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.

Rapid mapping of interactions between Human SNX-BAR proteins measured in vitro by AlphaScreen and single-molecule spectroscopy.

Protein dimerization and oligomerization is commonly used by nature to increase the structural and functional complexity of proteins. Regulated protein assembly is essential to transfer information in signaling, transcriptional, and membrane trafficking events. Here we show that a combination of cell-free protein expression, a proximity based interaction assay (AlphaScreen), and single-molecule fluorescence allow rapid mapping of homo- and hetero-oligomerization of proteins. We have applied this approach to the family of BAR domain-containing sorting nexin (SNX-BAR) proteins, which are essential regulators of membrane trafficking and remodeling in all eukaryotes. Dimerization of BAR domains is essential for creating a concave structure capable of sensing and inducing membrane curvature. We have systematically mapped 144 pairwise interactions between the human SNX-BAR proteins and generated an interaction matrix of preferred dimerization partners for each family member. We find that while nine SNX-BAR proteins are able to form homo-dimers, several including the retromer-associated SNX1, SNX2, and SNX5 require heteromeric interactions for dimerization. SNX2, SNX4, SNX6, and SNX8 show a promiscuous ability to bind other SNX-BAR proteins and we also observe a novel interaction with the SNX3 protein which lacks the BAR domain structure.

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.

Dysregulated G2 phase checkpoint recovery pathway reduces DNA repair efficiency and increases chromosomal instability in a wide range of tumours.

Defective DNA repair is being demonstrated to be a useful target in cancer treatment. Currently, defective repair is identified by specific gene mutations, however defective repair is a common feature of cancers without these mutations. DNA damage triggers cell cycle checkpoints that are responsible for co-ordinating cell cycle arrest and DNA repair. Defects in checkpoint signalling components such as ataxia telangiectasia mutated (ATM) occur in a low proportion of cancers and are responsible for reduced DNA repair and increased genomic instability. Here we have investigated the AURKA-PLK1 cell cycle checkpoint recovery pathway that is responsible for exit from the G2 phase cell cycle checkpoint arrest. We demonstrate that dysregulation of PP6 and AURKA maintained elevated PLK1 activation to promote premature exit from only ATM, and not ATR-dependent checkpoint arrest. Surprisingly, depletion of the B55α subunit of PP2A that negatively regulates PLK1 was capable of overcoming ATM and ATR checkpoint arrests. Dysregulation of the checkpoint recovery pathway reduced S/G2 phase DNA repair efficiency and increased genomic instability. We found a strong correlation between dysregulation of the PP6-AURKA-PLK1-B55α checkpoint recovery pathway with signatures of defective homologous recombination and increased chromosomal instability in several cancer types. This work has identified an unrealised source of G2 phase DNA repair defects and chromosomal instability that are likely to be sensitive to treatments targeting defective repair.

Multiple interaction nodes define the postreplication repair response to UV-induced DNA damage that is defective in melanomas and correlated with UV signature mutation load.

Ultraviolet radiation-induced DNA mutations are a primary environmental driver of melanoma. The reason for this very high level of unrepaired DNA lesions leading to these mutations is still poorly understood. The primary DNA repair mechanism for UV-induced lesions, that is, the nucleotide excision repair pathway, appears intact in most melanomas. We have previously reported a postreplication repair mechanism that is commonly defective in melanoma cell lines. Here we have used a genome-wide approach to identify the components of this postreplication repair mechanism. We have used differential transcript polysome loading to identify transcripts that are associated with UV response, and then functionally assessed these to identify novel components of this repair and cell cycle checkpoint network. We have identified multiple interaction nodes, including global genomic nucleotide excision repair and homologous recombination repair, and previously unexpected MASTL pathway, as components of the response. Finally, we have used bioinformatics to assess the contribution of dysregulated expression of these pathways to the UV signature mutation load of a large melanoma cohort. We show that dysregulation of the pathway, especially the DNA damage repair components, are significant contributors to UV mutation load, and that dysregulation of the MASTL pathway appears to be a significant contributor to high UV signature mutation load.

Combined use of subclinical hydroxyurea and CHK1 inhibitor effectively controls melanoma and lung cancer progression, with reduced normal tissue toxicity compared to gemcitabine.

Drugs such as gemcitabine that increase replication stress are effective chemotherapeutics in a range of cancer settings. These drugs effectively block replication and promote DNA damage, triggering a cell cycle checkpoint response through the ATR-CHK1 pathway. Inhibiting this signalling pathway sensitises cells to killing by replication stress-inducing drugs. Here, we investigated the effect of low-level replication stress induced by low concentrations (> 0.2 mm) of the reversible ribonucleotide reductase inhibitor hydroxyurea (HU), which slows S-phase progression but has little effect on cell viability or proliferation. We demonstrate that HU effectively synergises with CHK1, but not ATR inhibition, in > 70% of melanoma and non-small-cell lung cancer cells assessed, resulting in apoptosis and complete loss of proliferative potential in vitro and in vivo. Normal fibroblasts and haemopoietic cells retain viability and proliferative potential following exposure to CHK1 inhibitor plus low doses of HU, but normal cells exposed to CHK1 inhibitor combined with submicromolar concentrations of gemcitabine exhibited complete loss of proliferative potential. The effects of gemcitabine on normal tissue correlate with irreversible ATR-CHK1 pathway activation, whereas low doses of HU reversibly activate CHK1 independently of ATR. The combined use of CHK1 inhibitor and subclinical HU also triggered an inflammatory response involving the recruitment of macrophages in vivo. These data indicate that combining CHK1 inhibitor with subclinical HU is superior to combination with gemcitabine, as it provides equal anticancer efficacy but with reduced normal tissue toxicity. These data suggest a significant proportion of melanoma and lung cancer patients could benefit from treatment with this drug combination.

Mechanism of action of the third generation benzopyrans and evaluation of their broad anti-cancer activity in vitro and in vivo.

Successive rounds of chemical modification in three generations of benzopyran molecules have shown to select for different mechanisms of actions and progressive increases in anti-cancer activity. In this study, we investigated the mechanism of action of the third-generation benzopyran compounds, TRX-E-002-1 and TRX-E-009-1. High-content screening of a panel of 240 cancer cell lines treated with TRX-E-009-1 demonstrated it has broad anti-cancer potential. Within this screen, melanoma cell lines showed a range of sensitivities and subsequently a second independent panel of 21 melanoma 3D spheroid lines were assessed for their responses to both TRX-E-002-1 and TRX-E-009-1 compounds. Time-lapse microscopy illustrated both of these compounds caused mitotic delays in treated cells, resulting in either mitotic slippage or apoptosis. This finding along with immunostaining, in vitro polymerization assays, and animal experiments in both athymic and immunocompetent mice, demonstrates that these third-generation benzopyran compounds are potent tubulin polymerization inhibitors in vitro and in vivo, and this is the molecular basis of their anti-cancer activity in melanoma. These findings indicate these BP compounds may offer a novel anti-microtubule strategy for cancer intervention and provides the basis for further investigation into biomarkers of clinical sensitivity.

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.

Genome-Wide Overexpression Screen Identifies Genes Able to Bypass p16-Mediated Senescence in Melanoma.

Malignant melanomas often arise from nevi, which result from initial oncogene-induced hyperproliferation of melanocytes that are maintained in a CDKN2A/p16-mediated senescent state. Thus, genes that can bypass this senescence barrier are likely to contribute to melanoma development. We have performed a gain-of-function screen of 17,030 lentivirally expressed human open reading frames (ORFs) in a melanoma cell line containing an inducible p16 construct to identify such genes. Genes known to bypass p16-induced senescence arrest, including the human papilloma virus 18 E7 gene ( HPV18E7), and genes such as the p16-binding CDK6 with expected functions, as well as panel of novel genes, were identified, including high-mobility group box (HMGB) proteins. A number of these were further validated in two other models of p16-induced senescence. Tissue immunohistochemistry demonstrated higher levels of CDK6 in primary melanomas compared with normal skin and nevi. Reduction of CDK6 levels drove melanoma cells expressing functional p16 into senescence, demonstrating its contribution to bypass senescence.

Repositioning "old" drugs for new causes: identifying new inhibitors of prostate cancer cell migration and invasion.

The majority of prostate cancer (PCa) deaths occur due to the metastatic spread of tumor cells to distant organs. Currently, there is a lack of effective therapies once tumor cells have spread outside the prostate. It is therefore imperative to rapidly develop therapeutics to inhibit the metastatic spread of tumor cells. Gain of cell motility and invasive properties is the first step of metastasis and by inhibiting motility one can potentially inhibit metastasis. Using the drug repositioning strategy, we developed a cell-based multi-parameter primary screening assay to identify drugs that inhibit the migratory and invasive properties of metastatic PC-3 PCa cells. Following the completion of the primary screening assay, 33 drugs were identified from an FDA approved drug library that either inhibited migration or were cytotoxic to the PC-3 cells. Based on the data obtained from the subsequent validation studies, mitoxantrone hydrochloride, simvastatin, fluvastatin and vandetanib were identified as strong candidates that can inhibit both the migration and invasion of PC-3 cells without significantly affecting cell viability. By employing the drug repositioning strategy instead of a de novo drug discovery and development strategy, the identified drug candidates have the potential to be rapidly translated into the clinic for the management of men with aggressive forms of PCa.

A novel ATM-dependent checkpoint defect distinct from loss of function mutation promotes genomic instability in melanoma.

Melanomas have high levels of genomic instability that can contribute to poor disease prognosis. Here, we report a novel defect of the ATM-dependent cell cycle checkpoint in melanoma cell lines that promotes genomic instability. In defective cells, ATM signalling to CHK2 is intact, but the cells are unable to maintain the cell cycle arrest due to elevated PLK1 driving recovery from the arrest. Reducing PLK1 activity recovered the ATM-dependent checkpoint arrest, and over-expressing PLK1 was sufficient to overcome the checkpoint arrest and increase genomic instability. Loss of the ATM-dependent checkpoint did not affect sensitivity to ionizing radiation demonstrating that this defect is distinct from ATM loss of function mutations. The checkpoint defective melanoma cell lines over-express PLK1, and a significant proportion of melanomas have high levels of PLK1 over-expression suggesting this defect is a common feature of melanomas. The inability of ATM to impose a cell cycle arrest in response to DNA damage increases genomic instability. This work also suggests that the ATM-dependent checkpoint arrest is likely to be defective in a higher proportion of cancers than previously expected.

Genome-wide gain-of-function screen for genes that induce epithelial-to-mesenchymal transition in breast cancer.

Epithelial to mesenchymal transition (EMT) is a developmental program that has been implicated in progression, metastasis and therapeutic resistance of some carcinomas. To identify genes whose overexpression drives EMT, we screened a lentiviral expression library of 17000 human open reading frames (ORFs) using high-content imaging to quantitate cytoplasmic vimentin. Hits capable of increasing vimentin in the mammary carcinoma-derived cell line MDA-MB-468 were confirmed in the non-tumorigenic breast-epithelial cell line MCF10A. When overexpressed in this model, they increased the rate of cell invasion through Matrigel™, induced mesenchymal marker expression and reduced expression of the epithelial marker E-cadherin. In gene-expression datasets derived from breast cancer patients, the expression of several novel genes correlated with expression of known EMT marker genes, indicating their in vivo relevance. As EMT-associated properties are thought to contribute in several ways to cancer progression, genes identified in this study may represent novel targets for anti-cancer therapy.

Gateway-compatible vectors for high-throughput protein expression in pro- and eukaryotic cell-free systems.

Although numerous techniques for protein expression and production are available the pace of genome sequencing outstrips our ability to analyze the encoded proteins. To address this bottleneck, we have established a system for parallelized cloning, DNA production and cell-free expression of large numbers of proteins. This system is based on a suite of pCellFree Gateway destination vectors that utilize a Species Independent Translation Initiation Sequence (SITS) that mediates recombinant protein expression in any in vitro translation system. These vectors introduce C or N terminal EGFP and mCherry fluorescent and affinity tags, enabling direct analysis and purification of the expressed proteins. To maximize throughput and minimize the cost of protein production we combined Gateway cloning with Rolling Circle DNA Amplification. We demonstrate that as little as 0.1 ng of plasmid DNA is sufficient for template amplification and production of recombinant human protein in Leishmania tarentolae and Escherichia coli cell-free expression systems. Our experiments indicate that this approach can be applied to large gene libraries as it can be reliably performed in multi-well plates. The resulting protein expression pipeline provides a valuable new tool for applications of the post genomic era.

Aurora A Is Critical for Survival in HPV-Transformed Cervical Cancer.

Human papillomavirus (HPV) is the causative agent in cervical cancer. HPV oncogenes are major drivers of the transformed phenotype, and the cancers remain addicted to these oncogenes. A screen of the human kinome has identified inhibition of Aurora kinase A (AURKA) as being synthetically lethal on the background of HPV E7 expression. The investigational AURKA inhibitor MLN8237/Alisertib selectively promoted apoptosis in the HPV cancers. The apoptosis was driven by an extended mitotic delay in the Alisertib-treated HPV E7-expressing cells. This had the effect of reducing Mcl-1 levels, which is destabilized in mitosis, and increasing BIM levels, normally destabilized by Aurora A in mitosis. Overexpression of Mcl-1 reduced sensitivity to the drug. The level of HPV E7 expression influenced the extent of Alisertib-induced mitotic delay and Mcl-1 reduction. Xenograft experiments with three cervical cancer cell lines showed Alisertib inhibited growth of HPV and non-HPV xenografts during treatment. Growth of non-HPV tumors was delayed, but in two separate HPV cancer cell lines, regression with no resumption of growth was detected, even at 50 days after treatment. A transgenic model of premalignant disease driven solely by HPV E7 also demonstrated sensitivity to drug treatment. Here, we show for the first time that targeting of the Aurora A kinase in mice using drugs such as Alisertib results in a curative sterilizing therapy that may be useful in treating HPV-driven cancers.

Callose deposition during the interaction between cowpea (Vigna unguiculata) and the monokaryotic stage of the cowpea rust fungus (Uromyces vignae).

The interaction between the race 1 of the cowpea rust fungus (Uromyces vignae Barclay) and the resistant cowpea (Vigna unguiculata (L.) Walp.) ev. Queen Anne is characterized by the deposition of callose around intracellular fungal structures. Ultrastructural examination of the early stages of infection by basidiospores of the fungus revealed two types of deposits induced by intracellular hyphae: a non-callose collar at the penetration site and a callose encasement around the invasion hypha. The callose encasement was developed from the site where the fungus encountered the inside of the plant cell wall and was separated from the fungus by the extrahyphal membrane and an extension of plant plasma membrane. The incidence of encasements was reduced in plants treated with inhibitors of transcription (actinomycin D), protein synthesis (cycloheximide), protein glycosylation (tunicamycin) and microfilament polymerization (cytochalasin E). Inhibitors of Golgi-associated vesicle transfer (monensin, brefeldin A) and anti-microtubule agents (colchicine, oryzalin) had no effect. When the fungus was killed by heat treatment in either the resistant or the susceptible cultivar, callose was deposited at various locations along the fungus, mostly in the extrahyphal matrix. The data suggest that unlike the extrahaustorial membrane surrounding D-haustoria of this fungus, the extrahyphal membrane is capable of generating callose. Since this process does not normally occur in the susceptible or the resistant cv. even when callose is deposited in the latter by regions of the plasma membrane not associated with the fungus, we conclude that the deposition of callose by the extrahyphal membrane is inhibited by the living fungus.

Generation of a genome scale lentiviral vector library for EF1α promoter-driven expression of human ORFs and identification of human genes affecting viral titer.

The bottleneck in elucidating gene function through high-throughput gain-of-function genome screening is the limited availability of comprehensive libraries for gene overexpression. Lentiviral vectors are the most versatile and widely used vehicles for gene expression in mammalian cells. Lentiviral supernatant libraries for genome screening are commonly generated in the HEK293T cell line, yet very little is known about the effect of introduced sequences on the produced viral titer, which we have shown to be gene dependent. We have generated an arrayed lentiviral vector library for the expression of 17,030 human proteins by using the GATEWAY® cloning system to transfer ORFs from the Mammalian Gene Collection into an EF1alpha promoter-dependent lentiviral expression vector. This promoter was chosen instead of the more potent and widely used CMV promoter, because it is less prone to silencing and provides more stable long term expression. The arrayed lentiviral clones were used to generate viral supernatant by packaging in the HEK293T cell line. The efficiency of transfection and virus production was estimated by measuring the fluorescence of IRES driven GFP, co-expressed with the ORFs. More than 90% of cloned ORFs produced sufficient virus for downstream screening applications. We identified genes which consistently produced very high or very low viral titer. Supernatants from select clones that were either high or low virus producers were tested on a range of cell lines. Some of the low virus producers, including two previously uncharacterized proteins were cytotoxic to HEK293T cells. The library we have constructed presents a powerful resource for high-throughput gain-of-function screening of the human genome and drug-target discovery. Identification of human genes that affect lentivirus production may lead to improved technology for gene expression using lentiviral vectors.

A high-throughput platform for lentiviral overexpression screening of the human ORFeome.

In response to the growing need for functional analysis of the human genome, we have developed a platform for high-throughput functional screening of genes overexpressed from lentiviral vectors. Protein-coding human open reading frames (ORFs) from the Mammalian Gene Collection were transferred into lentiviral expression vector using the highly efficient Gateway recombination cloning. Target ORFs were inserted into the vector downstream of a constitutive promoter and upstream of an IRES controlled GFP reporter, so that their transfection, transduction and expression could be monitored by fluorescence. The expression plasmids and viral packaging plasmids were combined and transfected into 293T cells to produce virus, which was then used to transduce the screening cell line. We have optimised the transfection and transduction procedures so that they can be performed using robotic liquid handling systems in arrayed 96-well microplate, one-gene-per-well format, without the need to concentrate the viral supernatant. Since lentiviruses can infect both dividing and non-dividing cells, this system can be used to overexpress human ORFs in a broad spectrum of experimental contexts. We tested the platform in a 1990 gene pilot screen for genes that can increase proliferation of the non-tumorigenic mammary epithelial cell line MCF-10A after removal of growth factors. Transduced cells were labelled with the nucleoside analogue 5-ethynyl-2'-deoxyuridine (EdU) to detect cells progressing through S phase. Hits were identified using high-content imaging and statistical analysis and confirmed with vectors using two different promoters (CMV and EF1α). The screen demonstrates the reliability, versatility and utility of our screening platform, and identifies novel cell cycle/proliferative activities for a number of genes.