JAK2V617F Positive Myeloproliferative Neoplasms Patients Research Articles

Increase in Total Protein Level of Bruton's Tyrosine Kinase in Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are characterized by atherosclerosis and cardiovascular risk. The mechanisms that connect MPNs with atherothrombosis remain elusive. It is known that atherosclerotic plaque causes Glycoprotein VI (GPVI)-dependent platelet activation and hence activation of bruton kinase (Btk) and that Btk inhibitors in vivo and ex vivo suppress GPVI-mediated paltelet activation. Aim of this study is to measure the levels of GPVI and Btk. In addition, we measured the CXCL12 as marker of atherothrombosis. We studied 25 JAK2V617F positive MPNs patients (WHO criteria) (mean age 58 years) including PV 12, ET 8, and PMF 5. We measured platelet count by automated analyzer, GPVI by flow cytometry, total Btk (T-Btk) protein by immunoblot and total CXCL12 (T-CXCL12) protein by ELISA. All patients had thrombocytosis (630±40x109/L) and allele burden ≥ 50%. We found high GPVI expression (mean fluorescence intensity (MFI) 20±5.8) and high T-Btk (mean value 1.0) as well as high T-CXCL12 (15±2 μg/ml). A positive correlation there was between GPVI and Btk. We further investigated Btk mRNA tran script levels using RT-PCR assay in triplicate from each samples and found mean values ≥ 1.0. On the basis of these results the use of GPVI- and Btk- inhibiting agents as atherothrombosis-focused antiplatelet drugs is conceivable. Therefore, clinical trials are eagerly awaited.

Vascular Endothelial Dysfunction in Myeloproliferative Neoplasms and Gene Mutations.

Essential thrombocythemia (ET) and polycythemia vera (PV), are common Philadelphia-negative myeloproliferative neoplasms (MPN). Patients with MPN have a high rate of cardiovascular complications and often have acquired JAK2V617F and CALR genetic mutations. In this study, we aimed to analyze vascular endothelial function in patients with MPN.We evaluated 27 outpatients, including 10 patients diagnosed with MPN, flow-mediated dilatation (FMD), and nitroglycerin-mediated dilation (NMD), between September 2014 and August 2016. We measured serum adiponectin, which protects vascular endothelial function, and serum asymmetric dimethyl arginine (ADMA), which inhibits the production of adiponectin. The presence or absence of JAK2V617F and CALR mutations was evaluated in patients with MPN.Venous thrombosis was observed more frequently in patients with MPN than in those without. Seven MPN patients were diagnosed with PV, and 3 MPN patients were diagnosed with ET. JAK2V617F and CALR mutations were found in 5 and 3 MPN patients, respectively. FMD was significantly lower in JAK2V617F-positive MPN patients than in JAK2V617F-negative MPN patients, although NMD, adiponectin, and ADMA were similar in both groups. Adiponectin levels were higher and ADMA levels were lower in CALR-positive MPN patients than in CALR-negative MPN patients. There was no difference in FMD and NMD prevalence between the 2 groups. Furthermore, we had 3 representative MPN patients who were complicated with coronary spasm, possibly caused by MPN-related endothelial dysfunction.We found that patients with MPN presented with endothelial dysfunction, which was related to the presence of genetic mutations and was sometimes associated with cardiovascular disease.

Abstract 3806: Cigarette smoke extract permits survival of TET2-/- and JAK2V617F hematopoietic cells

Abstract Inactivating somatic mutations in the ten-eleven translocation 2 (TET2) gene is commonly found in clonal hematopoiesis (CH) and contributes to the pathogenesis of myeloid malignancies such as myeloproliferative neoplasms (MPN), myelodysplastic syndrome and acute myeloid leukemia. Incidentally, smoking behavior is associated with CH and we hypothesize that TET2 mutant hematopoietic cells are resistant to the deleterious effects of smoke exposure. Cigarette smoke extract (CSE) was prepared from mainstream cigarette smoke using a standard protocol and volume/volume percentage (v/v %) dilutions used for in vitro exposures. Lineage negative bone marrow cells isolated from TET2 wildtype (TET2+/+) and TET2 knockout (TET-/-) mice were exposed to CSE and cultured in methylcellulose containing semi-solid medium to assess myeloid colony formation. Cells were also grown in liquid culture under long-term culture conditions to demonstrate the effect of CSE on cell proliferation. Peripheral blood mononuclear cells from JAK2V617F positive MPN patients were treated with CSE for colony forming assays. TET2+/+ bone marrow cells showed decreased myeloid colony formation in the presence of CSE while TET2-/- cells were not significantly affected by CSE. Similarly in liquid cultures, TET2-/- lineage negative cells expanded significantly compared to their wildtype counterparts indicating resistance to the growth suppressive effects of CSE. The JAK2V617F mutation is the most common somatic mutation found in MPN patients and we observed that the suppressive effect of CSE on myeloid colony formation was more pronounced in normal subjects than JAK2V617F positive MPN subjects. This suggests that CH associated somatic mutations such as in TET2 promote resistance of bone marrow progenitors to environmental stressors like cigarette smoke, possibly via anti-apoptotic mechanisms. Future studies include studying the effects of inhalation exposure of combustible and electronic cigarette smoke on TET2-/- and JAK2V617F hematopoiesis in competitive bone marrow transplanted mice. Citation Format: Gajalakshmi Ramanathan, Brianna Craver, Rebecca J. Arechavala, David Herman, Michael T. Kleinman, Angela G. Fleischman. Cigarette smoke extract permits survival of TET2-/- and JAK2V617F hematopoietic cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3806.

TERT and JAK2 polymorphisms define genetic predisposition to myeloproliferative neoplasms in Japanese patients.

Myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are often characterized by specific somatic mutations in any of the three genes: JAK2, CALR, or MPL. A single nucleotide polymorphism (SNP), rs2736100, in the reverse transcriptase gene (TERT) and a germline JAK2 46/1 haplotype have been associated with MPNs in North American and European patients. We examined 201 Japanese MPN patients, including 52 with PV, 131 with ET, and 18 with PMF, as well as 366 control individuals for TERT rs2736100 and JAK2 rs10974944, a tagging SNP of the 46/1 haplotype. Furthermore, correlations between the JAK2 V617F allele burden at diagnosis and TERT rs2736100 or JAK2 rs10974944 were evaluated using a digital PCR assay for accurate quantitation. The JAK2 46/1 haplotype, but not the TERT rs2736100 SNP, was correlated to the JAK2 V617F mutant allele burden in JAK2 V617F-positive MPN patients. In conclusion, we demonstrated that both TERT rs2736100_C and JAK2 46/1 haplotype are predisposing factors for MPNs in Japanese patients. While TERT rs2736100_C tended to have a more general, non-specific effect on all MPNs, the JAK2 46/1 haplotype was essentially predisposed to the JAK2 V617F-positive MPNs.

JAK2 R1063H Variant Enhances V617F Constitutive Signaling and Favors Development of Essential Thrombocythemia with Increased Hemoglobin and Neutrophils

The clinical consequences of the highly prevalent JAK2 V617F mutation in Ph-negative myeloproliferative neoplasms (MPNs) are well studied. However, the impact on the MPN phenotype of co-occurring JAK2 variants is less well characterized. In this study, we focused on JAK2 R1063H, a variant previously reported in a pediatric case of hereditary erythrocytosis (Kapralova et al, Blood 2016) and in 3 out of 93 polycythemia vera patients that were JAK2 V617F-positive (Levine et al, Cancer Cell 2005). It has been described as a very rare polymorphism in population (frequency 0.004377).In order to gain insight into the clinical and functional relevance of coexisting JAK2 mutations, we tested a cohort of 390 JAK2 V617F-positive MPN patients for JAK2 R1063H and identified 14 carriers of both mutations. From the clinical perspective, the double-positive patients exhibited predominantly an essential thrombocythemia phenotype that was accompanied by significantly higher neutrophil granulocyte counts and hemoglobin values, when compared to those harboring only JAK2 V617F mutation. By employing targeted NGS for genes known to be involved in myeloid malignancies we found that the mutational profile and the number of additional somatic mutations in double-mutant patients seemed to be comparable to previous studies of JAK2 V617F-positive MPNs.Next, we used digital droplet PCR (ddPCR) for JAK2 R1063H and JAK2 V617F allele burden assessment. Quantification of JAK2 R1063H allele indicated that in 8 patients the variant was heterozygous, likely inherited (the percentage of the mutant allele being around 50%). In 3 patients with a high JAK2 V617F allelic burden a nearly homozygous status for R1063H was identified (a fractional abundance >80%) suggesting that one R1063H allele was inherited and the second one was acquired by uniparental disomy, as a result of mitotic recombination. In 3 other patients, the JAK2 R1063H mutation was most likely acquired due to the low percentage of the mutant allele (between 20.7% and 31.5%). For cis/trans configuration analysis of JAK2 V617F and R1063H mutations, we performed single-colony sequencing of subcloned JAK2 cDNA from 9 out of 14 patients. A cis configuration of the mutations was detected in 7 and a trans configuration in 2 cases. In one patient, the cis configuration of JAK2 mutations combined with results obtained by ddPCR (the allele burdens for R1063H and V617F were 31.5% and 52.8%, respectively) suggested that R1063H was acquired, following V617F acquisition.To assess the effect on JAK2 signaling of coexisting JAK2 V617F and R1063H mutations in cis or trans, we created human cDNA JAK2 mutants (V617F, R1063H and V617F/R1063H). STAT5 transcriptional activity of the JAK2 WT and JAK2 mutants in the presence of myeloid dimeric cytokine receptors (EPOR, given the higher hemoglobin levels, G-CSFR, given the observed neutrophilia in patients carrying JAK2 V617F and R1063H, and TPOR) measured in JAK2-deficient γ-2A cells by dual luciferase assay revealed a significantly higher constitutive activity of JAK2 V617F/R1063H (cis mutant) compared to that of JAK2 V617F, in both homozygous and heterozygous configurations and with either of the dimeric myeloid cytokine receptors. In the trans configuration of JAK2 V617F and JAK2 R1063H, we did not observe a significant difference compared to combination of JAK2 V617F and JAK2 WT. Western blot analysis assessing activated phosphorylated forms of JAK2, STAT5 and ERK1/2 demonstrated a higher level of constitutive activation of JAK2 and STAT5 generated by the double V617F/R1063H mutant versus JAK2 V617F and similar effect was shown also for ERK1/2 signaling.Our results suggest that either an acquisition of V617F in cis on a germline R1063H allele or a gain of additional JAK2 R1063H on JAK2 V617F will lead to an increase in downstream signaling by the driver JAK2 V617F. The oncogenic signaling of JAK2 V617F enhanced by the R1063H mutation has a clinical effect, leading to a significantly increased number of neutrophilic granulocytes and hemoglobin values, in agreement with higher levels of signaling via EPOR and G-CSFR.The work was supported by projects COP A1.1.4. ID: P_37_798, Contract 149/26.10.2016, (MySMIS2014+: 106774), MyeloAL, WELBIO F 44/8/5-MCF/UIG-10955, ARC: N° 16/21-073, by Ludwig Institute for Cancer Research, Salus Sanguinis Foundation, Fondation Les avions de Sébastien, Belgium and by GACR 17-05988S and MSMT LTAUSA17142. DisclosuresNo relevant conflicts of interest to declare.

Regulation of Ruxolitinib on matrix metalloproteinase in JAK2V617F positive myeloroliferative neoplasms cells

目的探讨JAK2抑制剂Ruxolitinib对JAK2V617F突变阳性的骨髓增殖性肿瘤（MPN）细胞内基质金属蛋白酶（MMP）调控的影响。方法①收集2012年1月到2015年12月保定市第一医院收治的40例未经治疗的JAK2V617F阳性MPN患者，以15名健康志愿者为对照组。免疫组化法检测两组骨髓活检组织中磷酸化JAK2（p-JAK2）、基质金属蛋白酶2（MMP-2）、MMP-9的表达水平。选取JAK2V617F阳性MPN患者骨髓细胞，体外应用Ruxolitinib干预，测定干预前后细胞迁移力和MMP-2、MMP-9表达。②不同浓度Ruxolitinib（0、50、100、250、500、1 000 nmol/L）作用于人红白血病细胞株HEL细胞不同时间后CCK-8检测细胞活力，Tanswell小室检测细胞迁移，荧光定量PCR检测JAK2、MMP-2、MMP-9 mRNA水平变化，Western blot检测p-JAK2、MMP-2、MMP-9蛋白表达。结果①MPN组p-JAK2、MMP-2、MMP-9蛋白表达均高于对照组[（78.56±24.55）%对（41.59±17.29）%、（48.25±18.74）%对（22.79±13.89）%、（53.29±19.28）%对（15.56±14.96）%，P值均<0.05]。Spearman相关分析显示MMP-2、MMP-9蛋白水平与JAK2V617F突变量呈正相关（r=0.526, P=0.001；r=0.543, P=0.001）。②Ruxolitinib能够呈时间和剂量依赖性抑制HEL细胞增殖。③迁移实验结果显示5 nmol/L Ruxolitinib作用MPN原代细胞及HEL细胞24 h后迁移至下室细胞数均少于无Ruxolitinib组（154.7±27.5对320.3±67.3，t=13.47，P=0.001；70.7±10.5对135.3±16.7，t=13.89，P=0.001）。④JAK2、MMP-2、MMP-9 mRNA及蛋白表达随Ruxolitinib剂量增加而降低。结论Ruxolitinib通过调控JAK2信号通路抑制MMP-2、MMP-9表达而抑制MPN细胞迁移能力。

Effects of the Sympathicomimetic Agonist Mirabegron on Disease Course, Mutant Allele Burden, Marrow Fibrosis, and Nestin Positive Stem Cell Niche in Patients with JAK2-Mutated Myeloproliferative Neoplasms. a Prospective Multicenter Phase II Trial SAKK 33/14

Myeloproliferative neoplasms (MPN) are clonal hematopoietic disorders characterized by aberrant proliferation of erythroid, megakaryocytic and myeloid lineages. They are associated with decreased survival, thromboembolic complications, hemorrhage and leukemic transformation. MPN can be subdivided into polycythemiavera(PV), essentialthrombocythemia(ET) and primary myelofibrosis (PMF). The JAK2-V617F mutation is present in 70-80% of all MPN patients. MPN is initiated and maintained by mutated hematopoietic stem and progenitor cells (HSPC). Bone marrow mesenchymal stem cells expressing the intermediate filament proteinnestin(nestin+ MSCs) that are innervated by sympathetic nerve fibers constitute an important component of the stem cell niche and regulate normal HSCs. Thesenestin+ MSCs are strongly reduced in bone marrow of JAK2-V617F positive MPN patients and in mice expressing JAK2-V617F due to damage of the sympathetic nerve fibers triggered by cytokines from the mutant cells. In a JAK2-V617F mouse model of MPN, treatment with a beta-3sympathicomimeticagonist corrected the damage inflicted by the MPN clones on their niches and ameliorated the MPN phenotype. To test the potentially beneficial effect on disease-control by modulating bone marrow niche cells in patients with MPN, we performed a phase II trial with the beta-3sympathicomimeticagonistmirabegron.Patients and Methods:The trial consisted ofmirabegrontreatment with 25 mg daily during the first week, followed by 50 mg daily for at least 24 weeks. Patients with acytohistologicallyconfirmed diagnosis of MPN and a JAK2-V617F allele burden >20% in granulocytes at study entry were eligible, if not treated with JAK2 inhibitors or interferon. Reduction of the JAK2-V617F mutant allele burden ³50% in granulocytes was defined as the primary end point. Secondary end points included changes in blood counts or MPN related symptoms. As a side study, bone marrow biopsies were quantified fornestin+ MSCs, fibrosis and CD34+ HSPCs. N=39 patients have been accrued in 10 institutions in Switzerland. Eight (21%) had ET, 22 (56%) PV, and 9 (23%) PMF. N=27 (69%) were male, the median age was 62 (Q1-Q3 53-72) years. Median mutated allele burden at study onset was 52% (Q1-Q3 33-73%). All patients had prior treatment, N=28 (72%) patients hadcytoreductivetreatment, the remaining patients hadantiaggregation, anticoagulation or phlebotomy.Results:No patient reached the primary endpoint of 50% reduction in allele burden, one patient achieved a 25% reduction by 24 weeks of treatment. Adverse events were mostly grade I or II on the CTCAE scale. Three patients had grade III events: two were considered to be at least possibly related to study medication.In the side study, 24 patients agreed to bone marrow biopsy prior to and at the end ofmirabegrontreatment and for 20 patients both measurements are available. In these patients an increase in thenestin+ MSCs cells from a median of 1.09 (Q1-Q3 0.38-3.27)/mm2 to 3.95 (Q1-Q3 1.98-8.79)/mm2 (p<0.0001, Wilcoxon signed-rank test) and a slight decrease of myelofibrosis from a median grade of 1.00 (Q1-Q3 0.50-3.00) to 0.75 (Q1-Q3 0.50-2.00) (p=0.02), were observed. The mean change in thenestin+ cells from baseline to week 24 was 3.52 (95% confidence interval 1.65-5.39)/mm2. Morphometric changes in thenestin+ MSCs were significant for PV (n=13, p=0.007) and PMF (n=5, p=0.04). Bone marrow CD34+ cells slightly increased from a median 2.50 (Q1-Q3 2.00-3.25) to 3.00 (Q1-Q3 2.00-3.75) (p=0.06).Conclusion:In this prospective phase II clinical trial treatment with the beta-3-sympathicomimetic agonistmirabegronfor 24 weeks failed to achieve the primary endpoint to reduce the JAK2-V617F mutant allele burden >50% in patients with MPN. However, an increase in thenestin+ MSCs in bone marrow and a slight decrease of myelofibrosis were found, which will be further investigated. [Display omitted] DisclosuresTheocharides:Novartis: Consultancy, Honoraria. Rüfer:Novartis: Consultancy, Speakers Bureau. Benz:Celgene: Consultancy. Tzankov:Novartis: Speakers Bureau; Abbott: Speakers Bureau. Skoda:Novartis: Consultancy, Speakers Bureau; Baxalta: Speakers Bureau; Shire: Consultancy, Speakers Bureau.

Expression of JAK2-V617F Kinase Profoundly Dysregulates the Functional Properties of β1 Integrins on Myeloid Cells

Introduction: Deregulated signal transduction pathways driven by the constitutively active JAK2-V617F kinase, play a central role in pathogenesis of the classical Philadelphia negative myeloproliferative neoplasms (MPNs). Polycythemia vera, essential thrombocythemia and primary myelofibrosis are characterized by clonal proliferation of myeloid cells and development of inflammatory syndrome (CRP elevation, fever, high proinflammatory cytokine plasma levels) particularly, in advanced phases of the disease. Integrins are heterodimeric cell surface receptors that mediate a wide range of anchorage dependent events fundamentally essential in adhesion and mobility of leukocytes during inflammatory states. An intricate network of intracellular signaling pathways triggers the activation of intergins by allosteric conformational changes and/or by altering lateral mobility to increase receptor clustering.Methods: We investigated the role of JAK2-V617F in the regulation of β1 integrin activation, function and expression in myeloid cells using in vitro cell culture models, in vivo JAK2-V617F knock-in model, murine bone marrow transplantation model and finally, clinical patient samples (collected after informed consent obtained in accordance with the Declaration of Helsinki). Integrin activation was studied using flow cytomery based soluble ligand binding assay and by analyzing the expression of 9EG7 activation epitope. The functional static adhesion assay was performed on ligand coated plates and data was acquired using a fluorescence microplate reader. Surface expression of integrins was investigated by flow cytometry.Results: To identify a potential biological impact of JAK2-V617F on β1 integrins, we generated a 32D murine myeloid progenitor cell line transfected with EpoR and either wildtype JAK2 or JAK2-V617F. Under serum starved conditions, the presence of the oncogenic JAK2-V617F mutation strongly induced activation of β1 integrins as revealed by a 6-fold rise in binding to its soluble ligand, VCAM-1-Fc and enhanced exposure of the 9EG7 high-affinity conformation-specific epitope. This also translated into increased adhesion on VCAM-1 in comparison to wildtype cells. Importantly, inhibition of the constitutive JAK2-V617F kinase activity by JAK inhibitor treatment, significantly downregulated β1 integrin activation as well as cellular adhesion. Furthermore, shRNA based knockdown of the endogenous JAK2-V617F expression in HEL (Human Erythroleukemia) cells, reduced adhesion on VCAM-1, thereby underscoring the functional role of the JAK2-V617F kinase in the regulation of β1 integrins. Interestingly, the cell surface expression of β1 integrins remains unaltered in the presence of JAK2-V617F or inhibitor treatment in the 32D cells as well as, following genetic inhibition in HEL cells. Additionally, in an in vivo retroviral bone marrow transplantation model in Balb/c mice, the overexpression of wildtype or JAK2-V617F, showed similar β1 integrin expression in granulocytes. Currently, in vivo studies in a JAK2-V617F knock-in murine model are under way. We then extended our study to investigate the role of constitutively active JAK2-V617F in integrin function in MPN patients. Again, no significant difference was found in β1 integrin expression on primary granulocytes from JAK2-V617F positive MPN patients and age-matched healthy donors. However, granulocytes from JAK2-V617F-positive patients showed higher activation of β1 integrins as exhibited by a 6-fold increase in binding to soluble VCAM-1-Fc when compared to healthy donors. In addition, preliminary analysis suggests stronger adhesion of JAK2-V617F positive granulocytes on VCAM-1. In an attempt to dissect the underlying molecular mechanism, we uncovered a previously undescribed role of JAK2 in the inside-out signaling of integrin activation in 32D myeloid cells. Overexpression of wildype JAK2 or V617F resulted in activation of the small GTPase Rap1, a key integrin regulator, and was subsequently downregulated upon treatment with JAK inhibitor.Conclusions: Taken together, our study highlights a unique and important role of JAK2-V617F in regulation of β1 integrin activation in myeloid cells. This may have functional consequences in pathophysiology of the disease, particularly, in dysregulation of leukocyte adhesion during thrombosis and in induction of inflammation. DisclosuresNo relevant conflicts of interest to declare.

Stathmin 1 Silencing Amplifies Ruxolitinib-Induced Apoptosis in JAK2V617F Cells

Introduction: The JAK/STAT pathway is constitutively activated in the myeloproliferative neoplasms (MPN), including essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). JAK/STAT pathway has been targeted by Ruxolitinib, a selective JAK1/2 inhibitor approved by FDA for intermediate and high-risk PMF treatment. Results from phase III clinical trials demonstrated that Ruxolitinib is well-tolerated, reduces inflammatory cytokines and splenomegaly, ameliorated constitutional symptoms; however, Ruxolitinib did not reverse bone marrow fibrosis, suggesting that the development of better approaches are necessary. In addition to the STAT3 transcriptional activity in the nucleus, activated STAT3 binds to and inhibits Stathmin 1 in the cytoplasm, resulting in microtubule stability. Stathmin 1 is an important cytoplasmic microtubule-destabilizing protein that plays critical roles in proliferation and accurate chromosome segregation through regulation of microtubule dynamics. Paclitaxel, an anti-neoplastic compound, is a microtubule-targeted drug that induces microtubule polymerization, cell-cycle block at the metaphase-anaphase transition and cell death, and inhibits Stathmin 1 activity. We hypothesized that an undesirable effect of Ruxolitinib treatment could be Stathmin 1 activation as a consequence of STAT3 inhibition; hence targeting Stathmin 1 in addition to JAK2 might be a reasonable approach in MPN.Aims: To investigate Stathmin 1 expression in CD34+ cells from MPN patients and the effects of Stathmin 1 silencing or Paclitaxel-induced microtubule stability on Ruxolitinib JAK2V617F-treated cells.Materials and Methods: CD34+ cells were obtained from a total of 104 peripheral blood (PB) samples collected from healthy donors (n=22) and patients with MPN (n=82; ET=34, PV=23, PMF=25; JAK2V617F=52, JAK2wt=30, CALRmut=19 and CALRwt=54). Gene and protein expressions were evaluated by qPCR and Western blot. In JAK2V617F-positive HEL cells, Stathmin 1 was stably knocked down with specific shRNA-expressing lentiviral vector, and cell growth, clonogenicity and apoptosis were evaluated by MTT assay, colony formation, and AnnexinV/PI and Caspase 3 activation by flow cytrometry, respectively. Alternatively, HEL cells were submitted or not to Paclitaxel treatment (5 and 10 nM). All conditions were evaluated in the presence or not of Ruxolitinib (100 and 300 nM). Statistical analyses were performed by Student’s t-test or Mann-Whitney test, as appropriate.Results: Stathmin 1 transcripts were significantly increased in PB CD34+ cells from PMF patients (2.73 [0.49-8.32]; p=.005) compared with healthy donors (1.08 [0.08-7.17]). No difference in Stathmin 1 expression was observed between healthy donors, ET and PV PB CD34+ cells. Stathmin 1 levels did not significantly differ between JAK2wtvs. JAK2V617F (p=.20); and CALRwtvs. CALRmut patients (p=.63). In HEL cells, Stathmin 1 silencing significantly reduced cell proliferation (p<.05) and clonal growth (p<.01). Although Stathmin 1 silencing itself did not modulate apoptosis, it increased apoptosis induced by Ruxolitinib treatment (p<.01). Stathmin 1 silencing resulted in microtubule stability (increased alpha-tubulin acetylation), whereas Ruxolitinib treatment induced microtubule instability (decreased alpha-tubulin acetylation) and inhibited JAK2 and STAT3 activity. Stathmin 1 silencing in association with Ruxolitinib prevented microtubule instability. In the attempt to pharmacologically inhibit Stathmin 1 activity, cells were treated with Paclitaxel, in association or not Ruxolitinib. Combined treatment resulted in a significantly increased apoptosis compared to monotherapy (p<.01). The ability of Paclitaxel of inhibiting Stathmin 1 was confirmed by increased phosphorylation of Stathmin 1 at serine 16 (an inhibitory site) in either Paclitaxel-treated cells and in Paclitaxel plus Ruxolitinib-treated cells, which was not observed in Ruxolitinib-treated cells.Conclusions:Stathmin 1 is highly expressed in PMF. Our study adds new insights for Stathmin 1 in hematological malignancies and indicates a possible role of Stathmin 1 in the aberrant JAK2V617F signaling pathway. Targeting Stathmin 1 or microtubule instability may improve Ruxolitinib response in JAK2V617F positive MPN patients. DisclosuresNo relevant conflicts of interest to declare.

JAK2V617F and Loss of Ezh2 in Hematopoietic Cells Contribute Synergistically to Myeloproliferative Neoplasm Initiation Potential, and Accelerate Progression of Disease

Background;The gain-of-function JAK2 mutation, JAK2V617F, is the most common molecular abnormality in myeloproliferative neoplasms (MPNs) and appears in patients with polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Ezh2 is a component of PRC2, catalyzing methylation of H3K27, and frequently mutated in MPN patients. Loss-of-function mutation of Ezh2 was reported as a poor prognostic marker in myelofibrosis patients. Many JAK2V617F-positive MPN patients harbor other mutations, but combination effects of JAK2V617F and Ezh2 mutation have not been analyzed.Methods & Results; To investigate the interaction between the two kinds of mutation in hematopoiesis, we studied transgenic mice with conditional expression of JAK2V617F, and inducible loss-of-function of Ezh2, both singly and in combination. Conditional expression of Cre was achieved using the SclCreER system with four weeks of tamoxifen injection, leading to excision of loxP-flanked alleles of Ezh2, and simultaneously induced expression of JAK2V617F, via the Flip-Flop recombination system (FF1). Mice with heterozygous deletion of Ezh2 showed no changes in peripheral blood, but homozygous deleted mice displayed slightly increased platelet counts compared to control mice. JAK2V617F-expressing (FF1) mice showed a typical PV phenotype, including erythrocytosis, thrombocytosis and neutrophilia. Mice expressing JAK2V617F with heterozygous deletion of Ezh2 (FF1;Ezh2+/-) also showed typical PV, but with more profound thrombocytosis and neutrophilia, and faster progression to fibrosis, than FF1 mice. Mice expressing JAK2V617F with homozygous deletion of Ezh2 (FF1;Ezh2-/-) had even shorter survival and showed more profound thrombocytosis and myelofibrosis than FF1;Ezh2+/- mice, but without erythrocytosis. These mice had dacryocytes and mobilized c-kit positive progenitor cells in peripheral blood. Bone marrow histology revealed mild fibrosis (grade: 1) in FF1 mice; more excessive fibrosis (grade: 1-2) in FF1;Ezh2+/- mice; and obvious fibrosis associated with collagen fiber formation and osteosclerosis (grade: 2) in FF1;Ezh2-/- mice. Cell compartment analysis in FF1 mice revealed expansion of hematopoietic stem cells (HSCs) and megakaryocyte progenitors (MegP) in bone marrow and spleen, while common myeloid progenitor (CMP), granulocyte monocyte progenitor (GMP) and megakaryocyte erythrocyte progenitor (MEP) were expanded principally in spleen. The expansion of HSCs and all progenitors were accelerated in FF1;Ezh2+/- mice and FF1;Ezh2-/- mice. Especially MegP and mature megakaryocytes were further expanded in FF1;Ezh2-/- mice compared to FF1;Ezh2+/-. To clearly estimate disease progression, transgenic bone marrow cells were transplanted to lethally irradiated recipient mice with WT competitor bone marrow cells and tamoxifen was administrated after transplantation. FF1 bone marrow cells clearly showed outcompeting potential to WT bone marrow cells. FF1;Ezh2+/- and FF1;Ezh2-/- bone marrow cells showed more stronger outcompeting potential than FF1 bone marrow cells. Finally, disease initiative potential was evaluated by limiting dilution transplantation. Total bone marrow cells, with heterozygous deletion of Ezh2 (Ezh2+/-), or expressing JAK2V617F (FF1), or the combination of the two mutations were transplanted (following Cre induction and recombination) to lethally irradiated recipient mice, mixed with competitor cells (control or Ezh2+/- bone marrow cells were transplanted by 1:100 dilution and FF1 or FF1;Ezh2+/- bone marrow cells were transplanted by 1:250 dilution against WT competitor cells). Bone marrow cells with Ezh2+/- showed higher reconstitution capacity than control (35% vs 10%; Ezh2+/- vs control) without MPN phenotype. Bone marrow cells with FF1;Ezh2+/- showed stronger reconstitution capacity (39% vs 15%; FF1;Ezh2+/- vs FF1), and greater disease initiative potential (50% vs 33%; FF1;Ezh2+/-vs FF1) than FF1.Summary; We clarified that loss-of-function of Ezh2 accelerated JAK2V617F-induced MPNs. Especially JAK2V617F with Ezh2 homozygous loss-of-function induced excessive megakaryopoiesis and resulted in PMF. JAK2V617F with heterozygous deletion of Ezh2 synergistically enhanced bone marrow reconstitution and disease initiative potential. DisclosuresSkoda:Novartis: Consultancy; Sanofi: Consultancy.

The Role Of p53 In JAK2V617F-Induced Myeloproliferative Neoplasms

Ph- myeloproliferative neoplasms (MPNs) are hematopoietic malignancies in which one or more myeloid lineages are abnormally amplified. These diseases represent a group of chronic conditions including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. MPNs mainly affect older people and have an average onset age of 55 years. Complications associated with MPNs include development of acute leukemia as well as thrombosis, hemorrhage, and myeloid metaplasia. JAK2V617F is found in the majority of patients with MPNs, and an overwhelming number of studies have demonstrated its pathogenicity. However, the precise action of JAK2V617F is not well defined, and other molecular defects involved in MPNs remain elusive. In earlier studies, we have generated transgenic mice expressing JAK2V617F in the hematopoietic system and demonstrated that JAK2V617F can cause MPN-like phenotypes in an age- and transgene dose-dependent manner. In this study, we address the involvement of tumor suppressor p53 in the progression of JAK2V617F-induced MPN phenotypes. We crossed our JAK2V617F transgenic mice with p53 knockout mice. Under the p53+/+ background, our JAK2V617F transgenic mice developed a mild MPN-like phenotypes in 15 weeks. However, under the heterozygous knockout p53-/+ condition, the mice developed a strong MPN-like phenotype in 8 weeks, manifested in higher blood cell counts, more severe splenomegaly, and earlier onset of myelofibrosis. This suggests that p53 affects the progression of MPNs, and reduced levels of p53 activity may be responsible for the heterogeneous phenotypes observed in MPN patients. Furthermore, when p53 was deleted from both chromosomes, JAK2V617F mice developed acute erythroleukemia, megakaryoblastic leukemia, or myeloid leukemia and died in 20 weeks with greatly enlarged spleen (>10 times the normal size) and liver (twice the normal size) infiltrated with leukemic cells. This indicates that loss of p53 in addition to JAK2V617F causes leukemic transformation. This is consistent with the earlier findings that p53 mutations exist in JAK2V617F-positive leukemia cell lines and JAK2V617F-positive MPNs patients who developed acute myeloid leukemia. Our study also suggests that targeting JAK2V617F and p53 simultaneously may provide effective treatment for MPNs. We employed nutlin-3 that disrupts the MDM2-p53 interaction thereby reducing degradation of p53. In vitro cell culture studies demonstrated that JAK2 inhibitors and nutlin-3 synergistically inhibited the growth of hematopoietic progenitor cells from JAK2V617F transgenic mice and MPN patients. Finally, from one of the p53-/- JAK2V617F transgenic mice, we derived an erythroleukemia cell line designated J53Z1. J53Z1 cells are CD71high and Ter119low and are dependent on erythropoietin for survival. They were effectively inhibited by known JAK2 inhibitors and should be useful in cell-based assays for screening of JAK2 inhibitors. Altogether, our study demonstrates that the level of p53 dictates the progression of JAK2V617F-induced MPNs and targeting p53 and JAK2V617F simultaneously may provide effective treatment for JAK2V617F-positive MNPs. Disclosures:No relevant conflicts of interest to declare.

Jak2V617F myeloproliferative neoplasm stem cells and interferon-alpha

The myeloproliferative neoplasms (MPNs) are clonal disorders of hematopoiesis that arise as a result of aberrant activation of tyrosine kinases and result in the proliferation and accumulation of mature myeloid cells in the blood, bone marrow and spleen. The prototypical MPN, chronic myeloid leukemia (CML) is caused by constitutive activation of ABL kinase occurring as a result of the BCR-ABL fusion. Importantly, targeted therapy to inhibit ABL kinase signaling, using imatinib, induces durable clinical responses in the majority of CML patients. The molecular pathogenesis of the BCR-ABL negative MPNs, polycythemia vera (too many red cells), essential thrombocythemia (too many platelets) and myelofibrosis, was elucidated in 2005 with the discovery of JAK2V617F, an activating mutation in the non-receptor tyrosine kinase, JAK2 [1]. The JAK2V617F mutation is present in more than 95% patients with polycythemia vera, and approximately 50% patients with essential thrombocythemia and myelofibrosis. In advanced disease, MPN may transform to acute leukaemia and this is associated with a dismal prognosis. In MPN, JAK2V617F is found in long-term hematopoietic stem cells (LT-HSCs), a population of rare quiescent cells present in the bone marrow and capable of long-term self-renewal. These somatically mutated LT-HSCs, referred to as MPN-propagating cells or MPN stem cells, maintain the MPN throughout the life of the patient and can serially transplant disease in animal models of Jak2V617F-mediated MPN [2, 3]. To definitively cure MPN will require complete eradication of the MPN disease-maintaining stem cell population. In contrast to the efficacy of imatinib in achieving molecular remissions in CML, JAK2 kinase inhibitors have shown only modest benefit in the treatment of myelofibrosis, namely in reducing splenomegaly and improving constitutional symptoms. Furthermore, the lack of an effect of the JAK2 inhibitors on reducing JAK2V617F allele burden in myelofibrosis patients [4] and the demonstration that MPN-propagating cells are insensitive to JAK2 kinase inhibition in vivo [2] suggests that MPN stem cells do not require JAK2 signaling for survival and that JAK2 inhibitor monotherapy will not cure MPN patients. The clinical efficacy of long-acting pegylated IFNα in MPN has renewed interest in understanding the mechanism of action of IFNα treatment in JAK2V617F-mediated MPN. Prolonged IFNα treatment results in normalization of blood counts in most patients with polycythemia vera, reduces JAK2V617F allele burden and in 15-40% of patients, the JAK2V617F mutant clone is rendered undetectable to sensitive molecular assays[5, 6]. These clinical results suggest that IFNα eradicates JAK2V617F-mutant LT-HSC, however the mechanism by which molecular remissions are achieved in MPN patients in response to treatment with IFNα have not been well defined. Figure 1 MPN stem cells are responsible for disease initiation and propagation in vivo To determine the efficacy of IFNα therapy on Jak2V617F-positive MPN stem cells in vivo, we used a murine model of Jak2V617F-mediated MPN [7]. We found that prolonged treatment of Jak2V617F mice with IFNα reversed the myeloproliferation phenotype (normalizing hemoglobin and white cell count and reducing extramedullary hematopoiesis). Importantly, we observed selective depletion of Jak2V617F over WT LT-HSC and this correlated with an inability of IFNα treated mice to propagate MPN into transplanted recipients. IFNα can directly activate the cell cycle in quiescent wild-type (WT) LT-HSCs and induce HSC proliferation in vivo [8]. We found that IFNα treatment induced enhanced cell-cycle activation of Jak2V617F-mutant LT-HSCs, as compared to WT LT-HSCs, explaining this preferential depletion over time. Using Ifnar1 knock-out mice (lacking the type 1 interferon receptor), we showed that the effects of IFNα on cell cycle are specifically mediated through type 1 interferon signaling and are intrinsic to Jak2V617F-mutant LT-HSCs. Using gene expression analysis we found an enrichment of cell cycle genes in Jak2V617F-mutant LT-HSC, both at baseline and after IFNα treatment, providing a potential explanation for their preferential sensitivity to IFNα treatment. This work suggests that IFNα achieves molecular remissions in JAK2V617F-positive MPN patients through the activation and consequent depletion of quiescent MPN disease-propagating cells. What are the clinical implications of this work? Firstly, these data support the rational design of clinical trials to test combinatorial therapeutic approaches using IFNα (to target quiescent MPN-stem cells) together with JAK2 kinase inhibitors or cytotoxic chemotherapy (to target actively cycling progenitor populations). Plausibly, the depletion of MPN stem cells in polycythemia vera could alter the natural history of the disease, through eliminating the malignant clone and thus preventing fibrotic and/or leukemic transformation, both devastating complications of MPN. Currently, IFNα is far from an ideal drug, and few patients are able to tolerate prolonged treatment due to its side effects. We have identified a number of novel targets that are differentially regulated in Jak2V617F and WT LT-HSCs following IFNα treatment. In ongoing studies, we are working to validate these targets with the goal of developing more specific, less toxic IFNα derivatives capable of selectively eliminating MPN stem cells, while sparing normal LT-HSCs and other organ systems.

The JAK2 46/1 haplotype is a risk factor for myeloproliferative neoplasms in Chinese patients

The presence of JAK2 V617F is associated with an inherited JAK2 46/1 haplotype, a risk factor for myeloproliferative neoplasms (MPN) in Caucasian populations. Whether the JAK2 46/1 haplotype is also a risk factor in the Chinese population is unknown. We assessed for the JAK2 46/1 haplotype and JAK2 V617F mutation in 225 MPN patients and 226 controls using a tagged SNP rs12340895. The allele frequencies of the JAK2 46/1 haplotype were distinct among different subtypes of MPN patients. The allele frequency was significantly higher in polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) than that in controls, with PV patients having the highest allele frequency (0.58, P = 6.00E-15). The distribution of rs12340895 genotypes in the JAK2 V617F mutated MPN patients was significantly different from that in controls (P = 1.67E-15). The percentage of GG genotype in controls was 2.2 %, but 31.0 % in JAK2 V617F-positive MPN patients. All the PV, ET, and PMF patients with the GG genotype also exhibited the V617F mutation. Compared to that of controls, the difference in genotype distribution in PV patients was the most significant (P = 4.83E-21), followed by ET (P = 2.07E-05) and PMF (P = 1.99E-04). Our results suggest that the JAK2 46/1 haplotype is a risk factor for MPN in the Chinese population, and patients with GG genotype in rs12340895 locus are susceptible to JAK2 V617F mutation.

The G allele of the JAK2 rs10974944 SNP, part of JAK2 46/1 haplotype, is strongly associated with JAK2 V617F-positive myeloproliferative neoplasms

Polycythemia vera, essential thrombocythemia, and primary myelofibrosis are myeloproliferative neoplasms, characterized in a majority of cases by a unique somatic point mutation, JAK2 V617F. Recently, it was shown that JAK2 V617F occurs more frequently on a specific JAK2 haplotype, named JAK2 46/1. We genotyped 149 myeloproliferative neoplasms patients (69 had polycythemia vera, 65 had essential thrombocythemia, and 15 had primary myelofibrosis) with a known JAK2 V617F mutational status and 150 controls for the JAK2 rs10974944 (C/G) single nucleotide polymorphism, in which the G allele tags the 46/1 haplotype. We found that the rs10974944 GG/CG genotypes were significantly enriched in patients compared to controls (p < 0.0001). After stratifying for the JAK2 V617F mutational status and for the mutant allele burden, we demonstrated that GG/CG genotypes were significantly more frequent in V617F positive compared to V617F negative patients (p = 0.001), but not in V617F negative patients compared to controls (p = 0.29). Similarly, the GG/CG genotypes were significantly enriched in V617F positive patients with a mutant allele burden >50% compared to those with a mutant allele burden <50% (p = 0.0006). Our results indicate that the G allele, part of the JAK2 46/1 haplotype, contributes significantly to the occurrence of JAK2 V617F-positive myeloproliferative neoplasms. Moreover, JAK2 46/1 seems to be associated with mutant allele burden >50% in JAK2 V617F-positive myeloproliferative neoplasms patients.

Reconciling Phenotype with Genotype in the MPN: Impact of SNP Array-Based Chromosomal Analysis.

Abstract 1893Poster Board I-916The diagnosis and accurate prognostication of the myeloproliferative neoplasms (MPN) is complicated by phenotypic mimicry and variable rates of disease evolution. The somatic mutation of JAK2 (JAK2V617F), identified in more than 90% of PV and in 50% of ET and PMF patients, is associated with acquired uniparental disomy (aUPD) on chromosome 9p, generating copy number-neutral loss of heterozygosity (CN-LOH) and results in homozygosity of JAK2V617F in roughly one third of JAK2V617F-positive patients. The molecular events upstream of the mitotic recombination that leads to somatic CN-LOH are essentially unknown. Intriguingly, JAK2V617F itself has been implicated in the generation of genomic instability (Plo et al 2008). Although these observations provide a rationale for how a single mutation could give rise to different clinical pathologies and downstream genomic instability in MPN, the JAK2V617F allele burden (AB) has not been systematically correlated with high resolution evaluation of amplifications, deletions, and CN-LOH in a large MPN cohort. We performed single nucleotide polymorphism arrays (SNP-A), a powerful karyotyping tool with the unique ability to detect CN-LOH on neutrophil DNA from 90 MPN patients. Published copy number variants (CNVs) and those identified in our internal cohort of 995 healthy controls were excluded as well as germline, non-clonal CN-LOH regions based on size criteria. Genomic results were correlated with the quantitative JAK2V617F AB, clinical phenotypes within and between disease classes, and in 21 paired longitudinal samples. SNP-A detected aUPD and/or chromosomal gains or losses in 81% of MPN patients. The prevalence of genomic lesions was lowest in ET compared to PV or PMF (40% of ET cases were lesion free), and lower still in JAK2V617F-negative ET (62% lesion free). aUPD was the most common genomic lesion, occurring in 59% of the MPN cases, and involving every chromosome except chromosomes 18 and 23. As expected, aUPD most commonly involved 9p (38 cases), encompassing JAK2. aUPD encompassing the TET2 gene at 4q24, recently implicated in MPN and other myeloid disorders, was seen in 3 of the 90 cases, with deletions spanning this gene in 2 additional cases. Five cases of aUPD at 2q were observed, and other recurrent regions were limited to 3 cases or fewer. The prevalence of chromosomal losses alone was far lower than UPD (22%), most commonly involved chromosome regions 20q, 13q and 17q, and was a feature primarily of PMF. The prevalence of aUPD in our MPN series of 59% was higher than in MDS (20%, p<0.001), MDS/MPN (35%, p=0.0074), and sAML (23%, p=0.0004) using the same arrays and analytical methods. Independent of MPN classification category, JAK2V617F positive patients had a higher prevalence of UPD than did JAK2V617F negative patients, although when excluding 9p UPD (24/59 vs 11/31) this was not significant. However, homozygous JAK2V617F patients had twice the prevalence of aUPD (again excluding 9P UPD) than did the heterozygous patients (8/25 vs 5/33), and twice the prevalence of multiple aUPD within individual cases. Of the 21 patients studied longitudinally over 5 years (range 2–9), 3 patients with chromosomal lesions present on the first sample were not present in the second: 9p UPD resolved in one PV patient who transformed to PMF, 1p UPD resolved in a JAK2V617F-negative ET patient, and a 20q deletion resolved in a heterozygous JAK2V617F-positive ET patient who transitioned to PV. Additional UPD regions occurred in only 1/8 heterozygous JAK2V617F positive MPN patients, while 5/11 JAK2V617F homozygous patients developed additional aUPD. In contrast, the acquisition of deletions occurred primarily in JAK2V617F heterozygous ET patients, all of whom had transitioned to PV or PMF. We conclude that aUPD is increased in the MPN compared to other myeloid disorders, and associates with the presence of JAK2 V617F in a gene dose-dependent manner. We hypothesize that aberrant JAK2 signaling contributes to the development of UPD but not chromosomal losses or gains. Furthermore, comparison of deletion, amplification, and UPD events in ET and PV relative to PMF suggest that PMF bears a closer genomic resemblance to MDS than to ET or PV. Thus SNP-A provides unique insight into MPN disease classification (PMF), disease progression, and genomic instability mechanisms. Disclosures:No relevant conflicts of interest to declare.

Clonal analyses define the relationships between chromosomal abnormalities and JAK2V617F in patients with Ph-negative myeloproliferative neoplasms

JAK2V617F occurs in approximately 93% of patients with polycythemia vera and approximately 50% of patients with either primary myelofibrosis or essential thrombocythemia. Chromosomal abnormalities are detected in 50% of patients with primary myelofibrosis, 29% with polycythemia vera, and 8% to 10% with essential thrombocythemia. The relationship between the presence of such chromosomal abnormalities and the JAK2V617 allele burden, and the role that each of these genetic events play in the origins and progression of the myeloproliferative neoplasms (MPNs), remain unclear. Individual hematopoietic colonies were assayed in vitro from the CD34(+) cells of six JAK2V617F-positive MPN patients with marker chromosomal abnormalities. Colonies were simultaneously analyzed for JAK2 genotype and chromosomal abnormalities. Among the 248 colonies assayed from cultures containing 500 CD34(+) cells, chromosomal abnormalities were detected in 5% of colonies with wild-type JAK2, 32% of JAK2V617F heterozygous colonies and 56% of JAK2V617F homozygous colonies. Overall, 92% of chromosomally abnormal colonies were also JAK2V617F homozygous. Although 54 colonies contained wild-type JAK2 exclusively, 4 of these colonies were characterized by chromosomal abnormalities. This study indicates that MPN hematopoietic progenitor cells do not necessarily always acquire genetic events in the same sequence. (Chromosomally abnormal progenitor cells are closely associated with JAK2V617F homozygosity; p=0.0001.). Chromosomal abnormalities such as +8, +9 can occasionally precede acquisition of JAK2V617F. These findings support the existence of earlier genetic events that precede JAK2V617F or cytogenetic abnormalities in MPN hematopoietic progenitor cells.

Involvement of mast cells by the malignant process in patients with Philadelphia chromosome negative myeloproliferative neoplasms

The Philadelphia chromosome negative myeloproliferative neoplasms (MPNs) are clonal hematologic malignancies frequently characterized by a mutation in JAK2 (JAK2V617F). Peripheral blood (PB) CD34(+) cells from patients with polycythemia vera (PV) and primary myelofibrosis (PMF) generated in vitro significantly fewer mast cells (MCs) than normal PB CD34(+) cells. The numbers of MC progenitors assayed from MPN CD34(+) cells were, however, similar to that assayed from normal CD34(+) cells. A higher percentage of the cultured MPN MCs expressed FcvarepsilonRIalpha, CD63 and CD69 than normal MCs, suggesting that cultured MPN MCs are associated with an increased state of MC activation. Further analysis showed that a higher proportion of cultured PV and PMF MCs underwent apoptosis in vitro. By using JAK2V617F, MplW515L and chromosomal abnormalities as clonality markers, we showed that the malignant process involved MPN MCs. JAK2V617F-positive MC colonies were assayable from the PB CD34(+) cells of each of the 17 JAK2V617F positive MPN patients studied. Furthermore, erlotinib, a JAK2 inhibitor, was able to inhibit JAK2V617F-positive PV MC progenitor cells, indicating that malignant MC progenitor cells are a potential cellular target for such JAK2 inhibitor-directed therapy.