In recent years, RNA-modifying enzymes have gained significant attention because their activities are relevant to cancer biology, and they are therefore potential targets for novel therapeutic intervention. Here, I target validated two RNA methyltransferases. I performed a late-stage study of METTL3, and I initiated and developed an early-stage analysis of THUMPD3.

Advanced validation of METTL3, a well characterised m⁶A RNA methyltransferase, was performed based on the availability of a specific small molecule inhibitor, METTL3i. I explored whether differences in sensitivities of lung cancer cell lines to METTL3i might be due to METTL3 binding specific gene promoters in a cell-dependent manner. My findings indicate that this is not the case. I also performed a synthetic lethality screen to identify epigenetic-related inhibitors that sensitise a METTL3i-resistant lung cancer cell line to the inhibitor. This identified potential pathways that could be targeted to overcome resistance to METTL3i-based therapy.

Independently of the above, an in-house bioinformatic analysis indicated a potential human RNA methyltransferase, THUMPD3, was linked to lung cancer biology. Furthermore, early in my project, THUMPD3 was reported to catalyse m²G in tRNAs. However, whether THUMPD3 also methylates non-tRNA substrates was not addressed. My project sought to address whether other RNAs are indeed m²G modified by THUMPD3 and to further investigate and validate the role of the enzyme in lung cancer biology.

Consistent with our bioinformatic data, depletion of THUMPD3 from lung cancer cells induced several notable effects; it negatively impacted cellular proliferation, and migration. It also induced apoptosis and disturbances in RNA splicing. Importantly, exogenous expression of THUMPD3 in normal lung fibroblasts stimulated their proliferation rate. Furthermore, transcriptome analysis indicated that changes in expression of proteins on the cell surface and the extra cellular matrix (ECM) may occur.

To identify potential RNA substrates containing THUMPD3-dependent m²G, I leveraged a newly invented method, PhOxi-seq, which is based upon blue light-induced photo-oxidation of certain modified nucleotides in RNA, including m²G. Initially, I further developed and optimised PhOxi-seq on tRNA, leading to the discovery of THUMPD3-dependent alterations in m²G6 tRNA modification. Subsequently, I broadened the scope of my screen by including purified human rRNA-depleted RNA from lung cancer cells as substrate in the photo- oxidation reaction. This approach unveiled a cohort of RNAs, mainly mRNAs, several of which are implicated in the ECM. This links in well with my phenotypic analysis on the same cells. Notably, this is the first time that m²G has been reported in human mRNAs, as well as other polyA+ RNAs. The implications of these findings are discussed.

In summary, my work extends the application of PhOxi-seq to identifying THUMPD3- dependent m²G sites (and potentially other oxidisable modifications induced by different enzymes) in various RNA types. My works also identifies a potential oncogenic role for THUMPD3 in lung cells. Thus, my findings highlight THUMPD3 as a potential therapeutic target, especially in a lung cancer setting.