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The Role of tRNA methyltransferases in Cancer https://doi.org/10.17863/CAM.120446 Michaelidou, Alexandra Kouzarides, Tony RNA modifications have emerged as pivotal regulators of cellular homeostasis, stress adapta- tion, and disease, particularly within the context of cancer. Among these, tRNA modifications play a crucial role in maintaining translational fidelity and supporting cancer cell survival under adverse conditions. This thesis explores the contribution of two tRNA methyltrans- ferases - TRMT1, responsible for catalysing the m2,2G modification at position 26 in tRNAs, and the METTL1/WDR4 complex, which catalyses the formation of m7G at position 46 in tRNAs - to cancer cell proliferation, stress responses, and adaptive mechanisms in various cancer types. The role of TRMT1 in colorectal and ovarian cancer models was first investigated. Phenotypic assays revealed that TRMT1 knockout resulted in a proliferation defect in DLD- 1 colorectal cancer cells, whereas no significant effect was observed in JHOC-5 ovarian cancer cells, underscoring a cell type specific dependency. Multi-omic analyses in DLD- 1 cells demonstrated that TRMT1 depletion likely triggers upregulation of an integrated stress response (ISR)-associated gene expression network indicative of impaired cellular homeostasis. These findings suggest that TRMT1 modulates colorectal cancer proliferation through stress adaptation, supporting its potential as a therapeutic target, although additional validation, is warranted. Alongside the above, I also examined cellular pathways perturbed by depletion of METTL1/WDR4 from OE21 oesophageal squamous cell carcinoma cells. As METTL1 knockout was found to be lethal in OE21 cells, as demonstrated by a postdoctoral researcher in the Kouzarides laboratory who was unable to obtain viable knockout clones, I focused on WDR4 knockout clones to explore adaptive mechanisms in m7G-deficient settings. This work identified divergent, cell line dependent responses to WDR4 loss. Whilst some WDR4 knock- out clonal cell lines displayed reduced proliferation accompanied by TE-derived dsRNA accumulation and innate immune activation, others clonal cell lines maintained wild-type-like growth. Transcriptomic and proteomic analyses of the various cell lines revealed that clones resistant to WDR4 targeting circumvented immune activation by suppressing TE expression and concurrently downregulating innate immune pathways. Furthermore, all clones exhibited enhanced cell adhesion, cytoskeletal remodelling, and autophagy-related gene expression programmes, likely promoting survival in the context of translational stress. While these adaptive networks were validated at the transcript and protein level, further assays will be required to confirm pathway functionality, particularly in autophagy and immune suppression. Together, my findings highlight the diverse strategies adopted by cancer cells in order to adapt to inhibition or loss of tRNA modification enzymes, such as TRMT1 and the METTL1/WDR4 complex. The work presented here underscores the complexity of targeting RNA modification pathways in cancer and emphasises the need to account for context-specific vulnerabilities and compensatory responses. By integrating phenotypic, transcriptomic, and proteomic data, these novel data strengthen our understanding of the interplay between RNA modifications, stress adaptation, and immune regulation in cancer biology and they bolster the therapeutic potential of targeting tRNA-modifying enzymes in the context of specific tumours. University of Cambridge 2025-03-27 Thesis Doctoral Doctor of Philosophy (PhD) https://www.repository.cam.ac.uk/handle/1810/387800 embargo 2026-08-01 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/12050b53-690d-4884-b74d-7db40ab61cb4/download 87eda9de84448d1f82354d60eee3eb5f https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/b6aad3d9-92b0-4fa2-9392-a37797c344e0/download 7388fef72399bda654a56f9938213ff4 http://purl.org/NET/rdflicense/allrightsreserved RNA modifications tRNA methyltransferases Cancer