Compensatory tRNA Modification by DUS3L Confers Resistance to METTL1 Loss in Oesophageal Cancer.

tRNA N7-methylguanosine (m7G46), installed by the METTL1/WDR4 methyltransferase, stabilizes the tRNA pool supporting high proliferative rates. Accordingly, many cancer cell lines are vulnerable to METTL1 loss, underscoring its potential as a therapeutic target. We identify a subset of cell lines insensitive to METTL1 depletion and use oesophageal lines as a model to explore the mechanism. The METTL1-resistant adenocarcinoma line OE33 preserves tRNA balance upon m7G46 deficiency by elevating dihydrouridylation at position 47 (D47). Depletion of DUS-L enzymes that catalyse D16/D17/D20/D47 on tRNAs sensitises OE33 to METTL1 loss. Concomitant loss of dihydrouridylation and m7G46 destabilizes the canonical L-shaped tRNA structure, disrupts coaxial helix stacking and anticodon-loop presentation, and reduces cellular fitness. Notably, METTL1-sensitive cell lines lack this compensatory pathway due to the deleterious effects of increasing their intrinsically low DUS3L levels. These findings reveal a DUS-L-mediated buffering mechanism that supports tRNA homeostasis in METTL1-resistant cancers and highlight DUS3L abundance as a potential biomarker for predicting METTL1 sensitivity. Moreover, they suggest that combined targeting of DUS-L and METTL1 may provide a therapeutic strategy for overcoming resistance in cancers, exemplified in cell lines such as OE33.