RESUMEN
BACKGROUND: Janus kinase (JAK) 2 plays pivotal roles in signaling by several cytokine receptors. The mutant JAK2 V617F is the most common molecular event associated with myeloproliferative neoplasms. Selective targeting of the mutant would be ideal for treating these pathologies by sparing essential JAK2 functions. OBJECTIVE: We characterize inhibitory strategies for JAK2 V617F and assess their effect on physiologic signaling by distinct cytokine receptors. METHODS: Through structure-guided mutagenesis, we assessed the role of key residues around F617 and used a combination of cellular and biochemical assays to measure the activity of JAKs in reconstituted cells. We also assessed the effect of several specific JAK2 V617F inhibitory mutations on receptor dimerization using the NanoBiT protein complementation approach. RESULTS: We identified a novel Janus kinase homology 2 (JH2) αC mutation, A598F, which is suggested to inhibit the aromatic stacking between F617 with F594 and F595. Like other JAK2 V617F inhibitory mutations, A598F decreased oncogenic activation and spared cytokine activation while preventing JAK2 V617F-promoted erythropoietin receptor dimerization. Surprisingly, A598F and other V617F-inhibiting mutations (F595A, E596R, and F537A) significantly impaired IFN-γ signaling. This was specific for IFN-γ because the inhibitory mutations preserved responses to ligands of a series of receptor complexes. Similarly, homologous mutations in JAK1 prevented signaling by IFN-γ. CONCLUSIONS: The JH2 αC region, which is required for JAK2 V617F hyperactivation, is crucial for relaying cytokine-induced signaling of the IFN-γ receptor. We discuss how strategies aiming to inhibit JAK2 V617F could be used for identifying inhibitors of IFN-γ signaling.
Asunto(s)
Janus Quinasa 2/genética , Receptores de Citocinas/metabolismo , Animales , Línea Celular , Humanos , Janus Quinasa 2/metabolismo , Ratones , Mutación , Transducción de SeñalRESUMEN
Three-part white blood cell differentials which are key to routine blood workups are typically performed in centralized laboratories on conventional hematology analyzers operated by highly trained staff. With the trend of developing miniaturized blood analysis tool for point-of-need in order to accelerate turnaround times and move routine blood testing away from centralized facilities on the rise, our group has developed a highly miniaturized holographic imaging system for generating lens-free images of white blood cells in suspension. Analysis and classification of its output data, constitutes the final crucial step ensuring appropriate accuracy of the system. In this work, we implement reference holographic images of single white blood cells in suspension, in order to establish an accurate ground truth to increase classification accuracy. We also automate the entire workflow for analyzing the output and demonstrate clear improvement in the accuracy of the 3-part classification. High-dimensional optical and morphological features are extracted from reconstructed digital holograms of single cells using the ground-truth images and advanced machine learning algorithms are investigated and implemented to obtain 99% classification accuracy. Representative features of the three white blood cell subtypes are selected and give comparable results, with a focus on rapid cell recognition and decreased computational cost.
Asunto(s)
Citometría de Flujo/métodos , Holografía/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Leucocitos/citología , Análisis de la Célula Individual/métodos , Algoritmos , Diseño de Equipo , Citometría de Flujo/instrumentación , Holografía/instrumentación , Humanos , Aprendizaje Automático , Miniaturización , Análisis de la Célula Individual/instrumentaciónRESUMEN
Safe, high-rate and cost-effective cell sorting is important for clinical cell isolation. However, commercial fluorescence-activated cell sorters (FACS) are expensive and prone to aerosol-induced sample contamination. Here we report a microfluidic cell sorter allowing high rate and fully enclosed cell sorting. The sorter chip consists of an array of micro heating hotspots. Pulsed resistive heating in the hotspots produces numerous micro vapor bubbles with short duration, which gives rise to a rapid jet flow for cell sorting. With this method, we demonstrated high sorting rate comparable to commercial FACS and the significant enrichment of rare cancer cells. This vapor bubble based cell sorting method can be a powerful tool for contamination-free and affordable clinical cell sorting such as circulating tumor cell isolation and cancer cell therapy.
Asunto(s)
Citometría de Flujo/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Línea Celular Tumoral , Diseño de Equipo , Citometría de Flujo/métodos , Humanos , Técnicas Analíticas Microfluídicas/métodosRESUMEN
The mechanisms by which JAK2 is activated by the prevalent pseudokinase (JH2) V617F mutation in blood cancers remain elusive. Via structure-guided mutagenesis and transcriptional and functional assays, we identify a community of residues from the JH2 helix αC, SH2-JH2 linker and JH1 kinase domain that mediate V617F-induced activation. This circuit is broken by altering the charge of residues along the solvent-exposed face of the JH2 αC, which is predicted to interact with the SH2-JH2 linker and JH1. Mutations that remove negative charges or add positive charges, such as E596A/R, do not alter the JH2 V617F fold, as shown by the crystal structure of JH2 V617F E596A. Instead, they prevent kinase domain activation via modulation of the C-terminal residues of the SH2-JH2 linker. These results suggest strategies for selective V617F JAK2 inhibition, with preservation of wild-type function.
Asunto(s)
Citocinas/farmacología , Janus Quinasa 2/química , Janus Quinasa 2/metabolismo , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Citometría de Flujo , Humanos , Janus Quinasa 2/genética , Mutación/genética , Fosforilación/efectos de los fármacos , Dominios Proteicos/genética , Dominios Proteicos/fisiologíaRESUMEN
A compelling clinical need exists for inexpensive, portable haematology analyzers that can be utilized at the point-of-care in emergency settings or in resource-limited settings. Development of a label-free, microfluidic blood analysis platform is the first step towards such a miniaturized, cost-effective system. Here we assemble a compact lens-free in-line holographic microscope and employ it to image blood cells flowing in a microfluidic chip, using a high-speed camera and stroboscopic illumination. Numerical reconstruction of the captured holograms allows classification of unlabeled leukocytes into three main subtypes: lymphocytes, monocytes and granulocytes. A scale-space recognition analysis to evaluate cellular size and internal complexity is also developed and used to build a 3-part leukocyte differential. The lens-free image-based classification is compared to the 3-part white blood cell differential generated by using a conventional analyzer on the same blood sample and is found to be in good agreement with it.
Asunto(s)
Citometría de Flujo/instrumentación , Dispositivos Laboratorio en un Chip , Leucocitos/citología , Técnicas Analíticas Microfluídicas , Voluntarios Sanos , Humanos , Técnicas Analíticas Microfluídicas/instrumentaciónRESUMEN
The discovery of the highly prevalent activating JAK (Janus kinase) 2 V617F mutation in myeloproliferative neoplasms, and of other pseudokinase domain-activating mutations in JAK2, JAK1 and JAK3 in blood cancers, prompted great interest in understanding how pseudokinase domains regulate kinase domains in JAKs. Recent functional and mutagenesis studies identified residues required for the V617F mutation to induce activation. Several X-ray crystal structures of either kinase or pseudokinase domains including the V617F mutant of JAK2 pseudokinase domains are now available, and a picture has emerged whereby the V617F mutation induces a defined conformational change around helix C of JH (JAK homology) 2. Effects of mutations on JAK2 can be extrapolated to JAK1 and TYK2 (tyrosine kinase 2), whereas JAK3 appears to be different. More structural information of the full-length JAK coupled to cytokine receptors might be required in order to define the structural basis of JH1 activation by JH2 mutants and eventually obtain mutant-specific inhibitors.
Asunto(s)
Neoplasias Hematológicas/enzimología , Quinasas Janus/metabolismo , Mutación , Trastornos Mieloproliferativos/enzimología , Humanos , Quinasas Janus/química , Quinasas Janus/genética , Modelos MolecularesRESUMEN
The association between somatic JAK2 mutation and myeloproliferative neoplasms (MPNs) is now well established. However, because JAK2 mutations are associated with heterogeneous clinical phenotypes and often occur as secondary genetic events, some aspects of JAK2 mutation biology remain to be understood. We recently described a germline JAK2V617I mutation in a family with hereditary thrombocytosis and herein characterize the hematopoietic and signaling impact of JAK2V617I. Through targeted sequencing of MPN-associated mutations, exome sequencing, and clonality analysis, we demonstrate that JAK2V617I is likely to be the sole driver mutation in JAK2V617I-positive individuals with thrombocytosis. Phenotypic hematopoietic stem cells (HSCs) were increased in the blood and bone marrow of JAK2V617I-positive individuals and were sustained at higher levels than controls after xenotransplantation. In signaling and transcriptional assays, JAK2V617I demonstrated more activity than wild-type JAK2 but substantially less than JAK2V617F. After cytokine stimulation, JAK2V617I resulted in markedly increased downstream signaling compared with wild-type JAK2 and comparable with JAK2V617F. These findings demonstrate that JAK2V617I induces sufficient cytokine hyperresponsiveness in the absence of other molecular events to induce a homogeneous MPN-like phenotype. We also provide evidence that the JAK2V617I mutation may expand the HSC pool, providing insights into both JAK2 mutation biology and MPN disease pathogenesis.
Asunto(s)
Mutación de Línea Germinal/fisiología , Hematopoyesis/genética , Janus Quinasa 2/genética , Adulto , Sustitución de Aminoácidos/fisiología , Animales , Células Cultivadas , Familia , Femenino , Hematopoyesis/fisiología , Humanos , Isoleucina/genética , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Persona de Mediana Edad , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/fisiopatología , Valina/genéticaRESUMEN
The constitutively active JAK2 V617F mutant is the major determinant of human myeloproliferative neoplasms (MPNs). We show that coexpression of murine JAK2 V617F and the murine thrombopoietin (Tpo) receptor (TpoR, c-MPL) in hematopoietic cell lines or heterozygous knock-in of JAK2 V617F in mice leads to down-modulation of TpoR levels. Enhanced TpoR ubiquitinylation, proteasomal degradation, reduced recycling, and maturation are induced by the constitutive JAK2 V617F activity. These effects can be prevented in cell lines by JAK2 and proteasome inhibitors. Restoration of TpoR levels by inhibitors could be detected in platelets from JAK2 inhibitor-treated myelofibrosis patients that express the JAK2 V617F mutant, and in platelets from JAK2 V617F knock-in mice that were treated in vivo with JAK2 or proteasome inhibitors. In addition, we show that Tpo can induce both proliferative and antiproliferative effects via TpoR at low and high JAK2 activation levels, respectively, or on expression of JAK2 V617F. The antiproliferative signaling and receptor down-modulation by JAK2 V617F were dependent on signaling via TpoR cytosolic tyrosine 626. We propose that selection against TpoR antiproliferative signaling occurs by TpoR down-modulation and that restoration of down-modulated TpoR levels could become a biomarker for the treatment of MPNs.
Asunto(s)
Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/fisiología , Inhibidores de Proteasoma , Inhibidores de Proteínas Quinasas/farmacología , Receptores de Trombopoyetina/genética , Sustitución de Aminoácidos/genética , Sustitución de Aminoácidos/fisiología , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Células HEK293 , Humanos , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación Missense/fisiología , Fenilalanina/genética , Receptor Cross-Talk/efectos de los fármacos , Receptor Cross-Talk/fisiología , Receptores de Trombopoyetina/metabolismo , Transducción de Señal/efectos de los fármacos , Valina/genéticaRESUMEN
BACKGROUND: Activating mutations in JAK1 and JAK2 have been described in patients with various hematologic malignancies including acute lymphoblastic leukemia and myeloproliferative neoplasms, leading to clinical trials with JAK inhibitors. While there has been a tremendous effort towards the development of specific JAK inhibitors, mutations conferring resistance to such drugs have not yet been observed. DESIGN AND METHODS: Taking advantage of a model of spontaneous cellular transformation, we sequenced JAK1 in selected tumorigenic BaF3 clones and identified 25 de novo JAK1 activating mutations, including 5 mutations already described in human leukemias. We further used this library of JAK1 mutation-positive cell lines to assess their sensitivity to ATP-competitive inhibitors. RESULTS: While most JAK1 mutants were sensitive to ATP-competitive JAK inhibitors, mutations targeting Phe958 and Pro960 in the hinge region of the kinase domain rendered JAK1 constitutively active but also resistant to all tested JAK inhibitors. Furthermore, mutation of the homologous Tyr931 in JAK2 wild-type or JAK2 V617F mutant found in patients with myeloproliferative neoplasms also conferred resistance to JAK inhibitors, such as INCB018424, which is currently in clinical use. CONCLUSIONS: Our data indicate that some activating mutations not only promote autonomous cell proliferation but also confer resistance to ATP-competitive inhibitors. In vivo, such a mutation can potentially occur as primary JAK-activating mutations but also as secondary mutations combining oncogenicity with drug resistance.
Asunto(s)
Adenosina Trifosfato/antagonistas & inhibidores , Resistencia a Antineoplásicos/genética , Janus Quinasa 1/genética , Janus Quinasa 2/genética , Mutación/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/fisiopatología , Animales , Unión Competitiva/efectos de los fármacos , Células CHO , Línea Celular Tumoral , Cricetinae , Cricetulus , Orden Génico , Janus Quinasa 1/antagonistas & inhibidores , Janus Quinasa 2/antagonistas & inhibidores , Ratones , Modelos Moleculares , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Estructura Secundaria de Proteína , Factor de Transcripción STAT5/antagonistas & inhibidores , Factor de Transcripción STAT5/metabolismo , Transducción de Señal/genéticaRESUMEN
The JAK2 V617F mutation present in over 95% of Polycythemia Vera patients and in 50% of Essential Thrombocythemia and Primary Myelofibrosis patients renders the kinase constitutively active. In the absence of a three-dimensional structure for the full-length protein, the mechanism of activation of JAK2 V617F has remained elusive. In this study, we used functional mutagenesis to investigate the involvement of the JH2 alphaC helix in the constitutive activation of JAK2 V617F. We show that residue F595, located in the middle of the alphaC helix of JH2, is indispensable for the constitutive activity of JAK2 V617F. Mutation of F595 to Ala, Lys, Val or Ile significantly decreases the constitutive activity of JAK2 V617F, but F595W and F595Y are able to restore it, implying an aromaticity requirement at position 595. Substitution of F595 to Ala was also able to decrease the constitutive activity of two other JAK2 mutants, T875N and R683G, as well as JAK2 K539L, albeit to a lower extent. In contrast, the F595 mutants are activated by erythropoietin-bound EpoR. We also explored the relationship between the dimeric conformation of EpoR and several JAK2 mutants. Since residue F595 is crucial to the constitutive activation of JAK2 V617F but not to initiation of JAK2 activation by cytokines, we suggest that small molecules that target the region around this residue might specifically block oncogenic JAK2 and spare JAK2 wild-type.
Asunto(s)
Janus Quinasa 2/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Secuencia de Aminoácidos , Línea Celular , Activación Enzimática , Humanos , Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/química , Janus Quinasa 2/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Homología de Secuencia de AminoácidoRESUMEN
The four mammalian Janus kinase (JAK) family members, JAK1, JAK2, JAK3 and TYK2, are non-receptor protein tyrosine kinases (PTKs) that are crucial for cytokine receptor signaling in blood formation and immune responses. Mutations and translocations in the JAK genes leading to constitutively active JAK proteins are associated with a variety of hematopoietic malignancies, including the myeloproliferative disorders (JAK2), acute lymphoblastic leukemia (JAK2), acute myeloid leukemia (JAK2, JAK1), acute megakaryoblastic leukemia (JAK2, JAK3) and T-cell precursor acute lymphoblastic leukemia (JAK1). In contrast, loss-of-function mutations of JAK3 and TYK2 lead to immunodeficiency. The role of JAKs as therapeutic targets is starting to expand, as more insights into their structure and activation mechanisms become available.
Asunto(s)
Neoplasias Hematológicas/metabolismo , Síndromes de Inmunodeficiencia/metabolismo , Isoenzimas/metabolismo , Quinasas Janus/metabolismo , Transducción de Señal/fisiología , Animales , Activación Enzimática , Humanos , Quinasas Janus/química , Quinasas Janus/genética , Mutación , Receptores de Citocinas/química , Receptores de Citocinas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Explaining the uniqueness of the acquired somatic JAK2 V617F mutation, which is present in more than 95% of polycythemia vera patients, has been a challenge. The V617F mutation in the pseudokinase domain of JAK2 renders the unmutated kinase domain constitutively active. We have performed random mutagenesis at position 617 of JAK2 and tested each of the 20 possible amino acids for ability to induce constitutive signaling in Ba/F3 cells expressing the erythropoietin receptor. Four JAK2 mutants, V617W, V617M, V617I, and V617L, were able to induce cytokine independence and constitutive downstream signaling. Only V617W induced a level of constitutive activation comparable with V617F. Also, only V617W stabilized tyrosine-phosphorylated suppressor of cytokine signaling 3 (SOCS3), a mechanism by which JAK2 V617F overcomes inhibition by SOCS3. The V617W mutant induced a myeloproliferative disease in mice, mainly characterized by erythrocytosis and megakaryocytic proliferation. Although JAK2 V617W would predictably be pathogenic in humans, the substitution of the Val codon, GTC, by TTG, the codon for Trp, would require three base pair changes, and thus it is unlikely to occur. We discuss how the predicted conformations of the activated JAK2 mutants can lead to better screening assays for novel small molecule inhibitors.
Asunto(s)
Janus Quinasa 2/metabolismo , Animales , Trasplante de Médula Ósea , Línea Celular , Proliferación Celular , Activación Enzimática , Regulación Enzimológica de la Expresión Génica , Humanos , Janus Quinasa 2/genética , Ratones , Mutación/genética , Fosfotirosina/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/genética , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Valina/genética , Valina/metabolismoRESUMEN
The aggregation of the presynaptic protein alpha-synuclein is associated with Parkinson's disease (PD). The details of the mechanism of aggregation, as well as the cytotoxic species, are currently not well understood. alpha-Synuclein has four tyrosine and no tryptophan residues. We introduced a tyrosine to tryptophan mutation at position 39 to create an intrinsic fluorescence probe and allow additional characterization of the aggregation process. Y39W alpha-synuclein had similar fibrillation kinetics (2-fold slower), pH-induced conformational changes, and fibril morphology to wild-type alpha-synuclein. In addition to intrinsic Trp fluorescence, acrylamide quenching, fluorescence anisotropy, ANS binding, dynamic light scattering, and FTIR were employed to monitor the kinetics of aggregation. These biophysical probes revealed the significant population of two classes of oligomeric intermediates, one formed during the lag period of fibrillation and the other present at the completion of fibrillation. As expected for a natively unfolded protein, Trp 39 was highly solvent-exposed in the monomer and is solvent-exposed in the two oligomeric intermediates; however, it is partially, but not fully, buried in the fibrils. These observations demonstrate the utility of Trp fluorescence labeled alpha-synuclein and demonstrate the existence of an oligomeric intermediate that exists as a transient reservoir of alpha-synuclein for fibrillation.