Non-canonical Peptides Research Articles

HPLC for at-line reaction monitoring and purification improves yield and purity of tRNA.

Engineered transfer RNA is an emerging therapeutic modality, particularly suited to treatment of diseases caused by genetic disorders based on premature termination codons, frameshifts, or missense mutations. It is also extensively used in reprogramming of in vitro translation systems to generate non-canonical amino acid-containing proteins and peptides, such as in mRNA display. Due to its length, chemical synthesis of tRNA is challenging and production of engineered tRNA at scale is currently limited to in vitro transcription from a DNA template. Previously, the highest reported in vitro transcription yield was 2.5 g/L, significantly below the industry standard for mRNA production of 7-10 g/L. To improve this process, we implemented monitoring of nucleoside triphosphate consumption and tRNA production during in vitro transcription, using at-line high-performance liquid chromatography, with a monolithic solid phase. This allowed for optimization of nucleoside triphosphate concentration, reduction of the in vitro transcription time to <4 h, and improvement of yield up to 4.7 g/L. A step-elution purification on a DEAE chromatographic monolith with >90% step yield was then developed. These improvements in the production and purification of tRNA represent an important step in facilitating production of tRNA for research purposes, and provide a method for purification of therapeutic tRNAs that is scalable and compatible with Good Manufacturing Practice requirements for clinical production.

MoPepGen: Rapid and Comprehensive Identification of Non-canonical Peptides.

Gene expression is a multi-step transformation of biological information from its storage form (DNA) into functional forms (protein and some RNAs). Regulatory activities at each step of this transformation multiply a single gene into a myriad of proteoforms. Proteogenomics is the study of how genomic and transcriptomic variation creates this proteomic diversity, and is limited by the challenges of modeling the complexities of gene-expression. We therefore created moPepGen, a graph-based algorithm that comprehensively generates non-canonical peptides in linear time. moPepGen works with multiple technologies, in multiple species and on all types of genetic and transcriptomic data. In human cancer proteomes, it enumerates previously unobservable noncanonical peptides arising from germline and somatic genomic variants, noncoding open reading frames, RNA fusions and RNA circularization. By enabling efficient detection and quantitation of previously hidden proteins in both existing and new proteomic data, moPepGen facilitates all proteogenomics applications. It is available at: https://github.com/uclahs-cds/package-moPepGen.

MHC-I-presented non-canonical antigens expand the cancer immunotherapy targets in acute myeloid leukemia

Identification of tumor neoantigens is indispensable for the development of cancer immunotherapies. However, we are still lacking knowledge about the potential neoantigens derived from sequences outside protein-coding regions. Here, we comprehensively characterized the immunopeptidome landscape by integrating multi-omics data in acute myeloid leukemia (AML). Both canonical and non-canonical MHC-associated peptides (MAPs) in AML were identified. We found that the quality and characteristics of ncMAPs are comparable or superior to cMAPs, suggesting ncMAPs are indispensable sources for tumor neoantigens. We further proposed a computational framework to prioritize the neoantigens by integrating additional transcriptome and immunopeptidome in normal tissues. Notably, 6 of prioritized 13 neoantigens were derived from ncMAPs. The expressions of corresponding source genes are highly related to infiltrations of immune cells. Finally, a risk model was developed, which exhibited good performance for clinical prognosis in AML. Our findings expand potential cancer immunotherapy targets and provide in-depth insights into AML treatment, laying a new foundation for precision therapies in AML.

DIPG-58. TARGETING DISORDERED DNA METHYLATION IN DIPG TO CONSTRAIN VARIABILITY AND INDUCE IMMUNE SIGNALING

Abstract BACKGROUND Diffuse Intrinsic Pontine Glioma (DIPG) is a universally fatal pediatric brain cancer characterized by the histone H3 K27M mutation. The known downstream consequences of this mutation include loss of repressive chromatin marks, global DNA hypomethylation, and altered gene expression. METHODS We sought to investigate the role of epigenetic variability as a basis of DIPG cellular heterogeneity and plasticity by using Whole Genome Bisulfite Sequencing (WGBS). Existing methods of analysis of WGBS are limited in that they average/smooth methylation data and fail to capture intrinsic variability. We performed (WGBS) on 23 primary patient samples of DIPG and applied an analysis that captures the variability of DNA methylation reads to characterize methylation stochasticity. We then sought to modulate the epigenome pharmacologically using a DNA methyltransferase inhibitor and assessed changes through RNA-Seq and immunopeptidomics. RESULTS We find that DIPG has a marked increase in methylation stochasticity (quantified as methylation entropy). Additionally, while DIPG is globally hypomethylated, it retains DNA methylation at specific regulatory regions, such as bivalent promoters, and key genes, such as FOXG1, CXCR4, and KLF4. We sought to reverse these changes using the DNA methyltransferase inhibitor, decitabine, in 4 DIPG neurosphere cell lines. Treatment with decitabine reversed the entropy seen in DIPG and induced innate immune signaling and interferon signaling in DIPG cells. Using RNA-seq, we found that decitabine treatment dramatically alters gene expression, including activation of endogenous retroviral elements, activation of interferon signaling, de-repression of hypermethylated targets, and expression of putative neoantigens, such as PRAME and DAZL. Immunopeptidomics profiling of MHC Class I bound canonical and non-canonical peptides of DIPG cell lines after decitabine treatment also revealed decitabine-induced differences. CONCLUSIONS Taken together, our study finds that the methylome of DIPG is highly disordered but is also highly responsive to pharmacologic modulation by hypomethylating agents. Furthermore, hypomethylating drugs can increase the immunogenicity of DIPG, thus offering the potential for future combination with immunotherapy.

NCPbook: A comprehensive database of noncanonical peptides.

Noncanonical peptides (NCPs) are a class of peptides generated from regions previously thought of as noncoding, such as introns, 5' UTRs, 3' UTRs, and intergenic regions. In recent years, the significance and diverse functions of NCPs have come to light, yet a systematic and comprehensive NCP database remains absent. Here, we developed NCPbook (https://ncp.wiki/ncpbook/), a database of evidence-supported NCPs, which aims to provide a resource for efficient exploration, analysis, and manipulation of NCPs. NCPbook incorporates data from diverse public databases and scientific literature. The current version of NCPbook includes 180,676 NCPs across 29 different species, evidenced by MS, ribosome profiling, or molecular experiments. These NCPs are distributed across kingdoms, comprising 123,408 from 14 plant species, 56,999 from 7 animal species, and 269 from 8 microbial species. Furthermore, NCPbook encompasses 9,166 functionally characterized NCPs playing important roles in immunity, stress resistance, growth, and development. Equipped with a user-friendly interface, NCPbook allows users to search, browse, visualize, and retrieve data, making it an indispensable platform for researching NCPs in various plant, animal, and microbial species.

Abstract CT112: An off-the-shelf multivalent vaccine containing cancer’s dark matter, DPV-001, combined with PD-1 +/- GITR in head &amp; neck cancer: safety, efficacy, and immunodynamics from the phase 1 GITRVax trial

Abstract Background: Immune responses to shared cancer antigens have correlated with durable clinical responses. DPV-001 is a novel microvesicle vaccine that contains &amp;gt;300 proteins for genes commonly overexpressed by cancer and is enriched for short-lived proteins (SLiPs), and defective ribosomal products (DRiPs), including non-canonical peptides, representing potential shared non-mutated alternative cancer neoantigens (dark matter). Preclinical studies identified increased therapeutic efficacy when this vaccine strategy was combined with anti-PD-1 and anti-GITR, leading to this clinical trial for patients with advanced or metastatic HNSCC. Methods: Following safety run-in, eligible pts were randomly assigned 1:1 to receive DPV-001 +/- GITR agonist mAb (INCAGN-1949; q2wks). All received sequenced PD-1 mAb (retifanlimab; q4wks) starting D15. Safety was evaluated as the primary endpoint and efficacy was evaluated as a secondary endpoint. Tumor biopsies were taken pre-treatment, Wk 2 and 8, and assessed by CITE-seq, scRNA-seq, BCR-seq, and TCR-seq. Multiplex immunofluoresence was performed on biopsies. Blood samples were taken pre-treatment and at multiple timepoints and analyzed by flow cytometry and seromics. Results: 18 pts received backbone therapy of DPV-001 + sequenced PD-1; of these, 9 pts also received GITR starting D1 (Arm 2). G3 irAE's included: hypothyroidism &amp; mucositis (Arm 1, same pt); pneumonitis and adrenal insufficiency (Arm 2, separate pts). No G4 toxicity was observed. Combining both arms, the response rate (RR) for PD-1 naïve pts was 55% (5/9), and 33% (3/9) for PD-1 experienced pts. Two CR's have been observed in Arm 2. Durability of response (DOR) and progression-free survival (PFS) will be reported at the meeting. DPV-001 induced increased Ki67+ CD4 EM (p&amp;lt;0.042) by D15, prior to first dose of PD-1. Addition of GITR at D1 further increased Ki67+ CD4 (p=0.008) &amp; CD8 EM cells (P=0.0006) by D15. Activated or proliferating CD4 and CD8 EM were significantly increased in both arms by D30 (p&amp;lt;0.025). Across above specified analyses, pts receiving GITR had the greatest increase. Evaluable pts had significantly (all p&amp;lt;0.032) increased numbers of TIL expressing IFN-γ and/or GZMB, LAG-3, and TIM3 in on-treatment biopsies. TCR, BCR, and seromic analyses, as well as studies to identify targets of anti-cancer immunity, are ongoing. Conclusions: This 18 pt trial of DPV-001 and sequenced PD-1 +/- GITR shows a promising RR and evidence of increased activation and expansion of effector T cells in PBL and tumor. Upregulation of LAG-3 and TIM3 by T cells that infiltrate the tumor and have increased in number, provide a rationale for including inhibitors for both in this treatment strategy. Current efforts include evaluating whether immune responses target shared non-canonical alternative neoantigens, or Dark Matter, contained in DPV-001, and whether synchronized antibody and cellular response is evidenced. Acknowledgements: Incyte, Providence Portland Medical Foundation, Steve and Cindy Harder, Trial Registry: NCT04470024, Ethics Approval: PSJH IRB# 2020000480 Citation Format: Rom S. Leidner, Christopher Paustian, Shawn M. Jensen, Tarsem Moudgil, Yoshinobu Koguchi, Noah Simons, Venkatesh Rajamanickam, Ryan Meng, Marcus A. Couey, Quyen Vu, Matthew H. Taylor, Anne Long, Abigail J. Staeck, Lakhvir Kaur, Tijana Jovanovic, Brady Bernard, Tanisha Christie, Noriko Iwamoto, Yuriko Minegishi, Koji Ueda, William Redmond, Lessli M. Rushforth, Takashi Shimada, Patrick Rethwisch, Ashish Patel, Richard B. Bell, Carlo B. Bifulco, Traci L. Hilton, Hong-Ming Hu, Brian Piening, Walter J. Urba, Bernard A. Fox. An off-the-shelf multivalent vaccine containing cancer’s dark matter, DPV-001, combined with PD-1 +/- GITR in head &amp; neck cancer: safety, efficacy, and immunodynamics from the phase 1 GITRVax trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT112.

Immunopeptidomics-based identification of naturally presented non-canonical circRNA-derived peptides

Circular RNAs (circRNAs) are covalently closed non-coding RNAs lacking the 5’ cap and the poly-A tail. Nevertheless, it has been demonstrated that certain circRNAs can undergo active translation. Therefore, aberrantly expressed circRNAs in human cancers could be an unexplored source of tumor-specific antigens, potentially mediating anti-tumor T cell responses. This study presents an immunopeptidomics workflow with a specific focus on generating a circRNA-specific protein fasta reference. The main goal of this workflow is to streamline the process of identifying and validating human leukocyte antigen (HLA) bound peptides potentially originating from circRNAs. We increase the analytical stringency of our workflow by retaining peptides identified independently by two mass spectrometry search engines and/or by applying a group-specific FDR for canonical-derived and circRNA-derived peptides. A subset of circRNA-derived peptides specifically encoded by the region spanning the back-splice junction (BSJ) are validated with targeted MS, and with direct Sanger sequencing of the respective source transcripts. Our workflow identifies 54 unique BSJ-spanning circRNA-derived peptides in the immunopeptidome of melanoma and lung cancer samples. Our approach enlarges the catalog of source proteins that can be explored for immunotherapy.

Widespread stable noncanonical peptides identified by integrated analyses of ribosome profiling and ORF features

Studies have revealed dozens of functional peptides in putative ‘noncoding’ regions and raised the question of how many proteins are encoded by noncanonical open reading frames (ORFs). Here, we comprehensively annotate genome-wide translated ORFs across five eukaryotes (human, mouse, zebrafish, worm, and yeast) by analyzing ribosome profiling data. We develop a logistic regression model named PepScore based on ORF features (expected length, encoded domain, and conservation) to calculate the probability that the encoded peptide is stable in humans. Systematic ectopic expression validates PepScore and shows that stable complex-associating microproteins can be encoded in 5’/3’ untranslated regions and overlapping coding regions of mRNAs besides annotated noncoding RNAs. Stable noncanonical proteins follow conventional rules and localize to different subcellular compartments. Inhibition of proteasomal/lysosomal degradation pathways can stabilize some peptides especially those with moderate PepScores, but cannot rescue the expression of short ones with low PepScores suggesting they are directly degraded by cellular proteases. The majority of human noncanonical peptides with high PepScores show longer lengths but low conservation across species/mammals, and hundreds contain trait-associated genetic variants. Our study presents a statistical framework to identify stable noncanonical peptides in the genome and provides a valuable resource for functional characterization of noncanonical translation during development and disease.

PXg: Comprehensive Identification of Noncanonical MHC-I–Associated Peptides From De Novo Peptide Sequencing Using RNA-Seq Reads

Discovering noncanonical peptides has been a common application of proteogenomics. Recent studies suggest that certain noncanonical peptides, known as noncanonical major histocompatibility complex-I (MHC-I)-associated peptides (ncMAPs), that bind to MHC-I may make good immunotherapeutic targets. De novo peptide sequencing is a great way to find ncMAPs since it can detect peptide sequences from their tandem mass spectra without using any sequence databases. However, this strategy has not been widely applied for ncMAP identification because there is not a good way to estimate its false-positive rates. In order to completely and accurately identify immunopeptides using de novo peptide sequencing, we describe a unique pipeline called proteomics X genomics. In contrast to current pipelines, it makes use of genomic data, RNA-Seq abundance and sequencing quality, in addition to proteomic features to increase the sensitivity and specificity of peptide identification. We show that the peptide-spectrum match quality and genetic traits have a clear relationship, showing that they can be utilized to evaluate peptide-spectrum matches. From 10 samples, we found 24,449 canonical MHC-I–associated peptides and 956 ncMAPs by using a target-decoy competition. Three hundred eighty-seven ncMAPs and 1611 canonical MHC-I–associated peptides were new identifications that had not yet been published. We discovered 11 ncMAPs produced from a squirrel monkey retrovirus in human cell lines in addition to the two ncMAPs originating from a complementarity determining region 3 in an antibody thanks to the unrestricted search space assumed by de novo sequencing. These entirely new identifications show that proteomics X genomics can make the most of de novo peptide sequencing’s advantages and its potential use in the search for new immunotherapeutic targets.

Comparative proteomic analysis of seed germination between allotetraploid cotton Gossypium hirsutum and Gossypium barbadense

Seed germination, a key initial event in the plant life cycle, directly affects cotton yield and quality. Gossypium barbadense and Gossypium hirsutum gradually evolved through polyploidization, resulting in different characteristics, and this interspecific variation lacks genetic and molecular explanation. This work aimed to compare the proteomes between G. barbadense and G. hirsutum during seed germination. Here, we identified 2740 proteins for G. barbadense and 3758 for G. hirsutum. In the initial state, proteins in two cotton involved similar bioprocess, such as sugar metabolism, DNA repairing, and ABA signaling pathway. However, in the post-germination stage, G. hirsutum expressed more protein related to redox homeostasis, peroxidase activity, and pathogen interactions. Analyzing the different expression patterns of 915 single-copy orthogroups between the two kinds of cotton indicated that most of the differentially expressed proteins in G. barbadense were related to carbon metabolism. In contrast, most proteins in G. hirsutum were associated with stress response. Besides that, by proteogenomic analysis, we found 349 putative non-canonical peptides, which may be involved in plant development. These results will help to understand the different characteristics of these two kinds of cotton, such as fiber quality, yield, and adaptability. Significance statementCotton is the predominant natural fiber crop worldwide; Gossypium barbadense and Gossypium hirsutum have evolved through polyploidization to produce differing traits. However, given their specific features, the divergence of mechanisms underlying seed germination between G. hirsutum and G. barbadense has not been discussed. Here, we explore what protein contributes to interspecific differences between G. barbadense and G. hirsutum during the seed germination period. This study helps to elucidate the evolution and domestication history of cotton polyploids and may allow breeders to understand their domestication history better and improve fiber quality and adaptability.

MARS an improved de novo peptide candidate selection method for non-canonical antigen target discovery in cancer

Understanding the nature and extent of non-canonical human leukocyte antigen (HLA) presentation in tumour cells is a priority for target antigen discovery for the development of next generation immunotherapies in cancer. We here employ a de novo mass spectrometric sequencing approach with a refined, MHC-centric analysis strategy to detect non-canonical MHC-associated peptides specific to cancer without any prior knowledge of the target sequence from genomic or RNA sequencing data. Our strategy integrates MHC binding rank, Average local confidence scores, and peptide Retention time prediction for improved de novo candidate Selection; culminating in the machine learning model MARS. We benchmark our model on a large synthetic peptide library dataset and reanalysis of a published dataset of high-quality non-canonical MHC-associated peptide identifications in human cancer. We achieve almost 2-fold improvement for high quality spectral assignments in comparison to de novo sequencing alone with an estimated accuracy of above 85.7% when integrated with a stepwise peptide sequence mapping strategy. Finally, we utilize MARS to detect and validate lncRNA-derived peptides in human cervical tumour resections, demonstrating its suitability to discover novel, immunogenic, non-canonical peptide sequences in primary tumour tissue.

Abstract A038: Non-canonical MHC class I-associated antigens in pancreatic cancer

Abstract Translation of the ostensibly non-coding genome in cancer can generate novel peptides capable of presentation by major histocompatibility complex class I (MHC-I, HLA-I in humans). These non-canonical peptide sources can broaden the landscape of potentially targetable antigens in low-to-intermediate mutational burden malignancies, typified by pancreatic ductal adenocarcinoma (PDA). However, non-canonical MHC-I-associated peptides (ncMAPs) have yet to be explored in pancreatic cancer. We purified the malignant compartment from low tumor cellularity PDA specimens using patient-derived organoids (PDOs) and developed a personalized proteogenomics pipeline coupled with high-depth immunopeptidomics to deeply characterize the repertoire of HLA:peptide targets presented specifically on malignant cells. We demonstrate that ncMAPs are abundant on PDA and predominate over mutation-derived neoepitopes in the detectable immunopeptidome. While emerging evidence in other tumor types has implicated dysregulated translation of non-canonical open reading frames (ncORFs), it is currently unknown to what extent these translation products are truly cancer-restricted and how effectively the resulting ncMAPs can elicit a cytolytic T lymphocyte (CTL) response. To investigate the cancer-specificity of non-canonical peptides, we developed a translation-centric analysis pipeline that examines ncORF expression across a range of healthy tissues, including healthy thymus. We provide evidence that ~30% of ncMAPs exhibit cancer-restricted translation patterns, and a substantial subset of these are shared among pancreatic cancer patients with the appropriate HLA. To investigate immunogenicity, we employed a highly sensitive ex vivo platform to prime and expand antigen-specific T cells. We demonstrate that PDA-restricted ncMAPs are highly immunogenic, on par with or even exceeding the immunogenicity observed with mutation-derived peptides or tumor-associated antigens assayed using the same platform. These findings uncover a critical role for dysregulated translation in pancreatic cancer as a potential source for recurrent cancer-restricted epitopes capable of recognition by cytotoxic T cells. We envision that this novel class of antigens will accelerate ongoing efforts to treat pancreatic cancer patients with vaccines and cell-based therapies. Citation Format: Zackery A. Ely, Zachary J. Kulstad, Eva K. Verzani, Jennifer G. Abelin, Sudarsana Addepalli, Karl R. Clauser, Marta Casarrubios, Kevin S. Kapner, Miles P. Agus, Connor J. Hennessey, Sine R. Hadrup, Susan Klaeger, Jennifer Su, Alex M. Jaeger, Brian M. Wolpin, Srivatsan Raghavan, Philip D. Greenberg, Andrew J. Aguirre, Steven A. Carr, Tyler Jacks, William A. Freed-Pastor. Non-canonical MHC class I-associated antigens in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A038.

Expedient and divergent synthesis of unnatural peptides through cobalt-catalyzed diastereoselective umpolung hydrogenation.

The development of a reliable method for asymmetric synthesis of unnatural peptides is highly desirable and particularly challenging. In this study, we present a versatile and efficient approach that uses cobalt-catalyzed diastereoselective umpolung hydrogenation to access noncanonical aryl alanine peptides. This protocol demonstrates good tolerance toward various functional groups, amino acid sequences, and peptide lengths. Moreover, the versatility of this reaction is illustrated by its successful application in the late-stage functionalization and formal synthesis of various representative chiral natural products and pharmaceutical scaffolds. This strategy eliminates the need for synthesizing chiral noncanonical aryl alanines before peptide formation, and the hydrogenation reaction does not result in racemization or epimerization. The underlying mechanism was extensively explored through deuterium labeling, control experiments, HRMS identification, and UV-Vis spectroscopy, which supported a reasonable CoI/CoIII catalytic cycle. Notably, acetic acid and methanol serve as safe and cost-effective hydrogen sources, while indium powder acts as the terminal electron source.

Abstract A076: Discovery and characterization of a novel, immunoproteasome activator that modulates the immunopeptidome, increases MHC class I antigenic presentation and enhances antitumor immunity

Abstract Evasion of immune destruction is a hallmark of cancer that thwarts the efficacy of immunotherapy and reduces patient benefit. The development of immunotherapies has sparked a revolution in oncology by releasing the power of the human immune system, but the identity of novel, actionable antigens exclusively presented on cancer cells remains elusive. Immune cells contain a highly specialized variant of proteasomes, known as immunoproteasomes, in which the three constitutive catalytic subunits are replaced by cytokine-inducible, homologous catalytic subunits. Immunoproteasomes are specially adapted for a role in MHC class I antigen processing and presentation to CD8+ T-cells. Here, we performed a high-through screen to detect novel molecular entities that activated proteasomal peptide-hydrolyzing activity. We identified a curated panel of molecules which not only enhanced proteasomal hydrolysis of the cell-permeable substrate LLVY-R110 but also enhanced the presentation of MHC class I antigens. E.G7-Ova is a mouse lymphoma cell line derived by electroporation of EL4 cells (C57BL/6, H-2b, T lymphoma) with chicken ovalbumin (OVA) cDNA. Proteasomes degrade OVA to generate the peptide SIINFEKL that is presented by MHC-class I molecules. Studies demonstrated that the hits identified in the screen increased SIINFEKL presentation on E.G7-Ova cells. The lead compound, compound A, dramatically enhanced the expression of the proteasome activator PA28a/b, expressed by PSME1/2. Treatment of multiple myeloma (MM) cells with compound A enhanced the hydrolysis of Ac-ANW-MCA, which is selectively cleaved by the immunoproteasome beta5-associated catalytic activity. Treatment of MM cells followed by mass spectrometry (MS) demonstrated that compound A modulated the relative presentation of individual antigenic peptides bound to MHC class I molecules on tumor cells. The identification of actionable tumor antigens is indispensable for the development of several cancer immunotherapies, including T cell receptor (TCR)–transduced T cells and patient-specific mRNA or peptide vaccines. MS-based immunopeptidomics enables the discovery of canonical and non-canonical antigens, i.e., antigens derived from sequences outside protein-coding regions or generated by non-canonical antigen-processing mechanisms. This method enables the rapid identification of non-canonical peptides, a fraction of which may be detected exclusively by cancer cells. Novel, immunoproteasome-selective therapeutics have the potential to contribute not only to CD8+ T-cell-mediated control of human cancers, but also intracellular infections, autoimmune diseases, neurodegenerative diseases and proteinopathies that are characterized by the pathologic accumulation of toxic, proteinaceous aggregates. Citation Format: James J. Driscoll, Priyanka S. Rana, James J. Ignatz-Hoover. Discovery and characterization of a novel, immunoproteasome activator that modulates the immunopeptidome, increases MHC class I antigenic presentation and enhances antitumor immunity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A076.

Nickel-Catalyzed Radical Mechanisms: Informing Cross-Coupling for Synthesizing Non-Canonical Biomolecules.

Nickel excels at facilitating selective radical chemistry, playing a pivotal role in metalloenzyme catalysis and modern cross-coupling reactions. Radicals, being nonpolar and neutral, exhibit orthogonal reactivity to nucleophilic and basic functional groups commonly present in biomolecules. Harnessing this compatibility, we delve into the application of nickel-catalyzed radical pathways in the synthesis of noncanonical peptides and carbohydrates, critical for chemical biology studies and drug discovery.We previously characterized a sequential reduction mechanism that accounts for chemoselectivity in cross-electrophile coupling reactions. This catalytic cycle begins with nickel(I)-mediated radical generation from alkyl halides, followed by carbon radical capture by nickel(II) complexes, and concludes with reductive elimination. These steps resonate with mechanistic proposals in nickel-catalyzed cross-coupling, photoredox, and electrocatalytic reactions. Herein, we present our insights into each step involving radicals, including initiation, propagation, termination, and the nuances of kinetics, origins of selectivity, and ligand effects.Radical generation from C(sp3) electrophiles via one-electron oxidative addition with low-valent nickel radical intermediates provides the basis for stereoconvergent and cross-electrophile couplings. Our electroanalytical studies elucidate a concerted halogen atom abstraction mechanism, where electron transfer is coupled with halide dissociation. Using this pathway, we have developed a nickel-catalyzed stereoselective radical addition to dehydroalanine, facilitating the synthesis of noncanonical peptides. In this application, chiral ligands modulate the stereochemical outcome through the asymmetric protonation of a nickel-enolate intermediate.The capture of the alkyl radical by nickel(II) expands the scope of cross-coupling, promotes reductive elimination through the formation of high-valent nickel(III) species, and governs chemo- and stereoselectivity. We discovered that nickel(II)-aryl efficiently traps radicals with a barrier ranging from 7 to 9 kcal/mol, followed by fast reductive elimination. In contrast, nickel(II)-alkyl captures radicals to form a nickel(III) species, which was characterized by EPR spectroscopy. However, the subsequent slow reductive elimination resulted in minimal product formation. The observed high diastereoselectivity of radical capture inspired investigations into C-aryl and C-acyl glycosylation reactions. We developed a redox auxiliary that readily couples with natural carbohydrates and produces glycosyl radicals upon photoredox activation. Nickel-catalyzed cross-coupling of the glycosyl radical with bromoarenes and carboxylic acids leads to diverse non-natural glycosides that can facilitate drug discovery.Stoichiometric studies on well-defined d8-nickel complexes have showcased means to promote reductive elimination, including ligand association, oxidation, and oxidative addition.In the final section, we address the influence of auxiliary ligands on the electronic structure and redox activity of organonickel intermediates. Synthesis of a series of low-valent nickel radical complexes and characterization of their electronic structures led us to a postulate that ligand redox activity correlates with coordination geometry. Our data reveal that a change in ligand redox activity can shift the redox potentials of reaction intermediates, potentially altering the mechanism of catalytic reactions. Moreover, coordinating additives and solvents may stabilize nickel radicals during catalysis by adjusting ligand redox activity, which is consistent with known catalytic conditions.

Proteogenomic analysis reveals RNA as a source for tumor-agnostic neoantigen identification

Systemic pan-tumor analyses may reveal the significance of common features implicated in cancer immunogenicity and patient survival. Here, we provide a comprehensive multi-omics data set for 32 patients across 25 tumor types for proteogenomic-based discovery of neoantigens. By using an optimized computational approach, we discover a large number of tumor-specific and tumor-associated antigens. To create a pipeline for the identification of neoantigens in our cohort, we combine DNA and RNA sequencing with MS-based immunopeptidomics of tumor specimens, followed by the assessment of their immunogenicity and an in-depth validation process. We detect a broad variety of non-canonical HLA-binding peptides in the majority of patients demonstrating partially immunogenicity. Our validation process allows for the selection of 32 potential neoantigen candidates. The majority of neoantigen candidates originates from variants identified in the RNA data set, illustrating the relevance of RNA as a still understudied source of cancer antigens. This study underlines the importance of RNA-centered variant detection for the identification of shared biomarkers and potentially relevant neoantigen candidates.

Synthesis of Fluorinated Non-Canonical Peptides from α-Fluorinated Carboxylic Acid

Key words fluorinated peptides - photochemistry - decarboxylation - α-fluorinated carboxylic acid

Glioblastoma stem cells express non-canonical proteins and exclusive mesenchymal-like or non-mesenchymal-like protein signatures.

Glioblastoma (GBM) cancer stem cells (GSCs) contribute to GBM's origin, recurrence, and resistance to treatment. However, the understanding of how mRNA expression patterns of GBM subtypes are reflected at global proteome level in GSCs is limited. To characterize protein expression in GSCs, we performed in-depth proteogenomic analysis of patient-derived GSCs by RNA-sequencing and mass-spectrometry. We quantified > 10 000 proteins in two independent GSC panels and propose a GSC-associated proteomic signature characterizing two distinct phenotypic conditions; one defined by proteins upregulated in proneural and classical GSCs (GPC-like), and another by proteins upregulated in mesenchymal GSCs (GM-like). The GM-like protein set in GBM tissue was associated with necrosis, recurrence, and worse overall survival. Through proteogenomics, we discovered 252 non-canonical peptides in the GSCs, i.e., protein sequences that are variant or derive from genome regions previously considered non-protein-coding, including variants of the heterogeneous ribonucleoproteins implicated in RNA splicing. In summary, GSCs express two protein sets that have an inverse association with clinical outcomes in GBM. The discovery of non-canonical protein sequences questions existing gene models and pinpoints new protein targets for research in GBM.

Mapping the structure–activity landscape of non-canonical peptides with MAP4 fingerprinting

Peptide structure–activity/property relationship (P-SA/PR) studies focus on understanding how the structural variations of peptides influence their biological activities and other functional properties.

Preparation of N-2-Nitrophenylsulfenyl Imino Peptides and Their Catalyst-Controlled Diastereoselective Indolylation.

N-2-Nitrophenylsulfenyl imino dipeptides bearing various functional groups were successfully prepared by MnO2 -mediated oxidation and then subjected to diastereoselective indolylation. Each diastereomer of the adduct was selectively obtained from the same substrates using the appropriate chiral phosphoric acid catalysts. These transformations would be useful for synthesizing non-canonical amino acid-containing peptides as novel drug candidates.