Development of mesothelioma-specific oncolytic immunotherapy enabled by immunopeptidomics of murine and human mesothelioma tumors.

Malignant pleural mesothelioma (MPM) is an aggressive tumor with a poor prognosis. As the available therapeutic options show a lack of efficacy, novel therapeutic strategies are urgently needed. Given its T-cell infiltration, we hypothesized that MPM is a suitable target for therapeutic cancer vaccination. To date, research on mesothelioma has focused on the identification of molecular signatures to better classify and characterize the disease, and little is known about therapeutic targets that engage cytotoxic (CD8+) T cells. In this study we investigate the immunopeptidomic antigen-presented landscape of MPM in both murine (AB12 cell line) and human cell lines (H28, MSTO-211H, H2452, and JL1), as well as in patients' primary tumors. Applying state-of-the-art immuno-affinity purification methodologies, we identify MHC I-restricted peptides presented on the surface of malignant cells. We characterize in vitro the immunogenicity profile of the eluted peptides using T cells from human healthy donors and cancer patients. Furthermore, we use the most promising peptides to formulate an oncolytic virus-based precision immunotherapy (PeptiCRAd) and test its efficacy in a mouse model of mesothelioma in female mice. Overall, we demonstrate that the use of immunopeptidomic analysis in combination with oncolytic immunotherapy represents a feasible and effective strategy to tackle untreatable tumors.

A novel immunopeptidomic-based pipeline for the generation of personalized oncolytic cancer vaccines.

Besides the isolation and identification of major histocompatibility complex I-restricted peptides from the surface of cancer cells, one of the challenges is eliciting an effective antitumor CD8+ T-cell-mediated response as part of therapeutic cancer vaccine. Therefore, the establishment of a solid pipeline for the downstream selection of clinically relevant peptides and the subsequent creation of therapeutic cancer vaccines are of utmost importance. Indeed, the use of peptides for eliciting specific antitumor adaptive immunity is hindered by two main limitations: the efficient selection of the most optimal candidate peptides and the use of a highly immunogenic platform to combine with the peptides to induce effective tumor-specific adaptive immune responses. Here, we describe for the first time a streamlined pipeline for the generation of personalized cancer vaccines starting from the isolation and selection of the most immunogenic peptide candidates expressed on the tumor cells and ending in the generation of efficient therapeutic oncolytic cancer vaccines. This immunopeptidomics-based pipeline was carefully validated in a murine colon tumor model CT26. Specifically, we used state-of-the-art immunoprecipitation and mass spectrometric methodologies to isolate >8000 peptide targets from the CT26 tumor cell line. The selection of the target candidates was then based on two separate approaches: RNAseq analysis and HEX software. The latter is a tool previously developed by Jacopo, 2020, able to identify tumor antigens similar to pathogen antigens in order to exploit molecular mimicry and tumor pathogen cross-reactive T cells in cancer vaccine development. The generated list of candidates (26 in total) was further tested in a functional characterization assay using interferon-γ enzyme-linked immunospot (ELISpot), reducing the number of candidates to six. These peptides were then tested in our previously described oncolytic cancer vaccine platform PeptiCRAd, a vaccine platform that combines an immunogenic oncolytic adenovirus (OAd) coated with tumor antigen peptides. In our work, PeptiCRAd was successfully used for the treatment of mice bearing CT26, controlling the primary malignant lesion and most importantly a secondary, nontreated, cancer lesion. These results confirmed the feasibility of applying the described pipeline for the selection of peptide candidates and generation of therapeutic oncolytic cancer vaccine, filling a gap in the field of cancer immunotherapy, and paving the way to translate our pipeline into human therapeutic approach.

Generation of ENSEMBL-based proteogenomics databases boosts the identification of non-canonical peptides.

SUMMARY: We have implemented the pypgatk package and the pgdb workflow to create proteogenomics databases based on ENSEMBL resources. The tools allow the generation of protein sequences from novel protein-coding transcripts by performing a three-frame translation of pseudogenes, lncRNAs and other non-canonical transcripts, such as those produced by alternative splicing events. It also includes exonic out-of-frame translation from otherwise canonical protein-coding mRNAs. Moreover, the tool enables the generation of variant protein sequences from multiple sources of genomic variants including COSMIC, cBioportal, gnomAD and mutations detected from sequencing of patient samples. pypgatk and pgdb provide multiple functionalities for database handling including optimized target/decoy generation by the algorithm DecoyPyrat. Finally, we have reanalyzed six public datasets in PRIDE by generating cell-type specific databases for 65 cell lines using the pypgatk and pgdb workflow, revealing a wealth of non-canonical or cryptic peptides amounting to >5% of the total number of peptides identified. AVAILABILITY AND IMPLEMENTATION: The software is freely available. pypgatk: https://github.com/bigbio/py-pgatk/ and pgdb: https://nf-co.re/pgdb. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Proteogenomics of non-small cell lung cancer reveals molecular subtypes associated with specific therapeutic targets and immune evasion mechanisms.

Despite major advancements in lung cancer treatment, long-term survival is still rare, and a deeper understanding of molecular phenotypes would allow the identification of specific cancer dependencies and immune evasion mechanisms. Here we performed in-depth mass spectrometry (MS)-based proteogenomic analysis of 141 tumors representing all major histologies of non-small cell lung cancer (NSCLC). We identified six distinct proteome subtypes with striking differences in immune cell composition and subtype-specific expression of immune checkpoints. Unexpectedly, high neoantigen burden was linked to global hypomethylation and complex neoantigens mapped to genomic regions, such as endogenous retroviral elements and introns, in immune-cold subtypes. Further, we linked immune evasion with LAG3 via STK11 mutation-dependent HNF1A activation and FGL1 expression. Finally, we develop a data-independent acquisition MS-based NSCLC subtype classification method, validate it in an independent cohort of 208 NSCLC cases and demonstrate its clinical utility by analyzing an additional cohort of 84 late-stage NSCLC biopsy samples.

PeptiCHIP: A Microfluidic Platform for Tumor Antigen Landscape Identification.

Identification of HLA class I ligands from the tumor surface (ligandome or immunopeptidome) is essential for designing T-cell mediated cancer therapeutic approaches. However, the sensitivity of the process for isolating MHC-I restricted tumor-specific peptides has been the major limiting factor for reliable tumor antigen characterization, making clear the need for technical improvement. Here, we describe our work from the fabrication and development of a microfluidic-based chip (PeptiCHIP) and its use to identify and characterize tumor-specific ligands on clinically relevant human samples. Specifically, we assessed the potential of immobilizing a pan-HLA antibody on solid surfaces via well-characterized streptavidin-biotin chemistry, overcoming the limitations of the cross-linking chemistry used to prepare the affinity matrix with the desired antibodies in the immunopeptidomics workflow. Furthermore, to address the restrictions related to the handling and the limited availability of tumor samples, we further developed the concept toward the implementation of a microfluidic through-flow system. Thus, the biotinylated pan-HLA antibody was immobilized on streptavidin-functionalized surfaces, and immune-affinity purification (IP) was carried out on customized microfluidic pillar arrays made of thiol-ene polymer. Compared to the standard methods reported in the field, our methodology reduces the amount of antibody and the time required for peptide isolation. In this work, we carefully examined the specificity and robustness of our customized technology for immunopeptidomics workflows. We tested this platform by immunopurifying HLA-I complexes from 1 × 106 cells both in a widely studied B-cell line and in patients-derived ex vivo cell cultures, instead of 5 × 108 cells as required in the current technology. After the final elution in mild acid, HLA-I-presented peptides were identified by tandem mass spectrometry and further investigated by in vitro methods. These results highlight the potential to exploit microfluidics-based strategies in immunopeptidomics platforms and in personalized immunopeptidome analysis from cells isolated from individual tumor biopsies to design tailored cancer therapeutic vaccines. Moreover, the possibility to integrate multiple identical units on a single chip further improves the throughput and multiplexing of these assays with a view to clinical needs.

Therapeutic Cancer Vaccination with Immunopeptidomics-Discovered Antigens Confers Protective Antitumor Efficacy.

Knowledge of clinically targetable tumor antigens is becoming vital for broader design and utility of therapeutic cancer vaccines. This information is obtained reliably by directly interrogating the MHC-I presented peptide ligands, the immunopeptidome, with state-of-the-art mass spectrometry. Our manuscript describes direct identification of novel tumor antigens for an aggressive triple-negative breast cancer model. Immunopeptidome profiling revealed 2481 unique antigens, among them a novel ERV antigen originating from an endogenous retrovirus element. The clinical benefit and tumor control potential of the identified tumor antigens and ERV antigen were studied in a preclinical model using two vaccine platforms and therapeutic settings. Prominent control of established tumors was achieved using an oncolytic adenovirus platform designed for flexible and specific tumor targeting, namely PeptiCRAd. Our study presents a pipeline integrating immunopeptidome analysis-driven antigen discovery with a therapeutic cancer vaccine platform for improved personalized oncolytic immunotherapy.

Charge Engineering Reveals the Roles of Ionizable Side Chains in Electrospray Ionization Mass Spectrometry.

In solution, the charge of a protein is intricately linked to its stability, but electrospray ionization distorts this connection, potentially limiting the ability of native mass spectrometry to inform about protein structure and dynamics. How the behavior of intact proteins in the gas phase depends on the presence and distribution of ionizable surface residues has been difficult to answer because multiple chargeable sites are present in virtually all proteins. Turning to protein engineering, we show that ionizable side chains are completely dispensable for charging under native conditions, but if present, they are preferential protonation sites. The absence of ionizable side chains results in identical charge state distributions under native-like and denaturing conditions, while coexisting conformers can be distinguished using ion mobility separation. An excess of ionizable side chains, on the other hand, effectively modulates protein ion stability. In fact, moving a single ionizable group can dramatically alter the gas-phase conformation of a protein ion. We conclude that although the sum of the charges is governed solely by Coulombic terms, their locations affect the stability of the protein in the gas phase.

Immediate Adaptation Analysis Implicates BCL6 as an EGFR-TKI Combination Therapy Target in NSCLC.

Drug resistance is a major obstacle to curative cancer therapies, and increased understanding of the molecular events contributing to resistance would enable better prediction of therapy response, as well as contribute to new targets for combination therapy. Here we have analyzed the early molecular response to epidermal growth factor receptor (EGFR) inhibition using RNA sequencing data covering 13,486 genes and mass spectrometry data covering 10,138 proteins. This analysis revealed a massive response to EGFR inhibition already within the first 24 h, including significant regulation of hundreds of genes known to control downstream signaling, such as transcription factors, kinases, phosphatases and ubiquitin E3-ligases. Importantly, this response included upregulation of key genes in multiple oncogenic signaling pathways that promote proliferation and survival, such as ERBB3, FGFR2, JAK3, and BCL6, indicating an early adaptive response to EGFR inhibition. Using a library of more than 500 approved and experimental compounds in a combination therapy screen, we could show that several kinase inhibitors with targets including JAK3 and FGFR2 increased the response to EGFR inhibitors. Further, we investigated the functional impact of BCL6 upregulation in response to EGFR inhibition using siRNA-based silencing of BCL6. Proteomics profiling revealed that BCL6 inhibited transcription of multiple target genes including p53, resulting in reduced apoptosis which implicates BCL6 upregulation as a new EGFR inhibitor treatment escape mechanism. Finally, we demonstrate that combined treatment targeting both EGFR and BCL6 act synergistically in killing lung cancer cells. In conclusion, or data indicates that multiple different adaptive mechanisms may act in concert to blunt the cellular impact of EGFR inhibition, and we suggest BCL6 as a potential target for EGFR inhibitor-based combination therapy.

Key Structural Motifs Balance Metal Binding and Oxidative Reactivity in a Heterobimetallic Mn/Fe Protein.

Heterobimetallic Mn/Fe proteins represent a new cofactor paradigm in bioinorganic chemistry and pose countless outstanding questions. The assembly of the active site defies common chemical convention by contradicting the Irving-Williams series, while the scope of reactivity remains unexplored. In this work, the assembly and C-H bond activation process in the Mn/Fe R2-like ligand-binding oxidase (R2lox) protein is investigated using a suite of biophysical techniques, including time-resolved optical spectroscopy, global kinetic modeling, X-ray crystallography, electron paramagnetic resonance spectroscopy, protein electrochemistry, and mass spectrometry. Selective metal binding is found to be under thermodynamic control, with the binding sites within the apo-protein exhibiting greater MnII affinity than FeII affinity. The comprehensive analysis of structure and reactivity of wild-type R2lox and targeted primary and secondary sphere mutants indicate that the efficiency of C-H bond activation directly correlates with the Mn/Fe cofactor reduction potentials and is inversely related to divalent metal binding affinity. These findings suggest the R2lox active site is precisely tuned for achieving both selective heterobimetallic binding and high levels of reactivity and offer a mechanism to examine the means by which proteins achieve appropriate metal incorporation.

Ribonucleotide reductase inhibitors suppress SAMHD1 ara-CTPase activity enhancing cytarabine efficacy.

The deoxycytidine analogue cytarabine (ara-C) remains the backbone treatment of acute myeloid leukaemia (AML) as well as other haematological and lymphoid malignancies, but must be combined with other chemotherapeutics to achieve cure. Yet, the underlying mechanism dictating synergistic efficacy of combination chemotherapy remains largely unknown. The dNTPase SAMHD1, which regulates dNTP homoeostasis antagonistically to ribonucleotide reductase (RNR), limits ara-C efficacy by hydrolysing the active triphosphate metabolite ara-CTP. Here, we report that clinically used inhibitors of RNR, such as gemcitabine and hydroxyurea, overcome the SAMHD1-mediated barrier to ara-C efficacy in primary blasts and mouse models of AML, displaying SAMHD1-dependent synergy with ara-C. We present evidence that this is mediated by dNTP pool imbalances leading to allosteric reduction of SAMHD1 ara-CTPase activity. Thus, SAMHD1 constitutes a novel biomarker for combination therapies of ara-C and RNR inhibitors with immediate consequences for clinical practice to improve treatment of AML.

A Mitochondrial LYR Protein Is Required for Complex I Assembly.

Complex I biogenesis requires the expression of both nuclear and mitochondrial genes, the import of proteins, cofactor biosynthesis, and the assembly of at least 49 individual subunits. Assembly factors interact with subunits of Complex I but are not part of the final holocomplex. We show that in Arabidopsis (Arabidopsis thaliana), a mitochondrial matrix protein (EMB1793, At1g76060), which we term COMPLEX I ASSEMBLY FACTOR 1 (CIAF1), contains a LYR domain and is required for Complex I assembly. T-DNA insertion mutants of CIAF1 lack Complex I and the Supercomplex I+III. Biochemical characterization shows that the assembly of Complex I is stalled at 650 and 800 kD intermediates in mitochondria isolated from ciaf1 mutant lines.I. Yeast-two-hybrid interaction and complementation assays indicate that CIAF1 specifically interacts with the 23-kD TYKY-1 matrix domain subunit of Complex I and likely plays a role in Fe-S insertion into this subunit. These data show that CIAF1 plays an essential role in assembling the peripheral matrix arm Complex I subunits into the Complex I holoenzyme.

Chemical flexibility of heterobimetallic Mn/Fe cofactors: R2lox and R2c proteins.

A heterobimetallic Mn/Fe cofactor is present in the R2 subunit of class Ic ribonucleotide reductases (R2c) and in R2-like ligand-binding oxidases (R2lox). Although the protein-derived metal ligands are the same in both groups of proteins, the connectivity of the two metal ions and the chemistry each cofactor performs are different: in R2c, a one-electron oxidant, the Mn/Fe dimer is linked by two oxygen bridges (µ-oxo/µ-hydroxo), whereas in R2lox, a two-electron oxidant, it is linked by a single oxygen bridge (µ-hydroxo) and a fatty acid ligand. Here, we identified a second coordination sphere residue that directs the divergent reactivity of the protein scaffold. We found that the residue that directly precedes the N-terminal carboxylate metal ligand is conserved as a glycine within the R2lox group but not in R2c. Substitution of the glycine with leucine converted the resting-state R2lox cofactor to an R2c-like cofactor, a µ-oxo/µ-hydroxo-bridged MnIII/FeIII dimer. This species has recently been observed as an intermediate of the oxygen activation reaction in WT R2lox, indicating that it is physiologically relevant. Cofactor maturation in R2c and R2lox therefore follows the same pathway, with structural and functional divergence of the two cofactor forms following oxygen activation. We also show that the leucine-substituted variant no longer functions as a two-electron oxidant. Our results reveal that the residue preceding the N-terminal metal ligand directs the cofactor's reactivity toward one- or two-electron redox chemistry, presumably by setting the protonation state of the bridging oxygens and thereby perturbing the redox potential of the Mn ion.

Voluntary exercise normalizes the proteomic landscape in muscle and brain and improves the phenotype of progeroid mice.

The accumulation of mitochondrial DNA (mtDNA) mutations is a suspected driver of aging and age-related diseases, but forestalling these changes has been a major challenge. One of the best-studied models is the prematurely aging mtDNA mutator mouse, which carries a homozygous knock-in of a proofreading deficient version of the catalytic subunit of mtDNA polymerase-γ (PolgA). We investigated how voluntary exercise affects the progression of aging phenotypes in this mouse, focusing on mitochondrial and protein homeostasis in both brain and peripheral tissues. Voluntary exercise significantly ameliorated several aspects of the premature aging phenotype, including decreased locomotor activity, alopecia, and kyphosis, but did not have major effects on the decreased lifespan of mtDNA mutator mice. Exercise also decreased the mtDNA mutation load. In-depth tissue proteomics revealed that exercise normalized the levels of about half the proteins, with the majority involved in mitochondrial function and nuclear-mitochondrial crosstalk. There was also a specific increase in the nuclear-encoded proteins needed for the tricarboxylic acid cycle and complex II, but not in mitochondrial-encoded oxidative phosphorylation proteins, as well as normalization of enzymes involved in coenzyme Q biosynthesis. Furthermore, we found tissue-specific alterations, with brain coping better as compared to muscle and with motor cortex being better protected than striatum, in response to mitochondrial dysfunction. We conclude that voluntary exercise counteracts aging in mtDNA mutator mice by counteracting protein dysregulation in muscle and brain, decreasing the mtDNA mutation burden in muscle, and delaying overt aging phenotypes.

Phosphorylation of G3BP1-S149 does not influence stress granule assembly.

Tourrière et al. (2013. J. Cell Biol. https://doi.org/10.1083/jcb.200212128) reported that G3BP1-S149 dephosphorylation promotes stress granule formation. We show that constructs used to establish this conclusion contain additional mutations causing these phenotypes, and that S149 phosphorylation status does not change upon stress.

Systematic assessment of antibody selectivity in plasma based on a resource of enrichment profiles.

There is a strong need for procedures that enable context and application dependent validation of antibodies. Here, we applied a magnetic bead assisted workflow and immunoprecipitation mass spectrometry (IP-MS/MS) to assess antibody selectivity for the detection of proteins in human plasma. A resource was built on 414 IP experiments using 157 antibodies (targeting 120 unique proteins) in assays with heat-treated or untreated EDTA plasma. For each protein we determined their antibody related degrees of enrichment using z-scores and their frequencies of identification across all IP assays. Out of 1,313 unique endogenous proteins, 426 proteins (33%) were detected in >20% of IPs, and these background components were mainly comprised of proteins from the complement system. For 45% (70/157) of the tested antibodies, the expected target proteins were enriched (z-score ≥ 3). Among these 70 antibodies, 59 (84%) co-enriched other proteins beside the intended target and mainly due to sequence homology or protein abundance. We also detected protein interactions in plasma, and for IGFBP2 confirmed these using several antibodies and sandwich immunoassays. The protein enrichment data with plasma provide a very useful and yet lacking resource for the assessment of antibody selectivity. Our insights will contribute to a more informed use of affinity reagents for plasma proteomics assays.

Breast cancer quantitative proteome and proteogenomic landscape.

In the preceding decades, molecular characterization has revolutionized breast cancer (BC) research and therapeutic approaches. Presented herein, an unbiased analysis of breast tumor proteomes, inclusive of 9995 proteins quantified across all tumors, for the first time recapitulates BC subtypes. Additionally, poor-prognosis basal-like and luminal B tumors are further subdivided by immune component infiltration, suggesting the current classification is incomplete. Proteome-based networks distinguish functional protein modules for breast tumor groups, with co-expression of EGFR and MET marking ductal carcinoma in situ regions of normal-like tumors and lending to a more accurate classification of this poorly defined subtype. Genes included within prognostic mRNA panels have significantly higher than average mRNA-protein correlations, and gene copy number alterations are dampened at the protein-level; underscoring the value of proteome quantification for prognostication and phenotypic classification. Furthermore, protein products mapping to non-coding genomic regions are identified; highlighting a potential new class of tumor-specific immunotherapeutic targets.

Metal-free ribonucleotide reduction powered by a DOPA radical in Mycoplasma pathogens.

Ribonucleotide reductase (RNR) catalyses the only known de novo pathway for the production of all four deoxyribonucleotides that are required for DNA synthesis1,2. It is essential for all organisms that use DNA as their genetic material and is a current drug target3,4. Since the discovery that iron is required for function in the aerobic, class I RNR found in all eukaryotes and many bacteria, a dinuclear metal site has been viewed as necessary to generate and stabilize the catalytic radical that is essential for RNR activity5-7. Here we describe a group of RNR proteins in Mollicutes-including Mycoplasma pathogens-that possess a metal-independent stable radical residing on a modified tyrosyl residue. Structural, biochemical and spectroscopic characterization reveal a stable 3,4-dihydroxyphenylalanine (DOPA) radical species that directly supports ribonucleotide reduction in vitro and in vivo. This observation overturns the presumed requirement for a dinuclear metal site in aerobic ribonucleotide reductase. The metal-independent radical requires new mechanisms for radical generation and stabilization, processes that are targeted by RNR inhibitors. It is possible that this RNR variant provides an advantage under metal starvation induced by the immune system. Organisms that encode this type of RNR-some of which are developing resistance to antibiotics-are involved in diseases of the respiratory, urinary and genital tracts. Further characterization of this RNR family and its mechanism of cofactor generation will provide insight into new enzymatic chemistry and be of value in devising strategies to combat the pathogens that utilize it. We propose that this RNR subclass is denoted class Ie.

Accumulation of endogenous peptides triggers a pathogen stress response in Arabidopsis thaliana.

The stepwise degradation of peptides to amino acids in plant mitochondria and chloroplasts is catalyzed by a network of oligopeptidases (presequence protease PreP, organellar oligopeptidase OOP) and aminopeptidases. In the present report, we show that the lack of oligopeptidase activity in Arabidopsis thaliana results in the accumulation of endogenous free peptides, mostly of chloroplastic origin (targeting peptides and degradation products). Using mRNA sequencing and deep coverage proteomics, allowing for the identification of 17 000 transcripts and 11 000 proteins, respectively, we uncover a peptide-stress response occurring in plants lacking PreP and OOP oligopeptidase activity. The peptide-stress response results in the activation of the classical plant defense pathways in the absence of pathogenic challenge. The constitutive activation of the pathogen-defense pathways imposes a strong growth penalty and a reduction of the plants reproductive fitness. Our results indicate that the absence of organellar oligopeptidases PreP1/2 and OOP results in the accumulation of peptides that are perceived as pathogenic effectors and activate the signaling pathways of plant-defense response.

Ether cross-link formation in the R2-like ligand-binding oxidase.

R2-like ligand-binding oxidases contain a dinuclear metal cofactor which can consist either of two iron ions or one manganese and one iron ion, but the heterodinuclear Mn/Fe cofactor is the preferred assembly in the presence of MnII and FeII in vitro. We have previously shown that both types of cofactor are capable of catalyzing formation of a tyrosine-valine ether cross-link in the protein scaffold. Here we demonstrate that Mn/Fe centers catalyze cross-link formation more efficiently than Fe/Fe centers, indicating that the heterodinuclear cofactor is the biologically relevant one. We further explore the chemical potential of the Mn/Fe cofactor by introducing mutations at the cross-linking valine residue. We find that cross-link formation is possible also to the tertiary beta-carbon in an isoleucine, but not to the secondary beta-carbon or tertiary gamma-carbon in a leucine, nor to the primary beta-carbon of an alanine. These results illustrate that the reactivity of the cofactor is highly specific and directed.

Publisher Correction: Discovery of coding regions in the human genome by integrated proteogenomics analysis workflow.

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