Abstract Immunotherapy interventions relies heavily on neoantigen availability. The human genome encodes non-canonical/mutant proteins that potentially contain neoantigenic peptides. Nevertheless, their typically low expression, potentially moderated by the Nonsense-Mediated Decay (NMD) pathway, restricts their therapeutic utility. In this study, we explored the NMD pathway influence on non-canonical/mutant protein expression, specifically focusing on UPF1 knockdown. We implemented proteogenomic approaches to ascertain if the encoding transcripts and their respective proteins were upregulated post-knockdown. Complementary to this, we conducted a comprehensive pan-cancer survey of UPF1 expression and an in vivo evaluation of UPF1 expression in Triple-Negative Breast Cancer (TNBC) tissue. Our empirical results delineated that UPF1 knockdown precipitates an increase in the transcription of non-canonical/mutant proteins, especially those originating from retained-introns, pseudogenes, long non-coding RNAs, and unannotated biotypes. Furthermore, the analysis revealed that UPF1 expression was conspicuously high across a range of neoplastic tissues, with protein levels notably amplified in patient derived TNBC tumours in comparison to adjacent tissues. Our study elucidates UPF1 functional role in attenuating transcriptional noise through the degradation of transcripts encoding non-canonical/mutant proteins. Interestingly, we observed an upregulation of the NMD pathway in cancer, potentially functioning as a “neoantigen masking” mechanism that subdues non-canonical/mutant protein expression. Suppressing this mechanism may unveil a new cadre of neoantigens accessible to the antigen presentation pathway. Our novel findings proffer a solid base for devising therapeutic strategies targeting UPF1 or the NMD pathway, given the pronounced presence of UPF1 in malignant cells, thus potentially augmenting immunotherapeutic responses in cancer.

Regulation of Non-Canonical Proteins Encoded by Small Open Reading Frames via the Nonsense-Mediated Decay Pathway.