This pilot study aims to explore the dynamics of T-cell receptor (TCR) and B-cell receptor (BCR) repertoires during the metastatic progression of non-small cell lung cancer (NSCLC) for three patients, from diagnosis to death, using high-throughput sequencing. By leveraging samples from the TRACERx and PEACE autopsy studies, we will profile immune repertoires in primary tumours, metastases, and longitudinal blood samples. This will help elucidate how the tumour and immune system co-evolve, particularly during treatment, and provide insights into the mechanisms behind immunotherapy resistance and metastasis. The study focuses on characterizing the diversity and clonality of TCR and BCR repertoires, identifying tumour-associated clonotypes, and correlating these findings with genomic tumour evolution and clinical outcomes.

Invasion and metastasis are fundamental factors that define prognosis. The study will be based on a new collaboration between Professor Nikolitsa Nomikou’s (UCL) and Professor Maddy Parsons’ (KCL) teams, and it focuses on the development of a novel preventive treatment approach against cancer progression and metastasis evolvement. The strategy is to generate a mechanistic insight into a novel cancer antiinvasive and antimetastatic approach based on a new class of therapeutics targeting polyglutamylation. More specifically, a co-polymer of glutamate and tyrosine, that has shown the potential to inhibit cancer cell invasion and metastasis, will be investigated with regards to its potential effect on the polyglutamylation of microtubules, focusing on pancreatic and prostate cancer.

The study will explore 3D model systems (spheroids) and high-resolution imaging, biochemical analysis of the tubulin polyglutamylation profile, and siRNA technology to assess and characterize the potential anti-invasive/antimetastatic effect of the co-polymer. MicroCT scans will be used to assess the level and profile of metastatic progression in animal models treated with this novel approach.

Whilst using genetically engineered immune cells to treat blood cancer has been highly successful, these results have been harder to deliver in the treatment of solid cancers.  One key challenge is that cancer cells often consume oxygen very quickly, meaning that the amount available for the immune cells is limited.  The Fisher laboratory specialises in engineering gamma-delta T cells, a type of immune cell which has many helpful properties in cancer immunotherapy.

This project focuses on finding out what happens to gamma delta T cells when they in environments with relatively low amounts of oxygen.  We will be exploring what happens to the cells at a behavioural and genetic level, comparing cells in hypoxic conditions to those with normal oxygen levels.  Once we have determined what changes occur, we will begin exploring opportunities for interfering with these systems with the intention of enhancing gamma delta T cell survival in this challenging environment.

Treating tumours at advanced stages is extremely challenging, making the identification and prevention of early-stage cancers essential for improving outcomes. Recent cancer screening programmes in the UK do reduce cancer mortality but deciphering the very early changes in normal tissue that increase cancer risk will further identify novel therapeutic targets for cancer prevention that could substantially reduce cancer-related deaths. Data from my laboratory and others indicates one key potential driver of early cancer development is the Apolipoprotein B Editing Complex 3 (APOBEC3) gene family. APOBEC3 (A3) genes are enzymes that function as part of the body’s defence against viruses. Unfortunately, A3 genes can also mutate host DNA and are linked with DNA damage in more than 70% of human cancer types.

Evidence suggests a connection between A3 genes and the early steps of cancer development. Specifically, A3 expression is correlated with breakage fusion bridge (BFBs) cycles, a DNA damage process that is implicated in cancer formation. Based on preliminary data I have gathered, I hypothesise that A3 enzymes drive BFB formation, inducing lung cancer. Using human lung cancer cell lines with or without A3 expression in addition to analysis of human data in collaboration with Professor Sam Janes laboratory at UCL we will explore this connection. This collaborative project embodies the CoLs Centre’s theme of early detection and prevention, as it will uncover a novel mechanism by which lung tumours are induced.

Our body’s immune system provides an important defence against cancer by finding and destroying cancer cells. However, various environmental factors can either weaken the immune system or change it in a way that it ends up promoting cancer. One such factor is western-style diet (WD) that is rich in fat and sugar. Chronic exposure to WD causes obesity that, in turn, is associated with increased incidence of many tumour types. Conversely, after diagnosis, cancer patients often change their dietary habits, typically towards healthy diets, but may also, to varying degrees, consume unhealthy comfort food. While there has been a great emphasis on the tumour-promoting effects of chronic WD consumption, to which extent short-term diet modifications influence tumour immunity remains poorly understood.

In this project, we will study the effects of short-term diet manipulations on the immune response against tumours in mice. We will also use these new experimental data to generate and train computational models that describe how nutrients are exchanged between cancer and immune cells and how diet composition alters these metabolic interactions. Ultimately, our investigations will point to ways of manipulating diet composition to strengthen the immune system’s ability to fight tumours.

We have published that genetic ablation of endothelial cell-focal adhesion kinase (EC-FAK) does not affect blood vessel function per se, but instead enhances DNA-damaging (doxorubicin or radiotherapy) treatment efficacy (Tavora et al.,Nature 2014). Thus, development of EC-targeting FAK-degrading agents could enhance the efficacy of standard of care DNA-damaging treatments.

This project is based on an established collaboration with Jamie Baker, Cliona MacMahon and Vijay Chudasama (UCL Chemistry Department) who have pioneered approaches to site-selective antibody-conjugate construction, including techniques specifically for antibody-PROTAC conjugation and Simon Boulton Crick (DNA-damaging expert) with Joanna Redmond and Chrystelle Soudy (Crick Chemists). This work was part of a RadNet PhD studentship (Rebecca Drake) and Barts Charity seed fund where the same EC-FAK PROTACs are being designed generated and tested in combination with radiotherapy in vitro. Funds are now requested to generate further EC-FAK PROTACs for initial testing in vivo in mouse models of cancer especially in combination with doxorubicin.

The integrin avb6 is weakly expressed or undetectable by immunohistochemistry on most tissues in the body. In contrast, over one third of carcinomas developing from these same tissues present high levels of avb6 on their cell surfaces, where it imparts increased growth and invasive propensities.  We have generated a human antibody (IgG) against avb6 that binds specifically to this single integrin and promotes the internalisation of the antibody into the cell upon binding to avb6.  If the antibody had a toxic drug attached it could kill avb6 -positive cancer cells.

Therefore we shall develop an antibody-drug-conjugate (ADC) from this antibody and test against avb6-positive versus avb6 -negative cancer cell lines in the lab, before assessing its capacity to stop the growth of and possibly even eliminate, avb6-positive tumours in mice.  Initial studies will use human tumours in mice allowing us to assess the off-target effects of the therapy as our antibody binds equally well to both human and mouse avb6. If we can find the correct dosing that balances therapeutic efficacy against off-target toxicity, we will seek funding for its translation into human studies.

T cell exhaustion is a critical barrier to the success of cell therapies in treating solid tumours due to the tumour microenvironment. A major challenge in the field is identifying genes that can be therapeutically targeted to protect against or reverse this exhaustion. Recent advancements in machine learning, along with the growing availability of single-cell data, are enabling a deeper understanding of gene regulatory networks and their associated cellular phenotypes. We have developed novel in silico approaches to identify potential CRISPR knock out gene targets to protect T cells from exhaustion by leveraging novel machine learning methodologies including graph neural networks and generative pretrain transformers. The CRUK-funded project will focus on experimentally validating these identified targets, with the ultimate goal of developing improved cell therapies for patients with solid tumours.

Neuroblastoma is an aggressive pediatric cancer mostly affecting babies and children under the age of five. Immunotherapy targeting the key antigen GD2 is standard of care for neuroblastoma, but is hampered by the ‘immunologically cold’ and immunosuppressive tumour microenvironment of this cancer. Copper depletion via chelation therapy has been shown to prime neuroblastoma microenvironment for immunotherapy, enhancing the efficacy of anti-GD2 antibody in immunocompetent mouse models. However, the dynamic of these processes in vivo is still unclear.

In this project we will assess the feasibility of using whole-body radionuclide imaging to quantify the effect of copper chelation on endogenous copper distribution, and GD2 expression in vivo, in clinically relevant models of neuroblastoma. By improving our understanding of how copper can modulate the immune response in neuroblastoma, we will inform further development and clinical translation of copper depletion/anti-GD2 immunotherapies combinations to enhance treatment efficacy for neuroblastoma patients.

Metastatic medulloblastoma poses a significant challenge. Our project uses advanced techniques, including spatial transcriptomics and metabolomics to explore how this cancer spreads within the tumour microenvironment. By understanding the processes behind its dissemination and distant implantation, our research has the potential to greatly improve our knowledge of tissue biology and disease mechanisms. This, in turn, will enable the development of novel therapeutic interventions, offering hope for better outcomes in paediatric oncology.

We recently discovered that colonic stem cells reside on a continuous differentiation trajectory, ranging from highly proliferative colonic stem cells (proCSCs) through to slow-cycling revival stem cells (revCSCs) (Qin et al., Cell, 2023). Through single-cell signalling analysis of >2,500 patient-derived organoids (PDOs) we then discovered that proCSCs are extremely chemosensitive whereas revCSCs are chemorefractory. Moreover, cancer associated fibroblasts (CAFs) can polarise chemosensitive proCSC to chemorefractory revCSC (Zapatero et al., Cell, 2023).

Due to their slow-cycling phenotype, revCSCs are resistant to traditional anti-mitotic cancer therapies and can survive treatment as drug tolerant presister cells (Tape, Trends in Cancer, 2023). In this CRUK City of London Centre Development Project will use ultra-sensitive mass-spectrometry (Mardakheh Lab, BCI) to identify novel revCSC-specific membrane proteins and then develop new anti-revCSC CAR-T therapies (Roddie Lab, UCL) to eradicate this chemorefractory cell-type in CRC (Roddie Lab, UCL).

Tumour-associated macrophages (TAMs) are key determinants of tumour growth, metastasis and response to treatment in Triple-negative breast cancer (TNBC), thus they provide an attractive therapeutic target. In this project, we aim to investigate how lipid metabolic pathways regulate the heterogeneity of TAMs within the tumor microenvironment and to provide mechanistic understanding on the pathways linking metabolism, TAM functions and tumour progression.

In this project, Drs Lucia Cottone (UCL), Maria Secrier (UCL) and Mirjana Efremova (QMUL) will join forces using their multi-disciplinary expertise to study osteosarcoma, the most common primary bone tumour affecting adolescents and young adults. Cutting-edge single cell sequencing technologies and in vitro model systems will be used to study how subpopulations of osteosarcoma cells dynamically change and ‘go to sleep’ to escape chemotherapy, which is designed to kill actively proliferating cancer cells.

Adjuvant radiotherapy (RT) is standard of care in most solid tumours. It is primarily given to eliminate residual tumour cells at the primary site and decrease risk of local recurrences. Nevertheless, some patients do still develop metastasis.  

We will study how the metastatic tissue is changed because of the systemic effects of RT. In particular we will study changes occurring in the immune infiltrate. Next, we will elucidate how these changes support the outgrowth of tumour cells that have disseminated at distant sites. This will help to identify combination therapies to give alongside with RT and reduce distant recurrences.  

Genes encoding for components of SWI/SNF chromatin remodelling complex are mutated in more than 20% of all human cancers, being among the most prominent tumour suppressors. Despite the well-studied impact of mutations on the SWI/SNF subunits, little is known how non-mutational mechanisms contribute to dysregulation of SWI/SNF chromatin remodelling complex during early lung cancer evolution. In this project we will explore how deregulation of RNA-based mechanisms lead to loss of SWI/SNF chromatin subunits and chromosome instability in lung cancer. The aim of this CRUK City of London Development Fund project is to combine expertise in RNA, cell and chromatin biology (Stojic lab) with Dr McGranahan lab’s expertise in bioinformatics and evolutionary methods to decipher lung cancer genome evolution. By charting the dynamics of non-mutational mechanisms leading to SWI/SNF impairment during early lung cancer evolution, will pave the way to development of new RNA-based strategies that can be used in cancer early detection and prevention.

Barrett’s oesophagus is the metaplastic replacement of the normal squamous oesophageal epithelium with a columnar phenotype likely caused by exposure of the distal oesophagus to chronic acid and bile reflux. It is a common condition and is the only known precursor of oesophageal adenocarcinoma. However, most patients with Barrett’s never progress to cancer yet are all enrolled into life-long, invasive, and expensive endoscopic surveillance programmes that every 2-5 years collect biopsies to detect cancer or dysplasia. There are no clinical biomarkers of future cancer risk, and our current approach is ‘wait and see’.

The use of machine learning (AI) algorithms has become popular in attempting to determine cancer risk in many premalignant conditions, however the success of these has been questionable. We believe this because the method of training these AI models employ has been restricted to feature extraction from tiles generated from Haemotoxylin and eosin(H&E)-stained tissue sections. We believe that while there is potential in this approach, the models are limited by general H&E features without knowing the exact cellular components. We are developing a machine learning model that combines H&E features of Barrett’s patients that are either have progressed or have not progressed to cancer with massive multiplex cellular phenotyping of these same samples. Knowing the identity and location of >98% of all cells within any given biopsy will provide significantly more information to train our model. Our aim is to determine if prior knowledge of the geographical location of all cells improves H&E machine learning predictive models in Barrett’s oesophagus.

Acute myeloid leukaemia (AML) in children remains a significant unmet, with high prevalence of specific gene fusions. We hypothesize that these fusion-proteins could be well presented by proteasome-generated spliced peptides, and recognised by CD8+ T cells. Their cytotoxicity could be used to attach AML by developing novel immunotherapies. The project will verify this hypothesis by applying a multidisciplinary strategy, which ranges from bioinformatics to clinics though biochemistry, molecular and cellular immunology.

The project is funded on the unique expertise of the applicants, who are located at King’s College London, University College London and Queen Mary University of London, and with the support of the Francis Crick Institute

One bottleneck in developing this combination therapy further is that many factors, which are central to its successful delivery, remain poorly understood. This is because the laboratory models that researchers have been using, do not adequately mimic the complexity of the human body. Consequently, it is now necessary to improve these models and use new techniques to obtain more and better research data from them. Here, we will pilot new analysis workflows, which will allow us to apply for more funding to develop this therapy combination.

Acute myeloid leukaemia (AML) is an aggressive haematological malignancy with unmet needs for improved treatment strategies. The leading cause of AML-related mortality remains treatment failure due to refractory or relapsed disease, resulting from resistance to chemotherapy of the rare leukaemic stem cells (LSCs) which repropagate the disease. Therefore, understanding LSC biology is the key to eradicate leukaemia.

Gene expression process that transmits genetic information from DNA to RNA to protein, determines cell fate decision, and as such dysregulation can lead to tumorigenesis of all cancers including AML. While multiple studies have investigated alterations leading to changes in RNAs, it is not well understood how dysregulation in protein synthesis contributes to malignant transformation. This is largely due to lack of approaches to measure translation process accurately and faithfully. To tackle, we will develop a mouse model inducibly expressing a ribosomal protein fused with an RNA-editing enzyme that can “label” translating RNAs. We will provide a proof-of-concept for its application to investigate alterations in protein synthesis during leukaemic transformation and to monitor translatomic responses in leukaemia cells upon chemotherapies. This organismic tool will provide new insights into AML cell and LSC biology which will reveal novel vulnerabilities for therapeutic development in leukaemia. Moreover, it will provide a novel approach to study translation regulation in other solid cancers.

We have developed a novel therapeutic concept in which CAR-T cells are rendered more proliferative and endowed with greater cytolytic activity when exposed to B-ALL blasts that have been pre-treated with a metabolic stressing agent that starves cells of arginine. With the support of the CRUK City of London Centre Award we will now analyse the molecular determinants of this phenomena using single cell proteomics via CyTOF, then expand to an in vivo testing platform to identify its potential pre-clinical benefit. Finally, we will test the portability of this effect to other tumour types to assess whether metabolic stress can be used more widely to enhance the anti-cancer effect of CAR-T.

Chimeric antigen receptor (CAR) T-cell immunotherapy represents a potentially powerful approach for treating neuroblastoma, however, immune suppressive macrophages in the microenvironment represent a potential barrier to robust clinical outcomes. This project will employ a screen of FDA-approved drugs to block the immune suppressive activity of macrophages on CAR T-cells. These drug candidates will be investigated using in vitro an in vivo models of neuroblastoma. As the drugs within the screen are FDA-approved, it will permit their rapid translation to the clinic for improving the efficacy of CAR T-cell immunotherapy in neuroblastoma.

Radiotherapy is a treatment that uses high-energy X-rays to kill cancer cells. We know from research that radiotherapy can sometimes help the human immune system to fight cancer, but unfortunately, this effect of radiotherapy is only rarely seen in patients. In recent years, researchers discovered that another type of therapy could improve this effect of radiotherapy when added to it. For patients whose cancer has already spread, it is very important that the immune system can battle their cancer everywhere in the body. Therefore, it is important for these patients to study this combined therapy further because it could lead to new treatment options for them.

One bottleneck in developing this combination therapy further is that many factors, which are central to its successful delivery, remain poorly understood. This is because the laboratory models that researchers have been using, do not adequately mimic the complexity of the human body. Consequently, it is now necessary to improve these models and use new techniques to obtain more and better research data from them. Here, we will pilot new analysis workflows, which will allow us to apply for more funding to develop this therapy combination.

The advent of immunotherapy has transformed the outlook of treatment in certain cancers, with a minority of late-stage patients even achieving complete cure. At the heart of immunotherapy lies the ability of our immune system to recognise and destroy cancer cells. This is mediated by tumour-

specific, as well as tumour-associated antigens, which get presented on the cell-surface to cytotoxic T-lymphocytes, in form of peptides associated with the Human Leukocyte Antigen-I (HLA-I). The majority of immunotherapy research has been focused on fully malignant tumours. However, some lines of evidence suggest that certain pre-malignant lesions can also become visible to and cleared by the immune system. Melanocytic naevi, benign pre-cancerous lesions formed of transformed melanocytes, are one such example. Several studies have shown that the immune system can actively detect and remove some naevi, but the molecular mechanisms as well as the antigens involved in this process remain unclear. Our preliminary results have revealed that oncogenic transformation triggers mis-presentation of diverse HLA-I-associated antigens on the surface of melanocytes, raising the possibility that this mis-presentation could be contributing towards the immunogenicity of naevi. Importantly, our findings indicate that this mis-presentation maybe linked to oncogene-induced senescence, a conserved tumour suppressive process which acts to arrest transformed cells and prevent their progression to full malignancy. In this project, we propose to further study the link between senescence and antigen mis-presentation, assessing whether mis-presentation is a general feature of all senescent cells. If true, our findings can lead to further studies aimed at harnessing the observed antigen mis-presentation for immunotherapeutic targeting of diverse types of pre-malignant cells.

Cell competition was initially discovered in studies performed in the fruit fly Drosophila, but is now recognised as crucial for tissue homeostasis and in cancer. Cell competition arises when neighboring cells display different relative fitness and frequently results in the elimination of the less fit cells, in a process resembling Darwinian evolution at the cellular scale. Similar events occur in cancer, whose development is associated with the selection of genetic chances that confer a survival advantage to cells. The precise mechanisms of cell competition regulation remain incompletely understood. Our project exploits a genetic system that allows generation of cell populations with distinct fitness to characterise the mechanisms of cell competition, with a particular focus on the genes differentially expressed in cells with different fates following the competitive process. We aim to reveal new mechanisms controlling cell fate during cancer development/progression.

Specific metabolic vulnerabilities, which can be leveraged therapeutically, have been identified in many malignancies. Our work has highlighted lipid biosynthesis, particularly the stearoyl-coA desaturase (SCD) enzyme, as a promising therapeutic target in acute myeloid leukemia (AML). However it remains unclear which patients would mostly benefit from targeting this pathway. This work aims to identify metabolic determinants of sensitivity to SCD inhibition (SCDi) in AML preclinical models thus allowing a more personalized use of SCDi in AML patients.

In this study we will use CAR-T cells in the solid tumour setting to test the hypothesis that fine tuning of gamma delta CAR-T cells to low oxygen conditions by upregulating CAR expression in low oxygen conditions, will lead to improved effector in the tumour microenvironment. We aim to show that modifications to the CAR design to be responsive to low oxygen concentration will decease CAR expression in T cells during manufacture leading to decreased tonic signaling and exhaustion. Secondly we will show that in the tumour environment, CAR-T will be expressed leading to greater effector function. Finally in normal tissues expressing a target antigen but with normal oxygen concentration, toxicity will be avoided since the CAR will not be induced.

Tumours are heterogeneous mixtures of different cell-types. In addition to mutated cancer cells themselves, the tumour microenvironment contains multiple stromal and immune cells that each contribute to a patient’s cancer. While we now understand the cellular composition of tumours relatively well, how different cell-types collectively integrate to drive cancer is poorly defined.

Our ignorance of cell-cell communication in the tumour microenvironment largely stems from our inability to simultaneously measure ligands (cues), receptors (transducers), and signals (effectors) across all cells in a tumour. Without such data it is incredibly challenging to ‘connect’ mutations in cancer cells with deregulated phenotypes in stromal and immune cells.

To address this, the UCL Cell Communication Laboratory (www.tape-lab.com) have developed a novel mass-cytometry (CyTOF) technology to measure single-cell post-translational modification (PTM) signalling in organoid models of the colorectal cancer (CRC) tumour microenvironment. While incredibly powerful for understanding PTM signalling, CyTOF data cannot describe the 1,000s of putative cell-type specific ligands or receptors responsible for transducing each signal. As a result, we cannot connect signals between cancer, stromal, and immune cells in the tumour microenvironment.

The aim of this CRUK City of London Development Fund project is to integrate our single-cell PTM technology (CyTOF) with single-cell ligand and receptor transcriptomics (10x Genomics). By simultaneously measuring cell-type specific ligand / receptor expression and PTM signalling in organoid models of the CRC tumour microenvironment (in collaboration with Vivian Li Lab, Francis Crick Institute), this will allow us to ‘connect’ cell-cell signalling across the tumour microenvironment.

Enhancing drug efficacy by manipulating the tumour microenvironment is a primary goal of our laboratory research. We have published that intravenous administration of the integrin-targetting agent, low dose Cilengitide (ldCil), increases tumor angiogenesis and drug delivery whilst reducing hypoxia and desmoplasia in gold standard preclinical models of lung and pancreatic (Kras^LSL-G12D/+;p53^R172H/+;PDXCre, KPC) cancers. Together this approach reduces tumour growth and metastasis, whilst extending survival (Reynolds et al., Nature Medicine, 2009; Wong et al., Cancer Cell 2015). We have also collaborated with medicinal chemists who have developed a second generation orally available agent, Agent X, currently under patent application. Our new data indicate that combination treatment including Agent X also reduces desmoplasia.

Magnetic resonance elastography (MRE) is an innovative non-invasive imaging technique that not only determines the presence of a tumour but also identifies the desmoplastic pathophysiological status of cancers and can be used to assess the anti-desmoplastic efficacy of cancer therapies. Our partner in this application, Ralph Sinkus’s has developed methods of improved MRE technologies across multiple cancer types has provided the opportunity to utilize MRE as a tool for therapy efficacy (Fovargue et al., NMR Biomed. 2018; Jamin et al., Cancer Res 2015).

Through this City of London Development Fund we now have the opportunity to join forces from Barts and Kings to examine the efficacy our new anti-cancer strategy using state of the art imaging thus bringing added-value to this cross institutional collaboration.

Despite progress in the histological classification of the different subtypes of sarcoma, the clinical outcome of patients with the majority of these subtypes has not improved over the last 40 years. To change this, there is a need to understand the molecular basis of sarcoma in the context of clinical presentation, outcome and response to therapies.

Deep sequencing of cancer genomes has revealed complex patterns of inter- and intra-tumour heterogeneity, providing insight into a tumour’s evolutionary trajectory and is starting to inform on patient prognosis and therapeutic decisions. The main clinical consequence of tumour heterogeneity and clonal evolution is the emergence of resistance to systemic drug therapy. Epigenetics has been shown to contribute to intra-tumour heterogeneity and evolution, as first reported in haematological malignancies, by measuring DNA methylation at single-nucleotide level using Reduced-Representation Bisulfite Sequencing (RRBS).

This study aims to generate a comprehensive profile at base pair resolution of the DNA methylation heterogeneity in osteosarcoma tumours and for the first time determine if locally disordered methylation exists in osteosarcoma. Building on the samples already collected and submitted to the 100,000 Genomes project, which includes whole genome sequencing of up to 8 samples per osteosarcoma, we will interrogate DNA methylation heterogeneity at a single nucleotide level using enhanced RRBS targeting over 4 million CpG dinucleotides in CpG-rich regions of the genome. Further, we will characterise the epigenetic tumour evolution in osteosarcoma tumours by looking at multiple samples from each tumour lesion, and determine associations between heterogeneity and the presence of particular driver mutations by using WGS data from the 100,000 Genomes Project.

Most cancer evolution studies, because they rely on bulk DNA sequencing data, are inevitably compromised by ambiguous estimates of tumour ploidy.

Aberrations in ploidy and in particular whole genome doubling are macro-evolutionary events that are associated with increased fitness of cancer cells and poor prognosis. The largest pan-cancer bulk DNA sequencing studies to date have relied on ploidy estimates derived in silico, which are ambiguous and often incorrect. The problem arises from the fact that, in theory, multiples of 2 times the ploidy are mathematically equivalent solutions to fit the data, and although hints in the data can help select between the various potential solutions, this exercise remains hand-wavy. In addition, tumours can show heterogeneity at the level of ploidy, containing multiple populations with very different ploidy values, which is not modelled by current copy number inference methods.

Hence, our current pan-cancer picture of heterogeneity at the level of copy number and ploidy is challenged, and, although previous and recent results strongly support the use of systematic experimental ploidy validation to inform sequencing-based copy number callers, this still has to become good practice in the field. This is especially relevant and timely for large whole genome sequencing programs such as the 100,000 genomes project, but also for all DNA-profiling-based studies of cancer evolution and intra tumour heterogeneity.

This is why we aim to develop standards for the field by benchmarking ploidy estimated from bulk sequencing against experimental ploidy using FACS, single cell sequencing and digital imaging; and study the extent and spatial mapping of intra-tumour heterogeneity at the level of ploidy. This will be done across multiple cancer types, with a particular focus on undifferentiated sarcomas, which were recently shown to display high amounts of inter- and intra-tumour heterogeneity at the level of ploidy.

Immunotherapy has revolutionised the outlook on treatment of various cancers in recent years. Several strategies have been developed for immunotherapy, majority of which ultimately depend on presence of neoantigens, tumour-specific antigens which arise from coding mutations that get translated and presented on the surface of the cells in form of peptides associated with Human Leukocyte Antigen-I (HLA-I). The ability to detect and monitor these neoantigens is critical, not only for better understanding the process of cancer cell detection by the immune system, but also for devising novel immunotherapies against cancer such as personalised vaccines. Despite years of research, however, neoantigen discovery and validation remains a daunting problem in the cancer biotherapeutics field.

The most common method for neoantigen discovery is in silico prediction of neoantigens from bulk tumour sequencing data. Although relatively easy to implement, the actual success rate of in silico methods for predicting neoantigens that can elicit an immune response remains very low. Direct identification of neoantigens by mass spectrometry (MS) represents an alternative to in silico based prediction. However, current available methods either suffer from lack of specificity, or are labour-intensive and require large quantities of input material, thus rendering their widespread use challenging. In this project, we propose to develop a novel approach that overcomes these major limitations, enabling rapid MS based identification of HLA-I-bound neoantigens, with high specificity, from a variety of live cancer models. As a proof-of-concept, we will then apply our method to analysis of neoantigens a panel of patient-derived colorectal cancer organoids grown in 3D culture.

Immunotherapy has provided new hope for many cancer patients, with the potential to treat or cure even advanced metastatic disease. Although considered a huge breakthrough, immunotherapy only currently works in a subset of patients. Identifying ways to predict which patients will respond and approaches to sensitise non-responders, are critical next steps to extend impact. In colon cancer, immunotherapy ‘checkpoint inhibitors’ have already been approved for use in a subset of cancer patients who show high mutation rates (MSI+ tumours). Immunoscore – a quantitative assessment of the immune infiltrate in tumours – is also emerging as a powerful indicator of response to immunotherapy in colon cancer. As a consequence, interventions which modulate the immune infiltrate are of huge interest as they could be used to expand the group of patients who could benefit.

Our multidimensional and in vivo data indicate that Rho effector kinases may suppress tumour growth and promote an anti-tumour immune landscape. In this project, we wish to assess the immune cell repertoire in colorectal tumours induced in a Rho effector knockout model, to assess a novel mechanism to predict or modulate immunotherapy responses. We will use state-of-the-art Mass Cytometry (CyTOF) and tumour phenotyping to examine a broad range of immune cell types in colon tumours from our novel in vivo models. Our preliminary data suggest that specific Rho effectors may promote infiltration of tumours with CD8+ T-cells; intervention by activating or inhibiting this pathway would be expected to modulate both the immune infiltrate and response to immune checkpoint inhibition. This study will lay the ground work for the development of novel therapies to use in conjunction with existing immunotherapy drugs.

Cancer genomes contain a plethora of genomic alterations, affecting single base pairs up to whole chromosomes. Recurrently altered genomic regions likely contain important cancer genes that could represent novel targets for cancer therapy. However, teasing out which of these regions represent important drivers from those that are more prone to alterations has remained a challenge. We aim to use single-cell data to investigate which copy number alterations are important in cancer.

Ferroptosis is a morphologically and biochemically distinct form of regulated cell death that results from the excessive peroxidation of polyunsaturated fatty acids. Ferroptotic cell death is catalysed by iron and if left unregulated results in a chain reaction of lipid-derived reaction oxygen species (ROS) and eventual membrane destruction. Glutathione metabolism and antioxidant capacity mediate the sensitivity of tumours to ferroptosis through the combined activity of glutathione peroxidase and the cystine/glutamate transporter, xCT. Recently it has been shown that that immunotherapy-activated CD8+ T-cells induce ferroptosis-specific lipid peroxidation in tumour cells. T-cell immunity was enhanced by immune checkpoint blockade through a mechanism of interferon gamma release and subsequent xCT downregulation.

We are currently evaluating a novel PET imaging agent, [18F]FSPG, as a non-invasive marker of the tumour redox microenvironment. [18F]FSPG is taken up by tumour cells via xCT, providing a non-invasive measure of its activity. In this City of London Centre Development Fund project, we will monitor the efficacy of T-cell immunotherapy with [18F]FSPG in in vivo models of lung cancer. In genetically-modified animals and in patient-derived xenografts, we will image tumour-induced ferroptosis with [18F]FSPG following checkpoint blockade. Tumour-associated T-cell infiltration, activation and dysfunction will be assessed ex vivo along side lipid peroxidation as a marker of ferroptosis. Together, this will provide a rapid, whole-body assessment of the treatment efficacy for these novel immunotherapies.

Immuno-surveillance is a critical tumour-suppressive mechanism that transformed cells need to evade to establish a full-blown cancer. Many immunoevasive strategies employed by cancer cells rely on aberrant transcriptional regulation of immunomodulatory molecules, implicating chromatin and DNA methylation in these processes.

Despite the recent success of immune checkpoint inhibitors, a significant fraction of patients do not respond to the therapy, suggesting that cancer cells likely exploit yet unidentified immunoevasive strategies. Using a unique experimental model, we have identified chromatin regulators whose inactivation favours the transition from an immunogenic to an immunoevasive phenotype.

We now propose to dissect the processes underpinning this transition, assess their prevalence in various cancer types, and provide a proof of principle that interfering with the identified mechanisms synergizes with other therapies.