The Zygosity of JAK2V617F Determines the Disease Entities of Myeloproliferative Neoplasms By Modulating Erythropoiesis but Not Megakaryopoiesis

Abstract Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs) consists of different clinical entities that include polycythemia vera (PV) and essential thrombocythemia (ET). Despite of differential clinical features, JAK2V617F mutation is found in both PV and ET, leaving the cause of differential biological phenotypes by an oncogene obscure. Previously, studies have shown that higher allele frequencies or expression of JAK2V617F are associated with PV symptom/features in patients or model animals, respectively, suggesting that the copy number of JAK2V617F modulates hematopoietic cell differentiation and thus exhibits differential clinical features. However, this remained elusive in human hematopoiesis. To examine the impact of the zygosity of JAK2V617F allele on hematopoietic differentiation in human cells, we established induced pluripotent stem cells (iPSCs) harboring heterozygous- and homozygous-JAK2V617F mutation using genome-editing techniques from normal iPSCs. The introduction of JAK2V617F mutation with one or two copies did not alter the pluripotency of iPSCs and their capacity to differentiate into hematopoietic stem/progenitor cells (HSPCs) in vitro. When we induced hematopoietic cell differentiation from HSPCs, factor-independent erythropoiesis and megakaryopoiesis were induced in both heterozygous and homozygous JAK2V617F-HSPCs. Furthermore, homozygous JAK2V617F-HSPCs showed higher potential for erythropoiesis compared to the ones with heterozygous JAK2V617F, while the zygosity of JAK2V617F showed less effect on megakaryopoiesis. To further understand the molecular mechanism of hematopoietic cell differentiation modulated by differential copy number of JAK2V617F, we analyzed the activation of JAK-STAT signal by immunoblot analysis. The activation of JAK-STAT signals was more prominent in HSPCs harboring homozygous JAK2V617F, than those with heterozygous JAK2V617F. This suggested that the level of JAK2 phosphorylation was positively correlated with the copy number of JAK2V617F. These observations implied followings: 1) heterozygous JAK2V617F was sufficient to promote the development of MPN by inducing the factor-independent erythropoiesis and megakaryopoiesis; 2) the zygosity of JAK2V617F determined the disease phenotypes of MPNs by modulating erythropoiesis but not megakaryopoiesis; and 3) the homozygous JAK2V617F increased JAK-STAT signaling in HSPCs, promoting an increased erythropoiesis. Disclosures No relevant conflicts of interest to declare.

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Evaluating Clonal Dominance in a Murine Knock-in Model of Jak2V617F MPN

Blood ◽

10.1182/blood.v118.21.614.614 ◽

2011 ◽

Vol 118 (21) ◽

pp. 614-614 ◽

Cited By ~ 1

Author(s):

Ann Mullally ◽

Luke Poveromo ◽

Kristina Brumme ◽

Fatima Al-Shahrour ◽

Steven W. Lane ◽

...

Keyword(s):

Polycythemia Vera ◽

Progenitor Cell ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Conditional Knockout ◽

Jak2v617f Mutation ◽

Loss Of Function ◽

Hematopoietic Stem ◽

The Impact

Abstract Abstract 614 Myeloproliferative neoplasms (MPN) are clonal disorders of hematopoiesis but the mechanisms of clonal dominance in these diseases are poorly understood. The JAK2V617F mutation is found in the majority of patients with MPN and is sufficient to confer the MPN phenotype. We recently described a Jak2V617F knock-in MPN model in which the mutation was expressed from the endogenous murine Jak2 promoter and the disease phenotype closely recapitulated human polycythemia vera (PV). In the model we found that the MPN-initiating population is contained within the CD150+CD48− LineagelowSca1+cKithigh(LSK) long-term hematopoietic stem cell (HSC) compartment, and showed that the MPN is cell autonomous and serially transplantable. In long-term competitive transplantation experiments we found that the Jak2V617F CD150+CD48− LSK population demonstrates gradual clonal expansion over time and we are now investigating the regulation of this primitive HSC population in vivo. Erythropoietin (EPO) signaling is reported to be the fundamental defect in polycythemia vera (PV). The JAK2V617F mutation is present in 95% PV patients and can be detected in the HSC compartment. We evaluated the role of EPO signaling in the JAK2V617F mutant HSC compartment using a conditional Jak2V617F knock-in murine model. Floxed Jak2+/VF mice were crossed with Vav Cre or erythropoietin receptor GFP Cre (ErGFPcre) mice resulting in Jak2V617F expression in all hematopoietic lineages or in erythroid restricted Jak2V617F expression respectively. Jak2V617F-ErGFPcre mice demonstrated elevated hematocrit, expanded committed erythroid progenitors and suppressed EPO levels but had an attenuated MPN phenotype as compared with Jak2V617F-Vavcre mice. Notably, the hematopoietic stem and progenitor cell (HSPC) compartment was not expanded in Jak2V617F-ErGFPcre mice and HSCs from both Jak2V617F-Vavcre and Jak2V617F-ErGFPcre mice did not activate phosphoStat5 signaling in response to EPO stimulation. These results indicate that expression of Jak2V617F in the HSC compartment is required for development of a full MPN phenotype and suggest that cytokine receptors other than the EPO receptor are important in mediating clonal dominance within the HSC compartment in JAK2V617F mediated MPN. TET2 is one of three TET gene family members that appear to play a role in DNA demethylation. Acquired TET2 deletions and loss-of-function mutations have been found across a broad spectrum of myeloid malignancies indicating that TET2 may drive a common pathogenic step in myeloid cancers, such as the establishment and/or enhancement of clonal dominance. Tet2 null HSCs have recently been shown to have a competitive repopulating advantage over wild-type HSCs in murine transplantation assays. TET2 loss-of-function mutations are found in approximately 12% of MPN patients, are often found co-mutated with JAK2V617F and have been associated with leukemic transformation of MPN. To evaluate the effects of loss of Tet2 function on the self-renewal and differentiation of Jak2V617F mutant HSCs, we crossed Jak2V617F knock-in mice with Tet2 conditional knockout mice. At 6 weeks of age, Jak2V617F/Tet2 +/− Vav Cre mice do not demonstrate significant differences in MPN phenotype as compared with Jak2V617F/Tet2+/+ Vav Cre mice, in terms of peripheral blood counts, hematopoietic stem and progenitor cell numbers or in colony formation. Additional studies using older mice and Jak2V617F/Tet2−/− Vav Cre animals are underway to further investigate the impact of loss of Tet2 function on Jak2V617F mutant HSCs. Understanding the mechanisms that contribute to clonal dominance in MPN will help facilitate the development of strategies to selectively target MPN stem cells therapeutically, and thereby advance the treatment of MPN patients. Disclosures: No relevant conflicts of interest to declare.

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Loss of Wild-Type Jak2 Allele Enhances Myeloid Cell Expansion and Accelerates Myelofibrosis in Jak2V617F Knock-in Mice

Blood ◽

10.1182/blood.v120.21.809.809 ◽

2012 ◽

Vol 120 (21) ◽

pp. 809-809

Author(s):

Hajime Akada ◽

Saeko Akada ◽

Dongqing Yan ◽

Robert Hutchison ◽

Golam Mohi

Keyword(s):

Polycythemia Vera ◽

Cell Expansion ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Philadelphia Chromosome ◽

Myeloid Cell ◽

Negative Regulator ◽

Jak2v617f Mutation ◽

Common Mutation ◽

Wild Type

Abstract Abstract 809 The activating JAK2V617F mutation is the most common mutation found in Philadelphia chromosome (Ph)-negative myeloproliferative neoplasms (MPNs), which include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Although a majority of MPN patients carry heterozygous JAK2V617F mutation, loss of heterozygosity (LOH) on chromosome 9p involving JAK2 has been observed in ∼30% of patients with MPNs particularly in PV and PMF. JAK2V617F homozygosity through 9pLOH has been linked to more severe MPN phenotype. However, the contribution of 9pLOH in the pathogenesis of MPNs remains unclear. To investigate the role of wild-type JAK2 in MPNs induced by JAK2V617F, we have utilized conditional Jak2 knock-out and Jak2V617F knock-in alleles and generated heterozygous, hemizygous and homozygous Jak2V617F mice. Whereas heterozygous Jak2V617F expression results in a polycythemia vera-like disease in mice, loss of wild-type Jak2 allele in hemizygous or homozygous Jak2V617F mice results in a significantly greater increase in reticulocytes, white blood cells, neutrophils and platelets in the peripheral blood and larger spleen size. We also have found that hemizygous or homozygous Jak2V617F expression significantly increased megakaryocyte-erythroid progenitors in the bone marrow and spleens and marked infiltration of neutrophils in the liver compared with heterozygous Jak2V617F. More importantly, hemizygous or homozygous Jak2V617F mice show accelerated myelofibrosis compared with heterozygous Jak2V617F-expressing mice. Thus, loss of wild type Jak2 allele increases myeloid cell expansion and enhances the severity of the MPN. Together, these results suggest that wild-type Jak2 serves as a negative regulator of MPN induced by Jak2V617F. Disclosures: No relevant conflicts of interest to declare.

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Prevalence and Prognostic Impact of Allelic Imbalances Associated with Leukemic Transformation of Philadelphia Chromosome-Negative Myeloproliferative Neoplasms.

Blood ◽

10.1182/blood.v114.22.1280.1280 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1280-1280

Author(s):

Nils Heinrich Thoennissen ◽

Utz O. Krug ◽

Dhong Hyun Lee ◽

Norohiko Kawamata ◽

Terra L Lasho ◽

...

Keyword(s):

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Philadelphia Chromosome ◽

Snp Array ◽

Chronic Phase ◽

Prognostic Impact ◽

Genomic Alterations ◽

Leukemic Transformation ◽

Blast Phase ◽

Hematopoietic Stem

Abstract Abstract 1280 Poster Board I-302 Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF) are defined as clonal hematopoietic stem cell disorders. These disorders show an inherent tendency for transformation into leukemia (MPN-blast phase) which is hypothesized to be accompanied by acquisition of additional genomic lesions. We, therefore, obtained a comprehensive profile of genomic alterations associated with leukemic transformation by using single-nucleotide polymorphism (SNP) array in 88 MPN patients, as well as 71 cases with MPN-blast phase, and correlated these findings with their clinical parameters. A relatively high number of genomic alterations was found in MPN after leukemic transformation with 4.6 ± 0.6 abnormalities per sample compared to only 1.4 ± 0.2 changes per patient in chronic phase (p<0.001). Compared to the cytogenetic data, SNP-chip analysis detected about 47% additional chromosomal changes in the MPN samples, and 31% more in the MPN-blast phase cases, whereas SNP-array allelokaryotyping practically captured all cytogenetic abnormalities in our study population. Several additionally altered regions were detected in patients with MPN-blast phase compared to chronic phase, including both deletion and copy-number neutral-loss of heterozygosity (CNN-LOH) on chromosome 12p (9%) and 21q (9%), involving ETV6 and RUNX1. Notably, deletion and CNN-LOH on 17p involving TP53 were diagnosed in 18% of MPN-blast phase samples, which was highly associated with preceding treatment with alkylating agents (p=0.016). Moreover, trisomy 8, as well as amplification of 8q24.21 involving the MYC gene, were detected in 13% of patients with MPN-blast phase who were almost exclusively negative for the JAK2V617F mutation. Genome-wide inspection of further critical regions with promising new candidate genes involved in the evolution to the MPN-leukemic phase included deletion and CNN-LOH on 7q22.1 (SH2B2) in 18%, duplication/amplification on 19p13.2 (PIN1, ICAM1, CDC37) in 13% and 21q22.2 (ERG) in 9% of MPN patients with blast crisis. In contrast, we detected a decreased frequency of JAK2V617F in MPN-blast phase samples (52%) compared to chronic phase (71%). Also, the percentage of patients with homozygous mutant JAK2 as a result of CNN-LOH was lower in the MPN-blast phase (43%) compared to the chronic phase (53%). Taken together, the data suggest that gain-of-function mutation of JAK2 is not a perquisite for leukemic transformation. Remarkably, CNN-LOH on either 7q or 9p was related to decreased survival after leukemic transformation (p=0.02 and p=0.012, respectively). Given the variety of allelic imbalances, our data suggest that MPN-blast phase appears to be a heterogeneous disease prone to have evolved multiple mechanisms to provide a proliferative advantage to the abnormal leukemic clone. Our analysis of MPN genomes in the chronic compared to the leukemic stage provided new prognostic insights, as well as novel causative genes which might be involved in the transformation to MPN-blast phase. Disclosures No relevant conflicts of interest to declare.

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Human Adenylate Kinase 2 Deficiency Inhibits Hematopoietic Cell Differentiation towards Neutrophil and T Lymphoid Lineages.

Blood ◽

10.1182/blood.v114.22.78.78 ◽

2009 ◽

Vol 114 (22) ◽

pp. 78-78

Author(s):

Emmanuelle M. Six ◽

Chantal Lagresle-Peyrou ◽

Corinne Demerens-deChappedelaine ◽

Frederic Rieux-Laucat ◽

Andrea Didati ◽

...

Keyword(s):

Cell Differentiation ◽

Cell Line ◽

Adenylate Kinase ◽

Conflicts Of Interest ◽

Adenine Nucleotides ◽

Severe Combined Immunodeficiency ◽

Mitochondrial Protein ◽

Hematopoietic Cell ◽

Hematopoietic Stem ◽

Neutrophil Differentiation

Abstract Abstract 78 Reticular dysgenesis (RD), an autosomal recessive form of human Severe Combined Immunodeficiency is characterized by the absence of blood neutrophils and T lymphocytes. This pathology is due to biallelic mutations in the adenylate kinase 2 (AK2) gene, resulting in the loss of AK2 protein expression. AK2 is a mitochondrial protein which regulates the homeostasis of cellular adenine nucleotides by converting ADP into ATP and AMP. In order to understand the precise role of AK2 in hematopoiesis, we have developed a RNA interference strategy through lentiviral-mediated gene transfer of AK2 short hairpin RNAs (shAK2). The knock-down of AK2 in human or murine hematopoietic stem cells (HSC) inhibits their capacity to form granulocyte colonies in methycellulose assays and prevents them to generate mature polynucleated cells in liquid culture in the presence of G-CSF. We also determine the ability of shAK2-transduced HSC to differentiate along the T lymphoid lineage after co-culture on a OP9Delta1 stroma cell line. Our data demonstrated that the apparition of CD4+CD8+ cells was profoundly reduced in the presence of shAK2. To delineate the mechanism involved in this defect, we also studied the neutrophil differentiation of the HL60 promyelocytic cell line, following retinoic acid treatment. In this system, the absence of AK2 expression led to an arrest of neutrophil differentiation process, increased cell apoptosis and disrupt the mitochondrial membrane potential. All these data suggest a novel mechanism regulating hematopoietic cell differentiation, and involved in one of the most severe human immunodeficiency syndromes. Disclosures: No relevant conflicts of interest to declare.

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Transgene Dose- and Age-Dependent Development of Myeloproliferative Neoplasm Phenotypes In JAK2V617F Transgene Mice

Blood ◽

10.1182/blood.v116.21.1980.1980 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1980-1980

Author(s):

Wanting Ho ◽

Wanming Zhao ◽

Zhizhuang Joe Zhao

Keyword(s):

Transgenic Mice ◽

Copy Number ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Gene Copy ◽

Cell Transplant ◽

Dependent Manner ◽

Hematopoietic Stem ◽

Cell Counts ◽

Age Dependent

Abstract Abstract 1980 Myeloproliferative neoplasms (MPNs) are heterogeneous hematologic disorders represented by three main phenotypes: polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The major molecular lesion in these diseases is JAK2V617F, which occurs in over 95% patients with PV and in over 50% of patients with ET or PMF. The pathogenic effects of JAK2V617F have been demonstrated by retrovirus-mediated gene transfer, transgenic, and knock-in mouse models, but the precise mode of JAK2V617F action is not clear. Interestingly, in the knock-in model, expression of JAK2V617F causes severe PV-like disease but not ET-like phenotype as seen in patients. To verify the pathogenic role of JAK2V617F, we further characterized the phenotypes of three lines of JAK2V617F transgenic mice generated by using the vav gene promoter which drives expression of transgenes in the hematopoietic system. These mice developed MPN-like phenotypes in a transgene dose- and age-dependent manner. Line A mice have a JAK2V617F gene copy number of 13; they develop MPN phenotype with marked increases in blood counts and enlarged spleens as early as 4–6 weeks after birth. In contrast, lines B and D mice have a transgene copy number of 2 and 1, respectively, and it takes nearly 70 weeks for these mice to show MPN-like phenotypes. The phenotype of line A mice is particularly noteworthy. Essentially all the hemizygous line A mice displayed an ET-like phenotype with marked elevations in platelet counts (usually over 4000×109/L by the age of 15 weeks), but only a slight increase in red cell and white cell counts. In contrast, all the homozygous mice exhibited a clear PV-like phenotype with elevations in all three types of blood cells, although their platelets hardly ever went over 4000×109/L. The hemizygous mice developed myelofibrosis after 30 weeks while the homozygous mice showed the symptom within only 10 weeks. As expected, the increased blood cell counts and formation of myelofibrosis are associated with mobilization of hematopoietic stem/progenitor cells to peripheral hematopoietic tissues (blood, spleen, and liver). By conducting stem cell transplant experiments, we further proved that JAK2V617F-induced ET and PV-like phenotypes are transplantable. Our study demonstrates that transgenic expression of JAK2V617F is capable of producing all three phenotypes of MPNs in a transgense dose- and age-dependent manner. Our transgenic mice thus represent an excellent model system to study MPNs. Disclosures: No relevant conflicts of interest to declare.

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Lack of Association Between the 46/1 JAK2 Haplotype and the Presence of JAK2V617F Mutation In Splanchnic Vein Thrombosis Patients

Blood ◽

10.1182/blood.v116.21.4120.4120 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4120-4120

Author(s):

Eirini G Kouroupi ◽

Bruno Cassinat ◽

Aurelie Plessier ◽

Sylvia Bellucci ◽

Christine Chomienne ◽

...

Keyword(s):

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Jak2v617f Mutation ◽

Fisher Exact Test ◽

Peripheral Vein ◽

Splanchnic Vein Thrombosis ◽

Vein Thrombosis ◽

Tag Snp ◽

Mutational Status ◽

The Impact

Abstract Abstract 4120 Aim: The majority of myeloproliferative neoplasms (MPN), i.e. Polycythemia vera (PV), Essential Thrombocytemia (ET) and Primary Myelofibrosis, are characterized by the presence of the acquired JAK2V617F gene mutation. Recent studies revealed that the 46/1 or “GGCC” haplotype located in the JAK2 gene is strongly associated with the development of a JAK2V617F positive MPN. However, this particular haplotype was also detected in excess in JAK2V617F negative MPN carrying mutations across JAK2 exon 12 or the MPL gene, suggesting that this germline genetic variation increases the risk of developing a MPN regardless of the acquisition of one particular mutation. A MPN is found in approximately half of the patients presenting with Splanchnic Vein Thrombosis (SVT) and JAK2V617F mutation is present in virtually all of these MPN patients. In this study we sought to clarify the impact of the JAK2 46/1 haplotype on the susceptibility to SVT. Patients and Methods: Peripheral blood was obtained after informed consent; following DNA extraction we proceeded to genotyping using commercially available TaqMan SNP genotyping assays for one tag SNP (rs10974944) which is in complete linkage disequilibrium with the 46/1 haplotype. Results were confirmed using another tag SNP (rs1234867). The study was performed on 170 patients with SVT, 58 patients with peripheral vein thrombosis and 31 patients with JAK2V617F positive PV. Results: In the 170 patients with SVT the frequency of G-allele that stands for the 46/1 haplotype was 0.28 (CC n=89; C/G n=67; G/G n=14), not significantly different from that of the published population controls (n=1500) from the Welcome Trust Case Control Consortium WTCCC (0.24; p=0.11 by the Fisher exact test). The frequency of the 46/1 haplotype in 58 patients with peripheral vein thrombosis was also similar to that of the WTCCC (0.24). However, the frequency of the 46/1 haplotype in SVT patients was significantly different from its frequency in a group of 31 patients with JAK2V617F positive PV (0.52; p=0.0005). When we analysed SVT patients according to their JAK2 mutational status, we found no difference in the frequency of the 46/1 haplotype between the JAK2V617F positive patients (0.27; n=75) and the JAK2V617F negative patients (0.28; n=95) (p=0.90). Of note, a JAK2 allele burden greater than 50% was observed in 11% of patients with JAK2V617F positive SVT and 45% of patients with JAK2V617F positive PV. Conclusions: In this large cohort of 170 patients with SVT the frequency of the 46/1 haplotype was not different from the cohort of controls of the WTCCC. This result suggests that the 46/1 haplotype is not a susceptibility locus for the development of SVT. Moreover, this haplotype was not overrepresented in the group of SVT patients harbouring a JAK2V617F mutation compared to JAK2V617F negative patients. This result is in apparent contradiction with the hypothesis that the 46/1 haplotype predisposes to the acquisition of JAK2V617F mutation or of a MPN, in agreement with recent studies reporting almost identical 46/1 frequencies between V617F-negative patients and V617F-positive patients with low mutation burden (i.e. <50%), which is the case of SVT patients in this series. Disclosures: No relevant conflicts of interest to declare.

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Targeted Cancer Exome Sequencing Discovers Novel Recurrent Mutations In MPN

Blood ◽

10.1182/blood.v122.21.4099.4099 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4099-4099

Author(s):

Elena Tenedini ◽

Isabella Bernardis ◽

Valentina Artusi ◽

Lucia Artuso ◽

Enrica Roncaglia ◽

...

Keyword(s):

Somatic Mutations ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Philadelphia Chromosome ◽

Jak2v617f Mutation ◽

Exome Capture ◽

Multiple Control ◽

Disease Evolution ◽

Recurrent Mutations

Abstract The discovery of the JAK2V617F mutation in 2005 [Kralovics R, N Engl J Med 2005] represented a major breakthrough in the understanding of the molecular pathogenesis of Philadelphia chromosome negative chronic myeloproliferative neoplasms (MPN). Nevertheless several observations suggest that the JAK2V617F mutation may not be the disease funding mutation, at least in most instances. Therefore, a great deal of effort is ongoing with the aim to identifying novel genetic lesions contributing to the disease pathogenesis. The two major theoretical and technical drawbacks to the identification of new somatic mutations are represented, respectively, by the huge number of genes potentially involved in tumorigenesis of MPN and by the availability of a “pure” germline control DNA. Buccal swabs and saliva have been generally considered as readily available sources of DNA of non-hematopoietic origin, but detection of the JAK2V617F mutation in at least some of these samples indeed suggested the presence of myeloid cell contamination [Levine RL, Cancer Cell 2005]. So, in order to discover novel mutations in MPN using upfront technologies based on next-generation sequencing (NGS) we designed a “cancer exome” capture panel of 2000 unique genes and microRNAs. This panel was used to capture libraries generated from genomic DNA extracted from granulocytes and in vitro expanded CD3+ T-lymphocytes as germline control, in a cohort of 20 MPN patients. These captured libraries were than massively sequenced using the Roche 454 FLX platform. DNA samples had been collected at the diagnosis of PV in 9 subjects and PMF in 6 subjects, while the remaining 5 DNA samples were from 5 of the 9 PV patients at the time they evolved to post-PV myelofibrosis. After extensive bioinformatics analysis and multiple control adjustments, we finally produced a list of 171 novel “true” somatic mutations occurring in genes and microRNAs coding regions of those MPN samples; some of these mutations have been already described in MPN, whereas novel variants represent the vast majority. Despite patients harbored different numbers of somatic mutations, spanning from four to twenty-one variants, only 22 genes appear recurrently mutated. It is worth of notice the acquisition of additional mutations and/or the occurrence of loss of some mutations at the time of disease evolution from PV to a post-PV Myelofibrosis in the five patients for whom samples were available at both disease phases. Some of them, either acquired (NTRK1, PRDM2, BRCA2 and BARD1) or lost (APC, CARS, MLL3 and FAT2) had been found also in another PV or PMF sample. To test the recurrence of these mutations, we screened a different cohort of 189 patients composed by PMF (91 samples), PV (50 patients) and post-PV Myelofibrosis (48 samples) by Ion AmpliSeq technology on an Ion Torrent PGM platform. Deep amplicon sequencing of granulocytes DNA achieved a sample median of 1000-fold coverage. Excluding JAK2, MPL, IDH2, ASXL1 known variants, for 7 genes (SCRIB, MIR662, BARD1, TCF12, FAT4, DAP3, NRAS) we demonstrated in MPN a global mutation frequency greater than the 3%. Whereas some new variants need functional validation to prove causal mechanisms, some other mutations have a well-known pathogenic role in solid cancers but here are described for the first time in MPN. Disclosures: No relevant conflicts of interest to declare.

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Dysregulation Of Bcl2 Family Proteins Induced By JAK2V617F Mutation Contributes To The Abnormal Expansion Of Neoplastic Initiating Cells

Blood ◽

10.1182/blood.v122.21.2852.2852 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2852-2852

Author(s):

Lorena Lobo Figueiredo-Pontes ◽

Robert S Welner ◽

Yuta Mishima ◽

Mihoko Yamamoto ◽

Ann Mullally ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Extramedullary Hematopoiesis ◽

Jak2v617f Mutation ◽

Cell Stage ◽

Hematopoietic Stem ◽

Jak2 Inhibition ◽

Jak2 Inhibitors

Abstract The treatment of myeloproliferative neoplasms (MPN) harboring JAK2V617F mutation remains non-curative with the current strategies despite the development of JAK2 inhibitors, which improve the symptoms of the disease but do not effectively reduce the allele burden of the mutation. In addition, it has been shown that, although JAK2 inhibitors can control the expansion of the myeloerythroid precursors that contribute to the clinical phenotype, they fail to eliminate the most primitive stem cells, which are responsible for the initiation of the disease. The differential expression of genes/proteins between the hematopoietic stem cells (HSC) and the more committed bone marrow progenitors may reveal, at least in part, the contribution of each cell type to the pathogenesis of the disease. We have shown that JAK2 inhibition induces down-regulation of the anti-apoptotic protein Bcl-xL and up-regulation of the pro-apoptotic BH3-only protein, Bim, in human cells harboring the JAK2V617F mutation, suggesting a key role of these Bcl-2 family proteins as potential targets for JAK2-mediated apoptosis. Therefore, we aimed to study the contribution of the Bcl-2 family proteins to MPNs by the use of a conditional JAK2V617F knock-in model, particularly focusing on MPN initiating cells. The heterozygous Jak2V617F expressing animals (Jak2+/VF) develop a lethal MPN characterized by an elevated hematocrit and splenomegaly due to extramedullary hematopoiesis, with a prominent expansion of early (Lin- CD71+ Ter119+) and late (Lin- CD71- Ter119+) erythroid cells in the spleen, and increased pre-megakaryocyte-erythrocyte (Lin- Sca1- cKIThi CD41- FcgRII/III- CD105- CD150+), megakaryocyte (Lin- Sca1- cKIThi CD41+ CD150+) and erythroid (Lin- Sca1- cKIThi CD41- FcgRII/III- CD105+ CD150+) precursors in the bone marrow. HSC numbers (Lin- Sca1- cKIThi CD105+ CD150+) are also increased in Jak2+/VF as compared to the Jak2+/+ controls. Gene expression analysis revealed that Bcl-xL was upregulated in sorted LSKs (Lin- Sca-1+ cKIThi) containing HSCs but not in the more committed progenitors (CMP, GMP, MEP). By contrast, the pro-apoptotic Bim was downregulated in CMPs and MEPs but not at the LSK cells containing HSCs. Expression of Bcl-2 showed no difference between Jak2+/VF and Jak2+/+ mice. These data suggest that signals from JAK2V617F mutation may be cell-stage specific, which may explain why JAK2 inhibitors fail to eliminate MPN initiating cells. Modulating the overexpression of Bcl2 antiapoptotic proteins at the MPN initiating cells level could potentially render these cells sensitive to the JAK2 inhibition. Disclosures: No relevant conflicts of interest to declare.

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Clinical Significance Of Circulating Microparticles In Ph- Myeloproliferative Neoplasms (MPN)

Blood ◽

10.1182/blood.v122.21.2368.2368 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2368-2368 ◽

Cited By ~ 2

Author(s):

Yue Han ◽

Shixiang Zhao ◽

Wenjuan Zhang ◽

Jiannong Cen ◽

Wei Zhang ◽

...

Keyword(s):

Plasma Levels ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Philadelphia Chromosome ◽

Jak2v617f Mutation ◽

Polycythaemia Vera ◽

Healthy Donors ◽

Significant Difference ◽

Thrombotic Complications

Abstract Background Microparticles (MPs) are small membrane vesicles that are classified as red blood cell MPs (RMPs), platelet-derived MPs (PMPs), tissue factor MPs (TF+MPs) and endothelial MPs (EMPs) based on their origins. Philadelphia chromosome-negative myeloproliferative neoplasms (Ph-MPN) are disorders characterized by abnormal hematopoiesis, thrombosis, JAK2V617F mutation. Although MPs are considered as biomarkers reﬂecting procoagulant state in cancer patients, their involvement in the patients with Ph-MPN remains unclear. Our objective in this study was to measure the alterations of the four MPs types in the patients with MPN and to evaluate their correlations with JAK2V617F mutation and some clinical complications, especially for thrombosis and splenomegaly. Methods Sixty-seven patients with MPN were enrolled in this study, including 12 polycythaemia vera (PV), 49 essential thrombocythemia (ET) and 6 primary myelofibrosis (PMF). 30 healthy donors were selected as normal controls. Venous blood was anticoagulated with sodium citrate (1:9). Using flow cytometry, plasma samples were measured for RMPs, PMPs, TF+MPs and EMPs with phycoerythrin (PE)-conjugated monoclonal antibodies CD235a, CD61, CD142, and CD62E, respectively. Forward scatter was set in scale using fluorescent microspheres of 0.8μm and standard fluorescent microbeads (0-0.8μm) in diameter were used to set the microparticle gate. Data were expressed as median (M) and interquartile range (IQR). Meanwhile, genomic DNA was extracted from mononuclear cells and amplified by allele speciﬁc polymerase chain reaction (PCR). Results (1) Patients with MPN showed significantly higher plasma levels for all four MPs compared with healthy donors (P<0.05), namely 49.0/μl (15.8-109.5/μl) vs 21.0/μl (13.8-32.6/μl) for RMPs, 181.2/μl(75.8-1111.6/μl) vs 74.9/μl (55.5-115.4/μl) for PMPs, 48.1/μl (13.1-72.4/μl) vs 31.0/μl (14.9-47.6/μl) for TF+MPs and 310.2/μl (128.6-1130.5/μl) vs 155.9/μl (100.3-227.6/μl) for EMPs. (2) Among different subtypes of MPN, PMPs were higher in patients with PMF than patients with PV and ET (P<0.05), but there was no significant difference between PV and ET group. No obvious difference was found in RMPs, TF+MPs and EMPs among different subtypes of MPN patients. (3) MPN patients with JAK2V617F mutation (n=34) were found to have higher plasma levels of TF+MPs and RMPs than those without mutation (P<0.05) and this difference was not found for PMPs and EMPs. (4) MPN patients with various thrombotic complications (n=10) showed higher levels of all four types of MPs than those without thrombotic complications (n=31) (P<0.05). Elevated MP levels were also found in patients with splenomegaly (n=19) compared to those without splenomegaly (n=14) (P<0.05). Conclusion Higher levels of MPs were observed in MPN patients compared with healthy controls, especially in patients complicated with thrombosis and splenomegaly, which reflects a prothrombotic state. Moreover, significantly increased TF+MPs and RMPs were found in MPN patients with JAK2V617F mutatioin. Disclosures: No relevant conflicts of interest to declare.

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Loss of EZH2 Inhibits Erythropoiesis and Accelerates the Development of Myelofibrosis in Jak2V617F Knock-in Mice

Blood ◽

10.1182/blood.v124.21.159.159 ◽

2014 ◽

Vol 124 (21) ◽

pp. 159-159

Author(s):

Yue Yang ◽

Hajime Akada ◽

Dipmoy Nath ◽

Robert E Hutchison ◽

Golam Mohi

Keyword(s):

Peripheral Blood ◽

Blood Cells ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Flow Cytometric Analysis ◽

Gene Set Enrichment Analysis ◽

Jak2v617f Mutation ◽

Interferon Response ◽

Hematopoietic Stem ◽

Erythroid Precursors

Abstract EZH2, a component of the polycomb repressive complex 2 (PRC2), catalyzes the trimethylation of histone H3 at lysine 27 (H3K27) to repress the transcription of target genes. Inactivating mutations of EZH2 have been found in myelodysplastic syndromes and myeloproliferative neoplasms (MPNs) including myelofibrosis (MF). EZH2 mutations are associated with poor prognosis in patients with MF. However, the contribution of EZH2 mutations in the pathogenesis of MF remains unknown. The JAK2V617F mutation has been found in a majority of cases of MPNs including ~50% patients with MF. However, it is not clear whether JAK2V617F mutation alone is sufficient to cause MF. Interestingly, inactivating EZH2 mutations co-exist with JAK2V617F mutation in significant cases of MF. To understand the role of JAK2V617F in MPNs, we previously generated a conditional Jak2V617F knock-in mouse, which exhibits all the features of human PV. To determine if EZH2 mutations cooperate with JAK2V617F mutation in MF, we crossed the conditional EZH2 knock-out mice with conditional Jak2V617F knock-in mice and assessed the effects of concomitant deletion of EZH2 and expression of heterozygous Jak2V617F in mice hematopoietic compartments. Whereas Jak2V617F expression resulted in significant increase in red blood cells (RBC), hemoglobin, hematocrit, white blood cells and platelets in the peripheral blood of the Jak2V617F knock-in mice, deletion of EZH2 significantly reduced the RBC, hemoglobin, and hematocrit parameters in Jak2V617F knock-in mice. Interestingly, platelet counts were further increased in EZH2-deleted Jak2V617F-expressing mice. Flow cytometric analysis showed significant increase in CD71+Ter119neg/lo early erythroid precursors and decrease in CD71+Ter119high late erythroid precursors in the bone marrow (BM) and spleens of EZH2-deleted Jak2V617F mice suggesting a defect in erythroid differentiation upon EZH2 deletion in Jak2V617F mice. Notably, megakaryocytic precursors (CD41+CD61+) were significantly increased in the BM and spleens of EZH2-deleted Jak2V617F mice consistent with increased number of platelets in the peripheral blood of these mice. Similar to human PV, Jak2V617F expression resulted in cytokine-independent CFU-E colonies in the BM and spleens of Jak2V617F knock-in mice. However, deletion of EZH2 markedly inhibited cytokine-independent CFU-E colonies in the BM and spleens of Jak2V617F knock-in mice. Histopathologic analysis revealed extensive fibrosis in the BM and spleens of EZH2-deleted Jak2V617F mice at 24 weeks after induction while heterozygous Jak2V617F knock-in mice BM and spleens showed very mild fibrosis at this age. Control and EZH2-deficient mice did not exhibit any fibrosis in their BM or spleens. In order to determine whether the effects of EZH2 deletion in Jak2V617F mice were cell autonomous, BM cells from pIpC induced control, EZH2-deficient, Jak2V617F knock-in and EZH2-deleted Jak2V617F-expressing mice were transplanted into lethally irradiated syngeneic recipient mice. Transplanted animals receiving EZH2-deleted Jak2V617F BM developed severe fibrosis in their BM and spleens within 8 weeks after transplantation. Furthermore, recipients of EZH2-deleted Jak2V617F BM exhibited severe anemia and became moribund by 8 weeks after transplantation. In contrast, transplanted animals receiving control, EZH2-deficient or Jak2V617F BM did not exhibit fibrosis at 8 weeks after transplantation. Thus, the phenotypes observed in EZH2-deficient Jak2V617F mice are hematopoietic cell-autonomous. Together, these data suggest that loss of EZH2 inhibits erythropoiesis, promotes megakaryopoiesis and accelerates the development of MF in mice expressing Jak2V617F. To gain insights into the mechanisms by which EZH2 deficiency accelerates the development of MF in Jak2V617F mice, we performed microarray gene expression analysis on purified long-term hematopoietic stem cells (LT-HSC; Lin-c-kit+Sca-1+CD34-Flk2-). Gene set enrichment analysis revealed that interferon response-related genes and the genes related to TNF signaling pathway were up-regulated in LT-HSC of EZH2-deficient Jak2V617F mice compared with Jak2V617F LT-HSC. Further studies will validate the targets of EZH2 that are de-repressed upon EZH2 deletion in MF induced by Jak2V617F. In conclusion, our studies show that loss of EZH2 cooperates with Jak2V617F mutation in the development of MF. Disclosures No relevant conflicts of interest to declare.

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