PO.ET02.09 · 实验与分子治疗
治疗和恶性进展重塑剪接图谱，在IDH突变型胶质瘤中产生共享的、肿瘤全域的新抗原
Therapy and malignant progression reshape the splicing landscape to generate shared, tumor-wide neoantigens in IDH-mutant gliomas
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摘要 Abstract
中文摘要
背景：胶质瘤的低突变负荷和高异质性限制了免疫治疗的疗效。异常RNA剪接可产生可靶向的新连接（NJ）（Kwok等，2025），然而治疗和恶性转化对这一图谱的影响仍不明确。我们在IDH突变型胶质瘤中研究这些效应，以鉴定保守、上调且潜在可靶向的剪接来源新抗原。
方法：来自原发和复发IDH突变型胶质瘤的配对bulk RNA-seq数据取自UCSF脑肿瘤中心的患者数据和AG-120处理的胶质瘤细胞系数据（Wu等，Science 2025）。使用SSNIP流程（Kwok等，Nature 2025）表征NJ及其后续肽序列。使用HLAthena、MHCflurry、NetMHCpan、MUNIS和HLApollo在五个HLA-I等位基因（A0101、A0201、A0301、A1101、A2402）中对高置信度n-mer序列进行优先排序。在≥3个平台上得分位于前1个百分位的肽通过FPKM×剪接读段频率进行筛选。使用DESeq2和GSEA进行差异基因表达分析。NJ位点附近的公开可用eCLIP测序数据鉴定潜在的顺式结合RNA结合调控因子。
结果：在接受标准放化疗的患者中（n=90），57,400个NJ中的1,806个（3.1%）显著上调（log₂[JPM]>1；p<0.05）。在AG120处理的IDH突变型胶质瘤细胞系中，5,520个NJ中的90个（1.6%）相对未处理对照显著上调。在配对样本中，NJ表达在复发时增加（p=0.033），而AG120处理的细胞系中不存在此趋势（p=0.7）。NJ升高主要与分级进展相关，差异基因表达分析显示内含子识别/排除通路富集。对放化疗队列的进一步分析鉴定出BCAN、PTPRZ1和EEF1A1的NJ来源靶点，分别为最佳HLA-A*11:01、HLA-A*24:02、HLA-A*11:01候选。值得注意的是，PTPRZ1和EEF1A1的NJ在瘤内保守，且比Kwok等报告的免疫原性GNAS NJ更为丰富。两个上调的AG120 NJ与放化疗队列的NJ重叠并生成蛋白。这两个NJ分别从S100A13和RANBP2生成独特的癌症9-mer，它们反复表达并被强烈预测可结合HLA-A*02:01。前沿分析显示IGF2BP3上调，eCLIP证实S100A13和RANBP2 NJ位点附近存在结合位点。HepG2细胞中IGF2BP3敲低导致独特的剪接改变而无整体缺失，而敲低后S100A13 NJ表达显著降低。
结论：我们表明治疗和恶性进展通过改变剪接调控因子表达重塑IDH突变型胶质瘤的剪接图谱。此外，这种重塑产生了保守的、肿瘤全域的、推测具有免疫原性的NJ。
查看英文原文 English abstract
Background: The low mutational burden and high heterogeneity of gliomas limit the efficacy of immunotherapy. Aberrant RNA splicing can generate targetable neojunctions (NJs) (Kwok et al., 2025), yet the impact of therapy and malignant transformation on this landscape remains unclear. We investigate these effects in IDH-mutant gliomas to identify conserved, upregulated, and potentially targetable splice-derived neoantigens.
Methods: Paired bulk RNA-seq data from primary and recurrent IDH-mutant gliomas were obtained from the UCSF Brain Tumor Center patient data and AG-120-treated glioma cell line data (Wu et al., Science 2025). NJs and their subsequent peptide sequences were characterized using the SSNIP pipeline (Kwok et al., Nature 2025). High confidence n-mer sequences were prioritized using HLAthena, MHCflurry, NetMHCpan, MUNIS, and HLApollo across five HLA-I alleles (A0101, A0201, A0301, A1101, A2402). Peptides scoring within the top first percentile on ≥ 3 platforms were filtered by FPKM × splice read frequency. DESeq2 and GSEA were used for differential gene expression analyses. Publicly available eCLIP sequencing near NJ sites identified potential cis-binding RNA-binding regulators.
Results: In patients treated with standard chemoradiation (n = 90), 1,806 of 57,400 NJs (3.1%) were significantly upregulated (log₂[JPM] > 1; p < 0.05). In AG120-treated IDH-mutant glioma cell lines, 90 of 5,520 NJs (1.6%) were significantly upregulated versus non-treated controls. Across paired samples, NJ expression increased at recurrence (p = 0.033), a trend absent with AG120-treated cell lines (p = 0.7). NJ elevation was primarily associated with grade progression and differential gene expression analysis showed enrichment of intron-recognition/exclusion pathways. Further analysis of the chemoradiation cohort identified BCAN, PTPRZ1 and EEF1A1 NJ-derived targets as top HLA-A*11:01, HLA-A*24:02, HLA-A*11:01 candidates, respectively. Notably, the PTPRZ1 and EEF1A1 NJs were intratumorally conserved, and were more abundant than the immunogenic GNAS NJ reported by Kwok et al. Two upregulated AG120 NJs overlapped with those from the chemoradiation cohort and generated proteins. These two NJs generated distinct cancer 9-mers from S100A13 and RANBP2 , which were recurrently expressed and strongly predicted to bind HLA-A*02:01. Leading-edge analysis showed IGF2BP3 upregulation, and eCLIP confirmed binding sites near the S100A13 and RANBP2 NJ sites. IGF2BP3 knockdown in HepG2 cells caused distinct splicing alterations without global loss, while S100A13 NJ expression decreased significantly post-KD.
Conclusion: We show that therapy and malignant progression remodel the splicing landscape of IDH-mutant gliomas via altered splicing regulator expression. Moreover, this remodeling produces conserved, tumor-wide, and putatively immunogenic NJs.
利益披露 Disclosure
T. Wu, None.