Activating Janus kinase pseudokinase domain mutations in myeloproliferative and other blood cancers

2013 ◽  
Vol 41 (4) ◽  
pp. 1048-1054 ◽  
Author(s):  
Stefan N. Constantinescu ◽  
Emilie Leroy ◽  
Vitalina Gryshkova ◽  
Christian Pecquet ◽  
Alexandra Dusa
Keyword(s):  
Structural Information ◽  
Myeloproliferative Neoplasms ◽  
Janus Kinase ◽  
Cytokine Receptors ◽  
Structural Basis ◽  
X Ray ◽  
Activating Mutations ◽  
Tyrosine Kinase 2 ◽  
V617f Mutation ◽  
Specific Inhibitors

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.

Should We Screen for Janus Kinase 2 V617F Mutation in Cerebral Venous Thrombosis?

2017 ◽  
Vol 44 (3-4) ◽  
pp. 97-104 ◽  
Author(s):  
Matthias Lamy ◽  
Paola Palazzo ◽  
Pierre Agius ◽  
Jean Claude Chomel ◽  
Jonathan Ciron ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Cerebral Venous Thrombosis ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Janus Kinase ◽  
Jak2 V617f Mutation ◽  
Janus Kinase 2 ◽  
Jak2 Mutation ◽  
Venous Thromboembolic Events ◽  
V617f Mutation

Background: The presence of Janus Kinase 2 (JAK2) V617F mutation represents a major diagnostic criterion for detecting myeloproliferative neoplasms (MPN) and even in the absence of overt MPN, JAK2 V617F mutation is associated with splanchnic vein thrombosis. However, the actual prevalence and diagnostic value of the JAK2 V617F mutation in patients with cerebral venous thrombosis (CVT) are not known. The aims of this study were to assess the prevalence of JAK2 V617F mutation in a large group of consecutive CVT patients, to detect clinical, biological, and radiological features associated with the mutation, and to determine the long-term venous thrombosis recurrence rate in CVT patients with JAK2 mutation but without overt MPN in order to recommend the best preventive treatment. Methods: This was a prospective study conducted on consecutive patients with a first-ever radiologically confirmed CVT. JAK2 V617F mutation analysis was assessed in all the study subjects. JAK2 V617F-positive patients were followed up to detect new venous thrombotic events. Results: Of the 125 included subjects, 7 were found to have JAK2 V617F mutation (5.6%; 95% CI 2.3-11.2). Older age (p = 0.039) and higher platelet count (p = 0.004) were independently associated with JAK2 V617F positivity in patients without overt MPN. During a mean follow-up period of 59 (SD 46) months, 2 JAK2 V617F-positive patients presented with 4 new venous thromboembolic events. Conclusions: Screening for the JAK2 V617F mutation in CVT patients seems to be useful even in the absence of overt MPN and/or in the presence of other risk factors for CVT because of its relatively high prevalence and the risk of thrombosis recurrence.

scholarly journals Structural basis of the dynamic human CEACAM1 monomer-dimer equilibrium

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Amit K. Gandhi ◽  
Zhen-Yu J. Sun ◽  
Walter M. Kim ◽  
Yu-Hwa Huang ◽  
Yasuyuki Kondo ◽  
...  
Keyword(s):  
Cell Adhesion Molecule ◽  
Nmr Assignment ◽  
Structural Information ◽  
Microbial Pathogens ◽  
Structural Basis ◽  
X Ray ◽  
X Ray Crystallography ◽  
Related Cell ◽  
Differential Scanning Fluorimetry ◽  
Cell Adhesion Molecule 1

AbstractHuman (h) carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) function depends upon IgV-mediated homodimerization or heterodimerization with host ligands, including hCEACAM5, hTIM-3, PD-1, and a variety of microbial pathogens. However, there is little structural information available on how hCEACAM1 transitions between monomeric and dimeric states which in the latter case is critical for initiating hCEACAM1 activities. We therefore mutated residues within the hCEACAM1 IgV GFCC′ face including V39, I91, N97, and E99 and examined hCEACAM1 IgV monomer-homodimer exchange using differential scanning fluorimetry, multi-angle light scattering, X-ray crystallography and/or nuclear magnetic resonance. From these studies, we describe hCEACAM1 homodimeric, monomeric and transition states at atomic resolution and its conformational behavior in solution through NMR assignment of the wildtype (WT) hCEACAM1 IgV dimer and N97A mutant monomer. These studies reveal the flexibility of the GFCC’ face and its important role in governing the formation of hCEACAM1 dimers and selective heterodimers.

scholarly journals JAK2-V617F mutation in patients with myeloproliferative neoplasms: Association with FLT3-ITD mutation

2010 ◽  
Vol 138 (9-10) ◽  
pp. 614-618
Author(s):  
Vesna Spasovski ◽  
Natasa Tosic ◽  
Tatjana Kostic ◽  
Sonja Pavlovic ◽  
Milica Colovic
Keyword(s):  
Cell Proliferation ◽  
Myeloproliferative Neoplasms ◽  
Myeloid Cell ◽  
Jak2 V617f ◽  
Signalling Pathway ◽  
Janus Kinase ◽  
Jak2 V617f Mutation ◽  
Polycythaemia Vera ◽  
Jak2 Mutation ◽  
V617f Mutation

Introduction. An acquired somatic mutation V617F in Janus kinase 2 gene (JAK2) is the cause of uncontrolled proliferation in patients with myeloproliferative neoplasms. It is known that uncontrolled myeloid cell proliferation is also provoked by alteration in other genes, e.g. mutations in receptor tyrosine kinase FLT3 gene. FLT3 represents the most frequently mutated gene in acute myeloid leukaemia. Interestingly, mutated FLT3- ITD (internal tandem duplication) protein is a member of the same signalling pathway as JAK2 protein, the STAT5 signalling pathway. STAT5 activation is recognized as important for selfrenewal of haematopoetic stem cells. Objective. The aim of this study was the detection of JAK2- V617F mutation in patients with myeloproliferative neoplasms. Additionally, we investigated the presence of FLT3-ITD mutation in JAK2-V617F-positive patients in order to shed the light on the hypothesis of a similar role of these two molecular markers in haematological malignancies. Methods. Using allele-specific PCR, 61 patients with known or suspected diagnosis of myeloproliferative neoplasms were tested for the presence of JAK2-V617F mutation. Samples that were positive for JAK2 mutation were subsequently tested for the presence of FLT3-ITD mutation by PCR. Results. Eighteen of 61 analysed patients were positive for JAK2-V617F mutation. Among them, 8/18 samples were diagnosed as polycythaemia vera, and 10/18 as essential thrombocythaemia. None of JAK2-V617F-positive patient was positive for FLT3-ITD mutation. Conclusion. This study suggests that one activating mutation is sufficient for aberrant cell proliferation leading to malignant transformation of haematopoetic stem cell.

scholarly journals CYT387, a novel JAK2 inhibitor, induces hematologic responses and normalizes inflammatory cytokines in murine myeloproliferative neoplasms

Blood ◽  
2010 ◽  
Vol 115 (25) ◽  
pp. 5232-5240 ◽  
Author(s):  
Jeffrey W. Tyner ◽  
Thomas G. Bumm ◽  
Jutta Deininger ◽  
Lisa Wood ◽  
Karl J. Aichberger ◽  
...  
Keyword(s):  
Cell Lines ◽  
Inflammatory Cytokines ◽  
Kinase Inhibitor ◽  
Myeloproliferative Neoplasms ◽  
Symptomatic Improvement ◽  
Janus Kinase ◽  
Jak2 Inhibitor ◽  
Cell Counts ◽  
Tyrosine Kinase 2 ◽  
Jak2 Inhibitors

Abstract Activating alleles of Janus kinase 2 (JAK2) such as JAK2V617F are central to the pathogenesis of myeloproliferative neoplasms (MPN), suggesting that small molecule inhibitors targeting JAK2 may be therapeutically useful. We have identified an aminopyrimidine derivative (CYT387), which inhibits JAK1, JAK2, and tyrosine kinase 2 (TYK2) at low nanomolar concentrations, with few additional targets. Between 0.5 and 1.5μM CYT387 caused growth suppression and apoptosis in JAK2-dependent hematopoietic cell lines, while nonhematopoietic cell lines were unaffected. In a murine MPN model, CYT387 normalized white cell counts, hematocrit, spleen size, and restored physiologic levels of inflammatory cytokines. Despite the hematologic responses and reduction of the JAK2V617F allele burden, JAK2V617F cells persisted and MPN recurred upon cessation of treatment, suggesting that JAK2 inhibitors may be unable to eliminate JAK2V617F cells, consistent with preliminary results from clinical trials of JAK2 inhibitors in myelofibrosis. While the clinical benefit of JAK2 inhibitors may be substantial, not the least due to reduction of inflammatory cytokines and symptomatic improvement, our data add to increasing evidence that kinase inhibitor monotherapy of malignant disease is not curative, suggesting a need for drug combinations to optimally target the malignant cells.

scholarly journals Structural basis of norepinephrine recognition and transport inhibition in neurotransmitter transporters

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Shabareesh Pidathala ◽  
Aditya Kumar Mallela ◽  
Deepthi Joseph ◽  
Aravind Penmatsa
Keyword(s):  
Chronic Pain ◽  
Drosophila Melanogaster ◽  
Binding Site ◽  
Biogenic Amine ◽  
Norepinephrine Transporter ◽  
Structural Basis ◽  
Pain Sensation ◽  
X Ray ◽  
Neurotransmitter Transporters ◽  
Specific Inhibitors

AbstractNorepinephrine is a biogenic amine neurotransmitter that has widespread effects on alertness, arousal and pain sensation. Consequently, blockers of norepinephrine uptake have served as vital tools to treat depression and chronic pain. Here, we employ the Drosophila melanogaster dopamine transporter as a surrogate for the norepinephrine transporter and determine X-ray structures of the transporter in its substrate-free and norepinephrine-bound forms. We also report structures of the transporter in complex with inhibitors of chronic pain including duloxetine, milnacipran and a synthetic opioid, tramadol. When compared to dopamine, we observe that norepinephrine binds in a different pose, in the vicinity of subsite C within the primary binding site. Our experiments reveal that this region is the binding site for chronic pain inhibitors and a determinant for norepinephrine-specific reuptake inhibition, thereby providing a paradigm for the design of specific inhibitors for catecholamine neurotransmitter transporters.

scholarly journals Megakaryocytic morphology in Janus kinase 2 V617F positive myeloproliferative neoplasm

2017 ◽  
Vol 06 (02) ◽  
pp. 075-078
Author(s):  
Shuchi Ghai ◽  
Sharada Rai
Keyword(s):  
Bone Marrow ◽  
Myeloproliferative Neoplasms ◽  
Myeloproliferative Neoplasm ◽  
Jak2 V617f ◽  
Janus Kinase ◽  
Jak2 V617f Mutation ◽  
Fisher Exact Test ◽  
Janus Kinase 2 ◽  
Bone Marrow Biopsies ◽  
V617f Mutation

Abstract Context: Alterations in megakaryocyte morphology are the hallmark of myeloproliferative neoplasms (MPNs). These neoplasm are also associated with Janus kinase 2 (JAK2) V617F mutation in nearly 95% patients with polycythemia vera (PV), 40% patients of essential thrombocythemia (ET) and 50% patients of myelofibrosis (MF). The utility of megakaryocyte morphology in these disorders in correlation with JAK2 V617F remains unresolved. Aims: The aim of the study was to assess the morphology of megakaryocytes in bone marrow aspirates (BMAs) and bone marrow biopsies of patients of BCR-ABL negative MPNs with JAK2 V617F mutation. Settings and Design: This study was a retrospective and prospective, hospital-based study undertaken for a period ranging from January 2011 to April 2015. Subjects and Methods: Assessment of morphological features of megakaryocytes in 15 BMAs and their respective biopsies which included seven cases of PV, three cases of ET, and five cases of MF with JAK2 V617F mutation. Statistical Analysis Used: Chi-square test and Fisher exact test were used to compare the different features of megakaryocytes. Software version SPSS 13.0 was used. Results: Megakaryocytes in ET were found to have characteristically large size with staghorn multinucleated nuclei and exhibiting large amount of cytoplasm. MF showed dense clustering of megakaryocytes with staghorn nucleus along with sinusoidal dilatation and intrasinusoidal hematopoiesis. PV showed loose and dense clustering of megakaryocytes with a predominance of cloud-like nuclei. Few of the megakaryocytic morphologic features showed overlap between MF and PV and between ET and early MF. Conclusions: Megakaryocytic morphology can aid in the accurate diagnosis of the different subcategories of MPNs. This would help in categorization of clinically suspicious patients of JAK2 V617F negative patients.

scholarly journals Structural basis of the dynamic human CEACAM1 monomer-dimer equilibrium

2020 ◽  
Author(s):  
Amit K. Gandhi ◽  
Zhen-Yu J. Sun ◽  
Walter M. Kim ◽  
Yu-Hwa Huang ◽  
Yasuyuki Kondo ◽  
...  
Keyword(s):  
Cell Adhesion Molecule ◽  
Nmr Assignment ◽  
Structural Information ◽  
Microbial Pathogens ◽  
Structural Basis ◽  
X Ray ◽  
X Ray Crystallography ◽  
Related Cell ◽  
Differential Scanning Fluorimetry ◽  
Cell Adhesion Molecule 1

AbstractHuman (h) carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) function depends upon IgV-mediated homodimerization or heterodimerization with host ligands, including hCEACAM5 and hTIM-3, and a variety of microbial pathogens. However, there is little structural information available on how hCEACAM1 transitions between monomeric and dimeric states which in the latter case is critical for initiating hCEACAM1 activities. We therefore mutated residues within the hCEACAM1 IgV GFCC’ face including V39, I91, N97 and E99 and examined hCEACAM1 IgV monomer-homodimer exchange using differential scanning fluorimetry, multi-angle light scattering, X-ray crystallography and/or nuclear magnetic resonance. From these studies, we describe hCEACAM1 homodimeric, monomeric and transition states at atomic resolution and its conformational behavior in solution through NMR assignment of the wildtype (WT) hCEACAM1 IgV dimer and N97A monomer. These studies reveal the flexibility of the GFCC’ face and its important role in governing the formation of hCEACAM1 dimers and potentially heterodimers.

scholarly journals Crystal Structure of a Putative Modulator of Gyrase (TldE) from Thermococcus kodakarensis

Crystals ◽  
2019 ◽  
Vol 9 (2) ◽  
pp. 107 ◽  
Author(s):  
Xin Zhang ◽  
Zhengqun Li ◽  
Yanxiang Zhao ◽  
Xilan Cheng ◽  
Yang Liu ◽  
...  
Keyword(s):  
Metal Ion ◽  
Structural Information ◽  
Sequence Similarity ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Diffusion Method ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Thermococcus Kodakarensis

TldD and TldE proteins interact and form a complex to degrade unfolded peptides. The gene Tk0499 from Thermococcus kodakarensis encoded a putative modulator of gyrase (TkTldE). Although TldE genes were common in bacteria and archaea, the structural basis on the evolution of proteins remained largely unknown. Here, the three-dimensional structure of TkTldE was determined by X-ray diffraction. Crystals were acquired by the sitting-drop vapor-diffusion method. X-ray diffraction data from crystals were collected at 2.35 Å. The space group and unit-cell parameters suggested that there were two molecules in the asymmetric unit. Our results showed that TkTldE forms a homodimer, which contained anti-parallel β-strands and a pair of α-helices. Comparison of the structures of TldE and TldD showed that despite their high sequence similarity, TldE lacked the conserved HExxxH and GxC motif in which two His and a Cys residues bound a metal ion. Taken together, these results provided insight into the structural information of this class of TldE/TldD.

scholarly journals Serum Has Higher Proportion of Janus Kinase 2 V617F Mutation Compared to Paired EDTA-Whole Blood Sample: A Model for Somatic Mutation Quantification Using qPCR and the 2-∆∆Cq Method

Diagnostics ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 153 ◽  
Author(s):  
Gustavo Barcelos Barra ◽  
Ticiane Henriques Santa Rita ◽  
Ana Luisa Santa Cruz Almeida ◽  
Rafael Henriques Jácomo ◽  
Lídia Freire Abdalla Nery
Keyword(s):  
Whole Blood ◽  
Mutant Allele ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Absolute Quantification ◽  
Janus Kinase ◽  
Molecular Diagnostic ◽  
Janus Kinase 2 ◽  
Cq Method ◽  
V617f Mutation

Detection of the Janus Kinase-2 (JAK2) V617F mutation is a diagnostic criterion for myeloproliferative neoplasms, and high levels of mutant alleles are associated with worse outcomes. This mutation is usually tested on blood DNA by allele-specific qPCR (AS-qPCR) and measured using absolute quantification. However, some automated DNA extractions co-extracts of PCR inhibitors from blood and qPCR absolute quantification need increased efforts in order to maintain standard curves. JAK2 V617F can also be detected in serum using droplet digital PCR (ddPCR), a specimen with less inhibitors and favorable to automated extractions, but ddPCR instruments are not wide available as qPCR thermocyclers. Here, we evaluate whether JAK2 V617F could be accurately quantified by AS-qPCR using the 2-∆∆Cq method on blood DNA and validate the assay using gold-standard molecular diagnostic protocols. Next, we apply the validated method to assess if the mutation could be reliably detected/quantified in serum. JAK2 V617F could be quantified by AS-qPCR using the 2-∆∆Cq method—the assay was highly accurate (bias of 1.91%) compared to a commercial kit, highly precise (total CV% of 0.40%, 1.92%, 11.12% for samples with 93%, 54%, and 2.5% of mutant allele), highly sensitive (limit of detection of 0.15%), and demonstrated a linear detection response from 1.1% to 99.9%. Serum presented a higher mutant allele burden compared to the paired whole blood (mean of 4%), which allows for an increased JAK2 mutant detection rate and favors increased JAK2 V617F high-throughput analysis.

Comprehensive Analysis of JAK2 Mutations: A Single Center Experience

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5568-5568
Author(s):  
Nahlah AlGhasham ◽  
Yasmeen Alnouri ◽  
Halah Abalkhail ◽  
Salem Khalil
Keyword(s):  
Methylenetetrahydrofolate Reductase ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Mefv Gene ◽  
Comprehensive Analysis ◽  
Janus Kinase ◽  
World Health ◽  
Jak2 Mutation ◽  
Mthfr Polymorphism ◽  
V617f Mutation

Abstract Mutations in Janus kinase 2 (JAK2) genes are the genetic hallmark of BCR-ABL1-negative myeloproliferative neoplasms (MPN). In 2005 several groups described a point mutation in codon 617 of the protein tyrosine kinase (JAK2 gene) in BCR-ABL1-negative MPN. The mutation was found in a vast majority of patients with polycythemia vera (PV), in approximately half of patients with essential thrombocythaemia (ET) and in myelofibrosis (MF). In 2007, an additional exon 12 mutation were found in a small percentage of PV patients. The 2008 World Health Organization classification of haematopoietic neoplasms includes JAK2 mutations as one of the strongest diagnostic criteria for PV and together with the MPLmutations for ET and PMF. In this study, we describe the JAK2 mutation profile in a series of 1811 samples collected over the period of 48-months (2010-2013). The samples were submitted from patients h thrombosis or clinically suspected for having MPN to the Molecular laboratory at King Faisal specialist Hospital & Research center, Riyadh, Saudi Arabia. A comprehensive analysis of exons 12-15 was carried using Sanger sequencing method. Of the 1811 samples, 1706 samples were available for analysis. JAK2 mutations was identified in 271 (16%) of the samples. We evaluated the positive cases for the following: age, gender, type of JAK2mutation, if the patients are evaluated for other genetic mutations and diagnosis. Of these 271 positive cases; 148 (54.6%) were females and 123 (45.4%) males. 103 (38%) cases were from inside the hospital while 168 (62%) cases were from different referring institutions in which the diagnosis was not available. Their age varied between 4 months and 97 years with a median of 54 years. A total of 262 (96.7%) patients were positive for JAK2 V617F; In the remaining 9 (3.3%) samples non V617F mutation were detected, in which 5 (1.8%) cases with JAK2 G571S, 1 case (0.3%) with JAK2 p.E543_D544del, 1 (0.3%) case with JAK2 p.Y570Y, 1 (0.3%) with JAK2 exon 12 deletion (p.R541_E543>K) and 1 (0.3%) with JAK2 del (540I - 542N), ins M. of the 271, 13 (5%) cases were associated with other cytogenetic or DNA Sequence abnormalities together with the JAK2mutation. Of the 13, 4 cases (1.4%) presented with Abnormal karyotype, 5 (1.8%) with a heterozygous Methylenetetrahydrofolate reductase (MTHFR) polymorphism, 3 (1.1%) cases with BCR/ABL1 and 1 case has a missense mutation p.V726A, in exon 10 of the MEFV gene. The positive in-house cases tested for JAK2mutation were 103 (38%). Clinical diagnosis included: 87 (84.4%) MPN cases of which 35 (40.2%) PV, 22 (25.3%) ET, 11 (12.6%) MF, 17 (19.5%) unclassifiable MPN, 2 (2.3%) CML. Moreover, 9 (8.7%) Budd-Chiari syndrome, 6 (5.8%) thrombosis (including DVT, PE and portal thrombosis) and 1 (0.9%) squamous cell carcinoma. In our study 1811 sample were tested in 4 years which means almost 1 sample is submitted daily for JAK2 testing in our institute. The Majority of the in-house patients tested were MPN patients with small percentage having thrombosis which could progress later to full MPN clone. Over 50 different non-V617F mutations have been identified in the literature and were presented by a small portion of our patients cohort. It was shown to have biologic effects similar to those caused by the V617F mutation; however, more research in this area is ongoing. Disclosures No relevant conflicts of interest to declare.

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