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  • ItemPublished versionOpen Access
    Germline pathogenic variants in PALB2 and other cancer-predisposing genes in families with hereditary diffuse gastric cancer without CDH1 mutation: a whole-exome sequencing study.
    (Elsevier BV, 2018-07) Fewings, Eleanor; Larionov, Alexey; Redman, James; Goldgraben, Mae A; Scarth, James; Richardson, Susan; Brewer, Carole; Davidson, Rosemarie; Ellis, Ian; Evans, D Gareth; Halliday, Dorothy; Izatt, Louise; Marks, Peter; McConnell, Vivienne; Verbist, Louis; Mayes, Rebecca; Clark, Graeme R; Hadfield, James; Chin, Suet-Feung; Teixeira, Manuel R; Giger, Olivier T; Hardwick, Richard; di Pietro, Massimiliano; O'Donovan, Maria; Pharoah, Paul; Caldas, Carlos; Fitzgerald, Rebecca C; Tischkowitz, Marc; Fewings, Eleanor [0000-0001-9012-3220]; Larionov, Alexey [0000-0001-6374-9391]; Goldgraben, Mae [0000-0002-1111-2804]; Chin, Suet-Feung [0000-0001-5697-1082]; Giger, Olivier [0000-0003-3390-6397]; Di Pietro, Massimiliano [0000-0003-4866-7026]; Pharoah, Paul [0000-0001-8494-732X]; Caldas, Carlos [0000-0003-3547-1489]; Fitzgerald, Rebecca [0000-0002-3434-3568]; Tischkowitz, Marc [0000-0002-7880-0628]
    BACKGROUND: Germline pathogenic variants in the E-cadherin gene (CDH1) are strongly associated with the development of hereditary diffuse gastric cancer. There is a paucity of data to guide risk assessment and management of families with hereditary diffuse gastric cancer that do not carry a CDH1 pathogenic variant, making it difficult to make informed decisions about surveillance and risk-reducing surgery. We aimed to identify new candidate genes associated with predisposition to hereditary diffuse gastric cancer in affected families without pathogenic CDH1 variants. METHODS: We did whole-exome sequencing on DNA extracted from the blood of 39 individuals (28 individuals diagnosed with hereditary diffuse gastric cancer and 11 unaffected first-degree relatives) in 22 families without pathogenic CDH1 variants. Genes with loss-of-function variants were prioritised using gene-interaction analysis to identify clusters of genes that could be involved in predisposition to hereditary diffuse gastric cancer. FINDINGS: Protein-affecting germline variants were identified in probands from six families with hereditary diffuse gastric cancer; variants were found in genes known to predispose to cancer and in lesser-studied DNA repair genes. A frameshift deletion in PALB2 was found in one member of a family with a history of gastric and breast cancer. Two different MSH2 variants were identified in two unrelated affected individuals, including one frameshift insertion and one previously described start-codon loss. One family had a unique combination of variants in the DNA repair genes ATR and NBN. Two variants in the DNA repair gene RECQL5 were identified in two unrelated families: one missense variant and a splice-acceptor variant. INTERPRETATION: The results of this study suggest a role for the known cancer predisposition gene PALB2 in families with hereditary diffuse gastric cancer and no detected pathogenic CDH1 variants. We also identified new candidate genes associated with disease risk in these families. FUNDING: UK Medical Research Council (Sackler programme), European Research Council under the European Union's Seventh Framework Programme (2007-13), National Institute for Health Research Cambridge Biomedical Research Centre, Experimental Cancer Medicine Centres, and Cancer Research UK.
  • ItemPublished versionOpen Access
    Compensation between CSF1R+ macrophages and Foxp3+ Treg cells drives resistance to tumor immunotherapy.
    (American Society for Clinical Investigation, 2018-06-07) Gyori, David; Lim, Ee Lyn; Grant, Francis M; Spensberger, Dominik; Roychoudhuri, Rahul; Shuttleworth, Stephen J; Okkenhaug, Klaus; Stephens, Len R; Hawkins, Phillip T; Roychoudhuri, Rahul [0000-0002-5392-1853]; Okkenhaug, Klaus [0000-0002-9432-4051]; Hawkins, Phillip Thomas [0000-0002-6979-0464]
    Redundancy and compensation provide robustness to biological systems but may contribute to therapy resistance. Both tumor-associated macrophages (TAMs) and Foxp3+ regulatory T (Treg) cells promote tumor progression by limiting antitumor immunity. Here we show that genetic ablation of CSF1 in colorectal cancer cells reduces the influx of immunosuppressive CSF1R+ TAMs within tumors. This reduction in CSF1-dependent TAMs resulted in increased CD8+ T cell attack on tumors, but its effect on tumor growth was limited by a compensatory increase in Foxp3+ Treg cells. Similarly, disruption of Treg cell activity through their experimental ablation produced moderate effects on tumor growth and was associated with elevated numbers of CSF1R+ TAMs. Importantly, codepletion of CSF1R+ TAMs and Foxp3+ Treg cells resulted in an increased influx of CD8+ T cells, augmentation of their function, and a synergistic reduction in tumor growth. Further, inhibition of Treg cell activity either through systemic pharmacological blockade of PI3Kδ, or its genetic inactivation within Foxp3+ Treg cells, sensitized previously unresponsive solid tumors to CSF1R+ TAM depletion and enhanced the effect of CSF1R blockade. These findings identify CSF1R+ TAMs and PI3Kδ-driven Foxp3+ Treg cells as the dominant compensatory cellular components of the immunosuppressive tumor microenvironment, with implications for the design of combinatorial immunotherapies.
  • ItemPublished versionOpen Access
    Phosphoinositide 3-kinase δ inhibition promotes antitumor responses but antagonizes checkpoint inhibitors.
    (American Society for Clinical Investigation, 2018-06-07) Lim, Ee Lyn; Cugliandolo, Fiorella M; Rosner, Dalya R; Gyori, David; Roychoudhuri, Rahul; Okkenhaug, Klaus; Roychoudhuri, Rahul [0000-0002-5392-1853]; Okkenhaug, Klaus [0000-0002-9432-4051]
    Multiple modes of immunosuppression restrain immune function within tumors. We previously reported that phosphoinositide 3-kinase δ (PI3Kδ) inactivation in mice confers resistance to a range of tumor models by disrupting immunosuppression mediated by regulatory T cells (Tregs). The PI3Kδ inhibitor idelalisib has proven highly effective in the clinical treatment of chronic lymphocytic leukemia and the potential to extend the use of PI3Kδ inhibitors to nonhematological cancers is being evaluated. In this work, we demonstrate that the antitumor effect of PI3Kδ inactivation is primarily mediated through the disruption of Treg function, and correlates with tumor dependence on Treg immunosuppression. Compared with Treg-specific PI3Kδ deletion, systemic PI3Kδ inactivation is less effective at conferring resistance to tumors. We show that PI3Kδ deficiency impairs the maturation and reduces the capacity of CD8+ cytotoxic T lymphocytes (CTLs) to kill tumor cells in vitro, and to respond to tumor antigen-specific immunization in vivo. PI3Kδ inactivation antagonized the antitumor effects of tumor vaccines and checkpoint blockade therapies intended to boost the CD8+ T cell response. These findings provide insights into mechanisms by which PI3Kδ inhibition promotes antitumor immunity and demonstrate that the mechanism is distinct from that mediated by immune checkpoint blockade.
  • ItemPublished versionOpen Access
    The VPS4 component of the ESCRT machinery plays an essential role in HPV infectious entry and capsid disassembly.
    (Springer Science and Business Media LLC, 2017-03-28) Broniarczyk, Justyna; Pim, David; Massimi, Paola; Bergant, Martina; Goździcka-Józefiak, Anna; Crump, Colin; Banks, Lawrence; Crump, Colin [0000-0001-9918-9998]
    Human Papillomavirus (HPV) infection involves multiple steps, from cell attachment, through endocytic trafficking towards the trans-Golgi network, and, ultimately, the entry into the nucleus during mitosis. An essential viral protein in infectious entry is the minor capsid protein L2, which engages different components of the endocytic sorting machinery during this process. The ESCRT machinery is one such component that seems to play an important role in the early stages of infection. Here we have analysed the role of specific ESCRT components in HPV infection, and we find an essential role for VPS4. Loss of VPS4 blocks infection with multiple PV types, suggesting an evolutionarily conserved critical step in infectious entry. Intriguingly, both L1 and L2 can interact with VPS4, and appear to be in complex with VPS4 during the early stages of virus infection. By using cell lines stably expressing a dominant-negative mutant form of VPS4, we also show that loss of VPS4 ATPase activity results in a marked delay in capsid uncoating, resulting in a defect in the endocytic transport of incoming PsVs. These results demonstrate that the ESCRT machinery, and in particular VPS4, plays a critical role in the early stages of PV infection.
  • ItemAccepted versionOpen Access
    Identification of F-box only protein 7 as a negative regulator of NF-kappaB signalling.
    (2012-09) Kuiken, Hendrik J; Egan, David A; Laman, Heike; Bernards, Rene; Beijersbergen, Roderick L; Dirac, Annette M; Laman, Heike [0000-0002-6089-171X]
    The nuclear factor κB (NF-κB) signalling pathway controls important cellular events such as cell proliferation, differentiation, apoptosis and immune responses. Pathway activation occurs rapidly upon TNFα stimulation and is highly dependent on ubiquitination events. Using cytoplasmic to nuclear translocation of the NF-κB transcription factor family member p65 as a read-out, we screened a synthetic siRNA library targeting enzymes involved in ubiquitin conjugation and de-conjugation for modifiers of regulatory ubiquitination events in NF-κB signalling. We identified F-box protein only 7 (FBXO7), a component of Skp, Cullin, F-box (SCF)-ubiquitin ligase complexes, as a negative regulator of NF-κB signalling. F-box protein only 7 binds to, and mediates ubiquitin conjugation to cIAP1 and TRAF2, resulting in decreased RIP1 ubiquitination and lowered NF-κB signalling activity.
  • ItemPublished versionOpen Access
    Sinus-like dilatations of the mammary milk ducts, Ki67 expression, and CD3-positive T lymphocyte infiltration, in the mammary gland of wild European rabbits during pregnancy and lactation.
    (Wiley, 2018-08) Hughes, Katherine; Watson, Christine J; Hughes, Katherine [0000-0002-3331-1249]
    Sinus-like dilatations of the mammary duct are recognisable in the mammary gland of pregnant and lactating wild European rabbits. These dilatations exhibit a bilaminar epithelial lining, with luminal epithelial cells expressing basal and lateral E-cadherin. Occasional binucleated mammary epithelial cells are present in the luminal layer. Underlying the luminal epithelial cells is a basal layer of cytokeratin 14-positive cells, supported by a thin layer of fibrous tissue. Multi-segmental epithelial proliferation, as indicated by Ki67 expression, is apparent in the luminal epithelial cells, suggesting a capacity for division during pregnancy and lactation. CD3-positive T lymphocytes are present both intraepithelially, suggesting exocytosis, and in foci subjacent to the ductular epithelium. We consider that sinus-like dilatations of the mammary duct may have the potential to give rise to a subset of the mammary gland neoplasms classified as ductal in origin. Milk accumulation in these sinus-like dilatations is likely to provide a niche for bacterial replication in cases of mastitis in rabbits. These structures are an important component of the innate immune system of the mammary gland, both as a physical barrier and as an interface between the milk and mammary immune cells.
  • ItemPublished versionOpen Access
    Expression of Fbxo7 in haematopoietic progenitor cells cooperates with p53 loss to promote lymphomagenesis.
    (Public Library of Science (PLoS), 2011) Lomonosov, Mikhail; Meziane, El Kahina; Ye, Hongtao; Nelson, David E; Randle, Suzanne J; Laman, Heike; Laman, Heike [0000-0002-6089-171X]
    Fbxo7 is an unusual F box protein that augments D-type cyclin complex formation with Cdk6, but not Cdk4 or Cdk2, and its over-expression has been demonstrated to transform immortalised fibroblasts in a Cdk6-dependent manner. Here we present new evidence in vitro and in vivo on the oncogenic potential of this regulatory protein in primary haematopoietic stem and progenitor cells (HSPCs). Increasing Fbxo7 expression in HSPCs suppressed their colony forming ability in vitro, specifically decreasing CD11b (Mac1) expression, and these effects were dependent on an intact p53 pathway. Furthermore, increased Fbxo7 levels enhanced the proliferative capacity of p53 null HSPCs when they were grown in reduced concentrations of stem cell factor. Finally, irradiated mice reconstituted with p53 null, but not wild-type, HSPCs expressing Fbxo7 showed a statistically significant increase in the incidence of T cell lymphoma in vivo. These data argue that Fbxo7 negatively regulates the proliferation and differentiation of HSPCs in a p53-dependent manner, and that in the absence of p53, Fbxo7 expression can promote T cell lymphomagenesis.
  • ItemPublished versionOpen Access
    Common and unique features of viral RNA-dependent polymerases.
    (Springer Science and Business Media LLC, 2014-11) te Velthuis, Aartjan JW; te Velthuis, Arend [0000-0002-5129-3953]
    Eukaryotes and bacteria can be infected with a wide variety of RNA viruses. On average, these pathogens share little sequence similarity and use different replication and transcription strategies. Nevertheless, the members of nearly all RNA virus families depend on the activity of a virally encoded RNA-dependent polymerase for the condensation of nucleotide triphosphates. This review provides an overview of our current understanding of the viral RNA-dependent polymerase structure and the biochemistry and biophysics that is involved in replicating and transcribing the genetic material of RNA viruses.
  • ItemPublished versionOpen Access
    Genomic positional conservation identifies topological anchor point RNAs linked to developmental loci.
    (Springer Nature, 2018-03-15) Amaral, Paulo P; Leonardi, Tommaso; Han, Namshik; Viré, Emmanuelle; Gascoigne, Dennis K; Arias-Carrasco, Raúl; Büscher, Magdalena; Pandolfini, Luca; Zhang, Anda; Pluchino, Stefano; Maracaja-Coutinho, Vinicius; Nakaya, Helder I; Hemberg, Martin; Shiekhattar, Ramin; Enright, Anton J; Kouzarides, Tony; De Paiva Rosa Amaral, Paulo [0000-0002-6696-5142]; Leonardi, Tommaso [0000-0002-4449-1863]; Han, Namshik [0000-0002-7741-6384]; Pandolfini, Luca [0000-0003-1444-8167]; Pluchino, Stefano [0000-0002-6267-9472]; Enright, Anton [0000-0002-6090-3100]; Kouzarides, Tony [0000-0002-8918-4162]
    BACKGROUND: The mammalian genome is transcribed into large numbers of long noncoding RNAs (lncRNAs), but the definition of functional lncRNA groups has proven difficult, partly due to their low sequence conservation and lack of identified shared properties. Here we consider promoter conservation and positional conservation as indicators of functional commonality. RESULTS: We identify 665 conserved lncRNA promoters in mouse and human that are preserved in genomic position relative to orthologous coding genes. These positionally conserved lncRNA genes are primarily associated with developmental transcription factor loci with which they are coexpressed in a tissue-specific manner. Over half of positionally conserved RNAs in this set are linked to chromatin organization structures, overlapping binding sites for the CTCF chromatin organiser and located at chromatin loop anchor points and borders of topologically associating domains (TADs). We define these RNAs as topological anchor point RNAs (tapRNAs). Characterization of these noncoding RNAs and their associated coding genes shows that they are functionally connected: they regulate each other's expression and influence the metastatic phenotype of cancer cells in vitro in a similar fashion. Furthermore, we find that tapRNAs contain conserved sequence domains that are enriched in motifs for zinc finger domain-containing RNA-binding proteins and transcription factors, whose binding sites are found mutated in cancers. CONCLUSIONS: This work leverages positional conservation to identify lncRNAs with potential importance in genome organization, development and disease. The evidence that many developmental transcription factors are physically and functionally connected to lncRNAs represents an exciting stepping-stone to further our understanding of genome regulation.
  • ItemPublished versionOpen Access
    The First Norovirus Longitudinal Seroepidemiological Study From Sub-Saharan Africa Reveals High Seroprevalence of Diverse Genotypes Associated With Host Susceptibility Factors
    (University of Chicago Press, 2018-04-18) Thorne, Lucy; Nalwoga, Angela; Mentzer, Alexander J; de Rougemont, Alexis; Hosmillo, Myra; Webb, Emily; Nampiija, Margaret; Muhwezi, Allan; Carstensen, Tommy; Gurdasani, Deepti; Hill, Adrian V; Sandhu, Manj S; Elliott, Alison; Goodfellow, Ian Gordon; Thorne, Lucy [0000-0001-7358-6047]; Nalwoga, Angela [0000-0001-8259-4735]; Hosmillo, Myra [0000-0002-3514-7681]; Goodfellow, Ian Gordon [0000-0002-9483-510X]
    Human noroviruses (HuNoVs) are a prominent cause of gastroenteritis, yet fundamental questions remain regarding epidemiology, diversity and immunity in sub-­Saharan African children. We investigated HuNoV seroprevalence and genetic and sociodemographic risk factors in Ugandan children.
  • ItemPublished versionOpen Access
    The First Norovirus Longitudinal Seroepidemiological Study From Sub-Saharan Africa Reveals High Seroprevalence of Diverse Genotypes Associated With Host Susceptibility Factors.
    (Oxford University Press (OUP), 2018-07-24) Thorne, Lucy; Nalwoga, Angela; Mentzer, Alexander J; de Rougemont, Alexis; Hosmillo, Myra; Webb, Emily; Nampiija, Margaret; Muhwezi, Allan; Carstensen, Tommy; Gurdasani, Deepti; Hill, Adrian V; Sandhu, Manj S; Elliott, Alison; Goodfellow, Ian; Hosmillo, Myra [0000-0002-3514-7681]; Sandhu, Manjinder [0000-0002-2725-142X]; Goodfellow, Ian [0000-0002-9483-510X]
    BACKGROUND: Human noroviruses (HuNoVs) are a prominent cause of gastroenteritis, yet fundamental questions remain regarding epidemiology, diversity, and immunity in sub-Saharan African children. We investigated HuNoV seroprevalence and genetic and sociodemographic risk factors in Ugandan children. METHODS: We randomly screened 797 participants of a longitudinal birth cohort (Entebbe, EMaBS) and 378 from a cross-sectional survey (rural Lake Victoria, LaVIISWA), for antibodies against HuNoV genotypes by ELISA. We used linear regression modeling to test for associations between HuNoV antibody levels and sociodemographic factors, and with the human susceptibility rs601338 FUT2 secretor SNP and histo-blood group antigens (A/B/O). RESULTS: Of EMaBS participants, 76.6% were seropositive by age 1, rising to 94.5% by age 2 years. Seroprevalence in 1 year olds of the rural LaVIISWA survey was even higher (95%). In the birth cohort, 99% of seropositive 2 year olds had responses to multiple HuNoV genotypes. We identified associations between secretor status and genogroup GII antibody levels (GII.4 P = 3.1 × 10-52), as well as ABO and GI (GI.2 P = 2.1 × 10-12). CONCLUSIONS: HuNoVs are highly prevalent in Ugandan children, indicating a substantial burden of diarrhea-associated morbidity with recurrent infections. Public health interventions, including vaccination, and increased surveillance are urgently needed.
  • ItemPublished versionOpen Access
    Functional Alleles of Chicken BG Genes, Members of the Butyrophilin Gene Family, in Peripheral T Cells.
    (Frontiers Media SA, 2018) Chen, Lei; Fakiola, Michaela; Staines, Karen; Butter, Colin; Kaufman, Jim; Kaufman, Jim [0000-0002-7216-8422]
    γδ T cells recognize a wide variety of ligands in mammals, among them members of the butyrophilin (BTN) family. Nothing is known about γδ T cell ligands in chickens, despite there being many such cells in blood and lymphoid tissues, as well as in mucosal surfaces. The major histocompatibility complex (MHC) of chickens was discovered because of polymorphic BG genes, part of the BTN family. All but two BG genes are located in the BG region, oriented head-to-tail so that unequal crossing-over has led to copy number variation (CNV) as well as hybrid (chimeric) genes, making it difficult to identify true alleles. One approach is to examine BG genes expressed in particular cell types, which likely have the same functions in different BG haplotypes and thus can be considered "functional alleles." We cloned nearly full-length BG transcripts from peripheral T cells of four haplotypes (B2, B15, B19, and B21), and compared them to the BG genes of the B12 haplotype that previously were studied in detail. A dominant BG gene was found in each haplotype, but with significant levels of subdominant transcripts in three haplotypes (B2, B15, and B19). For three haplotypes (B15, B19, and B21), most sequences are closely-related to BG8, BG9, and BG12 from the B12 haplotype. We found that variation in the extracellular immunoglobulin-variable-like (Ig-V) domain is mostly localized to the membrane distal loops but without evidence for selection. However, variation in the cytoplasmic tail composed of many amino acid heptad repeats does appear to be selected (although not obviously localized), consistent with an intriguing clustering of charged and polar residues in an apparent α-helical coiled-coil. By contrast, the dominantly-expressed BG gene in the B2 haplotype is identical to BG13 from the B12 haplotype, and most of the subdominant sequences are from the BG5-BG7-BG11 clade. Moreover, alternative splicing leading to intron read-through results in dramatically truncated cytoplasmic tails, particularly for the dominantly-expressed BG gene of the B2 haplotype. The approach of examining "functional alleles" has yielded interesting data for closely-related genes, but also thrown up unexpected findings for at least one haplotype.
  • ItemAccepted versionOpen Access
    Novel GPR34 and CCR6 mutation and distinct genetic profiles in MALT lymphomas of different sites.
    (Ferrata Storti Foundation (Haematologica), 2018-08) Moody, Sarah; Thompson, Joe Sneath; Chuang, Shih-Sung; Liu, Hongxiang; Raderer, Markus; Vassiliou, George; Wlodarska, Iwona; Wu, Fangtian; Cogliatti, Sergio; Robson, Alistair; Ashton-Key, Margaret; Bi, Yingwen; Goodlad, John; Du, Ming-Qing; Vassiliou, George [0000-0003-4337-8022]
    Mucosa-associated lymphoid tissue (MALT) lymphoma originates from a background of diverse chronic inflammatory disorders at various anatomic sites. The genetics underlying its development, particularly in those associated with autoimmune disorders, is poorly characterized. By whole exome sequencing of 21 cases of MALT lymphomas of the salivary gland and thyroid, we have identified recurrent somatic mutations in 2 G-protein coupled receptors (GPR34 and CCR6) not previously reported in human malignancies, 3 genes (PIK3CD, TET2, TNFRSF14) not previously implicated in MALT lymphoma, and a further 2 genes (TBL1XR1, NOTCH1) recently described in MALT lymphoma. The majority of mutations in GPR34 and CCR6 were nonsense and frameshift changes clustered in the C-terminal cytoplasmic tail, and would result in truncated proteins that lack the phosphorylation motif important for β-arrestin-mediated receptor desensitization and internalization. Screening of these newly identified mutations, together with previously defined genetic changes, revealed distinct mutation profiles in MALT lymphoma of various sites, with those of salivary gland characterized by frequent TBL1XR1 and GPR34 mutations, thyroid by frequent TET2, TNFRSF14 and PIK3CD mutations, and ocular adnexa by frequent TNFAIP3 mutation. Interestingly, in MALT lymphoma of the salivary gland, there was a significant positive association between TBL1XR1 mutation and GPR34 mutation/translocation (P=0.0002). In those of ocular adnexa, TBL1XR1 mutation was mutually exclusive from TNFAIP3 mutation (P=0.049), but significantly associated with IGHV3-23 usage (P=0.03) and PIK3CD mutation (P=0.009). These findings unravel novel insights into the molecular mechanisms of MALT lymphoma and provide further evidence for potential oncogenic co-operation between receptor signaling and genetic changes.
  • ItemAccepted versionOpen Access
    Human papillomavirus genome integration in squamous carcinogenesis: what have next-generation sequencing studies taught us?
    (Wiley, 2018-05) Groves, Ian J; Coleman, Nicholas; Groves, Ian J [0000-0001-8882-6701]; Coleman, Nicholas [0000-0002-5374-739X]
    Human papillomavirus (HPV) infection is associated with ∼5% of all human cancers, including a range of squamous cell carcinomas. Persistent infection by high-risk HPVs (HRHPVs) is associated with the integration of virus genomes (which are usually stably maintained as extrachromosomal episomes) into host chromosomes. Although HRHPV integration rates differ across human sites of infection, this process appears to be an important event in HPV-associated neoplastic progression, leading to deregulation of virus oncogene expression, host gene expression modulation, and further genomic instability. However, the mechanisms by which HRHPV integration occur and by which the subsequent gene expression changes take place are incompletely understood. The advent of next-generation sequencing (NGS) of both RNA and DNA has allowed powerful interrogation of the association of HRHPVs with human disease, including precise determination of the sites of integration and the genomic rearrangements at integration loci. In turn, these data have indicated that integration occurs through two main mechanisms: looping integration and direct insertion. Improved understanding of integration sites is allowing further investigation of the factors that provide a competitive advantage to some integrants during disease progression. Furthermore, advanced approaches to the generation of genome-wide samples have given novel insights into the three-dimensional interactions within the nucleus, which could act as another layer of epigenetic control of both virus and host transcription. It is hoped that further advances in NGS techniques and analysis will not only allow the examination of further unanswered questions regarding HPV infection, but also direct new approaches to treating HPV-associated human disease. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
  • ItemPublished versionOpen Access
    STAT3 activation by E6 is essential for the differentiation-dependent HPV18 life cycle.
    (Public Library of Science (PLoS), 2018-04) Morgan, Ethan L; Wasson, Christopher W; Hanson, Lucy; Kealy, David; Pentland, Ieisha; McGuire, Victoria; Scarpini, Cinzia; Coleman, Nicholas; Arthur, J Simon C; Parish, Joanna L; Roberts, Sally; Macdonald, Andrew; Wasson, Christopher W [0000-0001-6558-4738]; Arthur, J Simon C [0000-0002-8135-1958]; Parish, Joanna L [0000-0002-7111-4211]; Roberts, Sally [0000-0003-4653-9442]; Macdonald, Andrew [0000-0002-5978-4693]
    Human papillomaviruses (HPV) activate a number of host factors to control their differentiation-dependent life cycles. The transcription factor signal transducer and activator of transcription (STAT)-3 is important for cell cycle progression and cell survival in response to cytokines and growth factors. STAT3 requires phosphorylation on Ser727, in addition to phosphorylation on Tyr705 to be transcriptionally active. In this study, we show that STAT3 is essential for the HPV life cycle in undifferentiated and differentiated keratinocytes. Primary human keratinocytes containing high-risk HPV18 genomes display enhanced STAT3 phosphorylation compared to normal keratinocytes. Expression of the E6 oncoprotein is sufficient to induce the dual phosphorylation of STAT3 at Ser727 and Tyr705 by a mechanism requiring Janus kinases and members of the MAPK family. E6-mediated activation of STAT3 induces the transcription of STAT3 responsive genes including cyclin D1 and Bcl-xL. Silencing of STAT3 protein expression by siRNA or inhibition of STAT3 activation by small molecule inhibitors, or by expression of dominant negative STAT3 phosphorylation site mutants, results in blockade of cell cycle progression. Loss of active STAT3 impairs HPV gene expression and prevents episome maintenance in undifferentiated keratinocytes and upon differentiation, lack of active STAT3 abolishes virus genome amplification and late gene expression. Organotypic raft cultures of HPV18 containing keratinocytes expressing a phosphorylation site STAT3 mutant display a profound reduction in suprabasal hyperplasia, which correlates with a loss of cyclin B1 expression and increased differentiation. Finally, increased STAT3 expression and phosphorylation is observed in HPV positive cervical disease biopsies compared to control samples, highlighting a role for STAT3 activation in cervical carcinogenesis. In summary, our data provides evidence of a critical role for STAT3 in the HPV18 life cycle.
  • ItemPublished versionOpen Access
    Gamma-herpesvirus latency requires T cell evasion during episome maintenance.
    (Public Library of Science (PLoS), 2005-04) Bennett, Neil J; May, Janet S; Stevenson, Philip G; Stevenson, Philip [0000-0002-3520-5060]
    The gamma-herpesviruses persist as latent episomes in a dynamic lymphocyte pool. Their consequent need to express a viral episome maintenance protein presents a potential immune target. The glycine-alanine repeat of the Epstein-Barr virus episome maintenance protein, EBNA-1, limits EBNA-1 epitope presentation to CD8(+) T lymphocytes (CTLs). However, CTL recognition occurs in vitro, so the significance of such evasion for viral fitness is unclear. We used the murine gamma-herpesvirus-68 (MHV-68) to define the in vivo contribution of cis-acting CTL evasion to host colonisation. Although the ORF73 episome maintenance protein of MHV-68 lacks a glycine-alanine repeat, it was equivalent to EBNA-1 in conferring limited presentation on linked epitopes. This was associated with reduced protein synthesis and reduced protein degradation. We bypassed the cis-acting evasion of ORF73 by using an internal ribosome entry site to express in trans-a CTL target from the same mRNA. This led to a severe, MHC class I-restricted and CTL-dependent reduction in viral latency. Thus, despite MHV-68 encoding at least two trans-acting CTL evasion proteins, cis-acting evasion during episome maintenance was essential for normal host colonisation.
  • ItemOpen Access
    Regulation of Human γδ T Cells by BTN3A1 Protein Stability and ATP-Binding Cassette Transporters
    (Frontiers Media, 2018-04-04) Rhodes, DA; Chen, Hung-Chang; Williamson, James C; Hill, Alfred; Yuan, Jack; Smith, Sam; Rhodes, Harriet; Trowsdale, John; Lehner, Paul J; Herrmann, Thomas; Eberl, Matthias; Williamson, James [0000-0002-2009-189X]; Trowsdale, John [0000-0002-0150-5698]; Lehner, Paul [0000-0001-9383-1054]
    Activation of human Vγ9/Vδ2 T cells by ‘phosphoantigens’ (pAg), the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) and the endogenous isoprenoid intermediate isopentenyl pyrophosphate (IPP), requires expression of butyrophilin BTN3A molecules by presenting cells. However, the precise mechanism of activation of Vγ9/Vδ2 T cells by BTN3A molecules remains elusive. It is not clear what conformation of the three BTN3A isoforms transmits activation signals nor how externally delivered pAg accesses the cytosolic B30.2 domain of BTN3A1. To approach this problem we studied two HLA haplo-identical HeLa cell lines, termed HeLa-L and HeLa-M, which showed marked differences in pAg-dependent stimulation of Vγ9/Vδ2 T cells. Levels of IFN-γ secretion by Vγ9/Vδ2 T cells were profoundly increased by pAg loading, or by binding of the pan-BTN3A specific agonist antibody CD277 20.1, in HeLa-M compared to HeLa-L cells. IL-2 production from a murine hybridoma T cell line expressing human Vγ9/Vδ2 TCR transgenes confirmed that the differential responsiveness to HeLa-L and HeLa-M was TCR dependent. By tissue typing, both HeLa lines were shown to be genetically identical and full-length transcripts of the three BTN3A isoforms were detected in equal abundance with no sequence variation. Expression of BTN3A and interacting molecules such as periplakin or RhoB did not account for the functional variation between HeLa-L and HeLa-M cells, although evidence implicates a mechanism controlling BTN3A protein stability and trafficking. Plasma membrane profiling was used to identify proteins upregulated in HMB-PP treated HeLa-M. ABCG2, a member of the ABC transporter family was the most significant candidate, which crucially showed reduced expression in HeLa-L. Expression of a subset of ABC transporters, including ABCA1 and ABCG1, correlated with efficiency of T cell activation by cytokine secretion, although direct evidence of a functional role was not obtained by knockdown experiments. Our findings indicate a link between members of the ABC protein superfamily and the BTN3A-dependent activation of γδ T cells by endogenous and exogenous pAg.
  • ItemAccepted versionOpen Access
    Translating In Vivo Metabolomic Analysis of Succinate Dehydrogenase–Deficient Tumors Into Clinical Utility
    (American Society of Clinical Oncology (ASCO), 2018-11) Casey, Ruth T; McLean, Mary A; Madhu, Basetti; Challis, Benjamin G; ten Hoopen, Rogier; Roberts, Thomas; Clark, Graeme R; Pitfield, Deborah; Simpson, Helen L; Bulusu, Venkata R; Allinson, Kieren; Happerfield, Lisa; Park, Soo-Mi; Marker, Alison; Giger, Olivier; Maher, Eamonn R; Gallagher, Ferdia A; McLean, Mary [0000-0002-3752-0179]; Giger, Olivier [0000-0003-3390-6397]; Maher, Eamonn [0000-0002-6226-6918]; Gallagher, Ferdia [0000-0003-4784-5230]
    Purpose Mutations in the mitochondrial enzyme succinate dehydrogenase (SDH) subunit genes are associated with a wide spectrum of tumors, including pheochromocytomas and paragangliomas, GI stromal tumors, renal cell carcinomas, and pituitary adenomas. SDH-related tumorigenesis is believed to be secondary to accumulation of the oncometabolite succinate. Our aim was to investigate the potential clinical applications of proton-1 magnetic resonance spectroscopy (1H-MRS) in a range of suspected SDH-related tumors. Patients and Methods Fifteen patients were recruited to this study. Respiratory-gated single-voxel 1H-MRS was performed at 3T to quantify the content of succinate at 2.4 ppm and choline at 3.22 ppm. Results A succinate peak was seen in six patients, all of whom had germ line SDHx mutations or loss of SDHB by immunohistochemistry. Succinate peaks were also detected in two patients with metastatic wild-type GI stromal tumors and no detectable germ line SDHx mutations but with somatic epimutations in SDHC. Three patients without tumor succinate peaks retained SDHB expression, consistent with SDH functionality. In six patients with borderline or absent peaks, technical difficulties such as motion artifact rendered 1H-MRS difficult to interpret. Sequential imaging in a patient with a metastatic abdominal paraganglioma demonstrated loss of the succinate peak after four cycles of [177Lu]DOTATATE, with a corresponding biochemical response in normetanephrine. Conclusion This study has demonstrated the translation into clinical practice of in vivo metabolomic analysis using 1H-MRS in patients with SDH-deficient tumors. Potential applications include noninvasive diagnosis and disease stratification, as well as monitoring of tumor response to targeted treatments.
  • ItemPublished versionOpen Access
    ALK in Neuroblastoma: Biological and Therapeutic Implications.
    (MDPI AG, 2018-04-10) Trigg, Ricky M; Turner, Suzanne D; Trigg, Ricky M [0000-0001-9329-9344]; Turner, Suzanne D [0000-0002-8439-4507]
    Neuroblastoma (NB) is the most common and deadly solid tumour in children. Despite the development of new treatment options for high-risk NB, over half of patients relapse and five-year survival remains at 40-50%. Therefore, novel treatment strategies aimed at providing long-term disease remission are urgently sought. ALK, encoding the anaplastic lymphoma kinase receptor, is altered by gain-of-function point mutations in around 14% of high-risk NB and represents an ideal therapeutic target given its low or absent expression in healthy tissue postnatally. Small-molecule inhibitors of Anaplastic Lymphoma Kinase (ALK) approved in ALK fusion-positive lung cancer are currently undergoing clinical assessment in patients with ALK-mutant NB. Parallel pre-clinical studies are demonstrating the efficacy of ALK inhibitors against common ALK variants in NB; however, a complex picture of therapeutic resistance is emerging. It is anticipated that long-term use of these compounds will require combinatorial targeting of pathways downstream of ALK, functionally-related 'bypass' mechanisms and concomitant oncogenic pathways.
  • ItemPublished versionOpen Access
    A three-dimensional atlas of human dermal leukocytes, lymphatics, and blood vessels.
    (Elsevier BV, 2014-04) Wang, Xiao-Nong; McGovern, Naomi; Gunawan, Merry; Richardson, Connor; Windebank, Martin; Siah, Tee-Wei; Lim, Hwee-Ying; Fink, Katja; Yao Li, Jackson L; Ng, Lai G; Ginhoux, Florent; Angeli, Veronique; Collin, Matthew; Haniffa, Muzlifah; McGovern, Naomi [0000-0001-5200-2698]
    Dendritic cells (DCs), macrophages (Mφ), and T cells are major components of the skin immune system, but their interstitial spatial organization is poorly characterized. Using four-channel whole-mount immunofluorescence staining of the human dermis, we demonstrated the three-dimensional distribution of CD31(+) blood capillaries, LYVE-1(+) lymphatics, discrete populations of CD11c(+) myeloid DCs, FXIIIa(+) Mφ, and lymphocytes. We showed phenotypic and morphological differences in situ between DCs and Mφ. DCs formed the first dermal cellular layer (0-20 μm beneath the dermoepidermal junction), Mφ were located deeper (40-60 μm), and CD3(+) lymphocytes were observed throughout (0-60 μm). Below this level, DCs, T cells, and the majority of Mφ formed stable perivascular sheaths. Whole-mount imaging revealed the true extent of dermal leukocytes previously underestimated from cross-section views. The total area of apical dermis (0-30 μm) contained approximately 10-fold more myeloid DCs than the entire blood volume of an average individual. Surprisingly, <1% of dermal DCs occupied lymphatics in freshly isolated skin. Dermal DCs rapidly accumulated within lymphatics, but Mφ remained fixed in skin explants cultured ex vivo. The leukocyte architecture observed in normal skin was distorted in inflammation and disease. These studies illustrate the micro-anatomy of dermal leukocytes and provide further insights into their functional organization.