RESUMO
Lysine acylation can direct protein function, localization, and interactions. Sirtuins deacylate lysine towards maintaining cellular homeostasis, and their aberrant expression contributes to the pathogenesis of multiple pathological conditions, including cancer. Measuring sirtuins' activity is essential to exploring their potential as therapeutic targets, but accurate quantification is challenging. We developed 'SIRTify', a high-sensitivity assay for measuring sirtuin activity in vitro and in vivo. SIRTify is based on a split-version of the NanoLuc® luciferase consisting of a truncated, catalytically inactive N-terminal moiety (LgBiT) that complements with a high-affinity C-terminal peptide (p86) to form active luciferase. Acylation of two lysines within p86 disrupts binding to LgBiT and abates luminescence. Deacylation by sirtuins reestablishes p86 and restores binding, generating a luminescence signal proportional to sirtuin activity. Measurements accurately reflect reported sirtuin specificity for lysine acylations and confirm the effects of sirtuin modulators. SIRTify effectively quantifies lysine deacylation dynamics and may be adaptable to monitoring additional post-translational modifications.
RESUMO
Glycosaminoglycans (GAGs) play crucial roles in several biological processes including cell division, angiogenesis, anticoagulation, neurogenesis, axon guidance and growth, and viral and bacterial infections among others. The GAG cleaving hydrolases/lyases play a major role in the control of GAG structures, functions, and turn over. Dysregulation of GAG cleaving enzymes in vivo are linked to a number of human diseases including cancer, diabetes, atherosclerosis, arthritis, inflammation, and cardiovascular diseases. Several GAG cleaving enzymes are widely used for studying GAG glycobiology: heparitinases, chondroitinases, heparanases, hyaluronidases, and keratanases. Herein, we describe a method to synthesize four distinct nanometal surface energy transfer (NSET)-based gold-GAG-dye conjugates (nanosensors). Heparin, chondroitin sulfate, heparan sulfate, and hyaluronic acid are covalently linked with distinct fluorescent dyes and then immobilized on gold nanoparticles (AuNPs) to build nanosensors that serve as excellent substrates for GAG cleaving enzymes. Upon treatment of nanosensors with their respective GAG cleaving enzymes, dye-labeled oligosaccharides/disaccharides are released from AuNPs resulting in enhanced fluorescence recovery. These nanosensors have a great promise as diagnostic tools in various human pathophysiological conditions for detecting dysregulated expression of GAG cleaving enzymes and also as a sensitive analytical tool for assessing the quality control of pharmaceutical grade heparin polysaccharides that are produced in millions of small- and medium-sized animal slaughter houses worldwide.
Assuntos
Nanopartículas Metálicas , Animais , Sulfatos de Condroitina , Glicosaminoglicanos , Ouro , Heparina , Heparitina Sulfato , HumanosRESUMO
We discovered that the survival and growth of many primary acute myeloid leukemia (AML) samples and cell lines, but not normal CD34+ cells, are dependent on SIRT5, a lysine deacylase implicated in regulating multiple metabolic pathways. Dependence on SIRT5 is genotype-agnostic and extends to RAS- and p53-mutated AML. Results were comparable between SIRT5 knockdown and SIRT5 inhibition using NRD167, a potent and selective SIRT5 inhibitor. Apoptosis induced by SIRT5 disruption is preceded by reductions in oxidative phosphorylation and glutamine utilization, and an increase in mitochondrial superoxide that is attenuated by ectopic superoxide dismutase 2. These data indicate that SIRT5 controls and coordinates several key metabolic pathways in AML and implicate SIRT5 as a vulnerability in AML.
Assuntos
Leucemia Mieloide Aguda , Sirtuínas , Apoptose , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Lisina/metabolismo , Mitocôndrias/genética , Fosforilação Oxidativa , Sirtuínas/genéticaRESUMO
To map the cellular topography of the rare 3-O-sulfated structural motif of heparan sulfate (HS), we constructed quantum dot-based probes for antithrombin and FGF2, which reveal widely different distribution of the targeted HS motifs. The technology helps show that old and young aortic endothelia display widely different levels of the antithrombin-binding 3-O-sulfated HS motif.
Assuntos
Antitrombinas/química , Membrana Celular/metabolismo , Heparitina Sulfato/química , Sulfotransferases/metabolismo , Motivos de Aminoácidos , Animais , Células CHO , Membrana Celular/ultraestrutura , Cricetulus , Células Endoteliais , Fator 2 de Crescimento de Fibroblastos/química , Humanos , Camundongos Endogâmicos C57BL , Imagem Óptica , Ligação Proteica , Pontos Quânticos/químicaRESUMO
FLT3-ITD mutations occur in 20-30% of AML patients and are associated with aggressive disease. Patients with relapsed FLT3-mutated disease respond well to 2nd generation FLT3 TKIs but inevitably relapse within a short timeframe. In this setting, until overt relapse occurs, the bone marrow microenvironment facilitates leukemia cell survival despite continued on-target inhibition. We demonstrate that human bone marrow derived conditioned medium (CM) protects FLT3-ITD+ AML cells from the 2nd generation FLT3 TKI quizartinib and activates STAT3 and STAT5 in leukemia cells. Extrinsic activation of STAT5 by CM is the primary mediator of leukemia cell resistance to FLT3 inhibition. Combination treatment with quizartinib and dasatinib abolishes STAT5 activation and significantly reduces the IC50 of quizartinib in FLT3-ITD+ AML cells cultured in CM. We demonstrate that CM protects FLT3-ITD+ AML cells from the inhibitory effects of quizartinib on glycolysis and that this is partially reversed by treating cells with the combination of quizartinib and dasatinib. Using a doxycycline-inducible STAT5 knockdown in the FLT3-ITD+ MOLM-13 cell line, we show that dasatinib-mediated suppression of leukemia cell glycolytic activity is STAT5-independent and provide a preclinical rationale for combination treatment with quizartinib and dasatinib in FLT3-ITD+ AML.
Assuntos
Benzotiazóis/farmacologia , Dasatinibe/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos/genética , Metabolismo Energético , Duplicação Gênica , Técnicas de Silenciamento de Genes , Glicólise , Humanos , Fosforilação , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
BCR-ABL1 tyrosine kinase inhibitors (TKIs) are the cornerstone of treatment in chronic myeloid leukemia. Although there are now four TKIs approved for use in the front-line setting, acquired TKI resistance via secondary kinase domain mutations remains a problem for patients. K0706 is a novel BCR-ABL1 TKI currently under clinical investigation with structural elements similar to those of ponatinib and dasatinib. In this article, we functionally characterize the anti-leukemic activity of K0706 using cell proliferation assays in conjunction with drug resistance screening. We provide details from molecular modeling to support our in vitro findings and additionally describe our limited clinical experience with this drug in two patients treated on trial. We demonstrate that although K0706 retains efficacy against a large spectrum of clinically relevant mutations, it does not appear to have activity against BCR-ABL1T315I. Early trial experience suggests excellent tolerability, which may positively affect the place of K0706 within the ever-expanding chronic myeloid leukemia treatment paradigm.
Assuntos
Proliferação de Células/efeitos dos fármacos , Proteínas de Fusão bcr-abl , Leucemia Mielogênica Crônica BCR-ABL Positiva , Cromossomo Filadélfia , Inibidores de Proteínas Quinases/farmacologia , Animais , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/genética , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Masculino , CamundongosRESUMO
BCR-ABL1 point mutation-mediated resistance to tyrosine kinase inhibitor (TKI) therapy in Philadelphia chromosome-positive (Ph+) leukemia is effectively managed with several approved drugs, including ponatinib for BCR-ABL1T315I-mutant disease. However, therapy options are limited for patients with leukemic clones bearing multiple BCR-ABL1 mutations. Asciminib, an allosteric inhibitor targeting the myristoyl-binding pocket of BCR-ABL1, is active against most single mutants but ineffective against all tested compound mutants. We demonstrate that combining asciminib with ATP site TKIs enhances target inhibition and suppression of resistant outgrowth in Ph+ clinical isolates and cell lines. Inclusion of asciminib restores ponatinib's effectiveness against currently untreatable compound mutants at clinically achievable concentrations. Our findings support combining asciminib with ponatinib as a treatment strategy for this molecularly defined group of patients.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Imidazóis/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Niacinamida/análogos & derivados , Pirazóis/farmacologia , Piridazinas/farmacologia , Regulação Alostérica/efeitos dos fármacos , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/genética , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Imidazóis/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Camundongos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Terapia de Alvo Molecular/métodos , Mutação , Niacinamida/farmacologia , Niacinamida/uso terapêutico , Cultura Primária de Células , Pirazóis/uso terapêutico , Piridazinas/uso terapêuticoAssuntos
Proteínas Angiogênicas/genética , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Pré-Escolar , Evolução Fatal , Rearranjo Gênico , Humanos , Masculino , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
Angiogenesis, the sprouting of new blood vessels from existing vasculature, involves multiple complex biological processes, and it is an essential step for hemostasis, tissue healing and regeneration. Angiogenesis stimulants can ameliorate human disease conditions including limb ischemia, chronic wounds, heart disease, and stroke. The current strategies to improve the bioavailability of pro-angiogenic growth factors, including VEGF and FGF2, have remained largely unsuccessful. This study demonstrates that small molecules, termed click-xylosides, can promote angiogenesis in the in vitro matrigel tube formation assay and the ex ovo chick chorioallantoic membrane assay, depending on their aglycone moieties. Xyloside treatment enhances network connectivity and cell survivability, thereby, maintaining the network structures on matrigel culture for an extended period of time. These effects were achieved via the secreted xyloside-primed glycosaminoglycans (GAG) chains that in part, act through an ERK1/2 mediated signaling pathway. Through the remodeling of GAGs in the extracellular matrix of endothelial cells, the glycan approach, involving xylosides, offers great potential to effectively promote therapeutic angiogenesis.