LY2784544, a Novel JAK2 Inhibitor, Decreases In Vitro Growth of Hematopoietic Human Progenitors From JAK2 V617F Positive Polycythemia Vera Patients

Abstract Abstract 5054 Introduction: The discovery of JAK2 V617F mutation in patients with myeloproliferative disorders (MPD) has opened new perspectives for the development of targeted therapies. We have studied the efficacy of a novel molecule LY2784544 with JAK2 inhibitory activity in the in vitro growth of myeloid progenitors from JAK2 V617F-positive polycythemia vera (PV) patients. Objectives: To investigate the efficacy of LY2784544 in the inhibition of endogenous(e)BFU-E and CFU-GM growth in PV patients. Methods: In vitro cultures in semisolid media were performed from peripheral blood mononuclear cells (PBMC) of 6 PV patients who had never received cytoreductive treatment (4 patients with homozygous JAK2 V617F and 2 patients with heterozygous JAK2 V617F). PBMC were suspended in methylcellulose (Methocult. StemCell, Vancouver, Canada) without the addition of EPO and containing 0–30.0 μM LY2784544 drug. Concurrent plates containing EPO were plated as control cultures. The medium was distributed in multidishes and they were incubated at 37° with 5% CO2 and 95% humidity. Hemoglobinized colonies and granulomonocytic colonies were counted on day 14 by standard criteria (BFU-E defined by an aggregate of >50 hemoglobinized cells or three or more erythroid subcolonies and CFU-GM was defined by an aggregate of >50 cells). Each in vitro assay was performed in duplicate. DNA was obtained from peripheral blood granulocytes from each patient to quantify the JAK2 V617F allele burden at the time of culture assay. Results: LY2784544, at concentrations ranging from 0.03–30.0 μM, inhibited growth of unselected peripheral blood eBFU-E and CFU-GM from PV patients carrying the JAK2 V617F mutation in a dose-dependent manner, although without achieving complete inhibition of all colonies (fig.1). Conclusions: In vitro studies show that LY2784544 decreases the eBFU-E and CFU-GM growth in therapy-naive JAK2 V617F positive PV patients. Our data suggest that LY2784544 may be a candidate for the treatment of MPD carrying the JAK2 V617F mutation. Disclosures: Ma: Eli Lilly and Company: Employment. Walgren:Eli Lilly and Company: Employment.

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

Blood ◽

10.1182/blood.v106.11.3506.3506 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3506-3506 ◽

Cited By ~ 1

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

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WP1066 Inhibits Growth of Human Cells Carrying the JAK2 V617F Mutation.

Blood ◽

10.1182/blood.v108.11.4885.4885 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4885-4885

Author(s):

Taghi Manshouri ◽

Zeev Estrov ◽

Alfonso Quintas-Cardama ◽

Jorge Cortes ◽

Francis Giles ◽

...

Keyword(s):

Cell Line ◽

Anticancer Agents ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Jak2 V617f ◽

Janus Kinase ◽

Jak2 V617f Mutation ◽

Dependent Manner ◽

Hematopoietic Stem ◽

V617f Mutation

Abstract Myeloproliferative disorders (MPDs) are characterized by proliferation of one or more myeloid cell lineages in bone marrow and peripheral blood, with relatively preserved differentiation. Recent discovery of a dominant gain-of-function mutation in the Janus kinase 2 (JAK2) gene in patients with MPDs, involving the substitution of valine for phenylalanine at position 617 of the JAK2 protein (JAK2 V617F), represents the first acquired somatic mutation in hematopoietic stem cells described in these disorders. This discovery has opened new avenues for the development of targeted therapies for MPDs. WP1066 is a small molecule, a member of a novel class of anticancer agents whose development was based upon the backbone of AG490, a tyrphostin with activity against JAK2 V617F-expressing cell lines but limited in vivo activity. We investigated the inhibitory activity of the WP1066 against the JAK2 V617F-mutant expressing erythroid leukemia HEL cell line and peripheral blood mononuclear cells from patients with polycythemia vera (PV). WP1066 significantly inhibited the phosphorylation of JAK2 and downstream signal transduction proteins STAT3, STAT5, and ERK1/2 in a dose- and time-dependent manner. It induced a time- and dose-dependent antiproliferative and pro-apoptotic effects (activation of caspase 3, release of cytochrome c, and cleavage of PARP) in the JAK2 V617F-bearing HEL cell line in the low micromolar range. Pretreatment of cells with pan-caspase inhibitor Z-VAD abolished WP1066-induced apoptosis. The expression of apoptosis related proteins bcl-2, bax, and XIAP, however, was not changed. More important, WP1066 was effective in inhibiting cell growth in clonogenic assays of mononuclear cells harboring the JAK2 V617F mutation obtained from peripheral blood of patients with PV. We conclude that WP1066 is active both in vitro and ex vivo against cells carrying the JAK2 V617F mutation and represents a solid candidate for the treatment of JAK2 V617V-expressing MPDs.

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Relationship between JAK2 V617F Mutation Status, Granulocyte CD177 mRNA Expression and CD177 Soluble Protein Level in Patients with Polycythemia Vera.

Blood ◽

10.1182/blood.v106.11.2578.2578 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2578-2578

Author(s):

Daniela Pietra ◽

Alessandra Balduini ◽

Carmela Marseglia ◽

Matteo G. Della Porta ◽

Luca Malcovati ◽

...

Keyword(s):

Mrna Expression ◽

Polycythemia Vera ◽

Protein Level ◽

Mononuclear Cells ◽

Jak2 V617f ◽

Myeloproliferative Disorders ◽

Jak2 V617f Mutation ◽

Serum Samples ◽

Close Relationship ◽

V617f Mutation

Abstract A unique gain-of-function mutation of the Janus kinase 2 (JAK2) gene has been recently described in patients with polycythemia vera (PV), essential thrombocythemia and chronic idiopathic myelofibrosis [N Engl J Med. 2005 Apr 28;352(17):1779–90]. Although the currently available data clearly demonstrate that the JAK2 V617F mutation participates in the pathogenesis of myeloproliferative disorders, the mutation’s precise place in the hierarchical order of pathogenetic events remains to be established. We have recently reported that altered gene expression in myeloproliferative disorders correlates with activation of signaling by the V617F mutation of JAK2 (Blood. 2005 Aug 4; Epub ahead of print). Granulocyte CD177 (PRV1) mRNA overexpression has been initially reported as a potential marker of PV but later shown by us to rather be a marker of neutrophil activation [Br J Haematol. 2004 Sep;126(5):650–6]. In this study, we analyzed the relationship between JAK2 V617F mutation status, granulocyte CD177 mRNA expression and CD177 soluble protein level in 72 patients with PV. We also investigated the ontogeny of CD177 expression by hematopoietic cells with the aim of defining the stage of mRNA expression during myeloid, erythroid and megakaryocytic cell differentiation. Finally we studied the effect of soluble CD177 protein on hematopoietic cell proliferation and differentiation. Granulocyte CD177 mRNA expression and percentage of JAK2 V617F alleles were evaluated by quantitative Real Time PCR (qRT-PCR), while serum CD177 protein level was measured by a flow cytometry-based competitive antibody-binding assay. Liquid cultures were performed by culturing peripheral blood mononuclear cells obtained from healthy individuals and PV patients in the presence of high CD177-expressing, low CD177-expressing or CD177-depleted sera. After 12 days of culture, cells were collected, counted and evaluated for colony growth, and for flow cytometry analysis of myeloid, erythroid, megakaryocytic and CD34-positive cell subpopulations. qRT-PCR studies showed a close relationship between CD177 mRNA level and percentage of JAK2 V617F alleles (r=0.412, P<0.001). CD177 mRNA expression was almost undetectable in cell populations other than granulocytes. Studies of CFU-GM growth and differentiation indicated that CD177 mRNA expression is a late event restricted to the neutrophil stage of differentiation. Analysis of serum samples showed variable values for mean fluorescence intensity (MFI), indicating variable levels of the soluble CD177 protein in the patients studied. A very close relationship was found between granulocyte CD177 mRNA expression and soluble CD177 protein level (r=0.56, P=0.02). Incubation of mononuclear cells with serum samples showing high levels of soluble CD177 protein resulted in increased numbers of CD34-positive cells (P<0.02) and of erythroid progenitors (P<0.03). This effect was not detectable when low CD177-expressing or CD177-depleted sera were employed. These observations clearly indicate that the JAK2 V617F mutation is associated with enhanced granulocyte CD177 mRNA expression, and that this latter results in high levels of soluble CD177 protein. These elevated levels might contribute to the increased red cell production that characterizes polycythemia vera.

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JAK2 Mutation Status in Granulocytes, Platelets and Erythrocytes Differs Between Polycythemia Vera and Essential Thrombocythemia

Blood ◽

10.1182/blood.v112.11.5228.5228 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5228-5228

Author(s):

Kohtaro Toyama ◽

Norifumi Tsukamoto ◽

Akio Saito ◽

Hirotaka Nakahashi ◽

Yoko Hashimoto ◽

...

Keyword(s):

T Cells ◽

Polycythemia Vera ◽

Essential Thrombocythemia ◽

Mononuclear Cells ◽

Rna Extraction ◽

Jak2 V617f ◽

Janus Kinase ◽

Jak2 V617f Mutation ◽

Mutation Status ◽

V617f Mutation

Abstract Background The gain-of-function point mutation in Janus kinase 2 exon 14 gene (JAK2-V617F) influences the diagnosis of bcr/abl-negative chronic myeloproliferative disorders (CMPDs). We previously reported that analyzing platelets is advantageous in detecting the JAK2-V617F mutation, particularly in essential thrombocythemia (ET), when compared to granulocytes. However, there have been few reports analyzing the JAK2-V617F mutation in erythroid lineage cells, and comparing the mutation status in all three lineages. Method Study protocols were approved by the Institutional Review Board of Gunma University Hospital, and written informed consent was obtained from all the patients. Heparinized peripheral blood was obtained from 113 patients with CMPDs (82 with ET, 25 with polycythemia vera (PV), and 6 with primary myelofibrosis (PMF). After centrifugation, platelets were collected from the upper plasma layer. Remaining blood was mixed with Hank’s Balanced Salt Solution and was subjected to Ficoll-Hypaque density gradient centrifugation. Granulocytes were obtained from the pellet. Mononuclear cells were resuspended in RPMI 1640 medium; 5 × 105 cells were plated in duplicate in 1 ml of methylcellulose medium and cultured in a humidified atmosphere of 5 % of carbon dioxide at 37°C for 14 days in the presence of erythropoietin to obtain erythroid colonies (BFU-E). T-cells were obtained from the remaining mononuclear cells using anti-CD3 immunoconjugated magnetic beads. After extraction of DNA from granulocytes, T-cells and BFU-E, and RNA extraction from granulocytes and platelets, PCR amplification and sequencing of exon 14 of the Jak2 gene was performed to confirm the presence of JAK2-V617F mutations. To confirm the mutation status of granulocytes, T-cells and BFU-E, allele-specific PCR (AS-PCR) was performed. Results For ET, 57 out of 82 patients (69.5%) had the JAK2-V617F mutation. In the 57 patients with the JAK2-V617F mutation, 38 (67%) had the mutation in all three lineages, 5 had the mutation in granulocytes and platelets, 2 had the mutation in platelets and BFU-E, 10 patients had the mutation only in platelets and 2 patients had the mutation only in BFU-E. In contrast, for PV, 22/25 patients (88%) had the JAK2-V617F mutation. Of note, in 22 patients having JAK2-V617F mutation, 20 (91%) were JAK2-V617F mutation-positive in all three lineages; the remaining two patients had the mutation in either platelets or BFU-E. The frequency of JAK2-V617F in all three lineages was significantly higher in PV than in ET (p < 0.05). For PMF, 5 of 6 patients had the mutation in granulocytes, and 3 of these had it in all three lineages. Conclusion Among JAK2-V617F mutation-positive CMPDs, most PV patients had the JAK2-V617F mutation in all three lineages, thus suggesting that the JAK2-V617F mutation occurs in progenitor cell(s) common to granulocytes, platelets and erythrocytes. In contrast, only 67% of ET patients had the JAK2-V617F mutation in three lineages; in the remaining cases, not all of the three lineages have the mutation. This difference in lineages showing the JAK2-V617F mutation between the ET and PV may be related to the pathophysiological differences in ET and PV. Furthermore, the heterogeneous mutation status in ET may be related to its heterogeneous clinical manifestation.

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Increased death receptor resistance and FLIPshort expression in polycythemia vera erythroid precursor cells

Blood ◽

10.1182/blood-2005-07-3037 ◽

2006 ◽

Vol 107 (9) ◽

pp. 3495-3502 ◽

Cited By ~ 35

Author(s):

Ann Zeuner ◽

Francesca Pedini ◽

Michele Signore ◽

Giusy Ruscio ◽

Carlo Messina ◽

...

Keyword(s):

Polycythemia Vera ◽

Death Receptor ◽

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Erythroid Cells ◽

Jak2 Mutation ◽

Erythroid Precursor ◽

V617f Mutation ◽

Induced Apoptosis

Polycythemia vera (PV) is a clonal myeloproliferative disorder characterized by excessive erythrocyte production. Most patients with PV harbor an activating JAK2 mutation, but the molecular links between this mutation and erythrocyte overproduction are unknown. The interaction between death receptors and their ligands contributes to the physiological regulation of erythropoiesis through the inhibition of erythroblast proliferation and differentiation. With the use of an in vitro culture system to generate differentiating erythroid cells, we found that erythroblasts derived from patients with PV harboring the JAK2 V617F mutation were able to proliferate and generate higher numbers of mature erythroid cells in the presence of inhibitory signals delivered by CD95 (Fas/Apo-1) and TRAIL receptor stimulation. JAK2-mutated PV erythroblasts showed lower levels of CD95-induced caspase activation and incomplete caspase-mediated cleavage of the erythroid transcription factor GATA-1, which was entirely degraded in normal erythroblasts on CD95 stimulation. JAK2 mutation was associated in PV erythroblasts with cytokine-independent activation of the JAK2 effectors Akt/PKB and ERK/MAP and with a deregulated expression of c-FLIPshort, a potent cellular inhibitor of death receptor–induced apoptosis. These results show the presence in PV erythroblasts of proliferative and antiapoptotic signals that may link the JAK2 V617F mutation with the inhibition of death receptor signaling, possibly contributing to a deregulation of erythropoiesis.

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Differential Expression of miRNAs in Polycythemia Vera Peripheral Blood Cells.

Blood ◽

10.1182/blood.v110.11.262.262 ◽

2007 ◽

Vol 110 (11) ◽

pp. 262-262

Author(s):

Hana Bruchova ◽

Michaela Merkerova ◽

Eva Otahalova ◽

Josef T. Prchal

Keyword(s):

Gene Expression ◽

Polycythemia Vera ◽

Mirna Expression ◽

Peripheral Blood ◽

Expression Profiles ◽

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Aberrant Expression ◽

Qrt Pcr ◽

V617f Mutation

Abstract Polycythemia vera (PV) is a clonal myeloproliferative disorder caused by somatic mutation of a hematopoietic multipotent cell. PV hematopoiesis is characterized by an accumulation of phenotypically normal erythrocytes with overproduction of leukocytes and platelets. A somatic JAK2 V617F point mutation occurs in the majority (>95%) of PV patients. However, this mutation is also found in ∼50% of patients with either essential thrombocythemia (ET) or idiopathic myelofibrosis (MF). The non-specificity of this mutation, the presence of JAK2 V617F-negative PV patients, JAK2 V617F-negative and positive relatives, and evidence of both JAK2 V617F-negative and positive PV clones demonstrate that the JAK2 V617F mutation is not the initial and sole somatic event for the pathogenesis of PV. MicroRNAs (miRNAs) have been shown to be important regulators of hematopoiesis. To identify deregulated miRNAs involved in PV pathogenesis, we studied gene expression of miRNAs of in vitro expanded erythroid progenitors (EPs), peripheral blood mononuclear cells (MNCs), granulocytes, reticulocytes, and platelets from PV patients and healthy controls. Initially, we performed gene expression profiling in 5 PV patients and 5 control cells using CombiMatrix MicroRNA CustomArray with 326 probes. The array data were analyzed by Genesis software to determine differentially expressed miRNAs in PV. These miRNAs were tested in a larger set of samples (n=18) by qRT-PCR and their expression was correlated to the JAK2 V617F mutational level. Hierarchical clustering analysis defined miRNA expression profiles of particular cell lineage for normal and PV cells. Further, ANOVA identified 31 miRNAs differently (P<0.05) expressed in at least some PV lineage cells. Of these miRNAs, we confirmed downregulation of miR-150 in expanded EPs of all stages of maturation, downregulation of let7a and upregulation of miR-182 in PV granulocytes; upregulation of miR-143, miR-145 and miR-223 in PV MNCs; and down-regulation of miR-30b, miR-30c and miR-150 in PV reticulocytes by qRT-PCR. Correlation analysis of miRNA expression with JAK2 V617F mutational level showed a positive correlation of miR-143 (r=0.68) and a negative correlation of let7a (r =−0.63), miR-30c (r=−0.74), miR-342 (r=−0.66) and miR-150 (r=−0.89). To validate PV specificity, we compared expression levels of these miRNAs to other MPD disorders (MF and ET) in which the JAK2 V617F mutation occurs. Putative miRNA targets were predicted by TargetScan 4.0 and PicTar software, and transcript levels of selected target genes are being analyzed to determine their potential deregulation at the mRNA level. Downregulated miR-150 is predicted to the target MYB oncogene that plays an important role in erythropoiesis by maintaining proliferation at the early stages. The most potential target of let7a is HGMA2, whose aberrant expression may contribute to clonal hematopoiesis in PNH. The verification of these predictions is in process by use of miRNA inhibitors, protein levels, and functional studies of the progenitors. Our study demonstrates that the specific signatures of miRNAs define particular peripheral blood cell lineages. Furthermore, the deregulated miRNAs, whose expressions correlate with the JAK2 mutational level, may be associated with the PV phenotype. Understanding the role of deregulated miRNAs in PV should provide insight into the pathogenesis of PV and may lead to novel therapeutic strategies.

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Modulated Expression of BCL-xL and GATA-1 Genes Is a Common Feature in Myeloproliferative Disorders (MPD) Both in JAK2-V617F Positive and Negative Patients

Blood ◽

10.1182/blood.v110.11.1533.1533 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1533-1533

Author(s):

Sarah Pozzi ◽

Francesca Bertolotti ◽

Silvio Parodi ◽

Raffaella Ponassi ◽

Barbara Biasotti ◽

...

Keyword(s):

Mononuclear Cells ◽

Statistical Significance ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Myeloproliferative Disorders ◽

Expression Level ◽

Jak2 V617f Mutation ◽

Mutational Status ◽

V617f Mutation

Abstract JAK2-V617F mutation has been identified in more than 90% of patients with Polycythemia Vera (PV) and in 50 to 60% of patients with Essential Thrombocythemia (ET) or Primary Myelofibrosis (PMF). The mutation has reinforced the original idea that ET, PV, and PMF have a common background; however, some key questions remain open: why, in JAK2-V617F patients, only a proportion of progenitors is bearing the mutation and the other is wild type (wt); why patients with the same mutation have a different disease; what have in common patients JAK2-V617F positive (mutated) and wt with the same disease. We observed that a new synthetic peptide (072RB) able to bind Bcl-xL molecule, exerting an apoptotic effect, inhibited the in vitro cord blood (CB) mononuclear cells (MNC) growth. Moreover, this effect correlated with a high expression level of Bcl-xL messenger (RQ-PCR). Since Bcl-xL was involved in erythropoiesis, we extended the expression studies to bone marrow (BM) MNC from16 PV (13/16 mutated),15 ET (9/15 mutated) and peripheral blood (PB) MNC from 18 PMF (9/18 mutated). JAK2 mutational status was assigned by allele-specific-PCR (AS-PCR). MNC from PV-BM and PMF-PB showed a Bcl-xL level of expression significantly higher than in MNC from healthy donors (NBM and NPB) both in mutated and in wt patients (PV: p=0.01 and p=0.004; PMF: p=0.005 and p=0.05 respectively). In ET, the expression level of Bcl-xL tended to be elevated compared to controls but did not reach the statistical significance. Since other factors can modulate Bcl-xL expression independently from the constitutive activation of JAK2/STATs pathway induced by JAK2-mutated, we analysed GATA-1 gene, a transcription factor that binds the Bcl-xL promoter and that is involved in erythropoiesis and megakariocytopoiesis. We observed that GATA-1 was highly expressed in PV-BM and PMF-PB MNC both in mutated and in wt patients (PV: p=0.01 and p=0.05; PMF: p=0.001 and p=0.03). In ET-BM MNC, GATA-1 followed the Bcl-xL pattern of expression. The highest messengers levels were observed PMF-PB MNC and CB MNC that, after in vitro 072RB peptide treatments, showed a 25%–50% of cells growth inhibition with respect to untreated controls. The protein expression was confirmed by cytofluorimetric analyses. Our finding may indeed be compatible with reduced apoptosis both in mutated and wt patients. Thus, the elevated expression levels of Bcl-xL and GATA-1 genes may represent a common feature in MPD, independent from the presence of the JAK2-V617F mutation and supports the hypothesis of a “phenotypic continuum” in MPD. It is noteworthy that in patients bearing the mutation, a variable proportion of hematopoietic progenitors in PV, ET and PMF have been documented to be wt. In this context, our findings may explain why wt hematopoiesis is not overtaken by the mutated counterpart. These results could open a new insight in the field of MPD molecular characterization and may lead to new therapeutic approaches.

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Lineage Distribution of JAK2 Exon12 Mutations and JAK2-V617F in Patients with Polycythemia Vera

Blood ◽

10.1182/blood.v110.11.1527.1527 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1527-1527

Author(s):

Sai Li ◽

Robert Kralovics ◽

Gennaro De Libero ◽

Andre Tichelli ◽

Radek C. Skoda

Keyword(s):

T Cells ◽

B Cells ◽

Nk Cells ◽

Polycythemia Vera ◽

Peripheral Blood ◽

Jak2 V617f ◽

Lymphoid Cells ◽

Jak2 V617f Mutation ◽

V617f Mutation ◽

Lineage Distribution

Abstract Mutations in exon 12 of the JAK2 gene have been identified in a minority of patients with myeloproliferative disorders (MPD) and are associated with a selective increase in erythropoiesis resulting in polycythemia vera (PV) or idiopathic erythrocytosis. We compared the lineage distribution of JAK2 mutations in the peripheral blood of 8 PV patients with mutations in exon 12 and 21 PV patients with the JAK2-V617F mutation. Five different exon 12 mutations were observed in the 8 patients studied. Peripheral blood cells were fractionated to obtain granulocytes, platelets and mononuclear cells, which were further separated by FACS into T cells, B cells, NK cells and monocytes. Using a sensitive and quantitative assay to assess the percentages of chromosomes carrying exon 12 mutations, we detected exon 12 mutations in purified granulocytes, monocytes and platelets of all patients studied. A similar distribution was also found for the JAK2-V617F mutation. Exon 12 mutation was absent in sorted lymphoid cells of 5/8 patients, in 2/8 patients only NK cells were positive and in 1/8 patients also B cells carried the mutation. Exon 12 mutations were absent in T cells of all patients studied and JAK2-V617F was present in T cells of only 1/21 patients. Thus, inter-individual differences are notable in the involvement of lymphoid lineages for both exon 12 and JAK2-V617F mutations. To determine the presence of the JAK2 mutations in the erythroid lineage, we performed colony assays in methylcellulose, picked single erythroid colonies grown in the presence or absence of Epo and determined the allelic ratios for each individual colony. In 4/8 patients an exon 12 mutation (E543-D544del) was present in all EECs examined and similarly, in 8/12 patients the JAK2-V617F mutation was found in all EECs. In the remaining patients, we detected some EECs with only the wild type JAK2, suggesting that additional clonal events may also be present in some patients with exon 12 mutations. Interestingly, one patient carried exon 12 and JAK2-V617F mutations. None of the erythroid colonies in this patient carried both mutations simultaneously, indicating that the exon 12 mutation and JAK2-V617F represent two separate clones. One patient displayed erythroid colonies homozygous for the exon 12 mutation and an allelic ratio greater that 50% in granulocytes, indicating that progression to homozygosity can occur in some patients. The lineage distributions of exon 12 mutations and JAK2-V617F are similar and do not explain why exon 12 mutations are associated solely with PV phenotype, whereas JAK2-V617F can also cause essential thrombocythemia or primary myelofibrosis.

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