Upstream open reading frame translation enhances immunogenic peptide presentation in mitotically arrested cancer cells

Mitosis is a critical phase of the cell cycle and a vulnerable point where cancer cells can be effectively disrupted, leading to cell death and inhibition of tumor growth. However, challenges such as drug resistance remain significant in clinical applications. During mitosis, mRNA translation is generally downregulated, while non-canonical translation of specific transcripts proceeds. Here, we demonstrate that mitotic cancer cells redistribute ribosomes toward the 5' untranslated region (5' UTR) and the start of the coding sequence (CDS), enhancing the translation of thousands of upstream open reading frames (uORFs) and upstream overlapping open reading frames (uoORFs). This mitotic induction of uORF/uoORF enriches the presentation of immunopeptides at the surface of cancer cells following treatment with mitotic inhibitors. Functional assays indicate the potential of such neoepitopes to provoke cancer-cell killing by T cells. Altogether, our findings highlight the therapeutic potential of targeting uORF/uoORF-derived neoepitopes in combination with mitotic inhibitors to enhance immune recognition and tumor cell elimination. Overall design: We performed ribosome profiling in mitotically arrested U2OS. Cells were treated with Nocodazole (0.5 µM) for 16 hours.