Microsatellite-unstable (MSI) colorectal cancer is characterized by the accumulation of somatic insertion/deletion (InDel) mutations potentially generating tumor-specific, frameshifted protein sequences. Such mutations typically generate premature translation termination codons targeting the affected mRNAs to degradation by nonsense-mediated RNA decay (NMD), limiting the synthesis and HLA class I-mediated presentation of tumor-specific InDel neoepitopes. We reasoned that the NMD inhibitor 5-azacytidine (5AZA) could serve to increase the expression of NMD-sensitive neoepitopes and analyzed the immunopeptidome of MSI HCT-116 cells using a proteogenomic approach. After immunoprecipitation of HLA:peptide complexes, we identified more than 10,000 HLA class I-presented peptides by LC-MS/MS including five InDel neoepitopes. The InDel neoepitopes were verified on the genomic, transcriptomic, and peptidomic level. Treatment with 5AZA increased the expression of the corresponding frameshift- and premature termination codon-bearing mRNAs and enhanced the presentation of peptides originating from known NMD targets and one of the identified InDel neoepitopes. By analyzing an array of MSI colorectal cancer cell lines and patient samples, we found the underlying frameshift mutation to be highly recurrent and immunization with the corresponding neoepitope induced strong CD8+ T cell responses in an HLA-A*02:01 transgenic mouse model. Our data directly show that peptides originating from frameshifted open reading frames due to InDel mutations in mismatch repair-deficient cells are presented on the cell surface via HLA class I. Moreover, we demonstrate the utility of NMD inhibitor-enhanced HLA class I-mediated presentation of InDel neoepitopes as well as their immunogenicity, uncovering the clinical potential of NMD inhibition in anti-cancer immunotherapy strategies.

中文翻译：

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无意义介导的RNA衰变的药理抑制作用增强了MSI CRC中HLA I类介导的新表位的表达

微卫星不稳定（MSI）大肠癌的特征是体细胞插入/缺失（InDel）突变的积累，可能会产生肿瘤特异性的移码蛋白序列。这样的突变通常产生过早的翻译终止密码子，其靶向受影响的mRNA以通过无义介导的RNA衰变（NMD）降解，从而限制了肿瘤特异性InDel新表位的合成和HLA I类介导的表现。我们认为NMD抑制剂5-氮杂胞苷（5AZA）可以用于增加NMD敏感的新表位的表达，并使用蛋白质组学方法分析了MSI HCT-116细胞的免疫肽组。免疫沉淀HLA：肽复合物后，我们通过LC-MS / MS鉴定了10,000多种HLA I类呈递的肽，包括5个InDel新表位。在基因组，转录组和肽组水平上验证了InDel新表位。5AZA处理可增加相应的移码和带有提前终止密码子的mRNA的表达，并增强源自已知NMD靶标​​和已鉴定的InDel新表位之一的肽的呈递。通过分析一系列MSI大肠癌细胞系和患者样品，我们发现潜在的移码突变是高度复发的，并在HLA-A * 02：01转基因小鼠模型中用相应的新表位诱导的强CD8 + T细胞应答进行免疫。我们的数据直接表明，由于失配修复缺陷型细胞中InDel突变而导致的移码开放阅读框的肽段通过HLA I类呈现在细胞表面。