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1.
PLoS Pathog ; 20(3): e1012036, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38457376

RESUMO

Viruses actively reprogram the metabolism of the host to ensure the availability of sufficient building blocks for virus replication and spreading. However, relatively little is known about how picornaviruses-a large family of small, non-enveloped positive-strand RNA viruses-modulate cellular metabolism for their own benefit. Here, we studied the modulation of host metabolism by coxsackievirus B3 (CVB3), a member of the enterovirus genus, and encephalomyocarditis virus (EMCV), a member of the cardiovirus genus, using steady-state as well as 13C-glucose tracing metabolomics. We demonstrate that both CVB3 and EMCV increase the levels of pyrimidine and purine metabolites and provide evidence that this increase is mediated through degradation of nucleic acids and nucleotide recycling, rather than upregulation of de novo synthesis. Finally, by integrating our metabolomics data with a previously acquired phosphoproteomics dataset of CVB3-infected cells, we identify alterations in phosphorylation status of key enzymes involved in nucleotide metabolism, providing insight into the regulation of nucleotide metabolism during infection.


Assuntos
Cardiovirus , Infecções por Enterovirus , Enterovirus , Picornaviridae , Humanos , Enterovirus/fisiologia , Vírus da Encefalomiocardite/fisiologia , Replicação Viral , Enterovirus Humano B/fisiologia , Células HeLa
2.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1869(4): 159467, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38382574

RESUMO

Gram-negative bacteria possess an asymmetric outer membrane (OM) primarily composed of lipopolysaccharides (LPS) on the outer leaflet and phospholipids on the inner leaflet. The outer membrane functions as an effective permeability barrier to compounds such as antibiotics. Studying LPS biosynthesis is therefore helpful to explore novel strategies for new antibiotic development. Metabolic glycan labeling of the bacterial surface has emerged as a powerful method to investigate LPS biosynthesis. However, the previously reported methods of labeling LPS are based on radioactivity or difficult-to-produce analogs of bacterial sugars. In this study, we report on the incorporation of azido galactose into the LPS of the Gram-negative bacteria Escherichia coli and Salmonella typhi via metabolic labeling. As a common sugar analog, azido galactose successfully labeled both O-antigen and core of Salmonella LPS, but not E. coli LPS. This labeling of Salmonella LPS, as shown by SDS-PAGE analysis and fluorescence microscopy, differs from the previously reported labeling of either O-antigen or core of LPS. Our findings are useful for studying LPS biogenesis pathways in Gram-negative bacteria like Salmonella. In addition, our approach is helpful for screening for agents that target LPS biosynthesis as it allows for the detection of newly synthesized LPS that appears in the OM. Furthermore, this approach may also aid in isolating chemically modified LPS for vaccine development or immunotherapy.


Assuntos
Proteínas de Escherichia coli , Lipopolissacarídeos , Lipopolissacarídeos/metabolismo , Galactose/metabolismo , Antígenos O/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Antibacterianos
3.
Mol Oncol ; 2024 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-38275212

RESUMO

Metabolism plays a crucial role in regulating the function of immune cells in both health and disease, with altered metabolism contributing to the pathogenesis of cancer and many inflammatory diseases. The local microenvironment has a profound impact on the metabolism of immune cells. Therefore, immunological and metabolic heterogeneity as well as the spatial organization of cells in tissues should be taken into account when studying immunometabolism. Here, we highlight challenges of investigating metabolic communication. Additionally, we review the capabilities and limitations of current technologies for studying metabolism in inflamed microenvironments, including single-cell omics techniques, flow cytometry-based methods (Met-Flow, single-cell energetic metabolism by profiling translation inhibition (SCENITH)), cytometry by time of flight (CyTOF), cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq), and mass spectrometry imaging. Considering the importance of metabolism in regulating immune cells in diseased states, we also discuss the applications of metabolomics in clinical research, as well as some hurdles to overcome to implement these techniques in standard clinical practice. Finally, we provide a flowchart to assist scientists in designing effective strategies to unravel immunometabolism in disease-relevant contexts.

4.
Stem Cell Res Ther ; 14(1): 353, 2023 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-38072933

RESUMO

BACKGROUND: Ischemia/reperfusion injury is the leading cause of acute kidney injury (AKI). The current standard of care focuses on supporting kidney function, stating the need for more efficient and targeted therapies to enhance repair. Mesenchymal stromal cells (MSCs) and their secretome, either as conditioned medium (CM) or extracellular vesicles (EVs), have emerged as promising options for regenerative therapy; however, their full potential in treating AKI remains unknown. METHODS: In this study, we employed an in vitro model of chemically induced ischemia using antimycin A combined with 2-deoxy-D-glucose to induce ischemic injury in proximal tubule epithelial cells. Afterwards we evaluated the effects of MSC secretome, CM or EVs obtained from adipose tissue, bone marrow, and umbilical cord, on ameliorating the detrimental effects of ischemia. To assess the damage and treatment outcomes, we analyzed cell morphology, mitochondrial health parameters (mitochondrial activity, ATP production, mass and membrane potential), and overall cell metabolism by metabolomics. RESULTS: Our findings show that ischemic injury caused cytoskeletal changes confirmed by disruption of the F-actin network, energetic imbalance as revealed by a 50% decrease in the oxygen consumption rate, increased oxidative stress, mitochondrial dysfunction, and reduced cell metabolism. Upon treatment with MSC secretome, the morphological derangements were partly restored and ATP production increased by 40-50%, with umbilical cord-derived EVs being most effective. Furthermore, MSC treatment led to phenotype restoration as indicated by an increase in cell bioenergetics, including increased levels of glycolysis intermediates, as well as an accumulation of antioxidant metabolites. CONCLUSION: Our in vitro model effectively replicated the in vivo-like morphological and molecular changes observed during ischemic injury. Additionally, treatment with MSC secretome ameliorated proximal tubule damage, highlighting its potential as a viable therapeutic option for targeting AKI.


Assuntos
Injúria Renal Aguda , Vesículas Extracelulares , Células-Tronco Mesenquimais , Humanos , Secretoma , Isquemia/terapia , Isquemia/metabolismo , Vesículas Extracelulares/metabolismo , Injúria Renal Aguda/terapia , Injúria Renal Aguda/metabolismo , Metabolismo Energético , Oxirredução , Células-Tronco Mesenquimais/metabolismo , Trifosfato de Adenosina/metabolismo
5.
Methods Mol Biol ; 2669: 67-77, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37247055

RESUMO

Retinoids are light-sensitive molecules that are normally detected by UV absorption techniques. Here we describe the identification and quantification of retinyl ester species by high-resolution mass spectrometry. Retinyl esters are extracted by the method of Bligh and Dyer and subsequently separated by HPLC in runs of 40 min. The retinyl esters are identified and quantified by mass spectrometry analysis. This procedure enables the highly sensitive detection and characterization of retinyl esters in biological samples such as hepatic stellate cells.


Assuntos
Ésteres de Retinil , Vitamina A , Ésteres de Retinil/análise , Retinoides/análise , Espectrometria de Massas/métodos , Cromatografia Líquida de Alta Pressão/métodos
6.
Front Oncol ; 13: 1155621, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37091139

RESUMO

Multiple myeloma (MM) is characterized by the clonal expansion of malignant plasma cells in the bone marrow (BM). MM remains an incurable disease, with the majority of patients experiencing multiple relapses from different drugs. The MM tumor microenvironment (TME) and in particular bone-marrow stromal cells (BMSCs) play a crucial role in the development of drug resistance. Metabolic reprogramming is emerging as a hallmark of cancer that can potentially be exploited for cancer treatment. Recent studies show that metabolism is further adjusted in MM cells during the development of drug resistance. However, little is known about the role of BMSCs in inducing metabolic changes that are associated with drug resistance. In this Perspective, we summarize current knowledge concerning the metabolic reprogramming of MM, with a focus on those changes associated with drug resistance to the proteasome inhibitor Bortezomib (BTZ). In addition, we present proof-of-concept fluxomics (glucose isotope-tracing) and Seahorse data to show that co-culture of MM cells with BMSCs skews the metabolic phenotype of MM cells towards a drug-resistant phenotype, with increased oxidative phosphorylation (OXPHOS), serine synthesis pathway (SSP), TCA cycle and glutathione (GSH) synthesis. Given the crucial role of BMSCs in conveying drug resistance, insights into the metabolic interaction between MM and BMSCs may ultimately aid in the identification of novel metabolic targets that can be exploited for therapy.

7.
Eur Heart J Open ; 3(2): oead013, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36969380

RESUMO

Aims: Hyperlipidemia and T cell driven inflammation are important drivers of atherosclerosis, the main underlying cause of cardiovascular disease. Here, we detailed the effects of hyperlipidemia on T cells. Methods and results: In vitro, exposure of human and murine CD4+ T cells to very low-density lipoprotein (VLDL), but not to low-density lipoprotein (LDL) resulted in upregulation of Th1 associated pathways. VLDL was taken up via a CD36-dependent pathway and resulted in membrane stiffening and a reduction in lipid rafts. To further detail this response in vivo, T cells of mice lacking the LDL receptor (LDLr), which develop a strong increase in VLDL cholesterol and triglyceride levels upon high cholesterol feeding were investigated. CD4+ T cells of hyperlipidemic Ldlr-/- mice exhibited an increased expression of the C-X-C-chemokine receptor 3 (CXCR3) and produced more interferon-γ (IFN-γ). Gene set enrichment analysis identified IFN-γ-mediated signaling as the most upregulated pathway in hyperlipidemic T cells. However, the classical Th1 associated transcription factor profile with strong upregulation of Tbet and Il12rb2 was not observed. Hyperlipidemia did not affect levels of the CD4+ T cell's metabolites involved in glycolysis or other canonical metabolic pathways but enhanced amino acids levels. However, CD4+ T cells of hyperlipidemic mice showed increased cholesterol accumulation and an increased arachidonic acid (AA) to docosahexaenoic acid (DHA) ratio, which was associated with inflammatory T cell activation. Conclusions: Hyperlipidemia, and especially its VLDL component induces an atypical Th1 response in CD4+ T cells. Underlying mechanisms include CD36 mediated uptake of VLDL, and an altered AA/DHA ratio.

8.
Nat Commun ; 13(1): 5371, 2022 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-36100608

RESUMO

The importance of fatty acid (FA) metabolism in cancer is well-established, yet the mechanisms underlying metabolic reprogramming remain elusive. Here, we identify tetraspanin CD37, a prognostic marker for aggressive B-cell lymphoma, as essential membrane-localized inhibitor of FA metabolism. Deletion of CD37 on lymphoma cells results in increased FA oxidation shown by functional assays and metabolomics. Furthermore, CD37-negative lymphomas selectively deplete palmitate from serum in mouse studies. Mechanistically, CD37 inhibits the FA transporter FATP1 through molecular interaction. Consequently, deletion of CD37 induces uptake and processing of exogenous palmitate into energy and essential building blocks for proliferation, and inhibition of FATP1 reverses this phenotype. Large lipid deposits and intracellular lipid droplets are observed in CD37-negative lymphoma tissues of patients. Moreover, inhibition of carnitine palmitoyl transferase 1 A significantly compromises viability and proliferation of CD37-deficient lymphomas. Collectively, our results identify CD37 as a direct gatekeeper of the FA metabolic switch in aggressive B-cell lymphoma.


Assuntos
Antígenos de Neoplasias , Linfoma de Células B , Animais , Antígenos de Neoplasias/metabolismo , Ácidos Graxos/metabolismo , Linfoma de Células B/genética , Camundongos , Palmitatos , Tetraspaninas/genética , Tetraspaninas/metabolismo
9.
Nat Commun ; 13(1): 4578, 2022 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-35931688

RESUMO

Resistance to platinum-based chemotherapy represents a major clinical challenge for many tumors, including epithelial ovarian cancer. Patients often experience several response-relapse events, until tumors become resistant and life expectancy drops to 12-15 months. Despite improved knowledge of the molecular determinants of platinum resistance, the lack of clinical applicability limits exploitation of many potential targets, leaving patients with limited options. Serine biosynthesis has been linked to cancer growth and poor prognosis in various cancer types, however its role in platinum-resistant ovarian cancer is not known. Here, we show that a subgroup of resistant tumors decreases phosphoglycerate dehydrogenase (PHGDH) expression at relapse after platinum-based chemotherapy. Mechanistically, we observe that this phenomenon is accompanied by a specific oxidized nicotinamide adenine dinucleotide (NAD+) regenerating phenotype, which helps tumor cells in sustaining Poly (ADP-ribose) polymerase (PARP) activity under platinum treatment. Our findings reveal metabolic vulnerabilities with clinical implications for a subset of platinum resistant ovarian cancers.


Assuntos
Neoplasias Ovarianas , Platina , Carcinoma Epitelial do Ovário/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Recidiva Local de Neoplasia/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Platina/farmacologia , Platina/uso terapêutico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/farmacologia , Serina/farmacologia
10.
Redox Biol ; 55: 102403, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35863266

RESUMO

Defects in Coenzyme Q (CoQ) metabolism have been associated with primary mitochondrial disorders, neurodegenerative diseases and metabolic conditions. The consequences of CoQ deficiency have not been fully addressed, and effective treatment remains challenging. Here, we use mice with primary CoQ deficiency (Coq9R239X), and we demonstrate that CoQ deficiency profoundly alters the Q-junction, leading to extensive changes in the mitochondrial proteome and metabolism in the kidneys and, to a lesser extent, in the brain. CoQ deficiency also induces reactive gliosis, which mediates a neuroinflammatory response, both of which lead to an encephalopathic phenotype. Importantly, treatment with either vanillic acid (VA) or ß-resorcylic acid (ß-RA), two analogs of the natural precursor for CoQ biosynthesis, partially restores CoQ metabolism, particularly in the kidneys, and induces profound normalization of the mitochondrial proteome and metabolism, ultimately leading to reductions in gliosis, neuroinflammation and spongiosis and, consequently, reversing the phenotype. Together, these results provide key mechanistic insights into defects in CoQ metabolism and identify potential disease biomarkers. Furthermore, our findings clearly indicate that the use of analogs of the CoQ biosynthetic precursor is a promising alternative therapy for primary CoQ deficiency and has potential for use in the treatment of more common neurodegenerative and metabolic diseases that are associated with secondary CoQ deficiency.

11.
Front Immunol ; 13: 881166, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35844585

RESUMO

CD4+ conventional T cells (Tconvs) mediate adaptive immune responses, whereas regulatory T cells (Tregs) suppress those responses to safeguard the body from autoimmunity and inflammatory diseases. The opposing activities of Tconvs and Tregs depend on the stage of the immune response and their environment, with an orchestrating role for cytokine- and costimulatory receptors. Nutrient availability also impacts T-cell functionality via metabolic and biosynthetic processes that are largely unexplored. Many data argue that costimulation by Tumor Necrosis Factor Receptor 2 (TNFR2) favors support of Treg over Tconv responses and therefore TNFR2 is a key clinical target. Here, we review the pertinent literature on this topic and highlight the newly identified role of TNFR2 as a metabolic regulator for thymus-derived (t)Tregs. We present novel transcriptomic and metabolomic data that show the differential impact of TNFR2 on Tconv and tTreg gene expression and reveal distinct metabolic impact on both cell types.


Assuntos
Receptores Tipo II do Fator de Necrose Tumoral , Linfócitos T Reguladores , Citocinas/metabolismo , Imunidade , Contagem de Linfócitos , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo
12.
Mol Omics ; 18(1): 19-30, 2022 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-34879122

RESUMO

During the development of drug resistance, multiple myeloma (MM) cells undergo changes to their metabolism. However, how these metabolic changes can be exploited to improve treatment efficacy is not known. Here we demonstrate that targeting coenzyme Q10 (CoQ) biosynthesis through the mevalonate pathway works in synergy with the proteasome inhibitor bortezomib (BTZ) in MM. We show that gene expression signatures relating to the mitochondrial tricarboxylic acid (TCA) cycle and electron transport chain (ETC) predispose to clinical BTZ resistance and poor prognosis in MM patients. Mechanistically, BTZ-resistant cells show increased activity of glutamine-driven TCA cycle and oxidative phosphorylation, together with an increased vulnerability towards ETC inhibition. Moreover, BTZ resistance is accompanied by high levels of the mitochondrial electron carrier CoQ, while the mevalonate pathway inhibitor simvastatin increases cell death and decreases CoQ levels, specifically in BTZ-resistant cells. Both in vitro and in vivo, simvastatin enhances the effect of bortezomib treatment. Our study links CoQ synthesis to drug resistance in MM and provides a novel avenue for improving BTZ responses through statin-induced inhibition of mitochondrial metabolism.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Bortezomib , Mieloma Múltiplo , Ubiquinona , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Bortezomib/administração & dosagem , Bortezomib/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Humanos , Terapia de Alvo Molecular , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Sinvastatina/administração & dosagem , Sinvastatina/farmacologia , Ubiquinona/metabolismo
13.
Cells ; 10(9)2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34571936

RESUMO

Multiple myeloma (MM) is a hematological malignancy that exhibits aberrantly high levels of proteasome activity. While treatment with the proteasome inhibitor bortezomib substantially increases overall survival of MM patients, acquired drug resistance remains the main challenge for MM treatment. Using a combination treatment of docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) and bortezomib, it was demonstrated previously that pretreatment with DHA/EPA significantly increased bortezomib chemosensitivity in MM cells. In the current study, both transcriptome and metabolome analysis were performed to comprehensively evaluate the underlying mechanism. It was demonstrated that pretreating MM cells with DHA/EPA before bortezomib potently decreased the cellular glutathione (GSH) level and altered the expression of the related metabolites and key enzymes in GSH metabolism, whereas simultaneous treatment only showed minor effects on these factors, thereby suggesting the critical role of GSH degradation in overcoming bortezomib resistance in MM cells. Moreover, RNA-seq results revealed that the nuclear factor erythroid 2-related factor 2 (NRF2)-activating transcription factor 3/4 (ATF3/4)-ChaC glutathione specific gamma-glutamylcyclotransferase 1 (CHAC1) signaling pathway may be implicated as the central player in the GSH degradation. Pathways of necroptosis, ferroptosis, p53, NRF2, ATF4, WNT, MAPK, NF-κB, EGFR, and ERK may be connected to the tumor suppressive effect caused by pretreatment of DHA/EPA prior to bortezomib. Collectively, this work implicates GSH degradation as a potential therapeutic target in MM and provides novel mechanistic insights into its significant role in combating bortezomib resistance.


Assuntos
Biomarcadores Tumorais/metabolismo , Bortezomib/farmacologia , Ácidos Docosa-Hexaenoicos/farmacologia , Ácido Eicosapentaenoico/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glutationa/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Antineoplásicos/farmacologia , Biomarcadores Tumorais/genética , Humanos , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Transdução de Sinais , Células Tumorais Cultivadas
14.
Mol Cell ; 81(22): 4709-4721.e9, 2021 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-34562372

RESUMO

mRNA translation is a highly conserved and tightly controlled mechanism for protein synthesis. Despite protein quality control mechanisms, amino acid shortage in melanoma induces aberrant proteins by ribosomal frameshifting. The extent and the underlying mechanisms related to this phenomenon are yet unknown. Here, we show that tryptophan depletion-induced ribosomal frameshifting is a widespread phenomenon in cancer. We termed this event sloppiness and strikingly observed its association with MAPK pathway hyperactivation. Sloppiness is stimulated by RAS activation in primary cells, suppressed by pharmacological inhibition of the oncogenic MAPK pathway in sloppy cells, and restored in cells with acquired resistance to MAPK pathway inhibition. Interestingly, sloppiness causes aberrant peptide presentation at the cell surface, allowing recognition and specific killing of drug-resistant cancer cells by T lymphocytes. Thus, while oncogenes empower cancer progression and aggressiveness, they also expose a vulnerability by provoking the production of aberrant peptides through sloppiness.


Assuntos
Neoplasias/genética , Oncogenes , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Linfócitos T/citologia , Animais , Carcinogênese , Membrana Celular/metabolismo , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Mutação da Fase de Leitura , Mudança da Fase de Leitura do Gene Ribossômico , Humanos , Imunoterapia/métodos , Sistema de Sinalização das MAP Quinases , Melanoma/metabolismo , Camundongos , Neoplasias/metabolismo , Peptídeos/química , Inibidores de Proteínas Quinases , Ribossomos/metabolismo , Linfócitos T/metabolismo , Triptofano/química , Triptofano/metabolismo
15.
EMBO Mol Med ; 13(7): e13067, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34165243

RESUMO

Nephropathic cystinosis is a severe monogenic kidney disorder caused by mutations in CTNS, encoding the lysosomal transporter cystinosin, resulting in lysosomal cystine accumulation. The sole treatment, cysteamine, slows down the disease progression, but does not correct the established renal proximal tubulopathy. Here, we developed a new therapeutic strategy by applying omics to expand our knowledge on the complexity of the disease and prioritize drug targets in cystinosis. We identified alpha-ketoglutarate as a potential metabolite to bridge cystinosin loss to autophagy, apoptosis and kidney proximal tubule impairment in cystinosis. This insight combined with a drug screen revealed a bicalutamide-cysteamine combination treatment as a novel dual-target pharmacological approach for the phenotypical correction of cystinotic kidney proximal tubule cells, patient-derived kidney tubuloids and cystinotic zebrafish.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros , Cistinose , Sistemas de Transporte de Aminoácidos Neutros/genética , Anilidas , Animais , Cisteamina , Cistinose/tratamento farmacológico , Humanos , Nitrilas , Fenótipo , Compostos de Tosil , Peixe-Zebra
16.
Cancer Res ; 81(17): 4581-4593, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34158378

RESUMO

The HIV-protease inhibitor nelfinavir has shown broad anticancer activity in various preclinical and clinical contexts. In patients with advanced, proteasome inhibitor (PI)-refractory multiple myeloma, nelfinavir-based therapy resulted in 65% partial response or better, suggesting that this may be a highly active chemotherapeutic option in this setting. The broad anticancer mechanism of action of nelfinavir implies that it interferes with fundamental aspects of cancer cell biology. We combined proteome-wide affinity-purification of nelfinavir-interacting proteins with genome-wide CRISPR/Cas9-based screening to identify protein partners that interact with nelfinavir in an activity-dependent manner alongside candidate genetic contributors affecting nelfinavir cytotoxicity. Nelfinavir had multiple activity-specific binding partners embedded in lipid bilayers of mitochondria and the endoplasmic reticulum. Nelfinavir affected the fluidity and composition of lipid-rich membranes, disrupted mitochondrial respiration, blocked vesicular transport, and affected the function of membrane-embedded drug efflux transporter ABCB1, triggering the integrated stress response. Sensitivity to nelfinavir was dependent on ADIPOR2, which maintains membrane fluidity by promoting fatty acid desaturation and incorporation into phospholipids. Supplementation with fatty acids prevented the nelfinavir-induced effect on mitochondrial metabolism, drug-efflux transporters, and stress-response activation. Conversely, depletion of fatty acids/cholesterol pools by the FDA-approved drug ezetimibe showed a synergistic anticancer activity with nelfinavir in vitro. These results identify the modification of lipid-rich membranes by nelfinavir as a novel mechanism of action to achieve broad anticancer activity, which may be suitable for the treatment of PI-refractory multiple myeloma. SIGNIFICANCE: Nelfinavir induces lipid bilayer stress in cellular organelles that disrupts mitochondrial respiration and transmembrane protein transport, resulting in broad anticancer activity via metabolic rewiring and activation of the unfolded protein response.


Assuntos
Inibidores da Protease de HIV/farmacologia , Lipídeos de Membrana , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Nelfinavir/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Genoma , Glucose/metabolismo , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Lipidômica , Lipídeos/química , Fosfolipídeos/química , Fosforilação , Receptores de Adiponectina/metabolismo , Transdução de Sinais
17.
Nature ; 590(7845): 332-337, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33328638

RESUMO

Extensive tumour inflammation, which is reflected by high levels of infiltrating T cells and interferon-γ (IFNγ) signalling, improves the response of patients with melanoma to checkpoint immunotherapy1,2. Many tumours, however, escape by activating cellular pathways that lead to immunosuppression. One such mechanism is the production of tryptophan metabolites along the kynurenine pathway by the enzyme indoleamine 2,3-dioxygenase 1 (IDO1), which is induced by IFNγ3-5. However, clinical trials using inhibition of IDO1 in combination with blockade of the PD1 pathway in patients with melanoma did not improve the efficacy of treatment compared to PD1 pathway blockade alone6,7, pointing to an incomplete understanding of the role of IDO1 and the consequent degradation of tryptophan in mRNA translation and cancer progression. Here we used ribosome profiling in melanoma cells to investigate the effects of prolonged IFNγ treatment on mRNA translation. Notably, we observed accumulations of ribosomes downstream of tryptophan codons, along with their expected stalling at the tryptophan codon. This suggested that ribosomes bypass tryptophan codons in the absence of tryptophan. A detailed examination of these tryptophan-associated accumulations of ribosomes-which we term 'W-bumps'-showed that they were characterized by ribosomal frameshifting events. Consistently, reporter assays combined with proteomic and immunopeptidomic analyses demonstrated the induction of ribosomal frameshifting, and the generation and presentation of aberrant trans-frame peptides at the cell surface after treatment with IFNγ. Priming of naive T cells from healthy donors with aberrant peptides induced peptide-specific T cells. Together, our results suggest that IDO1-mediated depletion of tryptophan, which is induced by IFNγ, has a role in the immune recognition of melanoma cells by contributing to diversification of the peptidome landscape.


Assuntos
Apresentação de Antígeno , Mutação da Fase de Leitura , Melanoma/imunologia , Peptídeos/genética , Peptídeos/imunologia , Biossíntese de Proteínas/imunologia , Linfócitos T/imunologia , Linhagem Celular , Códon/genética , Mudança da Fase de Leitura do Gene Ribossômico/efeitos dos fármacos , Mudança da Fase de Leitura do Gene Ribossômico/genética , Mudança da Fase de Leitura do Gene Ribossômico/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Interferon gama/imunologia , Interferon gama/farmacologia , Melanoma/patologia , Peptídeos/química , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , Proteoma , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Triptofano/deficiência , Triptofano/genética , Triptofano/metabolismo
18.
Elife ; 92020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-33016874

RESUMO

The dependency of cancer cells on glutamine may be exploited therapeutically as a new strategy for treating cancers that lack druggable driver genes. Here we found that human liver cancer was dependent on extracellular glutamine. However, targeting glutamine addiction using the glutaminase inhibitor CB-839 as monotherapy had a very limited anticancer effect, even against the most glutamine addicted human liver cancer cells. Using a chemical library, we identified V-9302, a novel inhibitor of glutamine transporter ASCT2, as sensitizing glutamine dependent (GD) cells to CB-839 treatment. Mechanically, a combination of CB-839 and V-9302 depleted glutathione and induced reactive oxygen species (ROS), resulting in apoptosis of GD cells. Moreover, this combination also showed tumor inhibition in HCC xenograft mouse models in vivo. Our findings indicate that dual inhibition of glutamine metabolism by targeting both glutaminase and glutamine transporter ASCT2 represents a potential novel treatment strategy for glutamine addicted liver cancers.


Assuntos
Antineoplásicos/farmacologia , Glutamina/metabolismo , Neoplasias Hepáticas/metabolismo , Sistema ASC de Transporte de Aminoácidos/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Benzenoacetamidas/farmacologia , Proteínas de Transporte/antagonistas & inibidores , Linhagem Celular Tumoral , Sinergismo Farmacológico , Glutaminase/antagonistas & inibidores , Humanos , Camundongos , Antígenos de Histocompatibilidade Menor , Espécies Reativas de Oxigênio/metabolismo , Tiadiazóis/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
19.
EMBO J ; 38(21): e102147, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31523835

RESUMO

L-asparaginase (ASNase) serves as an effective drug for adolescent acute lymphoblastic leukemia. However, many clinical trials indicated severe ASNase toxicity in patients with solid tumors, with resistant mechanisms not well understood. Here, we took a functional genetic approach and identified SLC1A3 as a novel contributor to ASNase resistance in cancer cells. In combination with ASNase, SLC1A3 inhibition caused cell cycle arrest or apoptosis, and myriads of metabolic vulnerabilities in tricarboxylic acid (TCA) cycle, urea cycle, nucleotides biosynthesis, energy production, redox homeostasis, and lipid biosynthesis. SLC1A3 is an aspartate and glutamate transporter, mainly expressed in brain tissues, but high expression levels were also observed in some tumor types. Here, we demonstrate that ASNase stimulates aspartate and glutamate consumptions, and their refilling through SLC1A3 promotes cancer cell proliferation. Lastly, in vivo experiments indicated that SLC1A3 expression promoted tumor development and metastasis while negating the suppressive effects of ASNase by fueling aspartate, glutamate, and glutamine metabolisms despite of asparagine shortage. Altogether, our findings identify a novel role for SLC1A3 in ASNase resistance and suggest that restrictive aspartate and glutamate uptake might improve ASNase efficacy with solid tumors.


Assuntos
Asparaginase/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Transportador 1 de Aminoácido Excitatório/metabolismo , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Apoptose , Sistemas CRISPR-Cas , Proliferação de Células , Transportador 1 de Aminoácido Excitatório/antagonistas & inibidores , Transportador 1 de Aminoácido Excitatório/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias/enzimologia , Neoplasias/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Bioorg Med Chem Lett ; 29(18): 2626-2631, 2019 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-31362921

RESUMO

Pyrroline-5-carboxylate reductase 1 (PYCR1) is the final enzyme involved in the biosynthesis of proline and has been found to be upregulated in various forms of cancer. Due to the role of proline in maintaining the redox balance of cells and preventing apoptosis, PYCR1 is emerging as an attractive oncology target. Previous PYCR1 knockout studies led to a reduction in tumor growth. Accordingly, a small molecule inhibitor of PYCR1 could lead to new treatments for cancer, and a focused screening effort identified pargyline as a fragment-like hit. We report the design and synthesis of the first tool compounds as PYCR1 inhibitors, derived from pargyline, which were assayed to assess their ability to attenuate the production of proline. Structural activity studies have revealed the key determinants of activity, with the most potent compound (4) showing improved activity in vitro in enzyme (IC50 = 8.8 µM) and pathway relevant effects in cell-based assays.


Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Pargilina/farmacologia , Pirrolina Carboxilato Redutases/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Pargilina/síntese química , Pargilina/química , Pirrolina Carboxilato Redutases/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade
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