Identification of an acquired JAK2 mutation in polycythemia vera

Polycythemia vera (PV) is a human clonal hematological disorder. The molecular etiology of the disease has not been identified. PV hematopoietic progenitor cells exhibit hypersensitivity to growth factors and cytokines, suggesting possible abnormalities in protein-tyrosine kinases and phosphatases. By sequencing the entire coding regions of cDNAs of candidate enzymes, we identified a G:C--> T:A point mutation of the JAK2 tyrosine kinase in 20 of 24 PV blood samples but none in 12 normal samples. The mutation has varying degrees of heterozygosity and is apparently acquired. It changes conserved Val(617) to Phe in the pseudokinase domain of JAK2 that is known to have an inhibitory role. The mutant JAK2 has enhanced kinase activity, and when overexpressed together with the erythropoietin receptor in cells, it caused hyperactivation of erythropoietin-induced cell signaling. This gain-of-function mutation of JAK may explain the hypersensitivity of PV progenitor cells to growth factors and cytokines. Our study thus defines a molecular defect of PV.     

Erlotinib effectively inhibits JAK2V617F activity and polycythemia vera cell growth

JAK2(V617F), a mutant of tyrosine kinase JAK2, is found in most patients with polycythemia vera (PV) and a substantial proportion of patients with idiopathic myelofibrosis or essential thrombocythemia. The JAK2 mutant displays a much increased kinase activity and generates a PV-like phenotype in mouse bone marrow transplant models. This study shows that the anti-cancer drug erlotinib (Tarceva) is a potent inhibitor of JAK2(V617F) activity. In vitro colony culture assays revealed that erlotinib at micro-molar concentrations effectively suppresses the growth and expansion of PV hematopoietic progenitor cells while having little effect on normal cells. Furthermore, JAK2(V617F)-positive cells from PV patients show greater susceptibility to the inhibitor than their negative counterparts. Similar inhibitory effects were found with the JAK2(V617F)-positive human erythroleukemia HEL cell line. These data suggest that erlotinib may be used for treatment of JAK2(V617F)-positive PV and other myeloproliferative disorders.     

JAK1 and Tyk2 activation by the homologous polycythemia vera JAK2 V617F mutation: cross-talk with IGF1 receptor

The majority of polycythemia vera (PV) patients harbor a unique somatic mutation (V617F) in the pseudokinase domain of JAK2, which leads to constitutive signaling. Here we show that the homologous mutations in JAK1 (V658F) and in Tyk2 (V678F) lead to constitutive activation of these kinases. Their expression induces autonomous growth of cytokine-dependent cells and constitutive activation of STAT5, STAT3, mitogen-activated protein kinase, and Akt signaling in Ba/F3 cells. The mutant JAKs exhibit constitutive signaling also when expressed in fibrosarcoma cells deficient in JAK proteins. Expression of the JAK2 V617F mutant renders Ba/F3 cells hypersensitive to insulin-like growth factor 1 (IGF1), which is a hallmark of PV erythroid progenitors. Upon selection of Ba/F3 cells for autonomous growth induced by the JAK2 V617F mutant, cells respond to IGF1 by activating STAT5, STAT3, Erk1/2, and Akt on top of the constitutive activation characteristic of autonomous cells. The synergic effect on proliferation and STAT activation appears specific to the JAK2 V617F mutant. Our results show that the homologous V617F mutation induces activation of JAK1 and Tyk2, suggesting a common mechanism of activation for the JAK1, JAK2, and Tyk2 mutants. JAK3 is not activated by the homologous mutation M592F, despite the presence of the conserved GVC preceding sequence. We suggest that mutations in the JAK1 and Tyk2 genes may be identified as initial molecular defects in human cancers and autoimmune diseases.     

Evaluation of the JAK2-V617F gene mutation in Turkish patients with essential thrombocythemia and polycythemia vera

An activating mutation of Janus kinase 2 (JAK2-V617F) was previously described in chronic myeloproliferative disorders (MPD). In previously published studies, the frequency of the JAK2-V617F mutation was determined to be 80-90 % for patients with polycythemia vera (PV) and 40-70 % for essential thrombocythemia (ET). In this study, we analyzed the relationship between the JAK2-V617F mutation and clinical-hematological parameters in Turkish patients with MPD and compared these findings with published studies from other geographic regions. A total of 148 patients were studied; of which, 70 were diagnosed with PV and 78 with ET. The mutation status of JAK2 was determined using a tetra-primer polymerase chain reaction. We found that 80 % of the PV group and 42 % of the ET group were positive for the JAK2-V617F mutation. When all patients were analyzed, the levels of white blood cells, hemoglobin and splenomegaly were significantly different in patients with the JAK2-V617F mutation (p < 0.05). To our knowledge, this study is the first to evaluate the relationship between MPD and JAK2-V617F in Turkish patients. The JAK2-V617F mutation is frequently detected in the Turkish patients with MPD, and especially in patients with PV. Hence, it would be useful to include JAK2 mutation screening in the initial evaluation of patients suspected to have MPD.     

Identification of a novel inhibitor of JAK2 tyrosine kinase by structure-based virtual screening

Janus kinase 2 (JAK2) plays a crucial role in the pathomechanism of myeloproliferative disorders and hematologic malignancies. A somatic mutation of JAK2 (Val617Phe) was previously shown to occur in 98% of patients with polycythemia vera and 50% of patients with essential thrombocythemia and primary myelofibrosis. Thus, effective JAK2 kinase inhibitors may be of significant therapeutic importance. Here, we applied a structure-based virtual screen to identify novel JAK2 inhibitors. One JAK2 inhibitor in particular, G6, demonstrated remarkable potency as well as specificity, which makes it as a potential lead candidate against diseases related to elevated JAK2 tyrosine kinase activity.     

Development and inter-laboratory validation of unlabeled probe melting curve analysis for detection of JAK2 V617F mutation in polycythemia vera

Background

JAK2 V617F, a somatic point mutation that leads to constitutive JAK2 phosphorylation and kinase activation, has been incorporated into the WHO classification and diagnostic criteria of myeloid neoplasms. Although various approaches such as restriction fragment length polymorphism, amplification refractory mutation system and real-time PCR have been developed for its detection, a generic rapid closed-tube method, which can be utilized on routine genetic testing instruments with stability and cost-efficiency, has not been described.

Methodology/Principal Findings

Asymmetric PCR for detection of JAK2 V617F with a 3′-blocked unlabeled probe, saturate dye and subsequent melting curve analysis was performed on a Rotor-Gene® Q real-time cycler to establish the methodology. We compared this method to the existing amplification refractory mutation systems and direct sequencing. Hereafter, the broad applicability of this unlabeled probe melting method was also validated on three diverse real-time systems (Roche LightCycler® 480, Applied Biosystems ABI® 7500 and Eppendorf Mastercycler® ep realplex) in two different laboratories. The unlabeled probe melting analysis could genotype JAK2 V617F mutation explicitly with a 3% mutation load detecting sensitivity. At level of 5% mutation load, the intra- and inter-assay CVs of probe-DNA heteroduplex (mutation/wild type) covered 3.14%/3.55% and 1.72%/1.29% respectively. The method could equally discriminate mutant from wild type samples on the other three real-time instruments.

Conclusions

With a high detecting sensitivity, unlabeled probe melting curve analysis is more applicable to disclose JAK2 V617F mutation than conventional methodologies. Verified with the favorable inter- and intra-assay reproducibility, unlabeled probe melting analysis provided a generic mutation detecting alternative for real-time instruments.     

Towards a molecular understanding of polycythemia rubra vera.

Polycythemia rubra vera (PV) is one of four diseases collectively called the myeloproliferative disorders (MPDs). Each disorder leads to an increased production of one or several hematopoietic cell lineages. MPDs arise from acquired mutations in a pluripotent hematopoietic stem cell. However, the molecular mechanisms leading to the development of these diseases are poorly understood. This review will summarize and evaluate recent advances in our understanding of one particular MPD, PV.     

Optimization of a novel kinase inhibitor scaffold for the dual inhibition of JAK2 and FAK kinases

The elaboration of a novel scaffold for the inhibition of JAK2 and FAK kinases was targeted in order to provide a dual inhibitor that could target divergent pathways for tumor cell progression.     

Quantitation of sulfhydryl groups on erythrocytes in polycythemia vera

Summary The number of sulfhydryl groups on the surface of intact erythrocytes from patients with polycythemia vera is significantly less (34%) than those from control subjects. Sulfhydryl groups were measured indirectly by reversibly blocking-SH groups with the thiol reagent, 6,6-dithiodinicotinic acid, which forms stable mixed disulfides on the surface of erythrocytes. Glutathione was used to break the disulfides and release thione into the supernatant. Thione was then quantitated spectrophotometrically.Supported by NIH Biomedical Research Development Grant 1-S08-RR-09171-01     

Spontaneous coronary artery dissection: long stenting in a patient with polycythemia vera

Spontaneous coronary artery dissection is a rare cause of myocardial ischemia or sudden cardiac death. We describe a patient with polycythemia vera and a chronic spontaneous coronary artery dissection who was treated with successful angioplasty and long stenting.