Arginine deprivation enriches lung cancer proteomes with cysteine by inducing arginine-to-cysteine substitutants

Many types of human cancers suppress the expression of argininosuccinate synthase 1 (ASS1), a rate-limiting enzyme for arginine production. Although dependency on exogenous arginine can be harnessed by arginine-deprivation therapies, the impact of ASS1 suppression on the quality of the tumor proteome is unknown. We therefore interrogated proteomes of cancer patients for arginine codon reassignments (substitutants) and surprisingly identified a strong enrichment for cysteine (R&gt;C) in lung tumors specifically. Most R&gt;C events did not coincide with genetically encoded R&gt;C mutations but were likely products of tRNA misalignments. The expression of R&gt;C substitutants was highly associated with oncogenic kelch-like epichlorohydrin (ECH)-associated protein 1 (KEAP1)-pathway mutations and suppressed by intact-KEAP1 in KEAP1-mutated cancer cells. Finally, functional interrogation indicated a key role for R&gt;C substitutants in cell survival to cisplatin, suggesting that regulatory codon reassignments endow cancer cells with more resilience to stress. Thus, we present a mechanism for enriching lung cancer proteomes with cysteines that may affect therapeutic decisions.</p

Arginine deprivation enriches lung cancer proteomes with cysteine by inducing arginine-to-cysteine substitutants

Many types of human cancers suppress the expression of argininosuccinate synthase 1 (ASS1), a rate-limiting enzyme for arginine production. Although dependency on exogenous arginine can be harnessed by arginine-deprivation therapies, the impact of ASS1 suppression on the quality of the tumor proteome is unknown. We therefore interrogated proteomes of cancer patients for arginine codon reassignments (substitutants) and surprisingly identified a strong enrichment for cysteine (R&gt;C) in lung tumors specifically. Most R&gt;C events did not coincide with genetically encoded R&gt;C mutations but were likely products of tRNA misalignments. The expression of R&gt;C substitutants was highly associated with oncogenic kelch-like epichlorohydrin (ECH)-associated protein 1 (KEAP1)-pathway mutations and suppressed by intact-KEAP1 in KEAP1-mutated cancer cells. Finally, functional interrogation indicated a key role for R&gt;C substitutants in cell survival to cisplatin, suggesting that regulatory codon reassignments endow cancer cells with more resilience to stress. Thus, we present a mechanism for enriching lung cancer proteomes with cysteines that may affect therapeutic decisions.</p

Arginine deprivation enriches lung cancer proteomes with cysteine by inducing arginine-to-cysteine substitutants

Many types of human cancers suppress the expression of argininosuccinate synthase 1 (ASS1), a rate-limiting enzyme for arginine production. Although dependency on exogenous arginine can be harnessed by arginine-deprivation therapies, the impact of ASS1 suppression on the quality of the tumor proteome is unknown. We therefore interrogated proteomes of cancer patients for arginine codon reassignments (substitutants) and surprisingly identified a strong enrichment for cysteine (R>C) in lung tumors specifically. Most R>C events did not coincide with genetically encoded R>C mutations but were likely products of tRNA misalignments. The expression of R>C substitutants was highly associated with oncogenic kelch-like epichlorohydrin (ECH)-associated protein 1 (KEAP1)-pathway mutations and suppressed by intact-KEAP1 in KEAP1-mutated cancer cells. Finally, functional interrogation indicated a key role for R>C substitutants in cell survival to cisplatin, suggesting that regulatory codon reassignments endow cancer cells with more resilience to stress. Thus, we present a mechanism for enriching lung cancer proteomes with cysteines that may affect therapeutic decisions

Tryptophan depletion results in tryptophan-to-phenylalanine substitutants

Activated T cells secrete interferon-γ, which triggers intracellular tryptophan shortage by upregulating the indoleamine 2,3-dioxygenase 1 (IDO1) enzyme1–4. Here we show that despite tryptophan depletion, in-frame protein synthesis continues across tryptophan codons. We identified tryptophan-to-phenylalanine codon reassignment (W>F) as the major event facilitating this process, and pinpointed tryptophanyl-tRNA synthetase (WARS1) as its source. We call these W>F peptides ‘substitutants’ to distinguish them from genetically encoded mutants. Using large-scale proteomics analyses, we demonstrate W>F substitutants to be highly abundant in multiple cancer types. W>F substitutants were enriched in tumours relative to matching adjacent normal tissues, and were associated with increased IDO1 expression, oncogenic signalling and the tumour-immune microenvironment. Functionally, W>F substitutants can impair protein activity, but also expand the landscape of antigens presented at the cell surface to activate T cell responses. Thus, substitutants are generated by an alternative decoding mechanism with potential effects on gene function and tumour immunoreactivity

ABPEPserver: a web application for documentation and analysis of substitutants

Abstract Background Cancer immunotherapy is implemented by identifying antigens that are presented on the cell surface of cancer cells and illicit T-cell response (Schumacher and Schreiber, Science 348:69–74, 2015; Waldman et al., Nat Rev Immunol 20:651–668, 2020; Zhang et al., Front Immunol 12:672,356, 2021b). Classical candidates of such antigens are the peptides resulting from genetic alterations and are named “neoantigen" (Schumacher and Schreiber, Science 348:69–74, 2015). Neoantigens have been widely catalogued across several human cancer types (Tan et al., Database (Oxford) 2020;2020b; Vigneron et al., Cancer Immun 13:15, 2013; Yi et al., iScience 24:103,107, 2021; Zhang et al., BMC Bioinformatics 22:40, 2021a). Recently, a new class of inducible antigens has been identified, namely Substitutants, that are produced as a result of aberrant protein translation (Pataskar et al., Nature 603:721–727, 2022). Main Catalogues of Substitutant expression across human cancer types, their specificity and association to gene expression signatures remain elusive for the scientific community's access. As a solution, we present ABPEPserver, an online database and analytical platform that can visualize a large-scale tumour proteomics analysis of Substitutant expression across eight tumour types sourced from the CPTAC database (Edwards et al., J Proteome Res 14:2707–2713, 2015). Functionally, ABPEPserver offers the analysis of gene-association signatures of Substitutant peptides, a comparison of enrichment between tumour and tumour-adjacent normal tissues, and a list of peptides that serve as candidates for immunotherapy design. ABPEPserver will significantly enhance the exploration of aberrant protein production in human cancer, as exemplified in a case study. Conclusion ABPEPserver is designed on an R SHINY platform to catalogue Substitutant peptides in human cancer. The application is available at https://rhpc.nki.nl/sites/shiny/ABPEP/ . The code is available under GNU General public license from GitHub ( https://github.com/jasminesmn/ABPEPserver )

Tryptophan depletion results in tryptophan-to-phenylalanine substitutants

Activated T cells secrete interferon-γ, which triggers intracellular tryptophan shortage by upregulating the indoleamine 2,3-dioxygenase 1 (IDO1) enzyme1–4. Here we show that despite tryptophan depletion, in-frame protein synthesis continues across tryptophan codons. We identified tryptophan-to-phenylalanine codon reassignment (W>F) as the major event facilitating this process, and pinpointed tryptophanyl-tRNA synthetase (WARS1) as its source. We call these W>F peptides ‘substitutants’ to distinguish them from genetically encoded mutants. Using large-scale proteomics analyses, we demonstrate W>F substitutants to be highly abundant in multiple cancer types. W>F substitutants were enriched in tumours relative to matching adjacent normal tissues, and were associated with increased IDO1 expression, oncogenic signalling and the tumour-immune microenvironment. Functionally, W>F substitutants can impair protein activity, but also expand the landscape of antigens presented at the cell surface to activate T cell responses. Thus, substitutants are generated by an alternative decoding mechanism with potential effects on gene function and tumour immunoreactivity