Theses - Pathology

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Determining stromal evolution in response to immune checkpoint inhibition
Moody, William
Directing the immune system to recognise and eliminate developing tumours has been hypothesised for over a century, yet only recently has this concept translated to the clinic with the discovery of immune checkpoint inhibitors (ICIs) which reactivate anti-tumour T cells. However, their rapid translation into front-line treatment has left a number of the fundamental mechanisms of action of the drugs underexplored. One outstanding question is why a significant fraction of patients don’t respond to ICIs? Here, we begin to address this by investigating the response of non-immune components of the tumour microenvironment (TME) to ICI therapy, focusing on cancer-associated fibroblasts (CAFs). CAFs are an abundant and heterogeneous component of the TME, supporting tumourigenesis by secreting growth factors, cytokines, chemokines, extracellular matrix (ECM) and remodelling the physical environment. Indeed, these disparate functions have been shown to be performed by distinct subpopulations present in the TME. We observed significant alterations to the composition of the CAF compartment following ICI. ICI disrupted the evolutionary trajectory of CAF subpopulations, as ‘S1’ CAFs with increased immune modulatory capacity accumulated at the expense of ‘S3’ CAFs specialised in contractility. Single cell RNA-sequencing suggested S1 CAFs became more immune licenced following αPD-1 treatment, upregulating genes involved in MHC class I antigen presentation at early time points, and later in tumour development, ‘S2’ CAFs classically associated with ECM deposition also increased expression of antigen presentation genes. Alongside a decrease of immune-inhibitory receptors, we detected an increase in co-stimulatory molecules in CAFs from tumours responding to ICI. Our data indicates that these changes are driven by inflammatory mediators including IFN-γ, released by re-activated tumour infiltrating immune cells. Finally, we identified CAF-derived ECM as a potential barrier to ICI response. Combination of αPD-1 with losartan, a collagen synthesis inhibitor, showed a synergistic effect, increasing T cell infiltration and decreasing tumour growth. Together, these results show that the non-immune stroma plays an important role in determining ICI therapeutic efficacy, and is a source of novel biomarkers, as well as targets for new therapy combinations.
Embargo
Novel treatment strategy for ALK-positive Anaplastic Large Cell Lymphoma
Mun, Liew Jun
Anaplastic Lymphoma Kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) is a T-cell non-Hodgkin lymphoma characterised by a chromosomal rearrangement [t(2;5) (p23;q25)] involving the ALK and NPM1 genes on chromosomes 2 and 5 respectively. This translocation generates the NPM-ALK fusion protein, a hyperactive tyrosine kinase that is the driving oncogenic event in ALCL. Single-agent use of tyrosine kinase inhibitors (TKI) to inhibit ALK is a conventional second-line treatment and has been trialled for upfront use in combination with the ALCL99 therapeutic backbone. However, an extended duration of treatment is required as cessation of therapy can lead to rapid relapse although this is often salvageable with re-administration of the same or an alternative ALK TKI. In addition, for some patients, resistance can develop, and this tends to happen soon after starting treatment. Hence, whilst ALK TKIs hold much promise for the treatment of ALCL, there remain clinical issues that need to be addressed. One approach would be the introduction of a second targeted drug that has a different mechanism of action to be used in combination with ALK TKIs, which may potentiate these effects and could improve clinical outcomes. To identify alternative drug targets for ALK-positive ALCL in particular, those that may, when inhibited, have synergistic effects with ALK TKIs, cell lines were employed to investigate oncogenic activity of NPM-ALK in the nucleus of ALCL. Whilst NPM-ALK is known to be located in the cytoplasm where it drives the activity of several signalling pathways, its nuclear location and functions remain largely unexplored. Initially, I showed that NPM-ALK is in the chromatin fraction within the nucleus and that it binds to DNA, although likely not directly, but rather through a protein complex. Furthermore, by conducting NPM-ALK: DNA crosslinking followed by CUT&RUN- sequencing I showed that NPM-ALK, albeit indirectly, drives the expression of an RNA methyltransferase, N6-adenosine-methyltransferase 70 kDa subunit (METTL3). This enzyme catalyses N6-methyladenosine (m6A), the most abundant mRNA modification, through a complex with METTL14 and WTAP. Whilst METTL3 has been described as both an oncogene and a tumour suppressor gene in different cancers respectively, its expression and link to NPM-ALK activity in ALCL led me to investigate its oncogenic activities. Following exposure of cells to ALK TKIs or NPM-ALK knockdown, thereby inhibiting METTL3 activity, I showed by nucleoside mass spectrometry that cells contained reduced levels of m6A in polyA+ captured RNA. Furthermore, METTL3 was shown to be required for cell growth following shRNA-specific knockdown. A similar effect was observed by employing a novel, potent and selective first-in-class METTL3 inhibitor (METTL3i). Furthermore, inhibition of METTL3 activity with either the METTL3i, or knockdown of METTL3 combined with NPM-ALK inhibition showed significant additive activity in reducing cell viability. To validate this activity in a more clinically relevant model, a patient-derived xenograft (PDX) of ALCL adapted to culture in vitro was exposed to ALK and METTL3 inhibitors or METTL3 knockdown. Again, the combination of ALK and METTL3 inhibition showed a reduction in cell survival with additive effects. The mechanism for the observed reduction in cell viability was investigated further showing that the 3rd generation ALK TKI Lorlatinib led to cellular senescence, but that in addition to METTL3i, these cells underwent apoptosis. These data suggest that not only will the combination of ALK TKI and METTL3 inhibitor decrease viability, but it may also prevent residual senescent cells from reseeding tumour growth. Of interest, an additional PDX-derived cell line from an ALK TKI-resistant (Crizotinib) patient tumour was sensitive to METTL3 inhibition and showed near synergistic effects with the second-generation ALK TKI, Brigatinib. Subsequently, to identify alternative potential treatment options, drugs that affect epigenetic activities in the cell were investigated for their ability to impact ALCL cell survival in vitro. ALCL cell lines were exposed to METTL3i in combination with an ‘epigenetic drug’ library of 281 compounds that directly or indirectly affect epigenetic targets for 48 hours, and cell survival was monitored by a resazurin-based assay. This drug screen showed that METTL3i could potentiate several classes of ‘epigenetic drugs’ such as a G9a/GLP histone lysine methyltransferase inhibitor that was ineffective in reducing ALCL cell viability when used as a single agent. In conclusion, METTL3 is a novel downstream effector of NPM-ALK activity that acts in an oncogenic capacity to drive cell survival. Hence, inhibition of METTL3 represents a novel treatment approach that has additive activity with ALK TKIs and other ‘epigenetic drugs’ and may therefore prevent the development of ALK TKI resistance and long-term use of ALK inhibitors in the future.
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Assessing the reprogramming of somatic cells through nuclear transfer or extract incubation and the roles of chromatin remodeler Brg1
Barbosa Triana, Hector
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Investigating the Genomics and Heterogeneity of Childhood Non-Hodgkin Lymphoma
Matthews, Jamie; Matthews, Jamie [0000-0002-2980-8615]
The commonest childhood Non-Hodgkin Lymphomas (NHLs) form three major categories: the aggressive mature B-cell NHLs including Burkitt lymphoma (BL), Anaplastic Large Cell Lymphomas (ALCL), and lymphoblastic lymphomas. Most childhood NHL is high-grade and generally responds well to high-intensity chemotherapy. Unfortunately, many patients who relapse have a dismal prognosis, with long-term survival less than 30% for relapsed BL patients in particular. Furthermore, the side effects of chemotherapy on children are often severe, rendering intensive chemotherapy infeasible in less economically-developed regions. Significant progress has been made with the addition of Rituximab to BL therapy further improving overall survival, and the use of ALK inhibitors such as crizotinib have been used to successfully bridge relapsed or refractory ALCL patients to stem cell transplantation---however, resistance to both ALK inhibitors and Rituximab occurs in the clinic. Therefore, there is urgent need for less toxic therapies in primary NHL to improve the quality of life for curable patients, and to reduce the treatment-related mortality for patients without access to high-quality supportive care. Secondly, more effective therapies or strategies to treat relapsed/refractory disease are required to improve survival rates even in well-resourced settings. It is now recognised that genetic intratumour heterogeneity (ITH) contributes to drug resistance in malignant disease. However, many questions remain unresolved, and improved theoretical and experimental model systems will likely be required to resolve them. To this latter end, a novel relapsed BL patient-derived xenograft (PDX) was established---to our knowledge the first described in the literature---and the maintenance of genetic ITH through engraftment in immune-compromised mice was demonstrated through whole genome sequencing (WES). In addition, pervasive genetic ITH across time and space was detected by multiregion WES of BL cases, meaning that single biopsies cannot rule out the presence of a given variant, regardless of sequencing depth. Furthermore, analysis of allele frequencies in publicly-available data indicated that some BL patient samples harbour genetic evidence of recent subclonal selection, while others appear to have evolved neutrally or near-neutrally. These data highlight the pervasive nature of ITH and thus motivate the use of patient-derived xenograft models which better capture patient tumour heterogeneity than the long-established cell lines often used in preclinical studies. Similarly, a resource of relapsed ALCL PDX was generated and assessed for drug sensitivity. Two novel ALCL PDXs were established from patients at or before CNS replase, and a third was acquired from collaborators. All three patients had failed chemotherapy and crizotinib treatment, and two patients were resistant to crizotinib. WES of PDX revealed likely mechanisms of crizotinib resistance, with one patient harbouring concomitant ALK mutation and amplification, and the other showing amplification of several oncogenes including IRF4, which has previously been implicated in ALCL pathogenesis. Next, I tested the second generation ALK inhibitor brigatinib, demonstrating the utility of our models as a platform to test targeted therapies. Brigatinib was highly effective in the treatment of all three PDX, and superior to crizotinib in the crizotinib-sensitive PDX. Several brigatinib-treated mice demonstrated complete tumour remission after one to three weeks of treatment, pointing to brigatinib as a therapy option for high-risk patients. While increasing understanding of the molecular underpinnings of cancer is yielding promising new drug targets, translation of novel therapeutics into clinical practice is costly and time-consuming, with the 'bench to bedside' process rarely being completed within a decade. A major component of this process is testing for safety in animal and human subjects. Drug repurposing refers to the application of previously approved or well-understood drugs to new clinical settings, and has the potential to yield useful treatments in less time by selecting from drugs for which safety profiles have already been determined. Therefore, an /in vitro/ high-throughput screen using approximately 1400 regulatory body-approved drugs applied to BL and ALCL cell lines was undertaken, with several promising hits identified. However, the extent to which established cell lines faithfully represent patient disease is questionable, as /in vitro/ culture conditions can cause rapid divergence from the originating tumour, limiting translational relevance. Therefore, a proof-of-concept drug screen platform for BL PDX cells supported by a feeder cell line was established, offering a relatively low-cost, high-throughput platform to screen drugs for a given BL patient tumour. In conclusion, this thesis demonstrates extensive genetic ITH in BL, with implications for tumour sampling and resistance to treatment. Novel ALCL PDXs were generated, representing a patient group of unmet clinical need, and providing a resource to test drugs /in vivo/ with tumours of known genetic background. Finally, high-throughput drug screening was undertaken in order to identify known therapeutics with efficacy against NHL cell lines, and a co-culture system was established in order to extend screening to patient-derived cells.
Embargo
Understanding and Targeting Clearance of Persistent HPV Infections
Zheng, Ke
Given the great diversity of HPV types and tropism to epithelial sites (with associated diseases ranging from mild skin lesions to cancer), they share general genome organization and life strategy for long term infection after initial disease regression. Persistent infected cells form a viral reservoir, causing disease occurrence and are particularly problematic during carcinogenesis development for high-risk types; therefore, understanding HPV-mediated epithelial homeostasis is essential for understanding disease progression. In this study, I applied the multiplex high-content confocal imaging system to reveal the spatial heterogenicity of keratinocytes with and without exogenous HPV E6 expression. Two in vitro model systems were developed to mimick the basal/suprabasal layers of the epithelium and evaluate HPV E6 functions in modulating cellular environment based on either the expression of a real-time cell cycle indicator (FUCCI) and early differentiation marker (KRT10); or the competition between differently fluorescently labelled infected versus uninfected cells. Here, it is presented that HPV16E6 expressing keratinocytes exhibited increased proliferation, delayed contact inhibition and commitment to differentiation compared with normal keratinocytes. When the two populations are cultured together, HPV16E6 expressing cells have growth advantage over the adjacent uninfected neighbours in colonising the basal layer of epithelium. Functional-deficient mutants of HPV16E6 revealed PDZ-domain containing proteins contributes to the competition advantage independent of the p53-regulated cell cycle progression. Further RNAi on keratinocytes of established HPV16E6 PDZ targets suggested MAGI and hPAR3 are two important modulators in maintaining basal cell life span and facilitates the formation of viral reservoir. Similar phenotypes are also observed when keratinocytes expressing the E6 protein from different genera (α-HPV11/16/27, β-HPV8, γ-HPV65 and µ-HPV1), suggesting E6 is a conserved basal epithelial modulator which co-evolved with different epithelial sites for the establishment of local persistent HPV infections. These results provide new insights of the function diverted biology of HPV during disease development, which may fuel the understanding of basal epithelial homeostasis and the development of successful anti-viral therapies.
Open Access
Molecular biology of segment 8 of the influenza A virus genome
Arikainen, Artur
The single-stranded RNA genome of influenza A virus is divided into 8 segments, each of which encodes at least one polypeptide product. This work presents investigations into two aspects of the biology of the smallest (8th) segment: 1) a possible new gene product and 2) interactions of the viral non-structural protein translated from this segment – NS1 – with a host cell protein during infection. 1) The genome of influenza A virus is packaged into virions as negative sense viral RNA. To date, all known viral proteins are encoded in the positive sense. A large open reading frame exists on segment 8 of the influenza A genome in the opposite sense to, yet overlapping with, the established proteins expressed from that segment; this hypothetically encodes a protein named Negative Strand Protein or NSP. This thesis presents work that characterised the NSP polypeptide that would be translated from this open reading frame and the effect it may exert on viral replication. Using a reverse genetics system for the A/Puerto Rico/8/34 influenza A strain, mutant viruses were generated containing negative strand open reading frames of different lengths, as well as one with early stop codons that result in a severe truncation. All viruses replicated to similar titres in cell culture and produced similarly-sized plaques as the wild-type. The synthesis and distribution of the major viral structural proteins as well as the positive-strand segment 8 polypeptides were also not affected by alterations to the open reading frame. Negative strand polypeptide produced by in vitro translation was successfully immunoprecipitated by a specific antibody. However, no specific product could be detected from virus-infected cells. In conclusion, if this negative strand protein is indeed expressed by influenza A virus, it is not significant for virus replication in vitro, nor is it expressed to high levels. 2) The nucleolus is a dynamic hub of post-transcriptional RNA processing in eukaryotic cells. The influenza NS1 protein is a multi-functional inhibitor of many cellular anti-viral pathways, such as the interferon response. Prior to this work, a proteomics study found that influenza A virus infection induces the re-localisation of cellular protein ADAR1 to the nucleolus. ADAR1 is an RNA-editing enzyme that converts adenosine residues to inosines, which are then interpreted by cellular machinery as guanosine. There is a potential that ADAR1 activity may affect the influenza virus genome; either negatively through hypermutation, or positively via a specific coding sequence alterations. Evidence for non-specific editing of influenza RNA species has been reported, but the significance of this has not been demonstrated. For other virus families such as vesicular stomatitis, measles, human immunodeficiency and hepatitis delta viruses, ADAR1 has an apparently pro-viral role. The influenza A NS1 protein was determined to be both necessary and sufficient to induce the re-localisation of ADAR1 to cell nucleoli in several common cell lines. Analysis of viruses expressing mutant forms of the NS1 protein showed that lesions in interaction sites for binding to RNA or a cellular ubiquitin ligase – TRIM25 – prevented induction of this re-localisation. This correlated with the inability of these NS1 mutant proteins to suppress phosphorylation of cellular interferon response factor 3. It was also found, using pull-down assays, that ADAR1 interacted with influenza NS1 in an RNA-independent manner, but not with the NS1 mutant deficient in RNA binding. Additionally, a separate interaction was demonstrated between ADAR1 and components of the viral polymerase complex. In cells that had ADAR1 depleted by RNA interference, viral replication was reduced by 3-fold, thus not supporting the hypothesis that ADAR1 is a restriction factor for influenza A virus and instead suggesting a modest pro-viral function. In conclusion, the influenza A virus NS1 protein induces the nucleolar re-localisation of ADAR1, which does not act as an anti-viral factor for the virus.
Embargo
Multi-modal imaging of herpesvirus infection
Nahas, Kamal; Nahas, Kamal [0000-0003-3501-8473]
Herpes simplex virus (HSV)-1 is a large, enveloped DNA virus that undergoes a complex assembly pathway in the cell. Capsids assemble in the nucleus, cross the nuclear envelope into the cytoplasm by budding and fusion through the nuclear membranes, and acquire a proteinaceous ‘tegument’ layer in the cytoplasm before becoming enveloped at trans-Golgi network vesicles and/or recycling endosomes. Although the virus assembly pathway has been studied in detail using fluorescence and electron microscopy, several aspects of the process remain poorly understood. The remodelling of cellular organelles during HSV-1 infection has not been robustly quantitated using three dimensional (3D) volumetric analysis and the involvement of different cytoplasmic vesicles in envelopment remains to be differentiated. Furthermore, the precise roles of multiple viral gene products that are important for HSV- 1 assembly remain to be fully characterised. I used cryo-soft-X-ray tomography to image the ultrastructure of cryopreserved infected cells in 3D. In order to analyse these tomograms I developed Contour, a program to semi-automate the segmentation and quantitation of features from cryo-soft-X- ray tomograms. I characterised the role of different viral proteins in assembly by infecting cells with recombinant HSV-1 lacking expression of specific proteins and monitoring virion assembly in cells at late stages of infection. Super-resolution structured illumination microscopy allowed me to distinguish between unenveloped and enveloped virus particles in these cryopreserved samples. Tomographic X- ray imaging of HSV-1-infected cells allowed me to capture and characterise different stages of HSV-1 assembly in 3D. Additionally, confocal microscopy was used to probe the differential roles of TGN46- and transferrin receptor (TfR)-positive vesicles in the cytoplasmic envelopment of HSV-1 capsids. Striking changes occur to the morphology of mitochondria and the organisation of cytoplasmic vesicles and lipid droplets during HSV-1 infection. Comparative tomographic analysis offer insight into the roles of pUL16, pUL21, pUL34, VP16, and pUS3 in HSV-1 nuclear egress, and the roles of pUL11, VP16, pUL51, gK, and gE in cytoplasmic envelopment. Furthermore, this analysis indicates that envelopment occurs by budding of tegument-associated capsids into glycoprotein-containing patches of cellular vesicles, as opposed to wrapping of thin lamellae around capsids. The confocal microscopy data analysis suggests that TGN46+ vesicles are essential for envelopment but TfR+ vesicles are only sometimes involved. This multi-modal 3D imaging strategy has enabled elaboration of outstanding questions regarding the assembly pathway of HSV-1.
Embargo
Macrophage Diversity in the First Trimester Human Placenta: Phenotypes, Functions and Developmental Origins
Thomas, Jake
The placenta is the fetal-derived organ that forms the interface between mother and child during pregnancy and is essential for maintaining fetal and maternal health throughout gestation. The human placenta contains a population of tissue-resident macrophages called Hofbauer cells (HBC), however the functions and developmental origins of these cells remain poorly defined. Our attempts to profile HBC in the first trimester human placenta revealed the presence of maternally-derived cells in placental digests. We established reliable gating strategies for the isolation of HBC and placenta-associated maternal macrophages/monocytes (PAMM), allowing us to profile their functions at the steady state and in response to inflammatory stimuli. Our data suggest that HBC have roles in angiogenesis and tissue remodelling, whilst PAMM are involved in the repair of the placental surface. We also determined that HBC have the capacity to play a defensive role in protecting the fetus from pathogens, as they are responsive to Toll-like receptor stimulation and display microbicidal activity in vitro. Through transcriptomic analyses we show that HBC are likely derived from the first primitive wave of haematopoiesis in development, and analysis of publicly available datasets revealed that macrophages derived from primitive haematopoiesis can be specifically identified by a lack of HLA Class II expression. Finally, we identify a population of haematopoietic progenitors in the early first trimester placenta which display features of primitive erythro- myeloid progenitors. Using in vitro single cell culture experiments we show that these progenitors generate HBC-like cells which lack HLA-DR expression. These findings indicate that HBC are derived locally within the placenta via primitive haematopoiesis. Together this research provides a new framework for human placental immunology. By increasing our understanding of placental macrophages new insights will emerge about human placental development and how this underpins pregnancy disorders.
Embargo
Phenotypic and Functional Characterisation of Hofbauer Cells Across Gestation
Appios, Anna
The placenta is the first organ the fetus makes and is the interface between the mother and baby. A normal functioning placenta is crucial for a successful pregnancy. As the placenta develops, highly branched villous tree-like structures form which contain fibroblasts, immature capillaries and macrophages, termed Hofbauer cells (HBC). HBC are the only fetal immune cell population found in the healthy placenta throughout pregnancy. The roles of HBC at different stages of pregnancy are unclear, but they are likely to be important in placental development. We sought to provide an in-depth, high-dimensional characterisation of HBC within first trimester and full-term placenta, understand how changes in their function aid placental physiology, and investigate potential factors driving HBC characteristics. Whole-mount immunofluorescence imaging revealed that HBC morphology within placental villi changes during gestation; appearing as rounded cells in the first trimester compared to highly elongated and branched cells at full term. Live ex vivo imaging demonstrated that HBC were motile in the first-trimester placenta and sessile by full term. A high-dimensional mass cytometry (CyTOF) panel was developed and used to show that within the first trimester placenta HBC were a homogeneous population, lacking expression of the HLA class II marker HLA-DR. This contrasted with full-term placenta where HBC displayed heterogeneous HLA- DR expression. Analysis of publicly available 10X Genomics single-cell RNA-sequencing data determined that full-term HBC heterogeneity was restricted to variable HLA-DR expression. Furthermore, a robust bulk RNA-sequencing dataset was generated to profile placental macrophages from the first trimester and full-term placenta. This allowed for in-depth transcriptomic analyses which determined exceptional similarity between full-term HBC subsets. Functional enrichment analyses revealed that first trimester HBC were associated with placental processes such as hormone synthesis, trophoblast migration and vasculogenesis. Contrastingly, full-term HBC were associated with complement regulation, angiogenesis, and branching morphogenesis. An optimised in vitro culture system was established to show that the HLA-DR expression profile of HBC remains unchanged upon culture and HBC are resistant to IFNγ-inducible HLA class II expression. ATAC-seq revealed that chromatin accessibility around HLA class II genes and the master regulator transcription factor CIITA was reduced in first trimester HBC compared to full-term HBC and monocytes. Lastly, monocyte-derived macrophage cultures demonstrated the importance of M-CSF for driving key features of the HBC phenotype. In summary, the results presented in this thesis provide an in-depth characterisation of HBC and how they change across gestation to aid placental function in a healthy pregnancy.
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Therapeutically Enforced Differentiation: Analysis of a PCAF/GCN5 PROTAC in acute myeloid leukaemia
Abbas, Minaam
[Restricted]
Embargo
Investigating the mechanism of LolB, the Bacterial Outer Membrane Lipoprotein Receptor
Jepson, Abigail
In Gram-negative bacteria, the outer membrane constitutes the first line of defence against toxic compounds including antibiotics. Lipoproteins are vital structural elements of this barrier and crucial components of machineries underpinning its generation and maintenance. Proper localisation of outer membrane lipoproteins is essential for bacterial survival and requires the dedicated trafficking system LolABCDE. In the terminal stage of transport, LolB, the outer membrane receptor, receives lipoproteins from the periplasmic chaperone LolA and delivers them into the membrane. The essentiality and outer membrane location of LolB makes it an appealing target for the design of new antibiotics. However, this requires detailed understanding of how LolB performs two critical functions; the acceptance of lipoproteins bound to LolA, and their subsequent insertion into the inner leaflet of the outer membrane. I used structure-led mutagenesis, crystallography, biophysical approaches and functional assays to identify residues and regions of LolB essential for its mechanism. Using a LolA mutant with a higher affinity for LolB, I trapped a stable LolA-LolB intermediate and resolved its structure by X-ray crystallography. The structure reveals how the concave surface of LolA interacts with the convex LolB beta-barrel by means of hydrogen bonds and charge-charge interactions, involving three different regions of LolB. Mutation of key residues mediating the interaction was carried out, and the effect on interaction with LolA and lipoprotein acceptance was measured. I identified single mutations that abolished interaction with LolA, and abrogated lipoprotein transfer by targeting multiple charged residues, underlining their importance in LolA-LolB interaction and the transfer mechanism. LolB is likely to undergo conformational changes to accommodate lipoprotein, but how this occurs or where the lipoprotein binds have not been determined. To address this, I mutated residues located in potential hinge regions, and used a fluorescence-based assay to detect opening of the LolB cavity, before assessing the efficiency of lipoprotein transfer. LolB variant proteins which most efficiently accepted lipoprotein were isolated and screened in crystal trials. Following optimisation, the structure of a LolB variant in complex with a lipoprotein fragment was obtained, revealing how the LolB cavity expands to accommodate the three lipoprotein acyl chains and providing molecular details of the interaction. The final step of lipoprotein transport, the insertion activity of LolB, was dissected by developing an in vitro insertion assay using lipid coated silica beads in combination with mutagenesis of LolB residues. These efforts underlined the importance of a key loop and allied with my previous structural data, suggests models of how protein insertion can be achieved. Altogether the combination of structural, biochemical and biophysical approaches provides essential insight into all facets of LolB function. The data presented not only leads to a greater understanding of a fundamental bacterial transport system, but may also assist the rational design of new antibacterial molecules targeting LolB function.
Open Access
Sensing Foreign DNA: The Role of DNA-PKcs in Human Anti-viral Innate Immunity
Hristova, Dayana
Host cell pattern recognition receptors (PRRs) are a first line of defence against pathogens and function to generate a productive innate immune response. PRRs sense pathogen-associated molecular patterns (PAMPs), such as viral genomic DNA, which is a major PAMP during DNA virus infection. Viral DNA sensing leads to the activation of the STING-TBK1-IRF3 signalling axis and the production of type I interferon. Previously, our work identified the non-homologous end-joining protein DNA-PKcs, part of the DNA-dependent protein kinase (DNA-PK) complex, as an intracellular PRR for cytoplasmic viral DNA in murine cells. After screening several human cell lines, we established a robust system to dissect the DNA sensing pathway in human fibroblasts. In these human cells DNA-PKcs was found to be essential for the production of type I interferon via the STING pathway in response to DNA and DNA virus infection and we found that the kinase activity of DNA-PKcs was not required for this response. Many DNA viruses evade the immune response by inhibiting the pathway. We make use of attenuated Herpes simplex virus 1 (HSV- 1) and Vaccinia virus (VACV) that lack immunomodulatory proteins and drive type I interferon production. DNA-PKcs-/- cells have a defective immune response after infection with attenuated HSV and VACV. Furthermore, primary patient fibroblasts harbouring a mutation in DNA-PKcs showed a gain-of-function effect and an enhanced immune signaling to DNA and DNA virus infection. DNA-PKcs has also been linked to cell death during retrovirus integration although this has not been studied extensively. We carried out some preliminary work in this study, showing that DNA-PKcs-/- cells are more prone to cell death during HSV-1 infection and have reduced yields of virion production. This work demonstrates the role of DNA-PKcs as a viral DNA sensor in human cells and adds to the knowledge of the DNA sensing processes that are essential for anti-viral innate immunity.
Open Access
The transcriptional programme of natural killer (NK) cell functional maturation and maintenance
Imianowski, Charlotte
Natural killer (NK) cells are critical to immune surveillance against infections and cancer. Their role in immune surveillance requires that NK cells are present within tissues in a quiescent state. The functional maturation of NK cells is a tightly regulated process which is controlled by transcription factors (TFs), and multiple positive regulators of this process have been defined. However, mechanisms by which NK cells remain quiescent in tissues are incompletely elucidated. The transcriptional repressor BACH2 plays a critical role within the adaptive immune system but its function within innate lymphocytes has been unclear. The studies presented here show that BACH2 acts as an intrinsic negative regulator of NK cell maturation and function. BACH2 is expressed within developing and mature NK cells and promotes the maintenance of immature NK cells by restricting their maturation in the presence of tonic IL-15 signalling. Loss of BACH2 within NK cells results in accumulation of activated NK cells with unrestrained cytotoxic function and increased immune surveillance to pulmonary cancer metastasis. These findings establish a critical function of BACH2 as a negative regulator of innate cytotoxic function and tumour immune surveillance by NK cells.
Embargo
Ubiquitination of proteins involved in metabolism and immunomodulatory drug sensitivity in lymphocytes
Harris, Rebecca; Harris, Rebecca [0000-0002-5854-4700]
Proteins undergo post-translational modifications, such as ubiquitination and phosphorylation, which can alter their activity, localisation and stability, making a cell responsive to its internal and external environment. Ubiquitin ligases and kinases comprise large enzyme families which catalyse such reactions. The SCF-type E3 ubiquitin ligase sub-family utilise F-box proteins as the substrate targeting component. However, alongside promoting ubiquitination, the F-box protein Fbxo7 can also function as a scaffold and stabilise a subset of proteins, including Cdk6. Cdk6 is activated through binding the D-type cyclins, and historically, a key role has been as a cell cycle regulatory kinase that inactivates G1 checkpoint proteins. More recently, wider roles for Cdk6 have been identified, including as an inhibitor of glycolysis. Notably, Cdk6 has pro-survival activity in T acute lymphoblastic leukaemia (T-ALL) cells due to the phosphorylation and inhibition of glycolytic enzymes, including the rate-limiting gatekeeper, phosphofructokinase (PFKP). Our screens for Fbxo7-interacting partners identified a set of candidates that overlapped as cyclin D/Cdk6 substrates and included PFKP. Further study revealed that Fbxo7 promotes two post-translational modifications on PFKP, ubiquitination and phosphorylation, and specifically promotes Cdk6 activity. Analysis in T-ALL cells suggest that Fbxo7 inhibits the assembly of active PFKP complexes to ultimately inhibit glycolysis. This is confirmed in a murine model of reduced Fbxo7 expression, whose CD4+ T cells show higher levels of glycolytic flux, alongside various other metabolic defects including altered nucleotide biosynthesis and arginine metabolism. This places Fbxo7 as a negative regulator of glycolysis and unveils other diverse roles in metabolism, which may contribute to viability and activation defects observed in these Fbxo7-deficient murine T cells. Given that Fbxo7 negatively regulates glycolysis via PFKP, I also investigated how glucose regulates Fbxo7, as feedback loops in glucose signalling are commonplace in metabolic networks. I discovered Fbxo7 is a dose-dependent, glucose responsive protein in numerous cell types, which is both transcriptionally downregulated and targeted for autophagy in response to glucose starvation. Moreover, data suggest that Fbxo7 is responsive to other stresses, including oxidative stress, placing Fbxo7 as a nexus to link various cellular stress responses to metabolic reprogramming. In addition to PFKP, Fbxo7 has also been shown to recognise a protein called cereblon (CRBN), which is another E3 ubiquitin ligase. CRBN is of clinical relevance because its expression is required for the efficacy of immunomodulatory drugs (IMiDs) in multiple myeloma (MM), which primarily enable the recognition of neo-substrates by CRBN to elicit their anti-cancer effects. We sought to investigate a role for Fbxo7 in MM cells. We show that Fbxo7 promotes CRBN ubiquitination and propose that this targets CRBN for proteasomal degradation, which may have relevance for IMiD sensitivity. Together, these data identify two novel substrates for Fbxo7 ubiquitination and reveal a role for Fbxo7 in lymphocytes. We demonstrate that Fbxo7 expression is responsive to cellular stress and propose that Fbxo7 levels may fine-tune metabolism under different physiological and pathological conditions.
Open Access
Dedifferentiation of muscle cells during Trichinella spiralis infection
Nash, Bradley
The first stage larvae (L1) of the obligate parasitic nematode Trichinella spiralis invades host skeletal muscle cells and reside intracellularly. The host cell is dedifferentiated, losing its muscular specialisation, and re-entering the cell cycle transiently. Over a period of around 20 days post-invasion the host cell is converted into a “nurse cell” within which the L1 can remain for decades unimpeded. Coinciding with the establishment and maturation of the nurse cell is the development of the L1. The L1 develops a mature secretory organ, the stichosome, and a more complex cuticle and grows exponentially for weeks. The link between the development of a dedicated secretory organ, alongside the dedifferentiation of the host muscle cell is an area currently under investigation. The excretory/secretory products (ESPs) produced by the worm directly into the host cell are implicated as effectors of host cell dedifferentiation, among other nurse cell constructing processes. The identification of ESPs able to manipulated the differentiated state of muscle cells warrants investigation in developing treatments for muscle wasting diseases. The work undertaken in this thesis aims to enhance the current understanding of the process of dedifferentiation observed during infection and identify the parasite factors involved. ESPs produced by muscle released larvae are identified by proteomics and functionally annotated. Using this set of ESPs, a novel motif is determined that is enriched upstream of the Transcription Start Site (TSS) of ESP genes relative to non-ESP genes. Characterisation is undertaken of selected ESPs that have proposed involvement in the manipulation of cell cycle and differentiative processes. Finally, the transcriptomic events underpinning the dedifferentiation and redifferentiation of the host cells in early muscle infection are uncovered through a novel synchronous dual-species transcriptomic approach. Features associated with the ESPs, are used to predict ESPs from L1 genes differentially expressed during the early stages of infection. My work combines the independent analyses of L1 secretome data and synchronised dual-species transcriptomic data. This approach identifies a novel ESP associated motif, previously unreported ESPs collected from culture, stage specific parasite transcripts and an improved synchronous infection collection method to identify host processes and worm transcripts at high resolution. Ultimately, this thesis advances our scientific understanding of the genes from both the parasite and the host that contribute to the dedifferentiation of muscle cells during T. spiralis infection.
Embargo
A strategy to suppress STAT1 signalling conserved in pathogenic poxviruses and paramyxoviruses
Talbot-Cooper, Callum
Vaccinia virus (VACV) is a member of the poxviridae, a family of viruses with large double-stranded, linear DNA genomes that replicate in the cytoplasm of cells. The genome of vaccinia encodes over 200 proteins, many of which have been shown to function as inhibitors of the innate immune response. This includes at least four proteins that directly target interferon (IFN)-induced signalling. Here we report the first function of the uncharacterised, 60 aa 018 protein from VACV strain western reserve. The 018 protein was expressed early during infection and found to potently inhibit type I and type II IFN-induced signalling. Cellular protein STAT1, a crucial protein required for IFN-induced signal transduction, was identified as a direct binding partner of 018. Mapping experiment identified the SH2 domain of STAT1, a region important for STAT1 recruitment to IFN-receptors, as the site of 018 binding. In cells expressing 018, STAT1 failed to be phosphorylated, thereby preventing STAT1 activation. Taking the type II IFN pathway as a model, we demonstrated mechanistically, 018 was able to outcompete the binding of STAT1 to the activated IFN receptor. To gain further mechanistic insight, the co-crystal structure of the 018:STAT1 complexed was determined. This showed 018 forms a -hairpin fold whereby the two strands of the peptide augment the central -sheet of the SH2 domain. Unlike canonical SH2 ligand interactions, 018 did not bind into the pTyr pocket and thus presents a novel pTyr pocket binding independent mode of binding at an SH2 domain. To further study the role of 018 during infection, recombinant viruses lacking 018 were constructed and tested in an in vivo mouse model. Deletion 018 viruses were found to be attenuated despite the presence of addition viral IFN-induced signalling inhibitors, confirming the biological importance of 018 during infection. Comparison of the IFN antagonist V protein from Nipah virus, a member of the paramyxovirus family showed sequence similarity to the STAT1-binding region of 018. We showed, like 018, Nipah V protein blocks STAT1 association with the active IFN receptor. Furthermore, we provide mechanistic detail of the Nipah V:STAT1 interaction by solving the crystal structure of this protein complex.
Embargo
Structural and biochemical characterisation of pUL7 and pUL51, conserved tegument proteins required for efficient herpesvirus assembly
Butt, Benjamin; Butt, Benjamin [0000-0001-6718-0470]
Herpesviruses are ubiquitous viral pathogens that infect all known vertebrates. It is estimated that over 90% of adults worldwide are infected with at least one of the nine human herpesviruses, which cause a wide range of morbidity and mortality. These viruses share a conserved assembly pathway, during which genome-containing capsids acquire a lipid envelope in a process called secondary envelopment. Herpes simplex virus (HSV)-1 proteins pUL7 and pUL51 form a complex that is involved in secondary envelopment, although their molecular function is not well understood. Neither pUL7 nor pUL51 share appreciable sequence-level homology with any protein of known function, which has frustrated attempts to infer their function by analogy. Since viral proteins can exhibit structural homology to other proteins in the absence of appreciable sequence conservation, the pUL7:pUL51 complex was characterised using small angle X-ray scattering and X-ray crystallography. pUL7:pUL51 forms a 1:2 heterotrimer in solution that dimerises at high concentrations. The crystal structure of the core pUL7:pUL51 heterodimer revealed that pUL7 has a novel fold. Immunoprecipitation experiments showed that the interaction between pUL7 and pUL51 homologues is conserved across multiple human herpesviruses, although the precise molecular details of the interface have diverged. Immunocytochemistry and fluorescence microscopy showed that localisation of pUL7 and pUL51 homologues to trans-Golgi network membranes is conserved, but only HSV-1 pUL7:pUL51 is recruited to focal adhesions. HSV-1 pUL51 structurally resembles endosomal sorting complex required for transport (ESCRT)-III components, a class of cellular proteins involved in membrane scission. HSV-1 pUL51 binds lipids when recombinantly expressed, and forms filamentous and ring-shaped polymers that resemble those formed by ESCRT-III superfamily proteins when purified. Sequence analysis of pUL71, the human cytomegalovirus pUL51 homologue, identified a C-terminal motif that resembles those involved in recruitment of the ATPase vacuolar protein sorting-associated protein 4 (VPS4) by ESCRT-III proteins. Prediction of the pUL71:VPS4 complex structure suggested that pUL71 binds to VPS4 via a short helix and a linear motif, in a similar manner to cellular interactors. The predicted structure was computationally validated using molecular dynamics simulations, which defined the contribution of key residues to the pUL71:VPS4 interaction. While other viruses are known to recruit ESCRT components to mediate membrane scission during virion assembly, this is the first example of structural mimicry of an ESCRT-III component and direct recruitment of VPS4 by a virus. These results provide a structural framework for understanding the role of the conserved pUL7:pUL51 complex in herpesvirus secondary envelopment.
Embargo
Functional analysis of KLF2 and its lymphoma-derived mutants
Rust, Maria
Splenic marginal zone lymphoma is a low-grade B cell neoplasm, which originates from the splenic marginal zone B cells. Whole exome studies show recurrent somatic mutations in KLF2, and genes in the NOTCH2, NF-κB, BCR and TLR signalling pathways – pathways that are essential for the development and maintenance of marginal zone B cells. Of these, KLF2 mutations are the most frequent (20-42%) and are thought to lead to the inactivation of KLF2. KLF2 is a zinc finger transcription factor involved in a variety of cellular processes, including negative regulation of NF-κB activation. Functional assays show that KLF2 can suppress NF-κB activation, through both the canonical and non-canonical pathways. However, mutations in KLF2 lead to a loss in NF-κB suppression, which may promote the lymphomagenesis by facilitating differentiation of marginal zone B cells and their homing to the splenic marginal zone. We hypothesised that KLF2 may also modulate other pathways that are important in marginal zone B cell development, namely the NOTCH2 signalling pathway. In vitro reporter assays showed that wild type KLF2 suppresses the NOTCH2 signalling pathway. Introducing mutations that are recurrently seen in splenic marginal zone lymphoma patients into KLF2 resulted in aberrant regulation of NOTCH2 signalling. All three C-terminal truncation mutants and 8/10 lymphoma derived missense/ indel mutants showed a loss of NOTCH2 suppression. Interestingly, KLF2 C274Y, C279Y, H292Y, H296Y and S287P mutants displayed enhanced reporter activities, suggesting gain of function. Wild type KLF2 was also found to be capable of suppressing the activation of the MAPK/ ERK signalling pathway and both truncation and missense mutations in KLF2 resulted in a loss of this repression in a similar manner to the NOTCH2 pathway, with several mutants, including KLF2 C274Y, C279Y, H292Y and H296Y showing gain of function activity. This, along with previous studies, suggests that KLF2 may be acting as a global negative regulator of transcription. Mutations in KLF2 can lead to a loss of this repressive activity and, in the case of several missense mutants, additionally show a gain of function, which may contribute to lymphomagenesis. Confocal imaging analysis revealed remarkable differences in the subcellular localisation between wild type and mutant KLF2. While wild type KLF2 localises in the nucleus, we showed that truncations in KLF2 disrupted its nuclear localisation signals resulting in predominant localisation throughout the cytoplasm. Of the ten missense/ in frame deletion mutants investigated, seven displayed a “speckled” pattern of nuclear expression, with those that showed gain of function reporter activities producing this speckled pattern at a significantly higher rate. Further analysis suggested colocalisation between mutant KLF2 and SRSF2, a component of the spliceosome, suggesting that KLF2 may also be involved in splicing and RNA processing activities. Gene expression profiling showed that wild type and mutant KLF2 regulate many of the same genes that are involved in regulating B cell development and differentiation. Additionally, WNT signalling was identified as being differentially expressed when mutant KLF2 is expressed in comparison to wild type KLF2. Gene set enrichment analysis also showed that the WNT, BCR, chemokine, MAPK and JAK-STAT pathways were all significantly impacted by both wild type and mutant KLF2. Interestingly, KLF2 missense mutants showed additional effects on the TLR and NOTCH2 signalling pathways, in particular, many positive regulators of these pathways are upregulated with mutant KLF2 and downregulated with wild type KLF2. In conclusion, these studies have shown that KLF2 may be acting as a global negative regulator, particularly of pathways that are essential in the development of marginal zone B cells. Mutations in KLF2 not only abolish this suppression but, in several cases, lead to gain of function activities. These mutations caused aberrant localisation of KLF2, which suggested that KLF2 may be interacting with the spliceosome and its regulators to control gene expression.
Open Access
Structural and functional characterisation of pUL21, an α-herpesvirus phosphatase adaptor protein
Benedyk, Tomasz; Benedyk, Tomasz [0000-0001-6420-3665]
The current CoV-SARS-2 pandemic exemplifies the profound impact viruses can have upon our lives. Virus infection is a complex process where viral proteins cooperate to exploit host cell metabolism while evading immune responses. Herpes simplex virus 1 (HSV-1) is a very prevalent human herpesvirus that causes life-long infection and is known to dramatically remodel the cellular environment upon replication. This extensive manipulation is achieved using only a limited number of virus-encoded proteins, many of which possess numerous functions and exert their effects in multiple different subcellular compartments. HSV-1 pUL21 is an example of such multi-function protein: it is known to be important for assembly of new virus particles and viral cell-to-cell spread but its exact molecular functions remained unknown. Using a high throughput interactomics screen, previous members of the group identified potential cellular binding partners of pUL21: protein phosphatase 1 (PP1) and ceramide transfer protein (CERT). This thesis presents biophysical characterization of the direct interactions between pUL21 and these partners and describes the role of pUL21 as a novel viral phosphatase adaptor that recruits PP1 to multiple substrates, including CERT, to promote their dephosphorylation. Conservational and structural analyses led to the discovery of a non-canonical linear motif in pUL21, termed TROPPO, that is critical for PP1 binding and it is absolutely conserved across α-herpesviruses. In vitro evolution experiments using HSV-1 strains with mutated TROPPO motifs revealed that the phosphatase adaptor pUL21 antagonises the activity of the virus-encoded kinase pUS3. A correct balance of kinase and phosphatase activity is shown to be essential for correct subcellular localisation of the HSV-1 nuclear egress complex and for virus replication and dissemination. Using in vitro biochemical experiments, stable expression of pUL21 in cultured cells, and infection with wild-type HSV-1 or viruses expressing pUL21 with a mutated TROPPO motif, we confirmed that pUL21 stimulates PP1-dependent dephosphorylation of CERT and the viral nuclear egress complex component pUL31, plus additional as-yet unidentified proteins. The binding interface of the pUL21:CERT complex was determined using small-angle X-ray scattering, enabling the generation of pUL21 mutants where binding to CERT, but not to other substrates, was specifically disrupted. Generation of a CERT non-binding mutant facilitated a detailed characterization of the sphingolipid-modulatory role of pUL21 using ‘click chemistry’-based assays and revealed that pUL21-dependent upregulation of sphingomyelin turnover is required for the correct trafficking of maturating virions to the plasma membrane. In summary, this thesis presents structural and functional characterisation of HSV-1 pUL21, dissecting the multiple roles played by this protein during the replication of HSV-1. Furthermore, this study provides first insights into the modulation of sphingolipid homeostasis during virus infection, a critically understudied host:pathogen interaction.
Open Access
Investigating the importance of the Zika virus 3′ untranslated region to viral fitness
Storrie, Skye
The RNA binding protein Musashi 1 (MSI1) has been shown to bind to the Zika virus (ZIKV) 3′ untranslated region (3′UTR) and enhance viral replication. MSI1 is present in high levels in neural progenitor cells of the foetal brain, and previous work proposed that MSI1 could drive the pathogenesis caused by ZIKV, and therefore, could be linked to the cases of congenital microcephaly observed during the 2015/2016 epidemic. The work presented here utilises reverse genetics to investigate the mechanism of MSI1 enhancement of ZIKV replication by introducing putative MSI1 binding site mutations into the 3′UTR. Using U251 cells ± MSI1, it was shown that one of the site mutations (Site D) induced a MSI1 dependent reduction in viral replication, indicating that this site may be particularly important for MSI1 binding to the ZIKV 3′UTR. Following viral characterisation in Vero cells, several of the site mutants displayed varying levels of viral attenuation, in one case so severe it was not possible to rescue virus. Passage experiments revealed the importance of one site mutation (Site B), whereby the partial reversion of one of the two mutated nucleotides was observed, indicating the importance of a single nucleotide in the 3′UTR to the overall fitness of the virus. The structure of the flavivirus 3′UTR is well conserved, highlighting its importance to the viral life-cycle. To structurally map the 3′UTR of the different site mutants, a novel structural cassette was designed and employed, allowing, for the first time, in vitro selective 2′-hydroxyl acylation analysed by primer extension mapping of the full 3′UTR structure. This was used to study the contribution of specific nucleotides to the 3′UTR structure, revealing a disruption in RNA structure in the Site B mutant, and thus sfRNA production. Additionally, sfRNA production was found to be restored following the single nucleotide reversion. Further investigations linked the hampered sfRNA production to reduced virion production in IFN competent A549 cells, which was not observed in IFN deficient Vero cells, reaffirming the link between sfRNA production and immune evasion. Overall, this thesis highlights the importance of the ZIKV 3′UTR to viral fitness and provides useful tools for future investigations.

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