The ATM Ser49Cys Variant Effects ATM Function as a Regulator of Oncogene-Induced Senescence.

An apical component of the cell cycle checkpoint and DNA damage repair response is the ataxia-telangiectasia mutated (ATM) Ser/Thr protein kinase. A variant of ATM, Ser49Cys (rs1800054; minor allele frequency = 0.011), has been associated with an elevated risk of melanoma development; however, the functional consequence of this variant is not defined. ATM-dependent signalling in response to DNA damage has been assessed in a panel of patient-derived lymphoblastoid lines and primary human melanocytic cell strains heterozygous for the ATM Ser49Cys variant allele. The ATM Ser49Cys allele appears functional for acute p53-dependent signalling in response to DNA damage. Expression of the variant allele did reduce the efficacy of oncogene expression in inducing senescence. These findings demonstrate that the ATM 146C>G Ser49Cys allele has little discernible effect on the acute response to DNA damage but has reduced function observed in the chronic response to oncogene over-expression. Analysis of melanoma, naevus and skin colour genomics and GWAS analyses have demonstrated no association of this variant with any of these outcomes. The modest loss of function detected suggest that the variant may act as a modifier of other variants of ATM/p53-dependent signalling.

Melanoma mutations modify melanocyte dynamics in co-culture with keratinocytes or fibroblasts.

Melanocytic cell interactions are integral to skin homeostasis, and affect the outcome of multiple diseases, including cutaneous pigmentation disorders and melanoma. By using automated-microscopy and machine-learning-assisted morphology analysis of primary human melanocytes in co-culture, we performed combinatorial interrogation of melanocyte genotypic variants and functional assessment of lentivirus-introduced mutations. Keratinocyte-induced melanocyte dendricity, an indicator of melanocyte differentiation, was reduced in the melanocortin 1 receptor (MC1R) R/R variant strain and by NRAS.Q61K and BRAF.V600E expression, while expression of CDK4.R24C and RAC1.P29S had no detectable effect. Time-lapse tracking of melanocytes in co-culture revealed dynamic interaction phenotypes and hyper-motile cell states that indicated that, in addition to the known role in activating mitogenic signalling, MEK-pathway-activating mutations may also allow melanocytes to escape keratinocyte control and increase their invasive potential. Expanding this combinatorial platform will identify other therapeutic target mutations and melanocyte genetic variants, as well as increase understanding of skin cell interactions.

Skin Pigmentation Genetics for the Clinic.

Human pigmentation characteristics play an important role in the effects of sun exposure, skin cancer induction and disease outcomes. Several of the genes most important for this diversity are involved in the regulation and distribution of melanin pigmentation or enzymes involved in melanogenesis itself within the melanocyte cell present in the skin, hair and eyes. The single nucleotide polymorphisms and extended haplotypes within or surrounding these genes have been identified as risk factors for skin cancer, in particular, melanoma. These same polymorphisms have been under selective pressure leading towards lighter pigmentation in Europeans in the last 5,000-20,000 years that have driven the increase in frequency in modern populations. Although pigmentation is a polygenic trait, due to interactive and quantitative gene effects, strong phenotypic associations are readily apparent for these major genes. However, predictive value and utility are increased when considering gene polymorphism interactions. In melanoma, an increased penetrance is found in cases when pigmentation gene risk alleles such as MC1R variants are coincident with mutation of higher-risk melanoma genes including CDKN2A, CDK4 and MITF E318K, demonstrating an interface between the pathways for pigmentation, naevogenesis and melanoma. The clinical phenotypes associated with germline changes in pigmentation and naevogenic genes must be understood by clinicians, and will be of increasing relevance to dermatologists, as genomics is incorporated into the delivery of personalised medicine.

DCT protects human melanocytic cells from UVR and ROS damage and increases cell viability.

Dopachrome tautomerase (DCT) is involved in the formation of the photoprotective skin pigment eumelanin and has also been shown to have a role in response to apoptotic stimuli and oxidative stress. The effect of DCT on UVR DNA damage responses and survival pathways in human melanocytic cells was examined by knockdown experiments using melanoma cells, neonatal foreskin melanoblasts (MB) in monoculture and in co-culture with human keratinocytes. MB cell strains genotyped as either MC1R WT or MC1R RHC homozygotes, which are known to be deficient in DCT, were transduced with lentivirus vectors for either DCT knockdown or overexpression. We found melanoma cell survival was reduced by DCT depletion and by UVR over time. UVR-induced p53 and pp53-Ser15 levels were reduced with DCT depletion. Knockdown of DCT in MC1R WT and MC1R RHC MB cells reduced their survival after UVR exposure, whereas increased DCT protein levels enhanced survival. DCT depletion reduced p53 and pp53-Ser15 levels in WM266-4 melanoma and MC1R WT MB cells, while MC1R RHC MB cells displayed variable levels. Both MC1R WT and RHC genotypes of MB cells were responsive to UVR at 3 h with increases in both p53 and pp53-Ser15 proteins. MC1R WT MB cell strains in coculture with keratinocytes have an increased cell survival after UVR exposure when compared to those in monoculture, a protective effect which appears to be conferred by the keratinocytes.

Effect of MC1R variant allele status on MSH-ligand induction of dopachrome tautomerase in melanocytes co-cultured with keratinocytes.

A co-culture system of melanocytic cells and keratinocytes was used to examine dendricity and dopachrome tautomerase (DCT) responses in low penetrant 'r' homozygote and 'R/+' heterozygote MC1R variant allele expressing cells compared to that of wild-type (WT) cells. The V60L-/- homozygote r variant cells showed similar responses to ligand as WT MC1R strains, while V92M-/- homozygote r variant cells were generally shown to have greater dendricity and express higher DCT than the WT cells, even at basal levels. The R151C+/- heterozygote cells showed similar responses to WT cells, while the R160W+/- and D294H+/- variant cells were reduced in their responses to NDP-MSH, but still had an active cAMP response with forskolin treatment. These responses are consistent with the dominant negative effect of these alleles on the MC1R WT allele that has previously been demonstrated genetically and biochemically.

Endogenous Replication Stress Marks Melanomas Sensitive to CHEK1 Inhibitors In Vivo.

Purpose: Checkpoint kinase 1 inhibitors (CHEK1i) have single-agent activity in vitro and in vivo Here, we have investigated the molecular basis of this activity.Experimental Design: We have assessed a panel of melanoma cell lines for their sensitivity to the CHEK1i GNE-323 and GDC-0575 in vitro and in vivo The effects of these compounds on responses to DNA replication stress were analyzed in the hypersensitive cell lines.Results: A subset of melanoma cell lines is hypersensitive to CHEK1i-induced cell death in vitro, and the drug effectively inhibits tumor growth in vivo In the hypersensitive cell lines, GNE-323 triggers cell death without cells entering mitosis. CHEK1i treatment triggers strong RPA2 hyperphosphorylation and increased DNA damage in only hypersensitive cells. The increased replication stress was associated with a defective S-phase cell-cycle checkpoint. The number and intensity of pRPA2 Ser4/8 foci in untreated tumors appeared to be a marker of elevated replication stress correlated with sensitivity to CHEK1i.Conclusions: CHEK1i have single-agent activity in a subset of melanomas with elevated endogenous replication stress. CHEK1i treatment strongly increased this replication stress and DNA damage, and this correlated with increased cell death. The level of endogenous replication is marked by the pRPA2Ser4/8 foci in the untreated tumors, and may be a useful marker of replication stress in vivoClin Cancer Res; 24(12); 2901-12. ©2018 AACR.

Genetic variation in IRF4 expression modulates growth characteristics, tyrosinase expression and interferon-gamma response in melanocytic cells.

A SNP within intron4 of the interferon regulatory factor4 (IRF4) gene, rs12203592*C/T, has been independently associated with pigmentation and age-specific effects on naevus count in European-derived populations. We have characterized the cis-regulatory activity of this intronic region and using human foreskin-derived melanoblast strains, we have explored the correlation between IRF4 rs12203592 homozygous C/C and T/T genotypes with TYR enzyme activity, supporting its association with pigmentation traits. Further, higher IRF4 protein levels directed by the rs12203592*C allele were associated with increased basal proliferation but decreased cell viability following UVR, an etiological factor in melanoma development. Since UVR, and accompanying IFNγ-mediated inflammatory response, is associated with melanomagenesis, we evaluated its effects in the context of IRF4 status. Manipulation of IRF4 levels followed by IFNγ treatment revealed a subset of chemokines and immuno-evasive molecules that are sensitive to IRF4 expression level and genotype including CTLA4 and PD-L1.

Testing of viable human skin cell dilution cultures as an approach to validating microsampling.

Skin biopsies are a valuable technique in the diagnosis of cutaneous inflammatory and neoplastic conditions. We were interested to test the minimal size or equivalent volume by dilution of proteolytically disassociated skin tissue required to allow the isolation and propagation of cutaneous cells, for freezing, storage and biochemical analysis. It was possible to propagate with 100% efficiency fibroblast and melanocytic cells from a 0.1 to 0.5 mm3 equivalent neonatal foreskin sample using a combination of DispaseII and CollagenaseIV. The smallest tissue dilution that allowed melanocytic cell culture was 0.01 mm3, which equated to approximately 16 cells based on the average skin density of melanocytes. However, passaging of cells to create frozen stocks was achieved routinely only from 1 mm3 skin, equating to 1560 cells. Tissue-specific antigen expression of these cultures was tested by western blot of total protein extracts. There was no pigmentation antigen expression in fibroblast cultures; however, smooth muscle actin protein expression was high in fibroblast but absent from melanocytic cell strains. Melanocytic cells expressed pigmentation antigens and E-cadherin, but these were not detected in fibroblasts. Moreover, maturation of these melanocytic cells resulted in a decrease of Dopachrome Tautomerase antigen expression and induction of Tyrosinase protein consistent with the differentiation potential seen in cell cultures derived routinely from large sections of skin tissue.

Molecular analysis of common polymorphisms within the human Tyrosinase locus and genetic association with pigmentation traits.

We have compared the melanogenic activities of cultured melanocytes carrying two common TYR alleles as homozygous 192S-402R wild-type, heterozygous and homozygous variant. This includes assays of TYR protein, DOPAoxidase activity, glycosylation and temperature sensitivity of protein and DOPAoxidase levels. Homozygous wild-type strains on average had higher levels of TYR protein and enzyme activity than other genotypes. Homozygous 402Q/Q melanocytes produced significantly less TYR protein, displayed altered trafficking and glycosylation, with reduced DOPAoxidase. However, near wild-type TYR activity levels could be recovered at lower growth temperature. In a sample population from Southeast Queensland, these two polymorphisms were present on four TYR haplotypes, designated as WT 192S-402R, 192Y-402R and 192S-402Q with a double-variant 192Y-402Q of low frequency at 1.9%. Based on cell culture findings and haplotype associations, we have used an additive model to assess the penetrance of the ten possible TYR genotypes derived from the combination of these haplotypes.

MC1R variant allele effects on UVR-induced phosphorylation of p38, p53, and DDB2 repair protein responses in melanocytic cells in culture.

Variant alleles of the human melanocortin 1 receptor (MC1R) reduce the ability of melanocytes to produce the dark pigment eumelanin, with R alleles being most deficient. Cultured melanocytes of MC1R R/R variant genotype give reduced responses to [Nle(4), D-Phe(7)]α-melanocyte-stimulating hormone (NDP-MSH) ligand stimulation and lower levels of DNA repair than MC1R wild-type strains. p38 controls xeroderma pigmentosum (XP)-C recruitment to DNA damage sites through regulating ubiquitylation of the DNA damage-binding protein 2 (DDB2) protein, and p53 is implicated in the nuclear excision repair process through its regulation of XP-C and DDB2 protein expression. We report the effects of MC1R ligand treatment and UVR exposure on phosphorylation of p38 and p53, and DDB2 protein expression in MC1R variant strains. Wild-type MC1R melanocyte strains grown together with keratinocytes in coculture, when treated with NDP-MSH and exposed to UVR, gave synergistic activation of p38 and p53 phosphorylation, and were not replicated by R/R variant melanocytes, which have lower basal levels of phosphorylated forms of p38. Minor increases in p38 phosphorylation status in R/R variant melanocyte cocultures could be attributed to the keratinocytes alone. We also found that MC1R wild-type strains regulate DDB2 protein levels through p38, but MC1R R/R variant melanocytes do not. This work confirms the important functional role that the MC1R receptor plays in UVR stress-induced DNA repair.

Melanocortin MC1 receptor in human genetics and model systems.

The melanocortin MC(1) receptor is a G-protein coupled receptor expressed in the melanocytes of the skin and hair and is known for its key role in the regulation of human pigmentation. Melanocortin MC(1) receptor activation after ultraviolet radiation exposure results in a switch from the red/yellow pheomelanin to the brown/black eumelanin pigment synthesis within cutaneous melanocytes; this pigment is then transferred to the surrounding keratinocytes of the skin. The increase in melanin maturation and uptake results in tanning of the skin, providing a physical protection of skin cells from ultraviolet radiation induced DNA damage. Melanocortin MC(1) receptor polymorphism is widespread within the Caucasian population and some variant alleles are associated with red hair colour, fair skin, poor tanning and increased risk of skin cancer. Here we will discuss the use of mouse coat colour models, human genetic association studies, and in vitro cell culture studies to determine the complex functions of the melanocortin MC(1) receptor and the molecular mechanisms underlying the association between melanocortin MC(1) receptor variant alleles and the red hair colour phenotype. Recent research indicates that melanocortin MC(1) receptor has many non-pigmentary functions, and that the increased risk of skin cancer conferred by melanocortin MC(1) receptor variant alleles is to some extent independent of pigmentation phenotypes. The use of new transgenic mouse models, the study of novel melanocortin MC(1) receptor response genes and the use of more advanced human skin models such as 3D skin reconstruction may provide key elements in understanding the pharmacogenetics of human melanocortin MC(1) receptor polymorphism.