Viewed through a microscope, cells undergo a spectacular transformation as they enter mitosis, the phase of their existence just before they divide. The formation of the mitotic spindle, where the cell?s network of microtubule fibres is completely rearranged to span from either end to the chromosomes at the centre, is particularly striking. This spindle is a molecular machine that ensures the cell?s chromosomes are accurately distributed between its two daughter cells. Errors in the workings of the spindle are a known driving force of cancer and are also responsible for a congenital brain disease. Several control mechanisms ensure the mitotic spindle is normally assembled correctly. At an early stage in assembly, two proteins called TACC3 and ChTOG promote microtubule stability and hence promote assembly. These proteins are more effective when TACC3 is modified by a phosphate group: one phosphorous atom and three oxygen atoms that is commonly used by cells to alter the activity of their proteins. In the case of TACC3, the protein that adds the phosphate is called Aurora-A. Spindle assembly is thus controlled by the activity of Aurora-A, which is itself controlled by many other proteins under the influence of events within and outside the cell. We propose to investigate how the phosphate group influences the effectiveness of the TACC3/ChTOG partnership at the level of atoms. How this works is currently a mystery as the phosphate is only four atoms big, and yet it changes the activity of TACC3/ChTOG which total tens of thousands of atoms. We will use electron microscopy, a technique that allows us to see directly the shapes of proteins, to study the changes in TACC3 upon phosphorylation, and the effect on ChTOG. We will also use X-ray crystallography to determine the location of every atom within the proteins and to map the atoms by which TACC3 and ChTOG cooperate. This information will allow us to make a hypothesis for the details of how the TACC3/ChTOG partnership works and how phosphorylation enhances their effectiveness. We will use our protein structure models to design subtle modifications to TACC3 and ChTOG to test this hypothesis in human cells grown in culture. An overabundance of TACC3, ChTOG or Aurora-A have been linked with cancer, and TACC3 and Aurora-A are also important in brain development. These studies will provide the impetus for future investigations to understand the role of these proteins in human disease.

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Each human telomere is composed of 10-15 kb of repetitive DNA bound by a protein complex called shelterin, which forms a protective nucleoprotein cap at the chromosome end. In addition to shelterin, telomeric DNA is also wrapped around histone octamers in a closed, heterochromatic state that compacts telomeric DNA and represses transcription at the chromosome end (Tardat and Dejardin, 2018). Mutations that disrupt the assembly of chromatin at telomeres cause DNA damage and are found in essentially all 'ALT' type cancer cells (some 10-15 % of all tumour types), underlining the importance of chromatin in the function of telomeres. At non-telomeric sites, chromatin is assembled during S-phase when chromatin remodelling factors and histone chaperones disassemble nucleosomes in front of the replication fork and reassemble them on newly synthesised DNA (Hoek and Stillman, 2003). Although telomeric chromatin is also assembled during S-phase, genetic studies show that a distinct set of chromatin remodelling factors are required for this process, suggesting the replication and reassembly of nucleosomes at telomeres occurs through a distinct mechanism. The successful candidate will examine this mechanism using a combination of reconstitution biochemistry, biophysics and genetics. The starting point for the project is a reconstituted system for DNA replication that we have recently developed in the Telomere Biology lab. Combining this system with chromatinised DNA templates and purified chromatin remodelling factors, we will examine i) how chromatin affects the human replication fork ii) how telomere-specific chromatin remodelling factors allow replication and reassembly of nucleosomes on telomeric DNA and iii) the consequences of disrupting these processes within cells. As opportunities arise, we will also collaborate with other groups to characterise replication intermediates using cutting edge biophysical and structural techniq

Immune checkpoint inhibitor therapy is a new type of cancer treatment that has significantly improved the outcomes for many patients with widespread or locally advanced cancer. The immune system normally works to prevent cancer by recognising and destroying cancerous cells. However, many types of cancer use mechanisms to escape this immune surveillance. One way they do so is by reducing the activity of anti-cancer immune cells using cell-surface signalling molecules called immune checkpoints. When these immune checkpoints are engaged by cancer cells they render the immune cells inactive. This then allows tumours to grow unchecked. Immune checkpoint inhibitors block these immune checkpoints to reactivate anti-cancer immune cells. Given their success, these drugs have been licensed to treat an increasing range of cancer types. Their use is also expanding in the adjuvant setting, where patients receive immunotherapy after surgery to reduce the risk of their cancer returning. Thus, an ever-increasing number of patients receive these medications. Unfortunately, immune checkpoint inhibition is associated with potentially serious side effects. These are called immune-related adverse events (irAEs). They result from harmful overactivation of immune cells, which attack healthy tissues rather than cancer cells. irAEs affect up to 95% of cancer patients treated with the strongest form of immunotherapy, which combines two immune checkpoint inhibitors. Up to 50% of patients will experience irAEs that require either treatment with powerful immune-suppressing steroids or discontinuation of their immunotherapy. Both carry a risk of worse prognosis of their cancer, whilst steroids can also cause significant other side effects. We therefore require a better understanding of the mechanisms behind irAEs to develop improved treatments that target the irAE more specifically without compromising anti-cancer effects. Currently, how and why irAEs arise is very poorly understood. We do not know why some patients develop severe or even deadly irAEs whilst others develop no irAEs at all. We have also not identified the key types of immune cells and signalling molecules involved in irAEs. This is urgently required to identify potential new therapeutic targets. Our research project is designed to address these key issues. We will be working with blood and tissue samples from patients receiving immune checkpoint inhibitors. We are focussing on skin, as it is very commonly affected by irAEs. The first blood sample is taken before patients start treatment. We then take repeat samples during treatment and when irAEs develop. We will identify what types of immune cells and messaging molecules arise in the blood of irAE patients compared to non-irAE patients. One key hypothesis is that irAEs patients have a defect in specific immune cells, which control the activity of other "effector" immune cells. The controller immune cells we will assess are T-regulatory cells, which control cell-mediated immune responses, and follicular T-cells, which control antibody-mediated immune responses. We will measure their numbers and test whether cells from irAE patients are impaired in their ability to control immune activation. Skin biopsies from patients with and without skin irAEs will be analysed with a powerful technique called single cell RNA sequencing. This allows us to identify all the immune cells within the skin sample and what inflammatory messenger pathways are active in them. This can lead to the identification of novel therapeutic targets to switch the skin inflammation back off. Finally, we will explore whether it is possible to predict if an individual patient is likely to develop irAEs before they start immunotherapy. This has the potential to help personalise treatment. Patients at high risk of irAEs could be monitored more closely or potentially receive less intense treatment to avoid serious side effects.

See Section 9 of the report