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Abstract

Paclitaxel (PTX) is a widely used chemotherapeutic agent against cancer. The drug disrupts microtubule dynamics, causing mitotic arrest that activates apoptotic pathways. However, the anti-cancer efficacy of PTX is limited to specific cancer types, and its use is often accompanied by significant adverse effects. Thus, further investigation into the cellular effects of PTX is essential. In this study, I investigated the translational response induced by mitotic arrest. Using ribosome profiling (RiboSeq), I observed increased transcriptome-wide ribosome occupancy at 5’ un-translated regions (5'UTRs) and 5' ends of coding sequences in treated cancer cell lines. This effect was independent of the molecular mechanism of mitotic-arrest induction. Further computational analysis revealed an increased prediction rate of non-canonical open-reading frames (ncORFs), specifically upstream and upstream-overlapping ORFs (u/uoORFs) within the 5'UTR. Notably, translation rates for these genomic features were elevated. To enable further research, I compiled uORFs and uoORFs elements from various cancer cells into a comprehensive database. Building on these findings, I hypothesized that in vitro PTX treatment may lead to cell surface presentation of non-canonical peptides (nuPeptides) by HLA-I complexes. To test this hypothesis, state-of-the-art immunopeptidomics were employed to enrich HLA-I ligands and perform quantitative peptide detection via mass spectrometry in PTX- or DMSO-treated cells. This analysis revealed elevated levels of specific peptides originating from uORF or uoORF transcripts. Selected uORFs giving rise to nuPeptides were further validated in a complementary approach using uORF-SIINFEKL-reporter constructs and ex vivo CD8+ T cells. This demonstrated increased cytokine secretion and cancer cell killing capability upon PTX-induced mitotic arrest. In summary, my results demonstrate that PTX treatment provokes aberrant mRNA translation in the 5'UTR, resulting in nuPeptide synthesis. These ligands bind to HLA-I complexes and may trigger immune responses. My findings provide new insights into treatment-induced peptide biosynthesis from uORF and uoORF sequences to benefit future immunotherapies.