In the context of anti-tumor immunity, activated T cells secrete interferon-gamma (IFNγ) to induce indoleamine 2,3-dioxygenase (IDO1) in the target cancer cells. High IDO1 levels deplete tryptophan, leading to aberrant protein production due to tryptophan to phenylalanine codon reassignments (W>F substitutants) by tRNA misacylation1. Whether the generation of W>F substitutants is a controlled process contributing to cancer cell survival is unknown. Here, we set up a functional genetic screening approach to underpin the underlying mechanism of W>F substitutants. This has uncovered FTSJ1, an enzyme that 2’-O-methylates several tRNAs at positions 32 and 34 of their anticodon loop2. We show that FTSJ1 is highly expressed in cancer proteomes and that FTSJ1-mediated tRNAtrp modifications are required for the efficient production of W>F substitutants in the proteome and immunopeptidome of cancer cells. Functionally, FTSJ1 expression both stimulated cancer cell adaptability to IFNγ-mediated tryptophan shortage and induced T-cell activity against W>F neoepitopes. Thus, aberrant protein production is a regulated process with consequences for cell adaptation following stress.