is Required for the Efficacy of Anti-PD-1 Therapy.

Despite compelling rates of durable clinical responses to programmed cell death-1 (PD-1) blockade, advances are needed to extend these benefits to resistant tumors. We found that tumor-bearing mice deficient in the chemokine receptor CXCR3 responded poorly to anti-PD-1 treatment. CXCR3 and its ligand CXCL9 were critical for a productive CD8+ T cell response in tumor-bearing mice treated with anti-PD-1 but were not required for the infiltration of CD8+ T cells into tumors. The anti-PD-1-induced anti-tumor response was facilitated by CXCL9 production from intratumoral CD103+ dendritic cells, suggesting that CXCR3 facilitates dendritic cell-T cell interactions within the tumor microenvironment. CXCR3 ligands in murine tumors and in plasma of melanoma patients were an indicator of clinical response to anti-PD-1, and their induction in non-responsive murine tumors promoted responsiveness to anti-PD-1. Our data suggest that the CXCR3 chemokine system is a biomarker for sensitivity to PD-1 blockade and that augmenting the intratumoral function of this chemokine system could improve clinical outcomes.

A polymorphism in IRF4 affects human pigmentation through a tyrosinase-dependent MITF/TFAP2A pathway.

Sequence polymorphisms linked to human diseases and phenotypes in genome-wide association studies often affect noncoding regions. A SNP within an intron of the gene encoding Interferon Regulatory Factor 4 (IRF4), a transcription factor with no known role in melanocyte biology, is strongly associated with sensitivity of skin to sun exposure, freckles, blue eyes, and brown hair color. Here, we demonstrate that this SNP lies within an enhancer of IRF4 transcription in melanocytes. The allele associated with this pigmentation phenotype impairs binding of the TFAP2A transcription factor that, together with the melanocyte master regulator MITF, regulates activity of the enhancer. Assays in zebrafish and mice reveal that IRF4 cooperates with MITF to activate expression of Tyrosinase (TYR), an essential enzyme in melanin synthesis. Our findings provide a clear example of a noncoding polymorphism that affects a phenotype by modulating a developmental gene regulatory network.

Spatiotemporally resolved transcriptomics reveals the subcellular RNA kinetic landscape.

Spatiotemporal regulation of the cellular transcriptome is crucial for proper protein expression and cellular function. However, the intricate subcellular dynamics of RNA remain obscured due to the limitations of existing transcriptomics methods. Here, we report TEMPOmap-a method that uncovers subcellular RNA profiles across time and space at the single-cell level. TEMPOmap integrates pulse-chase metabolic labeling with highly multiplexed three-dimensional in situ sequencing to simultaneously profile the age and location of individual RNA molecules. Using TEMPOmap, we constructed the subcellular RNA kinetic landscape in various human cells from transcription and translocation to degradation. Clustering analysis of RNA kinetic parameters across single cells revealed 'kinetic gene clusters' whose expression patterns were shaped by multistep kinetic sculpting. Importantly, these kinetic gene clusters are functionally segregated, suggesting that subcellular RNA kinetics are differentially regulated in a cell-state- and cell-type-dependent manner. Spatiotemporally resolved transcriptomics provides a gateway to uncovering new spatiotemporal gene regulation principles.

Hyperactivation of sympathetic nerves drives depletion of melanocyte stem cells.

Empirical and anecdotal evidence has associated stress with accelerated hair greying (formation of unpigmented hairs)1,2, but so far there has been little scientific validation of this link. Here we report that, in mice, acute stress leads to hair greying through the fast depletion of melanocyte stem cells. Using a combination of adrenalectomy, denervation, chemogenetics3,4, cell ablation and knockout of the adrenergic receptor specifically in melanocyte stem cells, we find that the stress-induced loss of melanocyte stem cells is independent of immune attack or adrenal stress hormones. Instead, hair greying results from activation of the sympathetic nerves that innervate the melanocyte stem-cell niche. Under conditions of stress, the activation of these sympathetic nerves leads to burst release of the neurotransmitter noradrenaline (also known as norepinephrine). This causes quiescent melanocyte stem cells to proliferate rapidly, and is followed by their differentiation, migration and permanent depletion from the niche. Transient suppression of the proliferation of melanocyte stem cells prevents stress-induced hair greying. Our study demonstrates that neuronal activity that is induced by acute stress can drive a rapid and permanent loss of somatic stem cells, and illustrates an example in which the maintenance of somatic stem cells is directly influenced by the overall physiological state of the organism.

Lineage-specific transcriptional regulation of DICER by MITF in melanocytes.

DICER is a central regulator of microRNA maturation. However, little is known about mechanisms regulating its expression in development or disease. While profiling miRNA expression in differentiating melanocytes, two populations were observed: some upregulated at the pre-miRNA stage, and others upregulated as mature miRNAs (with stable pre-miRNA levels). Conversion of pre-miRNAs to fully processed miRNAs appeared to be dependent upon stimulation of DICER expression--an event found to occur via direct transcriptional targeting of DICER by the melanocyte master transcriptional regulator MITF. MITF binds and activates a conserved regulatory element upstream of DICER's transcriptional start site upon melanocyte differentiation. Targeted KO of DICER is lethal to melanocytes, at least partly via DICER-dependent processing of the pre-miRNA-17 approximately 92 cluster thus targeting BIM, a known proapoptotic regulator of melanocyte survival. These observations highlight a central mechanism underlying lineage-specific miRNA regulation which could exist for other cell types during development.

Protection from UV light is an evolutionarily conserved feature of the haematopoietic niche.

Haematopoietic stem and progenitor cells (HSPCs) require a specific microenvironment, the haematopoietic niche, which regulates HSPC behaviour1,2. The location of this niche varies across species, but the evolutionary pressures that drive HSPCs to different microenvironments remain unknown. The niche is located in the bone marrow in adult mammals, whereas it is found in other locations in non-mammalian vertebrates, for example, in the kidney marrow in teleost fish. Here we show that a melanocyte umbrella above the kidney marrow protects HSPCs against ultraviolet light in zebrafish. Because mutants that lack melanocytes have normal steady-state haematopoiesis under standard laboratory conditions, we hypothesized that melanocytes above the stem cell niche protect HSPCs against ultraviolet-light-induced DNA damage. Indeed, after ultraviolet-light irradiation, unpigmented larvae show higher levels of DNA damage in HSPCs, as indicated by staining of cyclobutane pyrimidine dimers and have reduced numbers of HSPCs, as shown by cmyb (also known as myb) expression. The umbrella of melanocytes associated with the haematopoietic niche is highly evolutionarily conserved in aquatic animals, including the sea lamprey, a basal vertebrate. During the transition from an aquatic to a terrestrial environment, HSPCs relocated into the bone marrow, which is protected from ultraviolet light by the cortical bone around the marrow. Our studies reveal that melanocytes above the haematopoietic niche protect HSPCs from ultraviolet-light-induced DNA damage in aquatic vertebrates and suggest that during the transition to terrestrial life, ultraviolet light was an evolutionary pressure affecting the location of the haematopoietic niche.

Functional genomics of human clear cell sarcoma: genomic, transcriptomic and chemical biology landscape for clear cell sarcoma.

BACKGROUND: Systemic therapy for metastatic clear cell sarcoma (CCS) bearing EWSR1-CREB1/ATF1 fusions remains an unmet clinical need in children, adolescents, and young adults. METHODS: To identify key signaling pathway vulnerabilities in CCS, a multi-pronged approach was taken: (i) genomic and transcriptomic landscape analysis, (ii) integrated chemical biology interrogations, (iii) development of CREB1/ATF1 inhibitors, and (iv) antibody-drug conjugate testing (ADC). The first approach encompassed DNA exome and RNA deep sequencing of the largest human CCS cohort yet reported consisting of 47 patient tumor samples and 8 cell lines. RESULTS: Sequencing revealed recurrent mutations in cell cycle checkpoint, DNA double-strand break repair or DNA mismatch repair genes, with a correspondingly low to intermediate tumor mutational burden. DNA multi-copy gains with corresponding high RNA expression were observed in CCS tumor subsets. CCS cell lines responded to the HER3 ADC patritumab deruxtecan in a dose-dependent manner in vitro, with impaired long term cell viability. CONCLUSION: These studies of the genomic, transcriptomic and chemical biology landscape represent a resource 'atlas' for the field of CCS investigation and drug development. CHK inhibitors are identified as having potential relevance, CREB1 inhibitors non-dependence of CCS on CREB1 activity was established, and the potential utility of HER3 ADC being used in CCS is found.

In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target.

Immunotherapy with PD-1 checkpoint blockade is effective in only a minority of patients with cancer, suggesting that additional treatment strategies are needed. Here we use a pooled in vivo genetic screening approach using CRISPR-Cas9 genome editing in transplantable tumours in mice treated with immunotherapy to discover previously undescribed immunotherapy targets. We tested 2,368 genes expressed by melanoma cells to identify those that synergize with or cause resistance to checkpoint blockade. We recovered the known immune evasion molecules PD-L1 and CD47, and confirmed that defects in interferon-γ signalling caused resistance to immunotherapy. Tumours were sensitized to immunotherapy by deletion of genes involved in several diverse pathways, including NF-κB signalling, antigen presentation and the unfolded protein response. In addition, deletion of the protein tyrosine phosphatase PTPN2 in tumour cells increased the efficacy of immunotherapy by enhancing interferon-γ-mediated effects on antigen presentation and growth suppression. In vivo genetic screens in tumour models can identify new immunotherapy targets in unanticipated pathways.

The Complex Interplay between Nevi and Melanoma: Risk Factors and Precursors.

One effort to combat the rising incidence of malignant melanoma is focused on early detection by the clinical and dermoscopic screening of melanocytic nevi. However, the interaction between nevi, which are congenital or acquired benign melanocytic proliferations, and melanoma is still enigmatic. On the one hand, the majority of melanomas are thought to form de novo, as only a third of primary melanomas are associated with a histologically identifiable nevus precursor. On the other hand, an increased number of melanocytic nevi is a strong risk factor for developing melanoma, including melanomas that do not derive from nevi. The formation of nevi is modulated by diverse factors, including pigmentation, genetic risk factors, and environmental sun exposure. While the molecular alterations that occur during the progression of a nevus to melanoma have been well characterized, many unanswered questions remain surrounding the process of nevus to melanoma evolution. In this review, we discuss clinical, histological, molecular, and genetic factors that influence nevus formation and progression to melanoma.

Skin pigmentation and its control: From ultraviolet radiation to stem cells.

In the light of substantial discoveries in epithelial and hair pigmentation pathophysiology, this review summarizes the current understanding of skin pigmentation mechanisms. Melanocytes are pigment-producing cells, and their key regulating transcription factor is the melanocyte-specific microphthalmia-associated transcription factor (m-MITF). Ultraviolet (UV) radiation is a unique modulator of skin pigmentation influencing tanning pathways. The delayed tanning pathway occurs as UVB produces keratinocyte DNA damage, causing p53-mediated expression of the pro-opiomelanocortin (POMC) gene that is processed to release α-melanocyte-stimulating hormone (α-MSH). α-MSH stimulates the melanocortin 1 receptor (MC1R) on melanocytes, leading to m-MITF expression and melanogenesis. POMC cleavage also releases ß-endorphin, which creates a neuroendocrine pathway that promotes UV-seeking behaviours. Mutations along the tanning pathway can affect pigmentation and increase the risk of skin malignancies. MC1R variants have received considerable attention, yet the allele is highly polymorphic with varied phenotypes. Vitiligo presents with depigmented skin lesions due to autoimmune destruction of melanocytes. UVB phototherapy stimulates melanocyte stem cells in the hair bulge to undergo differentiation and upwards migration resulting in perifollicular repigmentation of vitiliginous lesions, which is under sophisticated signalling control. Melanocyte stem cells, normally quiescent, undergo cyclic activation/differentiation and downward migration with the hair cycle, providing pigment to hair follicles. Physiological hair greying results from progressive loss of melanocyte stem cells and can be accelerated by acute stress-induced, sympathetic driven hyperproliferation of the melanocyte stem cells. Ultimately, by reviewing the pathways governing epithelial and follicular pigmentation, numerous areas of future research and potential points of intervention are highlighted.

Melanocortin 1 receptor is dispensable for acute stress induced hair graying in mice.

Stress is a risk factor for many skin conditions, but the cellular and molecular mechanisms of its impacts have only begun to be revealed. In mice, acute stress induces loss of melanocyte stem cells (MeSCs) and premature hair greying. Our previous work demonstrated that the loss of MeSCs upon acute stress is caused by the hyperactivation of the sympathetic nervous system. Stress also induces the secretion of stress hormones from the hypothalamic-pituitary-adrenal (HPA) axis; however, whether stress hormones are involved in the hair greying process has not been fully examined. In particular, the adrenocorticotropic hormone (ACTH) is released from the pituitary glands upon stress. ACTH is a ligand for the melanocortin 1 receptor (MC1R), which plays critical roles in regulating MeSC migration and skin pigmentation. We investigated whether the MC1R pathway is required for the stress-induced hair greying. We confirmed that MC1R is the major melanocortin receptor expressed in MeSCs. However, induction of acute stress via resiniferatoxin (RTX) injection still leads to hair greying in Mc1r mutant mice, suggesting that the ACTH-MC1R pathway is not a major contributor in acute stress-induced premature hair greying.

Stress-associated ectopic differentiation of melanocyte stem cells and ORS amelanotic melanocytes in an ex vivo human hair follicle model.

Hair greying depends on the altered presence and functionality of hair follicle melanocytes. Melanocyte stem cells (MelSCs) reside in the bulge of hair follicles and give rise to migrating and differentiating progeny during the anagen phase. Ageing, genotoxic stress, redox stress and multiple behaviour-associated acute stressors have been seen to induce hair greying by depleting the MelSC pool, a phenomenon which is accompanied by ectopic pigmentation of these cells, followed by their depletion from the stem cell niche. This aberrant differentiation produces a state from which a return to stem cell-like quiescence appears to be lost. The cellular features of stress-induced hair greying have been extensively studied in murine models. Here, we describe a method to assess and quantify human hair follicle MelSC differentiation by measuring ectopically pigmented MelSCs in isolated human hair follicles exposed to specific stress signal mediators. Ionizing radiation, hydrogen peroxide and noradrenaline have been shown to cause hair greying in mice. We demonstrate here that isolated, ex vivo cultured human hair follicles exposed to these treatments display similar ectopic pigmentation within the bulge area which is accompanied by induction of differentiated melanocytic markers. This study suggests that as in murine models, stress signalling induces closely matching phenotypic changes in human hair follicles which can be monitored and studied as a surrogate model for early steps in human hair greying.

PGC-1 coactivators regulate MITF and the tanning response.

The production of pigment by melanocytes tans the skin and protects against skin cancers. UV-exposed keratinocytes secrete α-MSH, which then activates melanin formation in melanocytes by inducing the microphthalmia-associated transcription factor (MITF). We show that PPAR-γ coactivator (PGC)-1α and PGC-1ß are critical components of this melanogenic system in melanocytes. α-MSH signaling strongly induces PGC-1α expression and stabilizes both PGC-1α and PGC-1ß proteins. The PGC-1s in turn activate the MITF promoter, and their expression correlates strongly with that of MITF in human melanoma cell lines and biopsy specimens. Inhibition of PGC-1α and PGC-1ß blocks the α-MSH-mediated induction of MITF and melanogenic genes. Conversely, overexpression of PGC-1α induces pigment formation in cell culture and transgenic animals. Finally, polymorphism studies reveal expression quantitative trait loci in the PGC-1ß gene that correlate with tanning ability and protection from melanoma in humans. These data identify PGC-1 coactivators as regulators of human tanning.

Neural crest state activation in NRAS driven melanoma, but not in NRAS-driven melanocyte expansion.

NRAS mutations are frequently found in many deadly malignancies and are the second most common oncogene driving malignant melanoma. Here, we generate a rapid transient transgenic zebrafish model of NRASQ61R-mutant melanoma. These fish develop extensive melanocytic proliferation in approximately 4 weeks. The majority of these lesions do not engraft upon transplantation and lack overt histologic features of malignancy. Our previous work demonstrated that activation of a neural crest cell transcriptional program is a key initiating event in zebrafish BRAF/p53-driven melanomas using the fluorescent reporter crestin:EGFP. By 8-12 weeks of age, some lesions progress to malignant melanoma and have cytologic atypia, destructive tissue invasion, and express neural crest progenitor markers, including crestin:EGFP. Our studies demonstrate that NRASQ61R induces extensive melanocyte expansion, which arise during zebrafish development and lack a transformed phenotype. These early lesions are highly predisposed to reactivate a neural crest progenitor fate and form malignant melanomas.

Melanoma: from mutations to medicine.

Melanoma is often considered one of the most aggressive and treatment-resistant human cancers. It is a disease that, due to the presence of melanin pigment, was accurately diagnosed earlier than most other malignancies and that has been subjected to countless therapeutic strategies. Aside from early surgical resection, no therapeutic modality has been found to afford a high likelihood of curative outcome. However, discoveries reported in recent years have revealed a near avalanche of breakthroughs in the melanoma field-breakthroughs that span fundamental understanding of the molecular basis of the disease all the way to new therapeutic strategies that produce unquestionable clinical benefit. These discoveries have been born from the successful fruits of numerous researchers working in many-sometimes-related, although also distinct-biomedical disciplines. Discoveries of frequent mutations involving BRAF(V600E), developmental and oncogenic roles for the microphthalmia-associated transcription factor (MITF) pathway, clinical efficacy of BRAF-targeted small molecules, and emerging mechanisms underlying resistance to targeted therapeutics represent just a sample of the findings that have created a striking inflection in the quest for clinically meaningful progress in the melanoma field.

Chemoprevention agents for melanoma: A path forward into phase 3 clinical trials.

Recent progress in the treatment of advanced melanoma has led to unprecedented improvements in overall survival and, as these new melanoma treatments have been developed and deployed in the clinic, much has been learned about the natural history of the disease. Now is the time to apply that knowledge toward the design and clinical evaluation of new chemoprevention agents. Melanoma chemoprevention has the potential to reduce dramatically both the morbidity and the high costs associated with treating patients who have metastatic disease. In this work, scientific and clinical melanoma experts from the national Melanoma Prevention Working Group, composed of National Cancer Trials Network investigators, discuss research aimed at discovering and developing (or repurposing) drugs and natural products for the prevention of melanoma and propose an updated pipeline for translating the most promising agents into the clinic. The mechanism of action, preclinical data, epidemiological evidence, and results from available clinical trials are discussed for each class of compounds. Selected keratinocyte carcinoma chemoprevention studies also are considered, and a rationale for their inclusion is presented. These data are summarized in a table that lists the type and level of evidence available for each class of agents. Also included in the discussion is an assessment of additional research necessary and the likelihood that a given compound may be a suitable candidate for a phase 3 clinical trial within the next 5 years.

Sun exposure and protection practices in children after allogeneic hematopoietic stem cell transplantation: A Survey-Based Cross-Sectional Cohort Study.

BACKGROUND/OBJECTIVE: Pediatric hematopoietic stem cell transplantation (HSCT) patients are at an increased risk for skin cancers. Sun exposure is a significant modifiable environmental risk factor. While patient education on sun protection and avoidance behaviors with regular dermatology evaluations are crucial for pediatric HSCT patients, the real-life practice of these sun-protection recommendations in this patient population compared to their peers is unknown. METHODS: A survey-based cross-sectional cohort study was performed in pediatric HSCT patients seen at the Dana-Farber Cancer Institute and Boston Children's Hospital over a 1.5-year period compared with age/sex/Fitzpatrick skin phototype-matched healthy controls. Study participants were surveyed using the validated Glanz survey for pediatric sun protection behavioral research. RESULTS: Eighty-five pediatric HSCT patients and 85 controls completed the study. Pediatric HSCT patients more frequently used sunscreen, hats, umbrellas, and sunglasses and obtained full-body skin exams compared to controls. No difference was observed in sun exposure during hours of peak sun intensity, frequency of purposeful tanning, tanning bed use, and the number of painful sunburns received between pediatric HSCT patients and controls. CONCLUSIONS: Although pediatric HSCT patients practice more sun protection behaviors, they experienced harmful sunburns and intentional tanning behaviors at the same rate as their peers. Patient-directed counseling and strategies to improve patient adherence to optimal sun protection behaviors could have a significant impact on the dermatology quality of life in pediatric HSCT patients.