Will Imitanib Be Useful for Patients with Polycythemia Vera?.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2601-2601 ◽  
Author(s):  
Amos S. Gaikwad ◽  
Srdan Verstovsek ◽  
Ko-Tung Chang ◽  
Donghoon Yoon ◽  
William Vainchenker ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Glucose Uptake ◽  
Polycythemia Vera ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Jak2 V617f ◽  
Erythroid Progenitors ◽  
Jak2 Mutation

Abstract Polycythemia vera (PV) is an acquired myeloproliferative, clonal stem cell disorder characterized by cytokine hypersensitivity. Several groups reported a clonal mutation in the pseudo kinase domain of the Janus kinase 2 (Jak2) protein, substituting phenylalanine at position 617 with valine (V617F) that causes constitutive activation of the JAK/STAT pathway in PV patients. Chronic myelogenous leukemia is another myeloproliferative disorder associated with aberrant tyrosine kinase (bcr-abl) that arises from t(9:22) translocation. Constitutive bcr-abl tyrosine kinase activity suppresses apoptosis leading to survival and proliferation of cells at low cytokine concentrations. Imitanib has been developed to inhibit the bcr-abl kinase activity and has had impressive therapeutic efficacy. However, imitanib also inhibits other tyrosine kinases. Clinical benefits of imitanib in PV have been reported. Mouse FDCP cells over-expressing either the wild-type Jak2 (JAK/W) or two cell lines with the V617F mutation (V617F), one with cytokine hypersensitivity and the other cytokine independent, were created. We examined the effect of imitanib by MTT proliferation assay and propidium iodide staining analysis. No appreciable changes in the proliferation and DNA content were observed in all three cell-lines after imitanib treatment at ~1μM (the concentration effective for the bcr-abl expressing cells and achievable in vivo in imitanib treated patients). However, after 72h of exposure, the cells expressing JAK2 V617F mutant showed 50% inhibition of growth at ~6μM imitanib with no significant effect on the growth of cells expressing JAK/W. To further understand the mechanism of growth inhibition of V617F cells by imitanib, we examined the metabolism of these cells since the constitutively active tyrosine kinases has been demonstrated to change the glucose metabolism. Imitanib treatment (5μM) for 72h caused about 30% decrease in the glucose uptake in V617F cells with only a marginal (~5%) decrease in glucose uptake was observed in the JAK/W cells. We then examined the effect of imitanib on in vitro expanded native human erythroid progenitors (CD71 and CD235a positive cells) from three normal and four PV individuals who expressed variable proportion of mutant JAK2 V617F alleles. In this small study, between 18–30% decrease in the proliferation of the cells from PV patients was seen with 1–2μM of imitanib compared to 8% seen with normal erythroid progenitors; however, the in vitro expansion was accompanied by a decrease of proportion of cells with JAK2 mutation (see abstract Prchal et. Al. at this meeting). Further, in ongoing clinical studies, one of 5 tested PV patients who showed an excellent clinical response to imitanib therapy had decrease of V617F mutation from 58 to 19% in circulating granulocytes. We conclude that high concentration of imitanib is required to achieve the cytotoxic effects in reporter cells transfected with JAK2 mutation that are not readily achievable in vivo; however, lower doses (but still significantly higher than those needed for bcr-abl expressing cells) are effective in native PV progenitors. These data do not exclude possible useful therapeutic effect of imitanib in PV either alone or in combination with other drugs. Amos Gaikwad, Ph.D. and Srdan Verstovsek, M.D. contributed equally to this study.

In Vitro Expansion of Polycythemia Vera Progenitors Favors Expansion of Erythroid Precursors without JAK2 V617F Mutation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3506-3506 ◽  
Author(s):  
Josef T. Prchal ◽  
Ko-Tung Chang ◽  
Jaroslav Jelinek ◽  
Yongli Guan ◽  
Amos Gaikwad ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Polycythemia Vera ◽  
Peripheral Blood ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Erythroid Progenitors ◽  
Erythroid Precursors ◽  
In Vitro Expansion ◽  
V617f Mutation

Abstract A single acquired point mutation of JAK2 1849G>T (V617F), a tyrosine kinase with a key role in signal transduction from growth factor receptors, is found in 70%–97% of patients with polycythemia vera (PV). In the studies of tyrosine kinase inhibitors on JAK2 1849G>T (see Gaikwad et all abstract at this meeting) we decided to study the possible therapeutic effect of these agents using native in vitro expanded cells from peripheral blood. To our surprise, the in vitro expansion of PV progenitors preferentially augmented cells without JAK2 1849G>T mutation. We used a 3 step procedure to amplify erythroid precursors in different stages of differentiation from the peripheral blood of 5 PV patients previously found to be homozygous or heterozygous for the JAK2 1849G>T mutation. In the first step (days 1–7), 106/ml MNCs were cultured in the presence of Flt-3 (50 ng/ml), Tpo (100 ng/ml), and SCF (100 ng/ml). In the second step (days 8–14), the cells obtained on day 7 were re-suspended at 106/ml in the same medium with SCF (50 ng/ml), IGF-1 (50 ng/ml), and 3 units/ml Epo. In the third step, the cells collected on day 14 were re-suspended at 106/ml and cultured for two more days in the presence of the same cytokine mixture as in the step 2 but without SCF. The cultures were incubated at 37oC in 5% CO2/95% air atmosphere and the medium renewed every three days to ensure good cell proliferation. The expanded cells were stained with phycoerythrin-conjugated anti-CD235A (glycophorin) and fluorescein isothiocyanate-conjugated anti-human-CD71 (transferrin receptor) monoclonal antibodies and analyzed by flow cytometry. The cells were divided by their differential expression of these antigens into 5 subgroups ranging from primitive erythroid progenitors (BFU-Es and CFU-Es) to polychromatophilic and orthochromatophilic erythroblasts; over 70% of harvested cells were early and late basophilic erythroblasts. The proportion of JAK2 1849G>T mutation in clonal PV granulocytes (GNC) before in vitro expansion and in expanded erythroid precursors was quantitated by pyrosequencing (Jelinek, Blood in press) and is depicted in the Table. These data indicate that in vitro expansion of PV progenitors favors expansion of erythroid precursors without JAK2 V617F mutation. Since three PV samples were from females with clonal granulocytes, erythrocytes, and platelets, experiments were underway to determine if the in vitro expanded erythroid cells were clonal PV cells without JAK2 V617F mutation, or derived from polyclonal rare circulating normal hematopoietic progenitors. The Proportion of JAK2 T Allele Patients GNC T Allele (%) Expanded Cells T Allele (%) PV1 (Female) 81 10 PV2 (Male) 77 28 PV3 (Male) 44 42 PV4 (Female) 78 19 PV5 (Female) 78 28

scholarly journals Insulin resistance in adult rat offspring associated with maternal dietary fat and alcohol consumption

2002 ◽  
Vol 173 (1) ◽  
pp. 63-71 ◽  
Author(s):  
CW Elton ◽  
JS Pennington ◽  
SA Lynch ◽  
FM Carver ◽  
SN Pennington
Keyword(s):  
Insulin Sensitivity ◽  
Tyrosine Kinase ◽  
Glucose Uptake ◽  
Dietary Fat ◽  
Kinase Activity ◽  
In Utero ◽  
Whole Body ◽  
Tyrosine Kinase Activity

Maternal diet during pregnancy has been reported to alter the offspring's ability to respond to a glucose challenge. The current studies report changes in basal and insulin-stimulated, in vitro glucose uptake in red (soleus) and white (extensor digitorum longus) muscle fiber types, as well as whole body insulin responsiveness of adult rat offspring associated with their mother's dietary fat and alcohol content during pregnancy. The offspring of Harlan-derived Sprague-Dawley female rats, dosed during pregnancy with ethanol (ETOH) via a liquid diet (35% of calories as ETOH) with either 12% or 35% of calories as fat, were compared with offspring from litters whose mothers were pair-fed an isocaloric amount of the liquid diet without ETOH. Maternal access to the liquid diets was terminated on day 20 of the pregnancies (sperm plug=day 0). The offspring were surrogate fostered within 48 h of birth to mothers which had consumed commercial chow throughout their pregnancy. Following weaning at 21 days of age, the offspring consumed only commercial rat chow and they were examined over the next 14 months for changes in glucose homeostasis as a consequence of in utero exposure to maternal dietary fat and/or alcohol. The 35% maternal fat diet resulted in both in vivo and in vitro decreases in insulin sensitivity. Thus, compared with adults whose mother's diet contained 12% fat, significant, in vitro muscle and in vivo whole body insulin resistance (measured by hyperinsulinemic-euglycemic clamping) was observed in adult rats whose mothers consumed 35% of dietary calories as fat. The addition of ethanol to the maternal 35% fat diet further reduced the offspring's red muscle tissues in vitro response to insulin, but did not affect whole body insulin sensitivity. Muscle basal and insulin-stimulated receptor tyrosine kinase activity were significantly decreased (approximately -50%) by the 35% fat maternal diet but there was no compensatory increase in serum insulin or glucose levels. Based upon both in vivo and in vitro data, these studies suggested that in utero exposure to 35% fat has a sustained effect on the adult offspring's glucose uptake/insulin sensitivity and that the effect is paralleled, at least in part, by decreased insulin receptor tyrosine kinase activity. In utero ETOH exposure resulted in the loss of basal and insulin-stimulated, in vitro glucose uptake in red muscle fibers but maternal dietary ETOH had no detectable effect on either in vivo insulin sensitivity or muscle tyrosine kinase activity.

Pure Heterozygous and Mixed JAK2 V617F Genotypes Result in Distinct Mechanisms of Erythrocytosis in Polycythemia Vera.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 662-662
Author(s):  
Sabrina Dupont ◽  
Aline Masse ◽  
Chloe James ◽  
Nicole Casadevall ◽  
William Vainchenker ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Progenitor Cells ◽  
Mononuclear Cells ◽  
Fold Increase ◽  
Jak2 V617f ◽  
Molecular Event ◽  
Erythroid Progenitors ◽  
Low Frequencies

Abstract The JAK2 V617F mutation is present in most patients with polycythemia vera (PV) and half with essential thrombocythemia (ET). Using real-time quantitative PCR, we analyzed the levels of JAK2 V617F in granulocytes and/or bone marrow mononuclear cells from 159 PV and 149 ET patients. High JAK2 V617F levels were correlated with higher leukocyte, granulocyte, hemoglobin values and higher endogenous erythroid colony formation. Thus, the phenotype of PV and ET may be closely linked to the JAK2 V617F level, which may reflect the clonal genotypic pattern of hematopoietic progenitor cells. It is thought that the occurrence of the mitotic recombination, which generates homozygous JAK2 V671F clones, is a key molecular event for the onset of PV. In this work, we aimed to study the consequences of the clonal JAK2 V617F genotype on the amplification properties and erythropoietin (EPO) hypersensitivity of PV (n=14) and ET (n=6) progenitors. Analysis of clonal genotypic patterns shows that ET patients harbor a mix of heterozygous and normal progenitors. Interestingly, we distinguish pure heterozygous PV profiles (3/14 patients) with no homozygous progenitors from homozygous PV profiles (11/14 patients) with normal, heterozygous and homozygous progenitors. Similar low frequencies of mutated immature progenitors, comprising long-term culture-initiating cells and lympho-myeloid progenitors, are found in ET and PV. In contrast, PV patients with pure heterozygous PV profiles have striking higher proportions (>90%) of mutated committed progenitors than other PV and ET patients. This result suggests a selective amplification of heterozygous cells in the early phases of hematopoiesis. Furthermore, by using increasing concentrations of EPO, homozygous mutated erythroid progenitors are demonstrated to be more sensitive to EPO than heterozygous ones, a majority of the former (69,5%) being EPO independent. Moreover, we demonstrate a two to three fold increase in in vitro amplification of ET and PV progenitor cells when compared to normal ones in serum free liquid culture containing IL3, Stem Cell Factor, Dexamethasone and 1 IU/mL EPO. In addition, the quantification of the mutated allele in immature CD34+CD38- cells, in CD34+CD38+ committed progenitor cells, in mature erythroblasts (GPA+) and in granulocytes shows a marked in vivo selective advantage of mutated cells in late stages of hematopoiesis. These results suggest that in PV, erythrocytosis results from two distinct mechanisms: a terminal erythroid amplification advantage triggered by homozygosity or a two-step process including the upstream amplification of heterozygous cells that may involve additional molecular event(s).

scholarly journals Identification of the human pim-1 gene product as a 33-kilodalton cytoplasmic protein with tyrosine kinase activity.

1988 ◽  
Vol 8 (4) ◽  
pp. 1498-1503 ◽  
Author(s):  
A Telerman ◽  
R Amson ◽  
R Zakut-Houri ◽  
D Givol
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Kinase Assay ◽  
In Vitro Translation ◽  
Tyrosine Kinase Activity

The human pim-1 gene was recently identified as a new putative oncogene located on chromosome 6p21, a region showing karyotypic abnormalities in particular leukemias. In the present work we characterized the pim protein product. In vitro translation of positively selected poly(A)+ mRNA indicates that this gene encodes a 33-kilodalton protein. Anti-pim antibodies were raised against a fused TrpE-pim protein induced in a bacterial expression vector. This antibody immunoprecipitated a 33-kilodalton protein from in vivo [35S]methionine-labeled K562 and KCl myelogenous origin cell lines. This protein was localized to the cytoplasm, and in vivo labeling as well as in vitro kinase assay suggests that it is a phosphoprotein with tyrosine kinase activity. This was further confirmed by performing autophosphorylation directly on a p33pim-containing gel band cut out after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results imply that the tyrosine kinase activity of pim can be recovered after boiling the pim-1 protein in sample buffer: a feature not described yet for this class of protein. These results suggest that pim-1 is a new member of the subgroup of oncogenes encoding tyrosine kinases.

scholarly journals Seven Cases of JAK2 Mutation Negative Chronic Polycythemia Patients with Elevated IGF-1R

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5389-5389
Author(s):  
Guanfang Shi ◽  
Rewais Morcus ◽  
Maksim Liaukovich ◽  
Ching Wong ◽  
Vladimir Gotlieb ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Polycythemia Vera ◽  
Mononuclear Cells ◽  
Jak2 V617f ◽  
Driver Mutations ◽  
Erythroid Progenitors ◽  
Jak2 Mutation ◽  
Minor Criteria

We previously reported that assaying blood MNC (Mononuclear cells) IGF-1 levels by Flow cytometry, will be helpful in differentiating Polycythemia vera (PV) from secondarypolycythemia (1). Patients with chronic polycythemia who are negative for JAK2 V617F or exon 12 mutation and who lack the typical bone marrow findings of polycythemia vera will remain a diagnostic enigma. We collected 7 cases of patients who had persistent chronic erythrocytosis ranging from 1-15 years with negative driver mutations, lacking the typical PV bone marrow findings and absence of secondary causes such as smoking or malignancies. Blood mononuclear cells (MNC ) were collected as well as blood DNA extracted for 237 genes including EPO-R, PHD2, VHL or HIF-1-alpha (Familial erythrocytosis genes)with Next generation sequence, performed by Genoptyx lab (Carisbad, CA) and assayed for IGF-1R by flow cytometry as described previously (1). The results are shown in Table 1. Conclusion. All these 7 patients with elevated IGF-1R who had no evidence of familial erythrocytosis gene mutation nor had any secondary cause for erythrocytosis, likely carried the diagnosis of PV. It was shown that EEC formation in PV is due to IGF-1 hypersensitivity (2), andsecondary polycythemia do not show significantly elevated IGF -1R (1). Hence the elevated IGF-1R in these 7 patients strongly suggests the diagnosis of PV, re-affirming our proposal that simple procedures to assay blood MNC cells for IGF-1R by flow cytometry will be helpful in the diagnosis of PV and to be added as one of the minor criteria in the diagnosis of PV. References 1. Wang JC, et al . Quantification of IGF-1 Receptor May Be Useful in Diagnosing Polycythemia Vera-Suggestion to Be Added to Be One of the Minor Criterion.PLoS One. 2016 Nov 3;11(11):e0165299. doi: 10.1371/journal.pone.0165299. 2. Correa PN, Eskinazi, D and Axelrad AA .Circulating Erythroid Progenitors in Polycythemia Vera Are Hypersensitive to Insulin-like Growth Factor-l In Vitro: Studies in an Improved Serum-Free Medium Blood, Vol83, No 1 (January l), 1994: pp 99-1 Disclosures Wang: Incyt: Research Funding.

Identification of the human pim-1 gene product as a 33-kilodalton cytoplasmic protein with tyrosine kinase activity

1988 ◽  
Vol 8 (4) ◽  
pp. 1498-1503
Author(s):  
A Telerman ◽  
R Amson ◽  
R Zakut-Houri ◽  
D Givol
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Kinase Assay ◽  
In Vitro Translation ◽  
Tyrosine Kinase Activity

The human pim-1 gene was recently identified as a new putative oncogene located on chromosome 6p21, a region showing karyotypic abnormalities in particular leukemias. In the present work we characterized the pim protein product. In vitro translation of positively selected poly(A)+ mRNA indicates that this gene encodes a 33-kilodalton protein. Anti-pim antibodies were raised against a fused TrpE-pim protein induced in a bacterial expression vector. This antibody immunoprecipitated a 33-kilodalton protein from in vivo [35S]methionine-labeled K562 and KCl myelogenous origin cell lines. This protein was localized to the cytoplasm, and in vivo labeling as well as in vitro kinase assay suggests that it is a phosphoprotein with tyrosine kinase activity. This was further confirmed by performing autophosphorylation directly on a p33pim-containing gel band cut out after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results imply that the tyrosine kinase activity of pim can be recovered after boiling the pim-1 protein in sample buffer: a feature not described yet for this class of protein. These results suggest that pim-1 is a new member of the subgroup of oncogenes encoding tyrosine kinases.

scholarly journals Mice Lacking the Inhibitory Collagen Receptor LAIR-1 Exhibit a Mild Thrombocytosis and Hyperactive Platelets

2017 ◽  
Vol 37 (5) ◽  
pp. 823-835 ◽  
Author(s):  
Christopher W. Smith ◽  
Steven G. Thomas ◽  
Zaher Raslan ◽  
Pushpa Patel ◽  
Maxwell Byrne ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Thrombus Formation ◽  
Physiological Role ◽  
Src Family Kinase ◽  
Glycoprotein Vi ◽  
Collagen Receptor ◽  
Deficient Mice

Objective— Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is a collagen receptor that belongs to the inhibitory immunoreceptor tyrosine-based inhibition motif–containing receptor family. It is an inhibitor of signaling via the immunoreceptor tyrosine-based activation motif–containing collagen receptor complex, glycoprotein VI-FcRγ-chain. It is expressed on hematopoietic cells, including immature megakaryocytes, but is not detectable on platelets. Although the inhibitory function of LAIR-1 has been described in leukocytes, its physiological role in megakaryocytes and in particular in platelet formation has not been explored. In this study, we investigate the role of LAIR-1 in megakaryocyte development and platelet production by generating LAIR-1–deficient mice. Approach and Results— Mice lacking LAIR-1 exhibit a significant increase in platelet counts, a prolonged platelet half-life in vivo, and increased proplatelet formation in vitro. Interestingly, platelets from LAIR-1–deficient mice exhibit an enhanced reactivity to collagen and the glycoprotein VI–specific agonist collagen-related peptide despite not expressing LAIR-1, and mice showed enhanced thrombus formation in the carotid artery after ferric chloride injury. Targeted deletion of LAIR-1 in mice results in an increase in signaling downstream of the glycoprotein VI–FcRγ-chain and integrin αIIbβ3 in megakaryocytes because of enhanced Src family kinase activity. Conclusions— Findings from this study demonstrate that ablation of LAIR-1 in megakaryocytes leads to increased Src family kinase activity and downstream signaling in response to collagen that is transmitted to platelets, rendering them hyper-reactive specifically to agonists that signal through Syk tyrosine kinases, but not to G-protein–coupled receptors.

TEL/PDGFβR Induces Hematologic Malignancies in Mice That Respond to a Specific Tyrosine Kinase Inhibitor

Blood ◽  
1999 ◽  
Vol 93 (5) ◽  
pp. 1707-1714 ◽  
Author(s):  
Michael H. Tomasson ◽  
Ifor R. Williams ◽  
Robert Hasserjian ◽  
Chirayu Udomsakdi ◽  
Shannon M. McGrath ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Transgenic Animals ◽  
Myeloid Lineage ◽  
Protein Tyrosine

Abstract The TEL/PDGFβR fusion protein is expressed as the consequence of a recurring t(5;12) translocation associated with chronic myelomonocytic leukemia (CMML). Unlike other activated protein tyrosine kinases associated with hematopoietic malignancies, TEL/PDGFβR is invariably associated with a myeloid leukemia phenotype in humans. To test the transforming properties of TEL/PDGFβR in vivo, and to analyze the basis for myeloid lineage specificity in humans, we constructed transgenic mice with TEL/PDGFβR expression driven by a lymphoid-specific immunoglobulin enhancer-promoter cassette. These mice developed lymphoblastic lymphomas of both T and B lineage, demonstrating that TEL/PDGFβR is a transforming protein in vivo, and that the transforming ability of this fusion is not inherently restricted to the myeloid lineage. Treatment of TEL/PDGFβR transgenic animals with a protein tyrosine kinase inhibitor with in vitro activity against PDGFβR (CGP57148) resulted in suppression of disease and a prolongation of survival. A therapeutic benefit was apparent both in animals treated before the development of overt clonal disease and in animals transplanted with clonal tumor cells. These results suggest that small-molecule tyrosine kinase inhibitors may be effective treatment for activated tyrosine kinase–mediated malignancies both early in the course of disease and after the development of additional transforming mutations.

scholarly journals Mutagenic analysis of the roles of SH2 and SH3 domains in regulation of the Abl tyrosine kinase.

1994 ◽  
Vol 14 (5) ◽  
pp. 2883-2894 ◽  
Author(s):  
B J Mayer ◽  
D Baltimore
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Sh3 Domain ◽  
Sh2 Domain ◽  
Sh3 Domains ◽  
Sh2 Domains ◽  
Abl Tyrosine Kinase ◽  
Transforming Activity

We have used in vitro mutagenesis to examine in detail the roles of two modular protein domains, SH2 and SH3, in the regulation of the Abl tyrosine kinase. As previously shown, the SH3 domain suppresses an intrinsic transforming activity of the normally nontransforming c-Abl product in vivo. We show here that this inhibitory activity is extremely position sensitive, because mutants in which the position of the SH3 domain within the protein is subtly altered are fully transforming. In contrast to the case in vivo, the SH3 domain has no effect on the in vitro kinase activity of the purified protein. These results are consistent with a model in which the SH3 domain binds a cellular inhibitory factor, which in turn must physically interact with other parts of the kinase. Unlike the SH3 domain, the SH2 domain is required for transforming activity of activated Abl alleles. We demonstrate that SH2 domains from other proteins (Ras-GTPase-activating protein, Src, p85 phosphatidylinositol 3-kinase subunit, and Crk) can complement the absence of the Abl SH2 domain and that mutants with heterologous SH2 domains induce altered patterns of tyrosine-phosphorylated proteins in vivo. The positive function of the SH2 domain is relatively position independent, and the effect of multiple SH2 domains appears to be additive. These results suggest a novel mechanism for regulation of tyrosine kinases in which the SH2 domain binds to, and thereby enhances the phosphorylation of, a subset of proteins phosphorylated by the catalytic domain. Our data also suggest that the roles of the SH2 and SH3 domains in the regulation of Abl are different in several respects from the roles proposed for these domains in the closely related Src family of tyrosine kinases.

Dysregulated Expression of miRNAs in Polycythemia Vera Erythroid Progenitors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3613-3613
Author(s):  
Hana Bruchova ◽  
Amos S. Gaikwad ◽  
Joshua Mendell ◽  
Josef T. Prchal
Keyword(s):  
Gene Expression ◽  
Polycythemia Vera ◽  
Jak2 V617f ◽  
Prediction Algorithm ◽  
Erythroid Cell ◽  
Molecular Defect ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Homogeneous Population

Abstract Polycythemia vera (PV), the most common myeloproliferative disorder, arises due to somatic mutation(s) of a single hematopoietic stem cell leading to clonal hematopoiesis. A somatic JAK2 V617F point mutation is found in over 80% of PV patients; however, it is not clear if the JAK2 V617F is the disease initiating mutation, sincethere are PV JAK2 V617F negative patients who have monoclonal hematopoiesis and erythropoietin independent erythropoiesis;in individual PV families, there are PV subjects with and without the JAK2 V617F mutation; andanalysis of clonal PV populations reveals the presence of <50 and >50% mutated JAK2 cells (Nussenzweig’ abstract this mtg), suggesting a mixed population of cells with regard to JAK2 status.In order to search for possible PV contributing molecular defect(s), we studied microRNAs (miRNAs) in a homogeneous population of in vitro expanded erythroid progenitors. MiRNAs are non-coding, small RNAs that regulate gene expression at the posttranscriptional level by direct mRNA cleavage, by translational repression, or by mRNA decay mediated by deadenylation. MiRNAs play an important regulatory role in various biological processes including human hematopoiesis. In vitro expanded erythroid progenitors were obtained from peripheral blood mononuclear cells of 5 PV patients (JAK2 V617F heterozygotes) and from 2 healthy donor controls. The cells were cultured in an erythroid-expansion medium for 21 days resulting in 70–80% homogenous erythroid cell population of identical differentiation stage. Gene expression profiling of miRNAs (Thomson, Nature Methods, 1:1, 2004) was performed using a custom microarray (Combimatrix) with 326 miRNA probes. Data were normalized by the global median method. The miRNAs with expression ratios greater than 1.5 or less than 0.5 were considered to be abnormal. Comparative analyses of controls versus PV samples revealed up-regulated expression of miR-let7c/f, miR-16, miR-451, miR-21, miR-27a, miR-26b and miR-320 and down-regulation of miR-150, miR-339 and miR-346 in PV. In addition, miR-27a, miR-26b and miR-320 were expressed only in PV. The putative targets of these miRNAs were predicted by TargetScan prediction algorithm. Up-regulated miR-let-7, miR-16 and miR-26b may modulate cyclin D2, which has an important role in G1/S transition and can be a target in the JAK2/STAT5 pathway (Walz, JBC, 281:18177, 2006). One of the putative targets of up-regulated miR-27a is EDRF1 (erythroid terminal differentiation related factor1), a positive regulator of erythroid differentiation. The BCL-6 gene is predicted to be the target of miR-339 and miR-346, and its activation blocks cellular differentiation. MiR-16 is known to be down-regulated in CLL, where it targets anti-apoptotic BCL-2; in contrast, we show that miR-16 is up-regulated in PV erythroid cells. We identified differentially expressed miRNAs in PV which target genes involved in the JAK/STAT pathway or genes that are modulated by JAK2 downstream molecules. This study indicates that miRNA dysregulation may play an important role in erythropoietic differentiation and proliferation in PV. Expression analyses of these miRNAs in a larger set of PV samples, using quantitative Real-Time-PCR, are in progress. Further, earlier erythroid and pluripotent hematopoietic progenitors are also being analyzed.

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