Cancer Biology
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Item Exploiting the Vulnerability of ARID1A Deficient Ovarian Cancers for Therapeutic Potential(Institute of Cancer Research (University Of London), 2024-09-26) Amin, N; Downs J; Downs, J; Amin, NowaThe switching defective/sucrose non-fermenting (SWI/SNF) chromatin remodelling complex is important for the cellular response to replication stress. 20% of cancers harbour modifications in SWI/SNF complex subunits. ARID1A, a key component of the SWI/SNF complex, is mutated across a variety of cancers, and notably in 35-57% of ovarian clear cell carcinomas (OCCC). Clinically applicable targeted therapies for this aggressive, chemo-resistant disease remains an unmet need. G-quadruplexes (G4s) are thermodynamically stable secondary DNA structures which are a consequence of folding of guanine-rich DNA sequences. Treatment with G4 stabilising ligands, some of which have entered clinical trials, leads to DNA double strand breaks (DSBs). This represents a therapeutic vulnerability for cancers with defects in the response to G4 ligands and resulting DNA DSBs. Here, isogenic cell line models were generated using CRISPR-Cas9 gene editing and genetic complementation of ARID1A to study the potential contribution of ARID1A to genotoxic stress. We found that ARID1A deficient cells show selective sensitivity to G4 stabilising ligands, and that there is evidence of delayed repair of DNA damage when ARID1A is deficient. Mechanistically, we discovered that NHEJ factors fail to mobilise onto chromatin after treatment with stabilising ligand PDS when ARID1A is deficient. Furthermore, we showed that inhibitor of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a protein ensuring effective NHEJ functions, when combined with PDS leads to synergistic decrease in cell viability in ARID1A deficient cells. These data provide new insights into G4 ligands-induced DNA damage and their repair in ARID1A-defective models. This knowledge could be exploited for a new therapeutic approach to treat ARID1A deficient ovarian cancer.Item Proteomic and immunopeptidomic characterisation of head and neck cancers(Institute of Cancer Research (University Of London), 2024-09-13) Melake, JM; Choudhary J; Choudhary, J; Melake, Johanna MiriamHead and neck squamous cell carcinoma (HNSCC) exhibits distinct clinical characteristics that can be associated with the initiation factors of carcinogens or HPV infection, the latter being associated with better survival outcomes following chemotherapy and radiation. Understanding the underlying molecular differences, particularly with regards to tumour recognition by the immune system, is crucial for advancing treatment options. Our study employs quantitative proteomics in HNSCC cell lines and identifies a HPV protein signature linked to TRIM28 chromatin regulation rather than solely HPV oncoprotein-driven protein deregulation. We further describe four cancer testis antigens (CTA) associated with HPV+ diseases. By treating cell lines with radiation and chemotherapy, we outline treatment response signatures unique to HPV+ and HPVcells and identify promising radiation-induced protein targets in HPV- cells with potential for combined targeted therapies. We implement a quantitative immunopeptidome workflow, and use it to characterise the HLA antigen landscape of HNSCC cell lines, finding no HPV-dependent differences in immunopeptidome size or functional enrichment of peptide source proteins. However, we discover tumour-associated, recurrent candidate peptides and CTA peptides from the SPAG and MAGE protein families highly recurrent in both HNSCC cell lines and patients. Radiation-driven modulation of cell line immunopeptidomes reveals a larger subset of upregulated peptides compared to downregulated ones, which are predominantly presented on HLAB. These radiation-induced peptides partially originate from stress-related proteins, linking protein-level to immunopeptidome-level modulation. Additionally, we investigate immunopeptide trajectories during radiotherapy in HNSCC patient blood plasma, mapping temporal peptide abundance changes in a pilot study of six patients. We discover recurrent radiation-induced peptides considered as prognostic biomarkers in various cancers and linked to poor disease outcomes. Overall, our study provides novel insights into protein and immunopeptidome regulation in HNSCC at baseline and posttreatment, uncovering tumour-associated, radiation-induced, or CTA protein and peptide targets that could ameliorate HNSCC treatment strategies. Declaration I confirm that: • the work present in this thesis is my own and where information has been derived from other sources, I confirm that this has been indicated in the thesis; • that the thesis does not exceed the prescribed word limit; and • that I will keep my contact details updated with the Library Theses Office and Registry throughout the examination process. Signature: Date:Item Deep cell phenotyping and spatial analysis of multiplexed imaging with TRACERx-PHLEX.(NATURE PORTFOLIO, 2024-06-15) Magness, A; Colliver, E; Enfield, KSS; Lee, C; Shimato, M; Daly, E; Moore, DA; Sivakumar, M; Valand, K; Levi, D; Hiley, CT; Hobson, PS; van Maldegem, F; Reading, JL; Quezada, SA; Downward, J; Sahai, E; Swanton, C; Angelova, M; Downward, Julian David HarryThe growing scale and dimensionality of multiplexed imaging require reproducible and comprehensive yet user-friendly computational pipelines. TRACERx-PHLEX performs deep learning-based cell segmentation (deep-imcyto), automated cell-type annotation (TYPEx) and interpretable spatial analysis (Spatial-PHLEX) as three independent but interoperable modules. PHLEX generates single-cell identities, cell densities within tissue compartments, marker positivity calls and spatial metrics such as cellular barrier scores, along with summary graphs and spatial visualisations. PHLEX was developed using imaging mass cytometry (IMC) in the TRACERx study, validated using published Co-detection by indexing (CODEX), IMC and orthogonal data and benchmarked against state-of-the-art approaches. We evaluated its use on different tissue types, tissue fixation conditions, image sizes and antibody panels. As PHLEX is an automated and containerised Nextflow pipeline, manual assessment, programming skills or pathology expertise are not essential. PHLEX offers an end-to-end solution in a growing field of highly multiplexed data and provides clinically relevant insights.Item Biocompatibility characterisation of CMOS-based Lab-on-Chip electrochemical sensors for in vitro cancer cell culture applications.(ELSEVIER ADVANCED TECHNOLOGY, 2024-10-15) Beykou, M; Bousgouni, V; Moser, N; Georgiou, P; Bakal, C; Bakal, ChristopherLab-on-Chip electrochemical sensors, such as Ion-Sensitive Field-Effect Transistors (ISFETs), are being developed for use in point-of-care diagnostics, such as pH detection of tumour microenvironments, due to their integration with standard Complementary Metal Oxide Semiconductor (CMOS) technology. With this approach, the passivation of the CMOS process is used as a sensing layer to minimise post-processing, and Silicon Nitride (Si3N4) is the most common material at the microchip surface. ISFETs have the potential to be used for cell-based assays however, there is a poor understanding of the biocompatibility of microchip surfaces. Here, we quantitatively evaluated cell adhesion, morphogenesis, proliferation and mechano-responsiveness of both normal and cancer cells cultured on a Si3N4, sensor surface. We demonstrate that both normal and cancer cell adhesion decreased on Si3N4. Activation of the mechano-responsive transcription regulators, YAP/TAZ, are significantly decreased in cancer cells on Si3N4 in comparison to standard cell culture plastic, whilst proliferation marker, Ki67, expression markedly increased. Non-tumorigenic cells on chip showed less sensitivity to culture on Si3N4 than cancer cells. Treatment with extracellular matrix components increased cell adhesion in normal and cancer cell cultures, surpassing the adhesiveness of plastic alone. Moreover, poly-l-ornithine and laminin treatment restored YAP/TAZ levels in both non-tumorigenic and cancer cells to levels comparable to those observed on plastic. Thus, engineering the electrochemical sensor surface with treatments will provide a more physiologically relevant environment for future cell-based assay development on chip.Item Mixed responses to targeted therapy driven by chromosomal instability through p53 dysfunction and genome doubling.(Springer Science and Business Media LLC, 2024-06-13) Hobor, S; Al Bakir, M; Hiley, CT; Skrzypski, M; Frankell, AM; Bakker, B; Watkins, TBK; Markovets, A; Dry, JR; Brown, AP; van der Aart, J; van den Bos, H; Spierings, D; Oukrif, D; Novelli, M; Chakrabarti, T; Rabinowitz, AH; Ait Hassou, L; Litière, S; Kerr, DL; Tan, L; Kelly, G; Moore, DA; Renshaw, MJ; Venkatesan, S; Hill, W; Huebner, A; Martínez-Ruiz, C; Black, JRM; Wu, W; Angelova, M; McGranahan, N; Downward, J; Chmielecki, J; Barrett, C; Litchfield, K; Chew, SK; Blakely, CM; de Bruin, EC; Foijer, F; Vousden, KH; Bivona, TG; TRACERx consortium; Hynds, RE; Kanu, N; Zaccaria, S; Grönroos, E; Swanton, C; Downward, Julian David HarryThe phenomenon of mixed/heterogenous treatment responses to cancer therapies within an individual patient presents a challenging clinical scenario. Furthermore, the molecular basis of mixed intra-patient tumor responses remains unclear. Here, we show that patients with metastatic lung adenocarcinoma harbouring co-mutations of EGFR and TP53, are more likely to have mixed intra-patient tumor responses to EGFR tyrosine kinase inhibition (TKI), compared to those with an EGFR mutation alone. The combined presence of whole genome doubling (WGD) and TP53 co-mutations leads to increased genome instability and genomic copy number aberrations in genes implicated in EGFR TKI resistance. Using mouse models and an in vitro isogenic p53-mutant model system, we provide evidence that WGD provides diverse routes to drug resistance by increasing the probability of acquiring copy-number gains or losses relative to non-WGD cells. These data provide a molecular basis for mixed tumor responses to targeted therapy, within an individual patient, with implications for therapeutic strategies.