Effect of a TNFα Blocker and Peginfa on Polycythemia Vera Clonal Hematopoiesis and Suppressed Normal Dormant Hematopoiesis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1820-1820
Author(s):  
Sabina Swierczek ◽  
Soo Jin Kim ◽  
Mohamed E Salama ◽  
William L. Heaton ◽  
Michael W. Deininger ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
X Chromosome ◽  
Mononuclear Cells ◽  
Myeloproliferative Neoplasms ◽  
Intracellular Cytokine Staining ◽  
Advisory Board ◽  
Erythroid Progenitors ◽  
The Impact ◽  
Allelic Burden

Abstract Polycythemia vera (PV) is a clonal disorder arising from a single stem cell while normal stem cells are present in the marrow but are suppressed by the PV clone by an unknown mechanism. Pegylated interferon alfa-2a (PegInfa), a better-tolerated form of Infa induces clinical remission, reduces JAK2V617F allelic burden, and may convert clonal to polyclonal hematopoiesis (EL Liu, Blood 2003). Tumor necrosis factor-α (TNFa) levels are increased in patients with myeloproliferative neoplasms, including PV (Fleischman, Blood 2011). We previously reported that transcripts of TNFα mRNA are higher in CD34+ cells compared to more differentiated cells. TNFa is markedly reduced in those PegInfa-treated patients with decreased JAK2V617F allelic burden and/or return of polyclonal hematopoiesis (detected in females by X-chromosome allelic usage ratio), while no such decrease was seen in PV with hydroxyurea-induced normalization of elevated hematocrit/platelets/leukocytes (Swierczek, ASH 2012). To directly interrogate the role of TNFα in inducing suppression of normal hematopoiesis, we used a TNFα blocking antibody (adalimumab) and examined its role on PV erythropoiesis using a 3-week liquid culture system characterized by synchronized differentiation of expanding erythroid progenitors (Bruchova, Exp Hemat, 2007). In vitro expanded PV erythroid progenitors were grown with or without adalimumab or PegInfa. We evaluated apoptosis, proliferation and differentiation at different stages of erythroid maturation and correlated these parameters with TNFα transcripts, JAK2V617F allelic burden and, in females, clonality. Although the initial mononuclear cells represented a heterogeneous population, the expansion process favors erythroid progenitors and results in their synchronized differentiation. The JAK2V617F allelic burden increased concomitant with erythroid expansion and reached its peak at day 11 (when the majority of cells are proerythroblasts and basophilic erythroblasts), indicating a preferential expansion of erythroid progenitors, and then declined progressively. The addition of adalimumab markedly reduced TNFα mRNA and JAK2V617F allelic burden compared to controls. We previously reported that in this in vitro liquid expansion system, PV erythroid progenitors exhibit accelerated differentiation at days 7-14 and increased proliferation at days 9-14, with a larger S-phase population (40%) than controls (20%) at day 11 (Bruchova Exp Hemat, 2007). Compared to controls, adalimumab increased proliferation and delayed differentiation at early stages of PV erythropoiesis, with the proportion of apoptotic cells consistently decreased compared to erythroid cells expanded without adalimumab. Furthermore, X-chromosome-based clonality assays revealed preferential expansion of normal progenitors in 1 informative female patient. We also measured the impact of TNFα inhibition with adalimumab on burst-forming units-erythroid (BFU-E) colonies from PV patients cultured ex vivo. As expected, JAK2WT, JAK2WT/V617F and JAK2V617F BFU-E colonies were detected in cultures performed in the absence of adalimumab, while the addition of adalimumab preferentially abrogated JAK2V617F homozygous BFU-Es. In analogous experiments, PegInfa markedly decreased TNFα mRNA and JAK2V617F allelic burden, and decreased differentiation, but unlike adalimumab, it decreased proliferation and had no demonstrable effect on apoptosis. Ongoing studies are being performed to correlate the TNFα mRNA expression changes seen with adalimumab treatment in erythroid progenitors with differences in TNFα protein levels using intracellular cytokine staining and flow cytometry. These data suggest that blocking TNFα can suppress the JAK2V617F clone, rescue normal dormant hematopoiesis, and provide a foundation for a combined PV therapy using TNFα blockers with either PegInfa or JAK2 inhibitors. Disclosures Deininger: BMS, Novartis, Celgene, Genzyme, Gilead: Research Funding; BMS, ARIAD, Novartis, Incyte, Pfizer: Advisory Board, Advisory Board Other; BMS, ARIAD, Novartis, Incyte, Pfizer: Consultancy.

Erythropoiesis in Polycythemia Vera Is Hyper-Proliferative and Has Accelerated Differentiation during In Vitro Erythroid Expansion and Is Associated with Higher Expressions of cMYB and EPOR at Early Erythroid Progenitors

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1549-1549
Author(s):  
Hana Bruchova ◽  
Donghoon Yoon ◽  
Archana Agarwal ◽  
Eva Otahalova ◽  
Hyojin Kim ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Mononuclear Cells ◽  
Early Stage ◽  
Severe Disease ◽  
Erythroid Differentiation ◽  
Erythroid Progenitors ◽  
Peripheral Blood Mononuclear ◽  
Healthy Donors ◽  
Differentiation Stage

Abstract Erythroid differentiation is a dynamic process leading to the production of mature red blood cells. Even small variations in this process may result in severe disease phenotype. To study this process, we used a three-phase erythroid expansion system to expand homogeneous erythroid progenitors (EPs) from peripheral blood mononuclear cells (PB-MNCs) (Bruchova H. et al, 2007, Exp. Hematology, in press). We then characterized the expanded EPs from polycythemia vera (PV) patients and healthy donors at various points of maturation comparing cell proliferation and differentiation stage. EPs from PV patients outgrew controls up to day 14 (∼12 fold for PV and ∼4 fold for control compared to day 1). Differentiation was analyzed using both FACS analysis (with CD71/CD235a staining) and morphological evaluation (Wright-Giemsa staining), and demonstrated a more rapid differentiation of PV EPs when compared to controls up to day 14. We then evaluated apoptosis/cell cycle analysis by propidium iodide staining. Although PV EPs contained larger S phase population (45%) than controls (34%) at day 11, the apoptosis proportion of PV EPs was increased ∼2 fold to control from day 14. To understand the molecular mechanism of these differences between PV and controls, we analyzed the gene expression of several known regulators in erythropoiesis - BCL2, EPOR, cMYB, p27. Two transcripts (EPOR and cMYB) showed unique profiles on PV EPs. The EPOR transcript increased earlier in PV; i.e. from day 7 until day 21 and reached a plateau at day 11, compared to day 9 until day 19 and plateau at day 14 in controls. In addition, PV EPs contained higher levels of EPOR transcripts than control on most of timepoints. Interestingly, cMYB, which is known to augment early progenitor proliferation, was highly expressed from day 7 in PV, through day 11. Control EPs also expressed cMYB from day 9 through day 11; however, cMYB levels from any stages of control EPs were markedly lower than PV EPs at day 7. In this study, we demonstrate that PV erythropoiesis has unique features of hyperproliferation and an accelerated differentiation. These features are associated with earlier and higher expressions of cMYB and EPOR at the early stage of erythropoiesis.

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

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

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

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

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

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

In Vitro Erythroid Cell Expansion Analysis in Polycythemia Vera.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4916-4916 ◽  
Author(s):  
Donghoon Yoon ◽  
Hana Bruchova ◽  
Archana M. Agarwal ◽  
Josef T. Prchal ◽  
Jaroslav F. Prchal
Keyword(s):  
Polycythemia Vera ◽  
Molecular Mechanism ◽  
Mononuclear Cells ◽  
Biological Behavior ◽  
Erythroid Cell ◽  
Erythroid Progenitors ◽  
Peripheral Blood Mononuclear ◽  
Expansion Protocol ◽  
Differentiation And Proliferation

Abstract Polycythemia vera (PV) is an acquired myeloproliferative clonal stem cell disorder characterized by cytokine hypersensitivity. The erythroid colony forming assay has been useful in PV diagnosis. However, studies aiming to elucidate molecular mechanism in PV pathogenesis often require large numbers of cells from a specific erythroid stage. For this purpose, we had adapted the mouse expansion assay described by Karur et al (Blood, 2006 in E-Pub) and differential display method described by Zhang et al (Blood, 2003, 102; 3938) to develop a human erythroid expansion protocol. In this study, we used peripheral blood mononuclear cells (PB-MNCs) from PV patients and healthy donors, and expanded them along erythroid lineage in 21 day culture. Through the culture, we took samples at 8 timepoints (days 1, 7, 9, 11, 14, 16, 19, 21) to evaluate differentiation patterns. We had generated 5 different regions using CD71 (transferrin receptor) and CD235a (glycophorin A) and characterized these regions by standard morphology analysis. This protocol allowed differentiation of PB-MNCs to all erythroid stages ending with late normoblast, reticulocyte, and mature erythrocytes (which do not survive well in this culture environment). The erythroid differentiation and proliferation patterns were different between PV and normal. In the first week of culture, there was no significant proliferation difference observed between PV and normal. In contrast, the differentiation progressed more rapidly in PV, likely reflecting the differentiation of Epo independent PV population (since the first week culture contains no Epo). In the second week of culture (Epo present), there was a markedly increased expansion of PV erythroid cells. However, the differentiation pattern of PV resembled that of normal. In conclusion, we demonstrated that our in vitro expansion method allows expansion of PB-MNCs along erythroid lineage with 60~80% stage homogeneity as to the stage of differentiation in both PV and normal. The differences of differentiation and proliferation pattern between PV and normal reflected the expected biological behavior of erythroid progenitors. Thus, the expansion protocol is useful to study molecular mechanism of PV pathogenesis, which requires large number of erythroid progenitors of various stages.

scholarly journals Diamond-blackfan anemia: in vitro response of erythroid progenitors to the ligand for c-kit

Blood ◽  
1991 ◽  
Vol 78 (9) ◽  
pp. 2198-2202 ◽  
Author(s):  
JL Abkowitz ◽  
KM Sabo ◽  
B Nakamoto ◽  
CA Blau ◽  
FH Martin ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Therapeutic Trial ◽  
Erythroid Progenitors ◽  
Primary Defect ◽  
Diamond Blackfan Anemia ◽  
Kit Receptor ◽  
In Vitro Response ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony

Abstract To provide insights into the pathogenesis of Diamond-Blackfan anemia, we examined the in vitro response of erythroid progenitors to the recently isolated ligand for c-kit (stem cell factor, SCF). For these studies, marrow or blood mononuclear cells from 10 Diamond-Blackfan patients were cultured with erythropoietin (Ep), Ep and interleukin-3, Ep and granulocyte-macrophage colony-stimulating factor, or Ep and lymphocyte conditioned media (LCM). These combinations were tested in the presence or absence of SCF. The mean number of cells per erythroid burst increased 5 to 50-fold in cultures containing SCF. Furthermore, many additional erythroid bursts were seen (mean increment 3.2 x baseline values). Although burst-forming unit-erythroid (BFU-E) from all patients responded, there were differences among individuals in the sensitivity of their BFU-E to SCF. In six patients and all control studies, plateau frequencies of erythroid bursts were achieved with less than or equal to 10 ng/mL SCF, whereas in studies from the other four patients, over 50 ng/mL SCF was required. These data invite speculation that the c-kit receptor/ligand axis is involved in the pathogenesis of Diamond-Blackfan anemia. More importantly and regardless of whether the observed patterns of response reflect the primary defect or an epiphenomenon, our data strongly support a therapeutic trial of SCF in patients with Diamond-Blackfan anemia.

scholarly journals Glucocorticoids and Catecholamines Affect in Vitro Functionality of Porcine Blood Immune Cells

Animals ◽  
2019 ◽  
Vol 9 (8) ◽  
pp. 545 ◽  
Author(s):  
Lena Reiske ◽  
Sonja Schmucker ◽  
Julia Steuber ◽  
Volker Stefanski
Keyword(s):  
Immune Cells ◽  
Lymphocyte Proliferation ◽  
Molecular Mechanisms ◽  
Mononuclear Cells ◽  
Intracellular Cytokine Staining ◽  
Stress Hormones ◽  
High Stress ◽  
Peripheral Blood Mononuclear ◽  
Indwelling Catheters

Stress hormones exert important modulating influences on the functionality of immune cells. Despite its major role as a livestock animal and its increasing use as an animal model, knowledge about this relationship in the domestic pig is rare. This study therefore aimed to characterize the effect of glucocorticoids and catecholamines on the proliferation and cytokine production of porcine peripheral blood mononuclear cells (PBMC). Blood was obtained from donor pigs equipped with indwelling catheters to exclude stress hormone exposition before in vitro testing. PBMC were stimulated in the presence of cortisol, adrenaline or noradrenaline at concentrations resembling low to high stress conditions. Proliferation was determined via 3H-thymidine incorporation, and TNFα producers were quantified by intracellular cytokine staining. Cortisol led to a decrease in mitogen-induced lymphocyte proliferation and the number of TNFα producing cells. In contrast, catecholamines increased proliferation while exerting repressive or no effects on the number of cytokine producers. Remarkably, in concentrations presumably found in lymphatic tissue in stress situations, noradrenaline suppressed lymphocyte proliferation completely. The shown repressive effects might especially have implications on health and welfare in pigs. The obtained results provide a preliminary database for extended studies on the molecular mechanisms of glucocorticoid and catecholamine actions on porcine immune cells.

scholarly journals Splenomegaly impacts prognosis in essential thrombocythemia and polycythemia vera: A single center study

2019 ◽  
Vol 11 (4) ◽  
Author(s):  
Vincenzo Accurso ◽  
Marco Santoro ◽  
Simona Raso ◽  
Angelo Davide Contrino ◽  
Paolo Casimiro ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Myeloproliferative Neoplasms ◽  
Clinical Manifestations ◽  
Primary Myelofibrosis ◽  
Thrombotic Risk ◽  
Single Center Study ◽  
The Impact ◽  
The Relationship

Splenomegaly is one of the major clinical manifestations of primary myelofibrosis and is common also in other chronic Philadelphia-negative myeloproliferative neoplasms, causing symptoms and signs and affecting quality of life of patients diagnosed with these diseases. We aimed to study the impact that such alteration has on thrombotic risk and on the survival of patients with essential thrombocythemia and patients with Polycythemia Vera (PV). We studied the relationship between splenomegaly (and its grade), thrombosis and survival in 238 patients with et and 165 patients with PV followed at our center between January 1997 and May 2019.

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

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

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

Dysregulated Expression of miRNAs in Polycythemia Vera Erythroid Progenitors.

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

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

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