Patients with advanced, metastatic solid tumours have an overall survival of less than five years. We and others have shown that tumours harbour extensive intra-tumour heterogeneity (ITH) leading to ongoing tumour evolution, diverse therapy resistance mechanisms and an almost infinite adaptability - a significant challenge to curative therapy. As ITH increases over time, it is critical to intervene early in the disease course when the disease burden is at its lowest, ideally targeting and limiting cancer initiation. We have recently shown that air pollution acts independently of DNA mutagenesis to promote lung cancer in never-smokers (LCINS), a disease more common in females, via an immune cell activating macrophage IL-1B axis. EGFR mutant alveolar type 2 cells, present in the aging lung, respond to macrophage-derived IL-1B by adopting a progenitor-like, proliferative cell state that promotes tumorigenesis. These results indicate that air pollution conforms to the Berenblum model of tumour promotion, where an initiator, the initial EGFR mutation, and a promoter, in this case air pollution, both are required for tumorigenesis. This mutation-independent mechanism of tumour promotion indicates that opportunities may exist to prevent cancer by targeting inflammatory mediators across tissues. This proposal aims to identify, model, and manipulate local and systemic processes and intrinsic and extrinsic risk factors that affect tumour initiation and promotion in LCINS. Using normal human lung tissue, novel and established animal models, single-cell RNA-sequencing and immune-phenotyping approaches we will develop models to identify, investigate, target and modulate tumour intrinsic and extrinsic factors contributing to tumour initiation and promotion and elucidate the underlying, currently unknown biological causes for the observed sex-differences in LCINS. Ultimately, we endeavour to identify actionable factors to prevent LCINS initiation without adverse systemic effects.

The Crick (a partnership between the Medical Research Council, Cancer Research UK, the Wellcome Trust and three leading universities: University College London, Imperial College London and King's College London) is a modern medical research institute hosting 120+ research laboratories. Central to The Crick's aspiration of becoming one of the world's leading research institutes is the delivery of cutting-edge science using state-of-the- art instrumentation. To achieve this, The Crick has established centralised Scientific Technology Platforms (STPs) that house this instrumentation and are supported by staff of specific expertise. This model allows operational efficiency and institute-wide consistent standards of the highest quality. The remarkable advances in proteomic technologies over the last 20 years means that mass spectrometry is now the preferred method for in-depth characterization of the protein components of biological systems. Indeed, recent technology developments are now enabling mass spectrometry-based proteomics to emerge as an essential approach for the analysis of clinical samples. The Proteomics STP collaborates with ~60 groups at The Crick, analysing upwards of 15,000 samples per year. Projects involve both Tandem Mass Tagging (TMT) isobaric tags and label free DDA and DIA for the quantitative analysis of protein expression. The technology within the STP is also used for the discovery and characterisation of post-translational modifications. To achieve this, the Proteomics STP currently houses three ThermoScientific Orbitrap instruments and a Bruker timsTOF Pro. The Orbitrap instruments are primarily used for qualitative and quantitative analysis of CoIP and proximity based labelling experiments using TMT, while the timsTOF Pro has been heavily used for the screening of small molecule fragment libraries against reactive cysteines. Demand for access to the timsTOF is now so high, that we seek to develop the capacity for label free analysis at The Crick. This proposal is for a fast-scanning mass spectrometer capable of performing high throughput DIA analysis. The unprecedented scan speed, mass resolution, chromatographic stability and speed that this system offers will enable studies that require high sensitivity, high throughput and high performance label free peptide and protein quantification.

Animals are composed of a myriad of cell types with unique transcriptomes and proteomes. Among these, neurons are arguably the most heterogeneous in terms of morphologies and functions. They achieve this diversity by refining their proteome, mostly through post-transcriptional processes such as RNA editing and alternative splicing. Another understudied mechanism that can increase proteomic diversity is the extension of protein products by translational stop codon readthrough. Research from the host lab, as well as others, have in fact shown that Drosophila neurons can decode stop codons as sense codons in a regulated manner and at a much higher rate than other cell types, potentially creating extended protein products of key functional significance. The goal of this proposal is to investigate the neuronal decoding of stop-codons and to understand: 1) how neurons regulate stop codon readthrough, 2) how it impacts on correct neuronal function, and 3) how universal it is. First, we will dissect the mechanism and cellular machinery involved in decoding by combining a variety of high-throughput methods with functional studies perturbing trans-acting factors. Next, we will build a map of stop codon decoding in the fly brain to uncover the hidden variability within neural cell types. This map will guide further functional studies addressing the contribution of these protein extensions in specific neuronal populations that mediate quantifiable behavioural outputs. Finally, we will reconstruct the evolutionary history of this non-standard genetic code and test whether their heterologous expression can promote translational readthrough in other systems such as disease-causing premature nonsense mutations in human cells.

Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.

Mapping the connectivity of neural circuits is essential for understanding their function in health and disease. However, our ability to map connectivity in polysynaptic circuits, i.e. between neurons separated by more than a single synaptic connection, remains limited in mammalian brains. Approaches such as electron microscopy and brain slice recordings can determine connectivity in microcircuits or small samples but cannot be used in live animals. Conversely, viral and non-viral trans-neuronal tracers can trace connections between brain areas, but either only cross a single synaptic connection or many in a poorly controlled way. To overcome these limitations, we propose to develop a viral approach that allows for tracing of polysynaptic circuits with control over the number and location of crossed synapses, as well as functional access to traced neurons in vivo. Our method will use a modified rabies virus that expresses an orthogonal receptor, which allows for selective re-delivery of the G-protein to presynaptic neurons. This will allow us to gain access to higher-order inputs to a specific neuronal population. If successful, we will apply this approach to analyse chemosensory inputs into hypothalamic neurons that regulate parental behaviour. This tracing approach has the potential to provide unprecedented access to polysynaptic circuits defined by connectivity and gene expression and could significantly advance our understanding of brain function.