Abstract 5625: Targeting the nonsense mediated mRNA decay pathway to prevent the degradation of highly immunogenic frameshift mutated transcripts

Abstract Introduction: Frameshift insertions/deletions (fs-indels) can lead to the generation of immunogenic non-self-peptides which contribute to anti-tumor immune responses. However, fs-indels often generate premature termination codons (PTCs), leading to mRNA degradation by the nonsense mediated mRNA decay (NMD) pathway. Importantly, some PTC-containing transcripts naturally escape NMD degradation and produce highly immunogenic neoantigens. Therefore, we hypothesize that NMD inhibition could be used to increase the cellular pool of fs-indels derived neoantigens in cancer cells, and enhance anti-tumor immunogenicity. NMD also regulates other cellular processes by degrading a subset of non-PTC-containing self-transcripts. Therefore, by focusing NMD inhibition towards the degradation of PTC-transcripts only, toxicity may be limited in a clinical setting. We investigate how targeting different NMD proteins affects the degradation of fs-transcripts across several cancers types. To assess the clinical relevance of NMD inhibition, we study how it specifically affects p53 fs-transcripts, since p53 is the most mutated gene in cancer and fs-mutant p53 has been shown to be highly immunogenic. Methods: Core NMD proteins were depleted across a panel of cell lines from different cancer types and transcriptomics, flow cytometry, qPCR and western blot were used to evaluate changes in the expression of NMD-targeted transcripts and NMD members. Proteomics and immunopeptidomics were used to study protein expression and HLA-peptide presentation arising from fs-transcripts. A Translating RNA Imaging by Coat protein Knock-off (TRICK) assay was developed to study the translation of NMD-targeted p53 mutants upon NMD inhibition. This assay can precisely quantify the translation of p53 mutant RNA upon different treatments and cellular stresses at single-molecule resolution. Results: We found an increase in expression of PTC-transcripts upon silencing of multiple NMD members. Our data suggests that some NMD members are better targets for inhibition of NMD PTC-surveillance, whilst others are more appropriate for inhibition of NMD-mediated gene expression regulation. These data support the recently proposed hypothesis that NMD is a branched pathway, where each branch targets specific subsets of transcripts depending on the cell type. These results are clinically relevant as the method by which NMD is inhibited could help optimize the generation of immunogenic neoantigens whilst minimizing toxicity. The TRICK assay has shown that inhibiting NMD surveillance reduces the degradation of p53 fs-transcripts and increases their protein expression. Conclusion: We have identified NMD pathway members that can be targeted to efficiently inhibit the surveillance role of NMD and prevent the degradation of fs-transcripts that could generate highly immunogenic neoantigens. Citation Format: Shanila Fernandez Patel, Roberto Vendramin, Danwen Qian, Yue Zhao, Lorena Ligammari, Krupa Thakkar, Jun Murai, Eva Grönroos, Jeremy Carlton, Kevin Litchfield. Targeting the nonsense mediated mRNA decay pathway to prevent the degradation of highly immunogenic frameshift mutated transcripts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5625.