The JAK2 V617F mutation occurs in hematopoietic stem cells in polycythemia vera and predisposes toward erythroid differentiation

Abstract Primary myelofibrosis (PMF) is a clonal stem cell disorder, characterized by deregulated proliferation of myelofibroblasts. Pathogenesis of PMF has been intensively investigated by analyzing clonality of the disease mainly by X-linked gene inactivation assays. These studies suggested that hematopoiesis in PMF is clonal, whereas fibroblasts are polyclonal not belonging to the PMF clone but they secondarily proliferate in response to cytokines produced by malignant megakaryocytes or monocytes. However, we have previously reported that PMF-initiating cells exist within the circulating CD34+CD38- fraction expressing CD45, whose phenotype is analogous to normal hematopoietic stem cells (HSCs), and this population can reconstitute myelofibrosis after xenotransplantation into NOD/SCID/IL2rg-null mouse newborns (Saito et al. ASH Annual Meeting 2007). In this assay, human PMF HSCs reconstitute PMF-like disease in mouse after transplantation, and strikingly the vast majority of myelofibroblasts were of human origin on FISH analysis, suggesting that myelofibroblasts in PMF can be generated directly from circulating PMF stem cells. We here tried to test this hypothesis by directly analyzing human PMF samples. We performed clonal analyses of fibrotic tissues in PMF patients at the single cell level by utilizing JAK2 V617F mutation as a clonal marker. Nine patients of PMF with JAK2 V617F mutation were enrolled in this study. The fibrotic bone marrow tissues were stained with vimentin, CD34 and KP1 to identify fibroblast-like cells, endothelial cells and myeloid cells, respectively. Single cells of fibrotic tissues were sampled by a laser micro-dissection system, and CD34+CD38-CD45+ HSCs were purified from the blood by FACS. Genomic DNA from single sampled cells was tested for JAK2 V617F mutation. This extensive analysis showed that ∼20-40% of single HSCs and granulocyte/monocyte progenitors (GMPs) had JAK2 V617F. Similar percentages of single vimentin+ fibroblast-like cells, CD34+ endothelial cells and KP-1+ myeloid cells had JAK2 V617F in each patient. Furthermore, each population also constituted similar percentages of heterozygous and homozygous JAK2 V617F as HSCs and GMPs did in each case. Because JAK2 V617F exists in each cell component of myelofibrotic tissues, whose frequency was almost equal to that of PMF HSCs, this study strongly suggests that the majority of myelofibrotic tissue cells belong to the PMF clone, and are differentiated directly from PMF HSCs. These data are compatible with our previous xenotransplantation data. Finally, it is also suggested that JAK2 V617F signaling is not necessary for PMF development. The new understanding of PMF pathophysiology shown here by our experiments might be useful to develop new treatment strategies for human PMF in future studies. Disclosures: Miyamoto: Kyushu University Hospital: Employment.

Download Full-text

Thrombopoietin Receptor (MPL) Genotype Modifies the Myeloproliferative Phenotype in a JAK2 V617F Transgenic Mouse Model of Polycythemia Vera.

Blood ◽

10.1182/blood.v114.22.964.964 ◽

2009 ◽

Vol 114 (22) ◽

pp. 964-964

Author(s):

Jerry L. Spivak ◽

Donna M Williams ◽

Brady L. Stein ◽

Ophelia Rogers ◽

Tsivia Hochman ◽

...

Keyword(s):

Stem Cells ◽

Mouse Model ◽

Hematopoietic Stem Cells ◽

Polycythemia Vera ◽

Transgenic Mouse ◽

Gene Dosage ◽

Jak2 V617f ◽

Transgenic Mouse Model ◽

Single Mutation ◽

Hematopoietic Stem

Abstract Abstract 964 The JAK2 V617F mutation is primarily associated with three chronic myeloproliferative disorders (MPD), polycythemia vera (PV), essential thrombocytosis (ET) and primary myelofibrosis (PMF) but how a single mutation could be responsible for three different disorders is still unresolved. A gene dosage effect was proposed based on the MPD phenotypes in mice with differential expression of a JAK2 V617F transgene, where low expression correlated with an ET phenotype and high expression with a PV phenotype. However, quantitative studies of JAK2 V617F expression in humans revealed significant overlap between PV and ET. Since JAK2 is the cognate tyrosine kinase for the erythropoietin (EPO) and thrombopoietin (TPO) receptors, and JAK2 V617F is expressed in pluripotent hematopoietic stem cells, PV is the ultimate clinical phenotype of the mutation. Furthermore, TPO but not EPO promotes the survival and proliferation of pluripotent hematopoietic stem cells, suggesting that the TPO receptor (Mpl) is essential not only for generating thrombocytosis, but also the stem cell expansion that is characteristic of PV. To examine the role of Mpl in the genesis of the JAK2 V617F MPD phenotype, we manipulated the MPL genotype in a transgenic mouse expressing 13 copies of JAK2 V617F (V617Ftg) (Blood 111:5109, 2009) by breeding these mice with MPL knockout mice (Science265:1445, 1994), which are hematologically normal except for profound thrombocytopenia, to create three genotypes: V617Ftg/MPL wild type (wt); V617Ftg/MPL heterozygote (het), and V617Ftg/MPL knockout (ko). We compared the blood counts, spleen weights, plasma TPO levels, and bone marrow and spleen histology of these three genotypes with each other and with MPL wt, MPL het and MPL ko mice over a 33 week period. Crossbreeding gave the expected genotypes, JAK2 V617F transgene expression was stable in all groups, platelet Mpl expression by immunoblotting correlated with MPL genotype, there was no unexpected mortality, and body weights were not different for any of the genotypes during the observation period. As expected, in V617Ftg/MPL wt mice there was a robust and persistent thrombocytosis (2087 +/− 641 × 106/μL vs 1005 +/− 176 × 106/μL, p<0.001), an erythrocytosis (hemoglobin, 18.3 +/− 1.1 gm % vs 14.9 +/− 0.72 gm %, p <0.001) that peaked at 14-16 weeks but then diminished, and a leukocytosis (16.3 +/− 5.1 × 106/μL vs 12.9 +/−3.4 ×106/μL, p = 0.043) as compared to MPL wt mice. By contrast, in V617Ftg/MPL ko mice, the PV phenotype was virtually abrogated in all cell types as compared to V617Ftg/MPL wt (hemoglobin, 16.1 +/− 0.87 vs 18.3 +/− 1.1, p< 0.001; leukocyte count, 11.3 +/− 2.8 vs 16. 3 +/− 5.1 , p= 0.003, and platelet count, 293 +/− 102 vs 2087 +/− 641, p< 0.001), and not different than their MPL ko counterparts except for a mild erythrocytosis (16.1 +/− 0.9 vs 14.9 +/−, p < 0.001), while in V617Ftg/MPL het mice, erythrocytosis was comparable to the V617Ftg/MPL wt mice and higher than in MPL het controls (17.9 +/− 1.4 gm% vs 14.9 +/− 0.9 gm % p <0.001), but there was only minimal thrombocytosis (1310 +/− 274 × 106/μL vs 1021+/− 241 × 106/μL, p< 0.001), and no leukocytosis (14.8 +/− 4.0 106/μL vs 14.1 +/− 3.7 × 106/μL, p=0.4 ) as compared to the MPL het mice. Marrow and spleen histology reflected the genotype and blood counts and spleen weight was increased equally in all three V617Ftg/MPL genotypes as compared to controls. Plasma TPO was elevated in MPL ko (5530 +/− 1334 pg/mL, p =0.006) and V617Ftg/MPL ko (4201 +/− 736 pg/mL, p = 0.001 ), but not in MPL het mice (723 +/− 720 pg/mL), compared to MPL wt mice (323 +/− 62 pg/mL), while in V617Ftg/MPL wt (163 +/− 52 pg/mL, p < 0.001) and V617Ftg/MPL het mice (176 +/− 56 pg/mL, p < 0.001) plasma TPO was lower than in MPL wt mice. Based on these data, we conclude that MPL genotype is an important modifier of the MPD phenotype in a JAK2 V617F transgenic mouse model of PV, not only for thrombopoiesis but, importantly, also for erythropoiesis and myelopoiesis. We also infer from these data that the impaired Mpl expression observed in human PV may also be a significant modifier of the JAK2 V617F phenotype, either by acting as a dominant-negative with respect to JAK2 V617F activity, or possibly through impaired plasma TPO regulation. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Description of a knock-in mouse model of JAK2V617F MPN emerging from a minority of mutated hematopoietic stem cells

Blood ◽

10.1182/blood.2019001163 ◽

2019 ◽

Vol 134 (26) ◽

pp. 2383-2387 ◽

Cited By ~ 8

Author(s):

Olivier Mansier ◽

Badr Kilani ◽

Amélie V. Guitart ◽

Alexandre Guy ◽

Virginie Gourdou-Latyszenok ◽

...

Keyword(s):

Stem Cells ◽

Mouse Model ◽

Hematopoietic Stem Cells ◽

Polycythemia Vera ◽

Jak2 V617f ◽

Jak2v617f Mutation ◽

Small Subset ◽

Hematopoietic Stem ◽

Key Points

Key Points PF4iCre;JAK2 V617F/WT mice develop a full MPN that mimics polycythemia vera. The PF4iCre system induces JAK2V617F mutation in a small subset of HSC.

Download Full-text

The study of Jak2 V617F mutation in polycythemia vera with zebrafish model

Cell Research ◽

10.1038/cr.2008.231 ◽

2008 ◽

Vol 18 (S1) ◽

pp. S141-S141

Author(s):

Alvin CH Ma ◽

Alice MS Cheung ◽

Alister C Ward ◽

Wing-Yan Au ◽

Yok-Lam Kwong ◽

...

Keyword(s):

Polycythemia Vera ◽

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Zebrafish Model ◽

V617f Mutation

Download Full-text

Ifnα Attenuates the Disease Phenotype and Extends Survival in Mouse Models of MPN

Blood ◽

10.1182/blood-2020-138813 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 53-53

Author(s):

Harini Nivarthi ◽

Andrea Majoros ◽

Eva Hug ◽

Ruochen Jia ◽

Sarada Achyutuni ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Mouse Models ◽

Jak2 V617f ◽

Type I ◽

Type I Ifn ◽

Hematopoietic Stem ◽

Exon 9 ◽

Ifn Treatment

The curative potential of Type I interferons for patients suffering from Myeloproliferative Neoplasms (MPNs) has been reported and these are the only class of drugs that can lead to reduction of the mutant allelic burden in patients. However, modelling IFN treatment in mice has been challenging. Here, we report the use of murine pegylated IFNα (murine ropeginterferon-a, mRopeg) developed by PharmaEssentia (Taipei, Taiwan) to model IFN treatment in transgenic MPN mouse models. We started treating JAK2V617Ff/+;vavCre and control vavCre mice (n=6-8) with PBS or mRopeg (600 ng/mouse/week), by subcutaneous injections from the time they were 4 weeks old. The mice were bled every 2 weeks from the facial vein and the blood parameters were monitored. We observed significant normalization of platelet and WBC counts in Jak2-V617F fl/+ vavCre mice to wild type levels. No effect on hematocrit and hemoglobin level was observed in the Jak2-V617F fl/+ vavCre mice. VavCre control animals showed no sign of negative effect such as cytopenia during the entire treatment course. We observed a highly significant prolongation of the survival of mRopeg treated JAK2V617Ff/+;vavCre mice over a duration of 80 days of treatment. While all the PBS treated JAK2V617Ff/+;vavCre mice died within 60 days, all the mRopeg treated mice were still alive till the end of the treatment duration. We also generated a novel transgenic mouse model that conditionally expresses hybrid mutant CALR protein (murine exons 1-8 and human CALR del52 exon9) from the endogenous murine Calr locus. We bred them into vavCre background (in both heterozyhous and homozygous states) to induce expression of CALR-del52 in hematopoietic cells. Upon Cre recombinase expression, the endogenous murine exon 9 is replaced by the human del52 exon 9 and the expression of the humanized Calr-del52 oncoprotein is detectable by Western blot analysis using mutant CALR specific antibodies. Calr-del52 animals develop an essential thrombocythemia (ET) like phenotype when expressed in a heterozygous state with elevated number of hematopoietic stem cells and megakaryocytes in the bone marrow. In the homozygous state, the thrombocythemia is more severe with splenomegaly and older animals show anemia with increased WBC. Bone marrow histology shows megakaryocytic hyperplasia with no sign of fibrosis up to age of one year. We treated a cohort of animals with 600 ng mRopeg/PBS once a week for 4 weeks. Peripheral blood counts were determined at baseline and at regular intervals during treatment. At the end of treatment, mice were sacrificed, and splenic and bone marrow cells were immunophenotyped and quantified by FACS. We observed correction of thrombocythemia in the homozygous Calr-del52 mice but no unspecific decrease of platelet count in the vavCre mRopeg treated animals. We observed significant specific reduction of the long-term hematopoietic stem cells (LT-HSCs/fraction A) in homozygous CALR-del52 mice. In conclusion, Type I IFN treatment significantly reduces platelet counts to normal levels in both JAK2 and CALR mutant driven MPN mouse models. The prolongation of survival of JAK2V617F transgenic mice upon Type I IFN treatment is particularly remarkable; as no survival data is reported until now in any clinical trials or other animal models. Further experiments are required to understand the mechanism of action of this phenomenon. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Relation between JAK2 (V617F) mutation status, granulocyte activation, and constitutive mobilization of CD34+ cells into peripheral blood in myeloproliferative disorders

Blood ◽

10.1182/blood-2005-09-3826 ◽

2006 ◽

Vol 107 (9) ◽

pp. 3676-3682 ◽

Cited By ~ 164

Author(s):

Francesco Passamonti ◽

Elisa Rumi ◽

Daniela Pietra ◽

Matteo G. Della Porta ◽

Emanuela Boveri ◽

...

Keyword(s):

Polycythemia Vera ◽

Gene Dosage ◽

Jak2 V617f ◽

Myeloproliferative Disorders ◽

Jak2 V617f Mutation ◽

Disease Category ◽

Mutation Status ◽

Cell Counts ◽

Cd34 Cells ◽

V617f Mutation

We studied the relationship between granulocyte JAK2 (V617F) mutation status, circulating CD34+ cells, and granulocyte activation in myeloproliferative disorders. Quantitative allele-specific polymerase chain reaction (PCR) showed significant differences between various disorders with respect to either the proportion of positive patients (53%-100%) or that of mutant alleles, which overall ranged from 1% to 100%. In polycythemia vera, JAK2 (V617F) was detected in 23 of 25 subjects at diagnosis and in 16 of 16 patients whose disease had evolved into myelofibrosis; median percentages of mutant alleles in these subgroups were significantly different (32% versus 95%, P < .001). Circulating CD34+ cell counts were variably elevated and associated with disease category and JAK2 (V617F) mutation status. Most patients had granulocyte activation patterns similar to those induced by administration of granulocyte colony-stimulating factor. A JAK2 (V617F) gene dosage effect on both CD34+ cell counts and granulocyte activation was clearly demonstrated in polycythemia vera, where abnormal patterns were mainly found in patients carrying more than 50% mutant alleles. These observations suggest that JAK2 (V617F) may constitutively activate granulocytes and by this means mobilize CD34+ cells. This exemplifies a novel paradigm in which a somatic gain-of-function mutation is initially responsible for clonal expansion of hematopoietic cells and later for their abnormal trafficking via an activated cell progeny.

Download Full-text

JAK1 and Tyk2 Activation by the Homologous Polycythemia Vera JAK2 V617F Mutation

Journal of Biological Chemistry ◽

10.1074/jbc.c500358200 ◽

2005 ◽

Vol 280 (51) ◽

pp. 41893-41899 ◽

Cited By ~ 107

Author(s):

Judith Staerk ◽

Anders Kallin ◽

Jean-Baptiste Demoulin ◽

William Vainchenker ◽

Stefan N. Constantinescu

Keyword(s):

Polycythemia Vera ◽

Jak2 V617f ◽

Jak2 V617f Mutation ◽

V617f Mutation

Download Full-text

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.

Download Full-text

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

Download Full-text

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.

Download Full-text