Integrative -omics and HLA-ligandomics analysis to identify novel drug targets for ccRCC immunotherapy

Abstract

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 eightyfive 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

RETRACTED ARTICLE: Downregulation of lncRNA ZNF582-AS1 due to DNA hypermethylation promotes clear cell renal cell carcinoma growth and metastasis by regulating the N(6)-methyladenosine modification of MT-RNR1

“…At present, radical surgical operation is the main treatment for patients with early ccRCC, and targeted therapy may prolong the survival time of patients with advanced or metastatic ccRCC [ 2 – 4 ]. Unfortunately, the prognosis of ccRCC patients is still poor, especially for patients with advanced and metastatic disease, the 5-year survival rate after diagnosis is only 12% [ 4 – 6 ]. Therefore, finding more effective and safer therapeutic targets has great potential value for improving the prognosis of ccRCC patients.…”

The Peptide Vaccine of the Future

“…A major drawback of such prediction-based approaches lies in the distorted correlation of gene expression with HLA-restricted antigen presentation, since the immunopeptidome is an independent complex layer formed by the antigen presentation machinery and therefore does not necessarily mirror the transcriptome or the proteome ( 21 , 32 , 33 , 34 , 35 ). In contrast to epitope prediction algorithms, the large-scale and in-depth direct identification of naturally HLA-presented peptides, known as HLA ligandome or immunopeptidome, from primary patient samples by mass spectrometry ( 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 ) is the only unbiased approach to comprehensively identify tumor rejection antigen candidates and validate the natural processing and presentation of these antigens ( 45 ). For the identification of tumor-associated unmutated self-peptides not presented on benign cells and tissues, the extensive screening of immunopeptidomes derived from benign tissues is of paramount importance.…”

The Origin and Immune Recognition of Tumor-Specific Antigens

“…The first approach postulates that a MAP is a TSA if it is found in cancer cells but not in an atlas of MAPs identified in normal tissue extracts [ 9 , 10 ]. The problem here is that this atlas does not contain the MAP repertoire of all cell types.…”