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Cancer remains the leading cause of death worldwide with approved oncology drugs continuing to have heterogenous patient responses and accompanied adverse effects (AEs) that limits effectiveness. Here, we examined >100 FDA-approved oncology drugs in the context of stemness using a surrogate model of transformed human pluripotent cancer stem cells (CSCs) vs. healthy stem cells (hSCs) capable of distinguishing abnormal self-renewal and differentiation. Although a proportion of these drugs had no effects (inactive), a larger portion affected CSCs (active), and a unique subset preferentially affected CSCs over hSCs (selective). Single cell gene expression and protein profiling of each drug's FDA recognized target provided a molecular correlation of responses in CSCs vs. hSCs. Uniquely, drugs selective for CSCs demonstrated clinical efficacy, measured by overall survival, and reduced AEs. Our findings reveal that while unintentional, half of anticancer drugs are active against CSCs and associated with improved clinical outcomes. Based on these findings, we suggest ability to target CSC targeting should be included as a property of early onco-therapeutic development.
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Epithelial ovarian cancer (EOC) has not significantly benefited from advances in immunotherapy, mainly because of the lack of well-defined actionable antigen targets. Using proteogenomic analyses of primary EOC tumors, we previously identified 91 aberrantly expressed tumor-specific antigens (TSAs) originating from unmutated genomic sequences. Most of these TSAs derive from non-exonic regions, and their expression results from cancer-specific epigenetic changes. The present study aimed to evaluate the immunogenicity of 48 TSAs selected according to two criteria: presentation by highly prevalent HLA allotypes and expression in a significant fraction of EOC tumors. Using targeted mass spectrometry analyses, we found that pulsing with synthetic TSA peptides leads to a high-level presentation on dendritic cells. TSA abundance correlated with the predicted binding affinity to the HLA allotype. We stimulated naïve CD8 T cells from healthy blood donors with TSA-pulsed dendritic cells and assessed their expansion with two assays: MHC-peptide tetramer staining and TCR Vß CDR3 sequencing. We report that these TSAs can expand sizeable populations of CD8 T cells and, therefore, represent attractive targets for EOC immunotherapy.
Assuntos
Antígenos de Neoplasias , Neoplasias Ovarianas , Humanos , Feminino , Antígenos de Neoplasias/imunologia , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/genética , Células Dendríticas/imunologia , Carcinoma Epitelial do Ovário/imunologia , Carcinoma Epitelial do Ovário/genética , Linfócitos T CD8-Positivos/imunologia , Imunoterapia/métodosRESUMO
Immunotherapy remains underexploited in acute myeloid leukemia (AML) compared to other hematological malignancies. Currently, gemtuzumab ozogamicin is the only therapeutic antibody approved for this disease. Here, to identify potential targets for immunotherapeutic intervention, we analyze the surface proteome of 100 genetically diverse primary human AML specimens for the identification of cell surface proteins and conduct single-cell transcriptome analyses on a subset of these specimens to assess antigen expression at the sub-population level. Through this comprehensive effort, we successfully identify numerous antigens and markers preferentially expressed by primitive AML cells. Many identified antigens are targeted by therapeutic antibodies currently under clinical evaluation for various cancer types, highlighting the potential therapeutic value of the approach. Importantly, this initiative uncovers AML heterogeneity at the surfaceome level, identifies several antigens and potential primitive cell markers characterizing AML subgroups, and positions immunotherapy as a promising approach to target AML subgroup specificities.
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Imunoterapia , Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/patologia , Imunoterapia/métodos , Proteínas de Membrana/metabolismoRESUMO
The hypomethylating agent 5-azacytidine (AZA) is the first-line treatment for AML patients unfit for intensive chemotherapy. The effect of AZA results in part from T-cell cytotoxic responses against MHC-I-associated peptides (MAPs) deriving from hypermethylated genomic regions such as cancer-testis antigens (CTAs), or endogenous retroelements (EREs). However, evidence supporting higher ERE MAPs presentation after AZA treatment is lacking. Therefore, using proteogenomics, we examined the impact of AZA on the repertoire of MAPs and their source transcripts. AZA-treated AML upregulated both CTA and ERE transcripts, but only CTA MAPs were presented at greater levels. Upregulated ERE transcripts triggered innate immune responses against double-stranded RNAs but were degraded by autophagy, and not processed into MAPs. Autophagy resulted from the formation of protein aggregates caused by AZA-dependent inhibition of DNMT2. Autophagy inhibition had an additive effect with AZA on AML cell proliferation and survival, increased ERE levels, increased pro-inflammatory responses, and generated immunogenic tumor-specific ERE-derived MAPs. Finally, autophagy was associated with a lower abundance of CD8+ T-cell markers in AML patients expressing high levels of EREs. This work demonstrates that AZA-induced EREs are degraded by autophagy and shows that inhibiting autophagy can improve the immune recognition of AML blasts in treated patients.
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Antimetabólitos Antineoplásicos , Autofagia , Azacitidina , Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Azacitidina/farmacologia , Autofagia/efeitos dos fármacos , Antimetabólitos Antineoplásicos/farmacologia , Antimetabólitos Antineoplásicos/uso terapêutico , Metilação de DNA/efeitos dos fármacos , Proliferação de Células , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologiaRESUMO
Transposable elements (TEs) are repetitive sequences representing ~45% of the human and mouse genomes and are highly expressed by medullary thymic epithelial cells (mTECs). In this study, we investigated the role of TEs on T-cell development in the thymus. We performed multiomic analyses of TEs in human and mouse thymic cells to elucidate their role in T-cell development. We report that TE expression in the human thymus is high and shows extensive age- and cell lineage-related variations. TE expression correlates with multiple transcription factors in all cell types of the human thymus. Two cell types express particularly broad TE repertoires: mTECs and plasmacytoid dendritic cells (pDCs). In mTECs, transcriptomic data suggest that TEs interact with transcription factors essential for mTEC development and function (e.g., PAX1 and REL), and immunopeptidomic data showed that TEs generate MHC-I-associated peptides implicated in thymocyte education. Notably, AIRE, FEZF2, and CHD4 regulate small yet non-redundant sets of TEs in murine mTECs. Human thymic pDCs homogenously express large numbers of TEs that likely form dsRNA, which can activate innate immune receptors, potentially explaining why thymic pDCs constitutively secrete IFN É/ß. This study highlights the diversity of interactions between TEs and the adaptive immune system. TEs are genetic parasites, and the two thymic cell types most affected by TEs (mTEcs and pDCs) are essential to establishing central T-cell tolerance. Therefore, we propose that orchestrating TE expression in thymic cells is critical to prevent autoimmunity in vertebrates.
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Proteína AIRE , Elementos de DNA Transponíveis , Camundongos , Humanos , Animais , Timo/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Timócitos/metabolismo , Células Epiteliais/metabolismo , Diferenciação Celular/genética , Camundongos Endogâmicos C57BLRESUMO
In a phenotypical screen of 56 acute myeloid leukemia (AML) patient samples and using a library of 10,000 compounds, we identified a hit with increased sensitivity toward SF3B1-mutated and adverse risk AMLs. Through structure-activity relationship studies, this hit was optimized into a potent, specific, and nongenotoxic molecule called UM4118. We demonstrated that UM4118 acts as a copper ionophore that initiates a mitochondrial-based noncanonical form of cell death known as cuproptosis. CRISPR-Cas9 loss-of-function screen further revealed that iron-sulfur cluster (ISC) deficiency enhances copper-mediated cell death. Specifically, we found that loss of the mitochondrial ISC transporter ABCB7 is synthetic lethal to UM4118. ABCB7 is misspliced and down-regulated in SF3B1-mutated leukemia, creating a vulnerability to copper ionophores. Accordingly, ABCB7 overexpression partially rescued SF3B1-mutated cells to copper overload. Together, our work provides mechanistic insights that link ISC deficiency to cuproptosis, as exemplified by the high sensitivity of SF3B1-mutated AMLs. We thus propose SF3B1 mutations as a biomarker for future copper ionophore-based therapies.
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Cobre , Leucemia Mieloide Aguda , Humanos , Cobre/metabolismo , Fatores de Processamento de RNA/genética , Mutação , Leucemia Mieloide Aguda/genética , Ionóforos/farmacologia , Fosfoproteínas/metabolismoRESUMO
Gene transcription is a highly regulated process, and deregulation of transcription factors activity underlies numerous pathologies including cancer. Albeit near four decades of studies have established that the E2F pathway is a core transcriptional network that govern cell division in multi-cellular organisms1,2, the molecular mechanisms that underlie the functions of E2F transcription factors remain incompletely understood. FOXK1 and FOXK2 transcription factors have recently emerged as important regulators of cell metabolism, autophagy and cell differentiation3-6. While both FOXK1 and FOXK2 interact with the histone H2AK119ub deubiquitinase BAP1 and possess many overlapping functions in normal biology, their specific functions as well as deregulation of their transcriptional activity in cancer is less clear and sometimes contradictory7-13. Here, we show that elevated expression of FOXK1, but not FOXK2, in primary normal cells promotes transcription of E2F target genes associated with increased proliferation and delayed entry into cellular senescence. FOXK1 expressing cells are highly prone to cellular transformation revealing important oncogenic properties of FOXK1 in tumor initiation. High expression of FOXK1 in patient tumors is also highly correlated with E2F gene expression. Mechanistically, we demonstrate that FOXK1, but not FOXK2, is specifically modified by O-GlcNAcylation. FOXK1 O-GlcNAcylation is modulated during the cell cycle with the highest levels occurring during the time of E2F pathway activation at G1/S. Moreover, loss of FOXK1 O-GlcNAcylation impairs FOXK1 ability to promote cell proliferation, cellular transformation and tumor growth. Mechanistically, expression of FOXK1 O-GlcNAcylation-defective mutants results in reduced recruitment of BAP1 to gene regulatory regions. This event is associated with a concomitant increase in the levels of histone H2AK119ub and a decrease in the levels of H3K4me1, resulting in a transcriptional repressive chromatin environment. Our results define an essential role of O-GlcNAcylation in modulating the functions of FOXK1 in controlling the cell cycle of normal and cancer cells through orchestration of the E2F pathway.
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The mechanistic target of rapamycin (mTOR) signaling is influenced by multiple regulatory proteins and post-translational modifications; however, underlying mechanisms remain unclear. Here, we report a novel role of small ubiquitin-like modifier (SUMO) in mTOR complex assembly and activity. By investigating the SUMOylation status of core mTOR components, we observed that the regulatory subunit, GßL (G protein ß-subunit-like protein, also known as mLST8), is modified by SUMO1, 2, and 3 isoforms. Using mutagenesis and mass spectrometry, we identified that GßL is SUMOylated at lysine sites K86, K215, K245, K261, and K305. We found that SUMO depletion reduces mTOR-Raptor (regulatory protein associated with mTOR) and mTOR-Rictor (rapamycin-insensitive companion of mTOR) complex formation and diminishes nutrient-induced mTOR signaling. Reconstitution with WT GßL but not SUMOylation-defective KR mutant GßL promotes mTOR signaling in GßL-depleted cells. Taken together, we report for the very first time that SUMO modifies GßL, influences the assembly of mTOR protein complexes, and regulates mTOR activity.
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Transdução de Sinais , Sumoilação , Serina-Treonina Quinases TOR , Humanos , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genética , Células HEK293 , Proteína SUMO-1/metabolismo , Proteína SUMO-1/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Homólogo LST8 da Proteína Associada a mTOR/metabolismo , Homólogo LST8 da Proteína Associada a mTOR/genética , Ubiquitinas/metabolismo , Ubiquitinas/genética , Lisina/metabolismoRESUMO
Hormone receptor-positive breast cancer (HR+) is immunologically cold and has not benefited from advances in immunotherapy. In contrast, subsets of triple-negative breast cancer (TNBC) display high leukocytic infiltration and respond to checkpoint blockade. CD8+ T cells, the main effectors of anticancer responses, recognize MHC I-associated peptides (MAPs). Our work aimed to characterize the repertoire of MAPs presented by HR+ and TNBC tumors. Using mass spectrometry, we identified 57,094 unique MAPs in 26 primary breast cancer samples. MAP source genes highly overlapped between both subtypes. We identified 25 tumor-specific antigens (TSAs) mainly deriving from aberrantly expressed regions. TSAs were most frequently identified in TNBC samples and were more shared among The Cancer Genome Atlas (TCGA) database TNBC than HR+ samples. In the TNBC cohort, the predicted number of TSAs positively correlated with leukocytic infiltration and overall survival, supporting their immunogenicity in vivo. We detected 49 tumor-associated antigens (TAAs), some of which derived from cancer-associated fibroblasts. Functional expansion of specific T cell assays confirmed the in vitro immunogenicity of several TSAs and TAAs. Our study identified attractive targets for cancer immunotherapy in both breast cancer subtypes. The higher prevalence of TSAs in TNBC tumors provides a rationale for their responsiveness to checkpoint blockade.
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Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/patologia , Antígenos de Neoplasias/genética , Imunoterapia/métodos , Linfócitos T CD8-Positivos/patologiaRESUMO
ABSTRACT: Acute megakaryoblastic leukemia (AMKL) is a rare, developmentally restricted, and highly lethal cancer of early childhood. The paucity and hypocellularity (due to myelofibrosis) of primary patient samples hamper the discovery of cell- and genotype-specific treatments. AMKL is driven by mutually exclusive chimeric fusion oncogenes in two-thirds of the cases, with CBFA2T3::GLIS2 (CG2) and NUP98 fusions (NUP98r) representing the highest-fatality subgroups. We established CD34+ cord blood-derived CG2 models (n = 6) that sustain serial transplantation and recapitulate human leukemia regarding immunophenotype, leukemia-initiating cell frequencies, comutational landscape, and gene expression signature, with distinct upregulation of the prosurvival factor B-cell lymphoma 2 (BCL2). Cell membrane proteomic analyses highlighted CG2 surface markers preferentially expressed on leukemic cells compared with CD34+ cells (eg, NCAM1 and CD151). AMKL differentiation block in the mega-erythroid progenitor space was confirmed by single-cell profiling. Although CG2 cells were rather resistant to BCL2 genetic knockdown or selective pharmacological inhibition with venetoclax, they were vulnerable to strategies that target the megakaryocytic prosurvival factor BCL-XL (BCL2L1), including in vitro and in vivo treatment with BCL2/BCL-XL/BCL-W inhibitor navitoclax and DT2216, a selective BCL-XL proteolysis-targeting chimera degrader developed to limit thrombocytopenia in patients. NUP98r AMKL were also sensitive to BCL-XL inhibition but not the NUP98r monocytic leukemia, pointing to a lineage-specific dependency. Navitoclax or DT2216 treatment in combination with low-dose cytarabine further reduced leukemic burden in mice. This work extends the cellular and molecular diversity set of human AMKL models and uncovers BCL-XL as a therapeutic vulnerability in CG2 and NUP98r AMKL.
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Antineoplásicos , Leucemia Megacarioblástica Aguda , Humanos , Criança , Pré-Escolar , Animais , Camundongos , Leucemia Megacarioblástica Aguda/tratamento farmacológico , Leucemia Megacarioblástica Aguda/genética , Leucemia Megacarioblástica Aguda/patologia , Proteômica , Fatores de Transcrição , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas RepressorasRESUMO
Identification of K-Ras and B-Raf mutations in colorectal cancer (CRC) is essential to predict patients' response to anti-EGFR therapy and formulate appropriate therapeutic strategies to improve prognosis and survival. Here, we combined parallel reaction monitoring (PRM) with high-field asymmetric waveform ion mobility (FAIMS) to enhance mass spectrometry sensitivity and improve the identification of low-abundance K-Ras and B-Raf mutations in biological samples without immunoaffinity enrichment. In targeted LC-MS/MS analyses, FAIMS reduced the occurrence of interfering ions and enhanced precursor ion purity, resulting in a 3-fold improvement in the detection limit for K-Ras and B-Raf mutated peptides. In addition, the ion mobility separation of isomeric peptides using FAIMS facilitated the unambiguous identification of K-Ras G12D and G13D peptides. The application of targeted LC-MS/MS analyses using FAIMS is demonstrated for the detection and quantitation of B-Raf V600E, K-Ras G12D, G13D, and G12V in CRC cell lines and primary specimens.
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Neoplasias Colorretais , Espectrometria de Massas em Tandem , Humanos , Cromatografia Líquida , Peptídeos/química , Proteínas Proto-Oncogênicas B-raf/genética , Mutação , Neoplasias Colorretais/genética , Íons/químicaRESUMO
MHC-I-associated peptides deriving from non-coding genomic regions and mutations can generate tumor-specific antigens, including neoantigens. Quantifying tumor-specific antigens' RNA expression in malignant and benign tissues is critical for discriminating actionable targets. We present BamQuery, a tool attributing an exhaustive RNA expression to MHC-I-associated peptides of any origin from bulk and single-cell RNA-sequencing data. We show that many cryptic and mutated tumor-specific antigens can derive from multiple discrete genomic regions, abundantly expressed in normal tissues. BamQuery can also be used to predict MHC-I-associated peptides immunogenicity and identify actionable tumor-specific antigens de novo.
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Neoplasias , Proteogenômica , Humanos , Antígenos de Neoplasias/genética , Antígenos de Histocompatibilidade Classe I , Neoplasias/genética , Peptídeos/genética , RNARESUMO
The identification of tumor-specific antigens (TSAs) is critical for developing effective cancer immunotherapies. Mass spectrometry (MS)-based immunopeptidomics has emerged as a powerful tool for identifying TSAs as physical molecules. However, current immunopeptidomics platforms face challenges in measuring low-abundance TSAs in a precise, sensitive, and reproducible manner from small needle-tissue biopsies (<1 mg). Inspired by recent advances in single-cell proteomics, microfluidics technology offers a promising solution to these limitations by providing improved isolation of human leukocyte antigen (HLA)-associated peptides with higher sensitivity. In this context, we highlight the challenges in sample preparation and the rationale for developing microfluidics technology in immunopeptidomics. Additionally, we provide an overview of promising microfluidic methods, including microchip pillar arrays, valved-based systems, droplet microfluidics, and digital microfluidics, and discuss the latest research on their application in MS-based immunopeptidomics and single-cell proteomics.
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Microfluídica , Neoplasias , Humanos , Espectrometria de Massas/métodos , Antígenos de Histocompatibilidade Classe I , Antígenos HLA , Antígenos de NeoplasiasRESUMO
Immunotherapeutic strategies aimed at enhancing tumor cell killing by tumor-specific T cells hold great potential for reducing tumor burden and prolonging survival of cancer patients. Although many potential tumor antigens have been described, identifying relevant targets when designing anti-cancer vaccines or targeted cell therapies remains a challenge. To identify novel, potentially immunogenic candidate tumor antigens, we performed integrated tumor transcriptomic, seromic, and proteomic analyses of high grade serous ovarian cancer (HGSC) patient tumor samples. We identified tumor neo-antigens and over-expressed antigens using whole exome and RNA sequencing and examined these in relation to patient-matched auto-antibody repertoires. Focusing on MHC class I epitopes recognized by CD8+ T cells, HLA-binding epitopes were identified or predicted from the highly expressed, mutated, or auto-antibody target antigen, or MHC-associated peptides (MAPs). Recognition of candidate antigenic peptides was assessed within the tumor-infiltrating T lymphocyte (TIL) population expanded from each patient. Known tumor-associated antigens (TAA) and cancer/testis antigens (CTA) were commonly found in the auto-antibody and MAP repertoires and CD8+ TILs recognizing epitopes from these antigens were detected, although neither expression level nor the presence of auto-antibodies correlated with TIL recognition. Auto-antibodies against tumor-mutated antigens were found in most patients, however, no TIL recognition of the highest predicted affinity neo-epitopes was detected. Using high expression level, auto-antibody recognition, and epitope prediction algorithms, we identified epitopes in 5 novel antigens (MOB1A, SOCS3, TUBB, PRKAR1A, CCDC6) recognized by HGSC patient TILs. Furthermore, selection of epitopes from the MAP repertoire identified 5 additional targets commonly recognized by multiple patient TILs. We find that the repertoire of TIL specificities includes recognition of highly expressed and immunogenic self-antigens that are processed and presented by tumors. These results indicate an ongoing autoimmune response against a range of self-antigens targeted by HGSC TILs.
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Linfócitos do Interstício Tumoral , Neoplasias Ovarianas , Masculino , Humanos , Feminino , Epitopos/metabolismo , Linfócitos T CD8-Positivos , Proteômica , Multiômica , Antígenos de Neoplasias , Peptídeos , Autoantígenos , Epitopos de Linfócito TRESUMO
Proteomic diversity in biological samples can be characterized by mass spectrometry (MS)-based proteomics using customized protein databases generated from sets of transcripts previously detected by RNA-seq. This diversity has only been increased by the recent discovery that many translated alternative open reading frames rest unannotated at unsuspected locations of mRNAs and ncRNAs. These novel protein products, termed alternative proteins, have been left out of all previous custom database generation tools. Consequently, genetic variations that impact alternative open reading frames and variant peptides from their translated proteins are not detectable with current computational workflows. To fill this gap, we present OpenCustomDB, a bioinformatics tool that uses sample-specific RNaseq data to identify genomic variants in canonical and alternative open reading frames, allowing for more than one coding region per transcript. In a test reanalysis of a cohort of 16 patients with acute myeloid leukemia, 5666 peptides from alternative proteins were detected, including 201 variant peptides. We also observed that a significant fraction of peptide-spectrum matches previously assigned to peptides from canonical proteins got better scores when reassigned to peptides from alternative proteins. Custom protein libraries that include sample-specific sequence variations of all possible open reading frames are promising contributions to the development of proteomics and precision medicine. The raw and processed proteomics data presented in this study can be found in PRIDE repository with accession number PXD029240.
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Proteínas , Proteômica , Humanos , Proteômica/métodos , Bases de Dados de Proteínas , Fases de Leitura Aberta , Proteínas/genética , Peptídeos/genética , Peptídeos/análiseRESUMO
Protein inhibitor of activated STAT (PIAS) proteins are E3 SUMO ligases playing important roles in protein stability and signaling transduction pathways. PIAS proteins are overexpressed in the triple-negative breast cancer cell line MDA-MB-231, and PIAS knockout (KO) results in a reduction in cell proliferation and cell arrest in the S phase. However, the molecular mechanisms underlying PIAS functions in cell proliferation and cell cycle remain largely unknown. Here, we used quantitative SUMO proteomics to explore the regulatory role of PIAS SUMO E3 ligases upon CRISPR/Cas9 KO of individual PIAS. A total of 1422 sites were identified, and around 10% of SUMO sites were regulated following KO of one or more PIAS genes. We identified protein substrates that were either specific to individual PIAS ligase or regulated by several PIAS ligases. Ki-67 and TOP2A, which are involved in cell proliferation and epithelial-to-mesenchymal transition, are SUMOylated at several lysine residues by all PIAS ligases, suggesting a level of redundancy between these proteins. Confocal microscopy and biochemical experiments revealed that SUMOylation regulated TOP2A protein stability, while this modification is involved in the recruitment of Ki-67 nucleolar proteins containing the SUMO interacting motif. These results provide novel insights into both the redundant and specific regulatory mechanisms of cell proliferation and cell cycle mediated by PIAS SUMO E3 ligases.
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Proteômica , Ubiquitina-Proteína Ligases , Antígeno Ki-67/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ciclo Celular , Proliferação de Células , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , SumoilaçãoRESUMO
DDX3X is a mammalian RNA helicase that regulates RNA metabolism, cancers, innate immunity and several RNA viruses. We discovered that herpes simplex virus 1, a nuclear DNA replicating virus, redirects DDX3X to the nuclear envelope where it surprisingly modulates the exit of newly assembled viral particles. DDX3X depletion also leads to an accumulation of virions in intranuclear herniations. Mechanistically, we show that DDX3X physically and functionally interacts with the virally encoded nuclear egress complex at the inner nuclear membrane. DDX3X also binds to and stimulates the incorporation in mature particles of pUs3, a herpes kinase that promotes viral nuclear release across the outer nuclear membrane. Overall, the data highlights two unexpected roles for an RNA helicase during the passage of herpes simplex viral particles through the nuclear envelope. This reveals a highly complex interaction between DDX3X and viruses and provides new opportunities to target viral propagation.
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Infecções por Herpesviridae , Herpesvirus Humano 1 , Animais , Herpesvirus Humano 1/genética , Proteínas Virais/metabolismo , Membrana Nuclear/metabolismo , Núcleo Celular/metabolismo , MamíferosRESUMO
Previous reports showed that mouse vaccination with pluripotent stem cells (PSCs) induces durable anti-tumor immune responses via T cell recognition of some elusive oncofetal epitopes. We characterize the MHC I-associated peptide (MAP) repertoire of human induced PSCs (iPSCs) using proteogenomics. Our analyses reveal a set of 46 pluripotency-associated MAPs (paMAPs) absent from the transcriptome of normal tissues and adult stem cells but expressed in PSCs and multiple adult cancers. These paMAPs derive from coding and allegedly non-coding (48%) transcripts involved in pluripotency maintenance, and their expression in The Cancer Genome Atlas samples correlates with source gene hypomethylation and genomic aberrations common across cancer types. We find that several of these paMAPs were immunogenic. However, paMAP expression in tumors coincides with activation of pathways instrumental in immune evasion (WNT, TGF-ß, and CDK4/6). We propose that currently available inhibitors of these pathways could synergize with immune targeting of paMAPs for the treatment of poorly differentiated cancers.
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Células-Tronco Pluripotentes Induzidas , Neoplasias , Células-Tronco Pluripotentes , Animais , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Camundongos , Neoplasias/metabolismo , Peptídeos/metabolismo , Células-Tronco Pluripotentes/metabolismoRESUMO
The sensitivity and depth of proteomic analyses are limited by isobaric ions and interferences that preclude the identification of low abundance peptides. Extensive sample fractionation is often required to extend proteome coverage when sample amount is not a limitation. Ion mobility devices provide a viable alternate approach to resolve confounding ions and improve peak capacity and mass spectrometry (MS) sensitivity. Here, we report the integration of differential ion mobility with segmented ion fractionation (SIFT) to enhance the comprehensiveness of proteomic analyses. The combination of differential ion mobility and SIFT, where narrow windows of â¼m/z 100 are acquired in turn, is found particularly advantageous in the analysis of protein digests and typically provided more than 60% gain in identification compared to conventional single-shot LC-MS/MS. The application of this approach is further demonstrated for the analysis of tryptic digests from different colorectal cancer cell lines where the enhanced sensitivity enabled the identification of single amino acid variants that were correlated with the corresponding transcriptomic data sets.
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Neoplasias do Colo , Proteogenômica , Cromatografia Líquida/métodos , Neoplasias do Colo/genética , Humanos , Íons , Proteoma , Proteômica/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
Genetically-encoded cyclic peptide libraries allow rapid in vivo screens for inhibitors of any target protein of interest. In particular, the Split Intein Circular Ligation of Protein and Peptides (SICLOPPS) system exploits spontaneous protein splicing of inteins to produce intracellular cyclic peptides. A previous SICLOPPS screen against Aurora B kinase, which plays a critical role during chromosome segregation, identified several candidate inhibitors that we sought to recapitulate by chemical synthesis. We describe the syntheses of cyclic peptide hits and analogs via solution-phase macrocyclization of side chain-protected linear peptides obtained from standard solid-phase peptide synthesis. Cyclic peptide targets, including cyclo-[CTWAR], were designed to match both the variable portions and conserved cysteine residue of their genetically-encoded counterparts. Synthetic products were characterized by tandem high-resolution mass spectrometry to analyze a combination of exact mass, isotopic pattern, and collisional dissociation-induced fragmentation pattern. The latter analyses facilitated the distinction between targets and oligomeric side products, and served to confirm peptidic sequences in a manner that can be readily extended to analyses of complex biological samples. This alternative chemical synthesis approach for cyclic peptides allows cost-effective validation and facile chemical elaboration of hit candidates from SICLOPPS screens.