scholarly journals New Paradigms for the Mechanisms of Thrombopoietin Receptor Activation and Dysregulation By the JAK2V617F Mutation

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2962-2962
Author(s):  
Stephan Wilmes ◽  
Maximillian Hafer ◽  
Tess A Stanly ◽  
Ignacio Moraga ◽  
Joni Vuorio ◽  
...  
Keyword(s):  
Single Molecule ◽  
Molecular Mechanisms ◽  
Myeloproliferative Neoplasms ◽  
Philadelphia Chromosome ◽  
Receptor Activation ◽  
Janus Kinase ◽  
Jak2v617f Mutation ◽  
Common Mutation ◽  
Receptor Dimerization ◽  
Group I

Janus kinase (JAK2)V617F is the most common mutation found in patients with Philadelphia chromosome negative myeloproliferative neoplasms (Ph- MPNs). The discovery of this mutation over 15 years ago revolutionised MPN diagnosis and inspired the development of JAK inhibitors as new therapeutic interventions. However, despite extensive structural and biophysical studies using JAK2 domains in isolation, the exact molecular mechanisms of JAK2V617F activation remains elusive. We have previously demonstrated that expression of the thrombopoietin (TPO) receptor, MPL, which interacts directly with JAK2, is essential for disease development in a mouse model of a JAK2V617F-positiveMPN (Blood 2014 124:3956-3963). Using total internal reflection fluorescence (TIRF) microscopy, we visualized MPL interaction dynamics in live cells on single molecule level. Effective cell surface MPL fluorescence labelling and dual-color imaging allowed us to determine the level of MPL dimerization under various experimental conditions. Using this assay, we clearly established that MPL is monomeric at physiologically relevant receptor densities. However, TPO stimulation results in significant dimerization of MPL (>50%) and an equilibrium between monomers and dimers. This counters the current dogma that MPL exists at the membrane as a pre-formed dimer. Strikingly, we found that JAK2V617F shifts this monomer-dimer equilibrium leading to significant TPO-independent MPL dimerization providing a novel mechanistic model of oncogenic JAK2 activation. To highlight the role of ligand-independent receptor dimerization in JAK2 activation, we compared three groups of autoactivating mutations in the PK domain covering the FERM-SH2 (FS2)-PK linker region (Group I), residues in the proximity of the αC helix (Group II) and at the autoinhibitory PK-TK interface (Group III). Consistent MPL dimerization was only observed for mutations in groups I and II. Mutations in these groups both localize to a potential homomeric PK/PK interface that has been implicated as a switch of JAK activation. Using MD simulations, we also found that the FERM domain of JAK2 strongly interacts with the inner leaflet of the lipid bilayer of the plasma membrane via a single hydrophobic residue (L224) surrounded by several positively charged residues that allows the region to act as a membrane anchor. This tight coupling to the membrane enforces an appropriate orientation between the JAKs within the receptor dimers required for optimal intermolecular PK/PK interaction that is critical for receptor dimerization. To interfere with membrane anchoring, we introduced a negative charge in this position (L224E). Strikingly, ligand-independent MPL dimerization and activation by JAK2V617F was dramatically reduced upon introducing L224E, supporting the vital importance of L224 for orienting JAK2 at the membrane to allow productive PK-PK interactions. Here, we demonstrate that JAK2V617F mutation acts by altering and strengthening the intermolecular interactions involving the PK/PK dimerization interface. In essence, these mutations drive cytoplasmic stabilization of receptor-JAK dimers, bypassing extracellular stabilization of dimers via cytokine binding. These results provide critical and entirely novel mechanistic insights into signal initiation in MPNs and readdress the roles of receptor-associated proteins. Disclosures Hubbard: Ajax Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Other: Co-Founder.

Loss of Wild-Type Jak2 Allele Enhances Myeloid Cell Expansion and Accelerates Myelofibrosis in Jak2V617F Knock-in Mice

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 809-809
Author(s):  
Hajime Akada ◽  
Saeko Akada ◽  
Dongqing Yan ◽  
Robert Hutchison ◽  
Golam Mohi
Keyword(s):  
Polycythemia Vera ◽  
Cell Expansion ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Philadelphia Chromosome ◽  
Myeloid Cell ◽  
Negative Regulator ◽  
Jak2v617f Mutation ◽  
Common Mutation ◽  
Wild Type

Abstract Abstract 809 The activating JAK2V617F mutation is the most common mutation found in Philadelphia chromosome (Ph)-negative myeloproliferative neoplasms (MPNs), which include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Although a majority of MPN patients carry heterozygous JAK2V617F mutation, loss of heterozygosity (LOH) on chromosome 9p involving JAK2 has been observed in ∼30% of patients with MPNs particularly in PV and PMF. JAK2V617F homozygosity through 9pLOH has been linked to more severe MPN phenotype. However, the contribution of 9pLOH in the pathogenesis of MPNs remains unclear. To investigate the role of wild-type JAK2 in MPNs induced by JAK2V617F, we have utilized conditional Jak2 knock-out and Jak2V617F knock-in alleles and generated heterozygous, hemizygous and homozygous Jak2V617F mice. Whereas heterozygous Jak2V617F expression results in a polycythemia vera-like disease in mice, loss of wild-type Jak2 allele in hemizygous or homozygous Jak2V617F mice results in a significantly greater increase in reticulocytes, white blood cells, neutrophils and platelets in the peripheral blood and larger spleen size. We also have found that hemizygous or homozygous Jak2V617F expression significantly increased megakaryocyte-erythroid progenitors in the bone marrow and spleens and marked infiltration of neutrophils in the liver compared with heterozygous Jak2V617F. More importantly, hemizygous or homozygous Jak2V617F mice show accelerated myelofibrosis compared with heterozygous Jak2V617F-expressing mice. Thus, loss of wild type Jak2 allele increases myeloid cell expansion and enhances the severity of the MPN. Together, these results suggest that wild-type Jak2 serves as a negative regulator of MPN induced by Jak2V617F. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Investigation of JAK2V617F Mutation Prevalence in Patients with Beta Thalassemia Major

2019 ◽  
Author(s):  
Zari Tahannejad Asadi ◽  
Reza Yarahmadi ◽  
Najmaldin Saki ◽  
Mohammad Taha Jalali ◽  
Ali Amin Asnafi ◽  
...  
Keyword(s):  
Genetic Disorder ◽  
Thalassemia Major ◽  
Janus Kinase ◽  
Jak2v617f Mutation ◽  
Beta Thalassemia ◽  
Common Mutation ◽  
Signal Transducers ◽  
Significant Difference ◽  
Polymerase Chain ◽  
Low Prevalence

AbstractBackgroundBeta (β)–thalassemia major is a genetic disorder with anemia and an increased level of erythropoietin by Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway. JAK plays an important role in cell signaling, and the common mutation in the JAK2 gene in myeloid disorders is called JAK2V617F.MethodsA total of 75 patients with beta (β)-thalassemia major patients, including 34 males (45%) and 41 females (55%), were enrolled in this study. The presence of the JAK2V617F mutation was assessed using the amplification-refractory mutation–polymerase chain reaction (ARMS-PCR) technique.ResultsAmong the 75 patients, 14 patients (19%) tested positive and 61 patients (81%) tested negative for JAK2V617F mutation. We observed no statistically significant difference in sex, age, genotype, and JAK2V617F mutation among patients (P> .05). However, a significant difference between blood-transfusion frequency and JAK2V617F mutation was observed (P <.05).ConclusionDue to the low prevalence of JAK2V617F mutation in thalassemia, using a larger population of the patients to investigate this mutation in ineffective erythropoiesis can be useful.

scholarly journals The Importance of Identification of M-BCRABL Oncogene and JAK2V617F Mutation in Myeloproliferative Neoplasms

2014 ◽  
Vol 60 (2) ◽  
pp. 44-48
Author(s):  
Annamária Szántó ◽  
Zsuzsanna Pap ◽  
Z Pávai ◽  
I Benedek ◽  
Judit Beáta Köpeczi ◽  
...  
Keyword(s):  
Molecular Markers ◽  
Molecular Biology ◽  
Myeloproliferative Neoplasms ◽  
Philadelphia Chromosome ◽  
Gene Mutations ◽  
Chronic Phase ◽  
Final Diagnosis ◽  
Jak2v617f Mutation ◽  
Myeloproliferative Disease

Abstract Background: The elucidation of the genetic background of the myeloproliferative neoplasms completely changed the management of these disorders: the presence of the Philadelphia chromosome and/or the BCR-ABL oncogene is pathognomonic for chronic myeloid leukemia and identification of JAK2 gene mutations are useful in polycytemia vera (PV), essential thrombocytemia (ET) and myelofibrosis (PMF). The aim of this study was to investigate the role of molecular biology tests in the management of myeloproliferative neoplasms. Materials and methods: We tested the blood samples of 117 patients between April 2008 and February 2013 at the Molecular Biology of UMF Târgu Mureș using RQ-PCR (for M-BCR-ABL oncogene) and/or allele-specific PCR (for JAK2V617F mutation). Results: Thirty-two patients presented the M-BCR-ABL oncogene, 16 of them were regularly tested as a follow-up of the administered therapy: the majority of chronic phase patients presented decreasing or stable values, while in case of accelerated phase and blast phase the M-BCR-ABL values increased or remained at the same level. Twenty patients were identified with the JAK2V617F mutation: 8 patients with PV, 4 with ET, 3 with PMF, 4 with unclassifiable chronic myeloproliferative disease and 1 patient with chronic myelomonocytic leukemia. There was no case of concomitant occurance of both molecular markers. Conclusions: Molecular biology testing plays an important role in the management of myeloproliferative neoplasms: identification of the molecular markers confirms the final diagnosis, excluding secondary causes of abnormal blood count parameters. Regular monitoring of MBCR- ABL expression level is useful in the follow-up of therapeutic efficiency.

Therapeutic Advances in Myeloproliferative Neoplasms: The Role of New-Small Molecule Inhibitors

2012 ◽  
pp. 406-410 ◽  
Author(s):  
Srdan Verstovsek
Keyword(s):  
Molecular Mechanisms ◽  
Therapeutic Potential ◽  
Myeloproliferative Neoplasms ◽  
Janus Kinase ◽  
Treatment Focus ◽  
Rational Combination ◽  
Short And Long Term ◽  
Jak2 Inhibitors

Overview: The discovery that a somatic point mutation (JAK2V617F) in the Janus kinase 2 ( JAK2) is highly prevalent in patients with myeloproliferative neoplasms (MPNs) has been a crucial breakthrough in our understanding of the underlying molecular mechanisms of these diseases. Therefore, preclinical and clinical research in recent years has focused intensely on the development of new therapies targeted to JAK2. These efforts culminated in recent approval of ruxolitinib as the first official therapy for patients with intermediate- or high-risk myelofibrosis (MF). Therapy with JAK2 inhibitors substantially improves quality of life and reduces organomegaly in MF with or without JAKV617F mutation. Recent results suggest that patients with advanced MF may live longer when receiving therapy with ruxolitinib. However, JAK2 inhibitors do not eliminate the disease and new medications are needed to expand on the benefits seen with JAK2 inhibitors. Although many agents are still in the early stages of development, the wealth of publications and presentations has continued to support our growing understanding of the pathophysiology of MF as well as the potential short- and long-term outcomes of these new and diverse approaches to treatment. Focus of ongoing efforts is particularly on the improvements in anemia and fibrosis, as well as on rational combination trials of JAK2 inhibitors and other potentially active agents. Therapeutic potential and limitations of JAK2 inhibitors and other novel medications in clinical studies are reviewed.

scholarly journals Molecular insights into regulation of JAK2 in myeloproliferative neoplasms

Blood ◽  
2015 ◽  
Vol 125 (22) ◽  
pp. 3388-3392 ◽  
Author(s):  
Olli Silvennoinen ◽  
Stevan R. Hubbard
Keyword(s):  
Molecular Characterization ◽  
Human Disease ◽  
Molecular Mechanisms ◽  
Myeloproliferative Neoplasms ◽  
Critical Role ◽  
Hematologic Malignancies ◽  
Janus Kinase ◽  
Constitutive Activation

Abstract The critical role of Janus kinase-2 (JAK2) in regulation of myelopoiesis was established 2 decades ago, but identification of mutations in the pseudokinase domain of JAK2 in myeloproliferative neoplasms (MPNs) and in other hematologic malignancies highlighted the role of JAK2 in human disease. These findings have revolutionized the diagnostics of MPNs and led to development of novel JAK2 therapeutics. However, the molecular mechanisms by which mutations in the pseudokinase domain lead to hyperactivation of JAK2 and clinical disease have been unclear. Here, we describe recent advances in the molecular characterization of the JAK2 pseudokinase domain and how pathogenic mutations lead to constitutive activation of JAK2.

scholarly journals Complex molecular genetic diagnostic algorithm in the diagnosis of myeloproliferative neoplasms

2014 ◽  
Vol 155 (52) ◽  
pp. 2074-2081 ◽  
Author(s):  
Tünde Krähling ◽  
Katalin Balassa ◽  
Nóra Meggyesi ◽  
András Bors ◽  
Judit Csomor ◽  
...  
Keyword(s):  
Triple Negative ◽  
Myeloproliferative Neoplasms ◽  
Philadelphia Chromosome ◽  
Molecular Techniques ◽  
Janus Kinase ◽  
Driver Mutations ◽  
Similar Distribution ◽  
Janus Kinase 2 ◽  
Chain Reactions ◽  
Thrombopoietin Receptor

Introduction: Mutations in Janus kinase 2, calreticulin and thrombopoietin receptor genes have been identified in the genetic background of Philadelphia chromosome negative, “classic” myeloproliferative neoplasms. Aim: The aim of the authors was to identify driver mutations in a large myeloproliferative cohort of 949 patients. Method: A complex array of molecular techniques (qualitative and quantitative allele-specific polymerase chain reactions, fragment analyzes, high resolution melting and Sanger sequencing) was applied. Results: All 354 patients with polycythemia vera carried Janus kinase 2 mutations (V617F 98.6%, exon 12: 1.4%). In essential thrombocythemia (n = 468), the frequency of V617F was 61.3% (n = 287), that of calreticulin 25.2% (n = 118), and that of thrombopoietin receptor mutations 2.1% (n = 10), while 11.3% (n = 53) were triple-negative. Similar distribution was observed in primary myelofibrosis (n = 127): 58.3% (n = 74) V617F, 23.6% (n = 30) calreticulin, 6.3% (n = 8) thrombopoietin receptor mutation positive and 11.8% (n = 15) triple-negative. Conclusions: The recent discovery of calreticulin gene mutations led to definite molecular diagnostics in around 90% of clonal myeloproliferative cases. Orv. Hetil., 2014, 155(52), 2074–2081.

Abstract 15575: The Clonal Hematopoiesis Mutation Jak2 V617F Aggravates NETosis and Endothelial Injury in Mouse Arteries With Erosion-like Intimas

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Roberto Molinaro ◽  
Rob S Sellar ◽  
Grasiele Sausen ◽  
Amelie Vromman ◽  
Eduardo Folco ◽  
...  
Keyword(s):  
Myeloproliferative Neoplasms ◽  
Janus Kinase ◽  
Endothelial Injury ◽  
Common Mutation ◽  
Flow Disturbance ◽  
Thrombotic Risk ◽  
Clonal Hematopoiesis ◽  
Endothelial Integrity ◽  
Superficial Erosion

Introduction: In the current era of intense LDL lowering, superficial erosion may be on the rise as a cause of ACS (25-30%). Experimental data on human atheromata support a role for neutrophils and the formation of neutrophil extracellular traps (NETs) in the pathogenesis of superficial erosion and subsequent thrombosis. The common mutation of Janus kinase 2 ( Jak2 V617F ) borne by clones of leukocytes derived from bone marrow stem cells sensitizes neutrophils to undergo NETosis and linked NET formation to increased thrombosis. Jak2 V617F associates with accelerated atherosclerosis and venous thrombosis in patients with myeloproliferative neoplasms (MPN) and in individuals harboring this mutation but without a demonstrable MPN. Hypothesis We hypothesized that i) the Jak2 V617F associated with clonal hematopoiesis and increased atherosclerotic and thrombotic risk in humans, predisposes to NETosis and subsequent endothelial injury and thrombosis at sites of flow disturbance in arteries with erosion-like intimas; and ii) the clinically approved Jak-1,2 inhibitor, ruxolitinib (Rux), can preserve endothelial integrity and reduce thrombosis in mice with myeloid Jak2 V617F . Methods: We generated cohorts of mice harboring Jak2 V617F or wild-type Jak2 . We used a surgical preparation recapitulating features of superficial erosion and assessed endothelial integrity, thrombosis, and NET formation in relation to genotype and in vivo Rux treatment. Results and Conclusions: Evans blue extravasation following introduction of flow disturbance at sites of erosion-prone intimas showed significant impairment of endothelial barrier in the group of mice bearing the Jak2 V617F mutation compared to control (WT) mice ( ** p<0.005). We further observed more thrombosis in mice bearing the Jak2 V617F mutation. Immunohistochemical evaluation of the regions of experimental superficial erosion revealed reduced endothelial continuity and increased NET accumulation in Jak2 V617F vs. WT mice. Rux treatment mitigated the adverse effects of Jak2 V617F on both NET accumulation and endothelial integrity, supporting the translational potential of these observations in individuals with clonal hematopoiesis due to Jak2 V617F and acute coronary syndromes.

Targeted Cancer Exome Sequencing Discovers Novel Recurrent Mutations In MPN

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4099-4099
Author(s):  
Elena Tenedini ◽  
Isabella Bernardis ◽  
Valentina Artusi ◽  
Lucia Artuso ◽  
Enrica Roncaglia ◽  
...  
Keyword(s):  
Somatic Mutations ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Philadelphia Chromosome ◽  
Jak2v617f Mutation ◽  
Exome Capture ◽  
Multiple Control ◽  
Disease Evolution ◽  
Recurrent Mutations

Abstract The discovery of the JAK2V617F mutation in 2005 [Kralovics R, N Engl J Med 2005] represented a major breakthrough in the understanding of the molecular pathogenesis of Philadelphia chromosome negative chronic myeloproliferative neoplasms (MPN). Nevertheless several observations suggest that the JAK2V617F mutation may not be the disease funding mutation, at least in most instances. Therefore, a great deal of effort is ongoing with the aim to identifying novel genetic lesions contributing to the disease pathogenesis. The two major theoretical and technical drawbacks to the identification of new somatic mutations are represented, respectively, by the huge number of genes potentially involved in tumorigenesis of MPN and by the availability of a “pure” germline control DNA. Buccal swabs and saliva have been generally considered as readily available sources of DNA of non-hematopoietic origin, but detection of the JAK2V617F mutation in at least some of these samples indeed suggested the presence of myeloid cell contamination [Levine RL, Cancer Cell 2005]. So, in order to discover novel mutations in MPN using upfront technologies based on next-generation sequencing (NGS) we designed a “cancer exome” capture panel of 2000 unique genes and microRNAs. This panel was used to capture libraries generated from genomic DNA extracted from granulocytes and in vitro expanded CD3+ T-lymphocytes as germline control, in a cohort of 20 MPN patients. These captured libraries were than massively sequenced using the Roche 454 FLX platform. DNA samples had been collected at the diagnosis of PV in 9 subjects and PMF in 6 subjects, while the remaining 5 DNA samples were from 5 of the 9 PV patients at the time they evolved to post-PV myelofibrosis. After extensive bioinformatics analysis and multiple control adjustments, we finally produced a list of 171 novel “true” somatic mutations occurring in genes and microRNAs coding regions of those MPN samples; some of these mutations have been already described in MPN, whereas novel variants represent the vast majority. Despite patients harbored different numbers of somatic mutations, spanning from four to twenty-one variants, only 22 genes appear recurrently mutated. It is worth of notice the acquisition of additional mutations and/or the occurrence of loss of some mutations at the time of disease evolution from PV to a post-PV Myelofibrosis in the five patients for whom samples were available at both disease phases. Some of them, either acquired (NTRK1, PRDM2, BRCA2 and BARD1) or lost (APC, CARS, MLL3 and FAT2) had been found also in another PV or PMF sample. To test the recurrence of these mutations, we screened a different cohort of 189 patients composed by PMF (91 samples), PV (50 patients) and post-PV Myelofibrosis (48 samples) by Ion AmpliSeq technology on an Ion Torrent PGM platform. Deep amplicon sequencing of granulocytes DNA achieved a sample median of 1000-fold coverage. Excluding JAK2, MPL, IDH2, ASXL1 known variants, for 7 genes (SCRIB, MIR662, BARD1, TCF12, FAT4, DAP3, NRAS) we demonstrated in MPN a global mutation frequency greater than the 3%. Whereas some new variants need functional validation to prove causal mechanisms, some other mutations have a well-known pathogenic role in solid cancers but here are described for the first time in MPN. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Single-molecule imaging reveals transforming growth factor-β-induced type II receptor dimerization

2009 ◽  
Vol 106 (37) ◽  
pp. 15679-15683 ◽  
Author(s):  
Wei Zhang ◽  
Yaxin Jiang ◽  
Qiang Wang ◽  
Xinyong Ma ◽  
Zeyu Xiao ◽  
...  
Keyword(s):  
Growth Factor ◽  
Single Molecule ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Receptor Activation ◽  
General Mechanism ◽  
Type Ii ◽  
Serine Threonine Kinase ◽  
Receptor Dimerization ◽  
Threonine Kinase

Transforming growth factor-β (TGF-β) elicits its signals through two transmembrane serine/threonine kinase receptors, type II (TβRII) and type I receptors. It is generally believed that the initial receptor dimerization is an essential event for receptor activation. However, previous studies suggested that TGF-β signals by binding to the preexisting TβRII homodimer. Here, using single molecule microscopy to image green fluorescent protein (GFP)-labeled TβRII on the living cell surface, we demonstrated that the receptor could exist as monomers at the low expression level in resting cells and dimerize upon TGF-β stimulation. This work reveals a model in which the activation of serine-threonine kinase receptors is also accomplished via dimerization of monomers, suggesting that receptor dimerization is a general mechanism for ligand-induced receptor activation.

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