BACKGROUND Clear cell renal cell carcinoma (ccRCC) is the dominant subtype of renal cancer. With currently available therapies, cure of advanced and metastatic ccRCC is achieved only in rare cases. Here, we developed a workflow integrating different -omics technologies to identify ccRCC-specific HLA-presented peptides as potential drug targets for ccRCC immunotherapy. METHODS We analyzed HLA-presented peptides by MS-based ligandomics of 55 ccRCC tumors (cohort 1), paired non-tumor renal tissues, and 158 benign tissues from other organs. Pathways enriched in ccRCC compared to its cell type of origin were identified by transcriptome and gene set enrichment analyses in 51 tumor tissues of the same cohort. To retrieve a list of candidate targets with involvement in ccRCC pathogenesis, ccRCC-specific pathway genes were intersected with the source genes of tumor-exclusive peptides. The candidates were validated in an independent cohort from The Cancer Genome Atlas (TCGA KIRC, n = 452). DNA methylation (TCGA KIRC, n = 273), somatic mutations (TCGA KIRC, n = 392), and gene ontology (GO) and correlations with tumor metabolites (cohort 1, n = 30) and immune-oncological markers (cohort 1, n = 37) were analyzed to characterize regulatory and functional involvements. CD8+ T cell priming assays were used to identify immunogenic peptides. The candidate gene EGLN3 was functionally investigated in cell culture. RESULTS A total of 34,226 HLA class I- and 19,325 class II-presented peptides were identified in ccRCC tissue, of which 443 class I and 203 class II peptides were ccRCC-specific and presented in ≥ 3 tumors. One hundred eighty-five of the 499 corresponding source genes were involved in pathways activated by ccRCC tumors. After validation in the independent cohort from TCGA, 113 final candidate genes remained. Candidates were involved in extracellular matrix organization, hypoxic signaling, immune processes, and others. Nine of the 12 peptides assessed by immunogenicity analysis were able to activate naïve CD8+ T cells, including peptides derived from EGLN3. Functional analysis of EGLN3 revealed possible tumor-promoting functions. CONCLUSIONS Integration of HLA ligandomics, transcriptomics, genetic, and epigenetic data leads to the identification of novel functionally relevant therapeutic targets for ccRCC immunotherapy. Validation of the identified targets is recommended to expand the treatment landscape of ccRCC.

中文翻译：

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整合组学和HLA配体组学分析可确定ccRCC免疫疗法的新型药物靶标。

背景技术透明细胞肾细胞癌（ccRCC）是肾癌的主要亚型。使用当前可用的疗法，仅在极少数情况下才能治愈晚期和转移性ccRCC。在这里，我们开发了一个集成了不同组学技术的工作流程，以识别ccRCC特异性HLA呈递的肽作为ccRCC免疫疗法的潜在药物靶标。方法我们通过基于MS的配体组学分析了55个ccRCC肿瘤（队列1），成对的非肿瘤肾组织和其他器官的158个良性组织，通过HLA呈现的肽进行了分析。通过转录组和基因组富集分析，在同一队列的51个肿瘤组织中鉴定了ccRCC与来源细胞类型相比富集的途径。要检索参与ccRCC发病机制的候选靶点列表，ccRCC特定途径基因与肿瘤专用肽的源基因相交。候选人在来自癌症基因组图谱（TCGA KIRC，n = 452）的独立队列中得到验证。DNA甲基化（TCGA KIRC，n = 273），体细胞突变（TCGA KIRC，n = 392），基因本体论（GO）以及与肿瘤代谢产物的相关性（第1组，n = 30）和免疫肿瘤标记物（第1组） n = 37）进行了分析，以表征监管和功能参与。CD8 + T细胞启动试验用于鉴定免疫原性肽。在细胞培养中对候选基因EGLN3进行了功能研究。结果在ccRCC组织中共鉴定了34,226种HLA I类肽和19,325种II类肽，其中443种I类和203类II类肽是ccRCC特异的，并存在于≥3个肿瘤中。499个相应的源基因中有185个参与了ccRCC肿瘤激活的途径。在来自TCGA的独立队列中进行验证后，剩下113个最终候选基因。候选者参与细胞外基质的组织，低氧信号传导，免疫过程等。通过免疫原性分析评估的12种肽中有9种能够激活幼稚的CD8 + T细胞，包括衍生自EGLN3的肽。EGLN3的功能分析显示可能的肿瘤促进功能。结论HLA配体组学，转录组学，遗传和表观遗传学数据的整合导致了ccRCC免疫疗法的新型功能相关治疗靶标的鉴定。建议验证已确定的目标，以扩大ccRCC的治疗范围。