Polycythemia Vera Erythroid Progenitors Research Articles

Phosphoproteomic Landscaping Identifies Non-canonical cKIT Signaling in Polycythemia Vera Erythroid Progenitors.

Although stem cell factor (SCF)/cKIT interaction plays key functions in erythropoiesis, cKIT signaling in human erythroid cells is still poorly defined. To provide new insights into cKIT-mediated erythroid expansion in development and disease, we performed phosphoproteomic profiling of primary erythroid progenitors from adult blood (AB), cord blood (CB), and Polycythemia Vera (PV) at steady-state and upon SCF stimulation. While AB and CB, respectively, activated transient or sustained canonical cKIT-signaling, PV showed a non-canonical signaling including increased mTOR and ERK1 and decreased DEPTOR. Accordingly, screening of FDA-approved compounds showed increased PV sensitivity to JAK, cKIT, and MEK inhibitors. Moreover, differently from AB and CB, in PV the mature 145kDa-cKIT constitutively associated with the tetraspanin CD63 and was not endocytosed upon SCF stimulation, contributing to unrestrained cKIT signaling. These results identify a clinically exploitable variegation of cKIT signaling/metabolism that may contribute to the great erythroid output occurring during development and in PV.

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

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. DisclosuresDeininger:BMS, Novartis, Celgene, Genzyme, Gilead: Research Funding; BMS, ARIAD, Novartis, Incyte, Pfizer: Advisory Board, Advisory Board Other; BMS, ARIAD, Novartis, Incyte, Pfizer: Consultancy.

Extent of hematopoietic involvement by TET2 mutations in JAK2V617F polycythemia vera

TET2 mutations are found in polycythemia vera and it was initially reported that there is a greater TET2 mutational burden than JAK2(V617F) in polycythemia vera stem cells and that TET2 mutations precede JAK2(V617F). We quantified the proportion of TET2, JAK2(V617F) mutations and X-chromosome allelic usage in polycythemia vera cells, BFU-Es and in vitro expanded erythroid progenitors and found clonal reticulocytes, granulocytes, platelets and CD34(+) cells. We found that TET2 mutations may also follow rather than precede JAK2(V617F) as recently reported by others. Only a fraction of clonal early hematopoietic precursors and largely polyclonal T cells carry the TET2 mutation. We showed that in vitro the concomitant presence of JAK2(V617F) and TET2 mutations favors clonal polycythemia vera erythroid progenitors in contrast with non-TET2 mutated progenitors. We conclude that loss-of-function TET2 mutations are not the polycythemia vera initiating events and that the acquisition of TET2 somatic mutations may increase the aggressivity of the polycythemia vera clone.

TET2 Mutations in Polycythemia Vera (PV) in Some Cases Follow Rather Than Precede JAK2 V617F Mutation, Are Not a Disease-Initiating Event, Affect Mainly Erythropoiesis, and Contribute to Increased Aggressivity of PV Clone.

Abstract 3913Poster Board III-849The Ten-Eleven Translocation (TET) 2 gene is a tumor suppressor gene. Its mutations of which are frequently found in PV and other myeloid malignancies. The published evidence suggests that a TET2 mutational burden is present in a higher proportion than JAK2V617F in PV stem cells; its mutations typically precede the JAK2V617F mutation, and are preferentially expressed in myeloid cells. We studied 40 PV patients. Two had known TET2 mutation, one with an intronic mutation (3954+2T>A) predicted to cause aberrant splicing, and the other with a deletion of a single nucleotide in exon 3 (3138delT) leading to the predicted truncation of the TET2 peptide. We quantitated the mutational burden of JAK2V617F and TET2 and the clonality of blood lineages using X-chromosome allelic usage ratios in the blood cells and BFU-E colonies. We also followed the mutational burden of the JAK2V617F and TET2 somatic mutations and expression of TET2 mRNA and monitored the proportion of polyclonal cells, in in vitro expanded erythroid progenitors. These data were compared to the PV patients without known TET2 mutations. Using an X-chromosome-based transcriptional clonality assay, the PV patients had predominantly clonal reticulocytes, granulocytes, platelets and, in those available, CD34-positive cells. Studies of individual BFU-E found that in two PV patients, the TET2 mutations followed rather than preceded the JAK2V617F mutation. We report that only a fraction of clonal CD34+ cells carry the TET2 mutation, and demonstrate that a small proportion of largely polyclonal T cells also carry the TET2 mutation. We report that the presence of both JAK2V617 and TET2 mutations favors accumulation of the mutated erythroid progenitors, while in similar conditions the PV JAK2V617-positive and TET2-negative cells are at a proliferative disadvantage compared to normal erythroid progenitors. We also examined the clonality of these in vitro-expanded erythroid progenitors to determine if the dormant minor populations of nonclonal hematopoietic cells are preferentially expanded along with those belonging to the PV clone. We found that some PV erythroid progenitors with JAK2V617F but no known TET2 mutations became polyclonal after in vitro erythroid expansion, while two PV patients with JAK2V617F and TET2 mutations remained clonal, suggesting that the TET2 mutated clonal progenitors retained their proliferative advantage. Lastly, compared to normal erythroid progenitors wherein TET2 mRNA increases with erythroid maturation, it decreases in PV erythroid progenitors regardless of the presence of a TET2 mutation. As predicted, the intronic TET2 mutation causing aberrant splicing had decreased TET2 expression compared to controls and other PV samples in all cells examined. However, the TET2 mRNA transcript in peripheral blood granulocytes and platelets in JAK2V617F positive PV, regardless of TET2 mutations, was significantly increased compared with normal controls. We conclude that loss-of-function TET2 mutations in the two studied PV subjects are not the PV initiating events. Our data suggest that these TET2 mutations in PV preferentially affect the erythroid lineage, contribute to increased erythroid proliferation, and cause relative inhibition of PV granulopoiesis and megakaryopoiesis. However, in aggregate, these in vitro data also suggest that the acquisition of the TET2 somatic mutations increases the aggressivity of the PV clone. Disclosures:No relevant conflicts of interest to declare.

The Effects of SB1518, a Novel Oral JAK2 Inhibitor, On Ex Vivo Expanded PV Erythroid Progenitors Correlate with Clinical Observations.

Abstract 2913Poster Board II-889 Introduction:Polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) in which the patients' erythroblastic lineage acquires a hyper-proliferative potential but retains normal maturation, leading to elevated hematocrit and its attendant risks. The discovery of an activating mutation (JAK2V617F) in almost all PV patients and about half of the other Ph-negative MPNs prompted the development of small-molecule JAK2 inhibitors as novel therapeutics. In preclinical translational studies, these compounds have been tested against primary hematopoietic cells in ex vivo assays such as the endogenous erythroid colony (EEC) assay or proliferation assay with erythroid progenitors (EP). Results have been mixed, with some compounds showing selective activity against JAK2V617F-bearing cells while others do not discriminate between wild-type and mutant. SB1518 is a JAK2 inhibitor being evaluated in Phase I/II studies on myelofibrosis patients. We report here the effects of SB1518 on ex vivo expanded EPs from PV patients and healthy volunteers. Patients and methods:Peripheral blood mononuclear cells (PBMCs) were isolated from 11 PV patients and 8 healthy volunteers. EPs were expanded ex vivo from the PBMCs using a published protocol which involves culturing in an erythropoietin (EPO)-containing medium over a 12-day period [Cancer Sci. (2008) Vol 99, 1265]. Flow cytometry for co-expression of CD71 and CD235a confirmed that >98% of the expanded cells belonged to the basophilic erythroblast lineage. The EPs were treated with SB1518 and other JAK2 inhibitors to determine the effects of JAK2 inhibition on downstream signaling, cell viability and JAK2V617F allele frequency. Results:SB1518 inhibited phospho-STAT5 levels in a dose-dependent manner (IC50 < 200 nM) and reduced the viability of expanded erythroid progenitors from all sources; normal volunteers with JAK2WT (mean IC50 = 260 nM) or PV patients with JAK2V617F (mean IC50 = 230 nM). The difference in IC50 between the 2 groups was not statistically significant. Moreover, SB1518 treatment had no effect on the JAK2V617F allele frequency in the EPs from PV patients, indicating similar drug sensitivity for EPs from the same patient, regardless of the presence of mutation. This suggests that the constitutively activated JAK2 mutant and the EPO-activated wild-type JAK2 exert equivalent quantitative impact on proliferative pathways in EPs. Similar effects were observed with other JAK2 inhibitors of different chemical classes and selectivity profiles. These data are consistent with current observations in JAK2 clinical trials, namely that clinical benefit is not dependent on JAK2 genotype and there is little or no effect on allele frequency in blood cells. Conclusions:In summary, we demonstrated that current JAK2 candidates, being inhibitory against both WT and mutant JAK2 kinases, will exert similar effects in biological systems sustained by JAK2 activation, whether constitutive or ligand-stimulated. Our data are consistent with current experience in myelofibrosis trials where clinical benefit is attainable independent of JAK2 mutation status and indicate that activation of JAK2 signaling either by an activating mutation or by other means, contributes to the symptoms of the disease. Disclosures:Goh:S*BIO PTE LTD: Employment. Hart:S*BIO PTE LTD: Employment, Equity Ownership. Tan:S*BIO PTE LTD: Employment. Chithra:S*BIO PTE LTD: Employment. Wood:S*BIO PTE LTD: Employment.

Erythropoiesis in polycythemia vera is hyper-proliferative and has accelerated maturation

Polycythemia vera (PV) is an acquired myeloproliferative clonal disorder, characterized by augmented erythropoiesis. To better define PV pathogenesis, we performed an in vitro erythroid expansion from peripheral blood mononuclear cells of controls and PV patients and evaluated the cells for proliferation, apoptosis, erythroid differentiation, and morphology at the defined time points. PV erythroid progenitors exhibited increased proliferation at days 9–14 and accelerated maturation at days 7–14, with a larger S-phase population (40%) than controls (20%) at day 11; however, the proportion of apoptotic cells was comparable to controls. Previously, we have identified PV-specific dysregulation of several microRNAs (i.e. miR-150, 451, 222, 155, 378). We had analyzed expression profiles of selected target genes of these microRNAs based on in silico prediction and their known function pertinent to the observed PV-specific erythropoiesis differences. p27, cMYB and EPOR showed differential expression in PV erythroid progenitors at the specific stages of erythroid differentiation. In this study, we identified accelerated maturation and hyper-proliferation at early stages of PV erythropoiesis. We speculate that aberrant expression of p27, c-MYB, and EPOR may contribute to these abnormal features in PV erythropoiesis.

Plasma Detection of JAK2 Mutation Is Not Suitable as a Clinical Test

The 1849G>T (V617F) JAK2 gene activating point mutation is identified in a substantial number of chronic myeloproliferative neoplasms (MPN) and the mutant allele burden correlates with some of the complications of MPN, justifying the introduction of quantitative JAK2 assays in clinical practice. Recent reports suggested a higher detection rate of the JAK2 mutation using plasma rather than by cell analysis and even suggested its use to detect zygosity. In this study we questioned the biologic basis of this suggestion and tested its validity by comparing JAK2 mutation allele burden in paired cell and plasma samples over time using a highly sensitive quantitative assay. Peripheral blood samples were collected from known patients with a positive JAK2 mutation as detected in their purified granulocytes. Separation of plasma and granulocytes (GNC) were carried on day 0,3,5,7,9,10 after sample collection. Quantitative assessment of JAK2 was performed on a total of 63 samples from 7 patients using allele-specific PCR amplification of JAK2-exon14 in genomic DNA isolated from the paired purified peripheral blood granulocytes and plasma samples. The detection of wild and mutant JAK2 alleles was achieved by highly sensitive and discriminatory allele-specific PCR utilizing allele specific primers that incorporate a mismatched nucleotide and synthetic ”locked nucleic acid” (Nussenzveig Expl Hemat. 35: 32–38, 2007). Quantitative assessment of the sample is achieved by real-time monitoring amplification from each allele (in separate reactions). The values for each reaction are then used to determine the frequency of the T-allele relative to the G-allele in the sample and statistical calculations were performed using SAS software, Version 9.1 of the SAS System Copyright © 2002–2003 SAS Institute Inc, Cary, NC, USA. Quantitative measurements of JAK2 V617F mRNA in these samples were also analyzed. The JAK2 V617F range was (0.2–90%) in GNC and (0–88%) in plasma with no concordance of corresponding values in any patient at any given time point. We identified a progressive increase in plasma JAK2 V617F DNA values accompanied by a progressive reciprocal decrease of JAK2 V617F in paired GNC samples in all cases over time, suggesting preferential lysis of the GNC bearing the JAK2 V617F mutation. Similar data were obtained using in vitro expansion of JAK2 V617F polycythemia vera erythroid progenitors. A repeated-measures ANOVA was calculated to determine whether there was interaction between GNC vs. plasma and time. The rate of change in the value of JAK2 did indicate an interaction and was significantly different (p=.003) between GNC and plasma. In one patient, the JAK2 V617F was detected in GNC (0.7%) but not in plasma sample on day 0. We conclude that detection of JAK2 V617F in plasma is due to decreased viability of the JAK2 bearing cells, which leads to progressive increase in plasma detection. There is no correlation between JAK2 V617F allelic burden values in GNC and plasma at any given point. JAK2 quantitation in plasma is not suitable as a clinical test.

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

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.

Study of two tyrosine kinase inhibitors on growth and signal transduction in polycythemia vera

An activating somatic mutation of Janus kinase 2 V617F (JAK2V617F) is present in most polycythemia vera (PV) patients. We studied efficacy of two potent tyrosine kinase inhibitors (TKI), AEE788 and AMN107, in vitro on cells bearing this mutation. We employed reporter cells expressing wild-type JAK2 and mutant JAK2V617F, human erythroleukemic cells (HEL) carrying JAK2V617F)to study the efficacy of these TKIs by cell proliferation assay, Annexin-V/propidium iodide staining, and on relevant cell-signaling and apoptotic events. These data were compared to ex vivo expanded native human erythroid PV progenitor cells grown in liquid cultures. AEE788 showed a time- and dose-dependent growth inhibitory effect that was greater in FDCP cells expressing JAK2V617F and HEL cells than in cells expressing wild-type JAK2. AEE788 caused dephosphorylation of Akt(S243) and signal transducer and activator of transcription 5(Y694) proteins, increase in Annexin-V binding and caspase-3 cleavage, suggesting induction of apoptosis. We also observed AEE788-mediated decrease in heat shock protein 70 and 90 antiapoptotic proteins. Similarly, native PV erythroid progenitors showed more sensitivity to AEE788 than normal erythroid progenitors. AEE788 also exerted dose-dependent inhibition of PV-specific erythroid colonies. Nilotinib (AMN107) however, lacked specificity and required high (>8 microM) concentrations to inhibit growth of JAK2V617F-carrying cells. Our data suggest that AEE788 exerts its apoptotic activity via downregulation of proliferative and antiapoptotic regulatory proteins. To our knowledge, this is the first report demonstrating the effect of AEE788 on PV erythroid progenitors. Differential effects on PV and normal progenitor cells suggest AEE788 has potential in the treatment of PV and other JAK2V617F positive hematologic malignancies.

Imatinib effect on growth and signal transduction in polycythemia vera

An activating mutation of Janus kinase 2 (JAK2) in majority of polycythemia vera (PV) and other myeloproliferative disorders was reported. As imatinib inhibits several tyrosine kinases, we studied its effect in PV. We employed FDCP reporter cells expressing wild-type JAK2 and mutant JAK2(V617F) to study the efficacy of imatinib by cell proliferation assay and its effect on several cell-signaling events. Imatinib's efficacy was also examined on in vitro expanded native human erythroid progenitors. In addition, analysis of the percent JAK2 T-allele and phospho-signal transducer and activator of transcription-5 (STAT5) in granulocytes of PV patients following imatinib therapy was assessed. Imatinib showed a specific time- and dose-dependent growth inhibitory effect on FDCP cells expressing JAK2(V617F), wherein we observed imatinib's inactivation of JAK2, STAT5 and cKIT proteins. In vitro expanded human PV erythroid progenitors were more sensitive to imatinib than normal erythroid progenitors and FDCP cells expressing JAK2(V617F), with growth inhibition at concentrations attainable in vivo. In an ongoing clinical study, a PV patient showed strong correlation between the percent JAK2 T-allele and his responsiveness to imatinib therapy. Our data elucidate the therapeutic benefit of imatinib seen in some PV patients. Our data suggest that JAK2/STAT5 and cKIT activation may be integrated. To our knowledge, this is the first report demonstrating imatinib's effect on PV erythroid progenitors. These studies underscore the limitation of experiments using cell lines expressing the gene of interest.

In vitro expansion of erythroid progenitors from polycythemia vera patients leads to decrease in JAK2 V617F allele

A G>T transversion in a tyrosine kinase JAK2 (V617F) was reported in over 80% of patients with polycythemia vera (PV). Current evidence suggests that JAK2(V617F) somatic mutation is involved in the pathogenesis of PV, as it confers erythropoietin-independent proliferation to erythroid progenitor cells. However, several unanswered questions regarding the essential role of JAK2(V617F) arose as 1) it is not a dominant mutation, 2) it is not PV-specific as it is found in several myeloproliferative disorders, and 3) some ( approximately 20%) PV patients lack the JAK2(V617F) mutation. We investigated the relative frequency of JAK2(V617F) in in vitro-expanded PV progenitors. In vitro expansion of erythroid progenitors from mononuclear cells was optimized. Frequency of JAK2(V617F) allele was measured by using allele-specific real-time polymerase chain reaction. Clonality was performed using established procedure. In vitro expansion of PV erythroid progenitors and differentiated dendritic cells resulted in a decrease of the frequency of JAK2(V617F) allele compared with granulocytes or CD235(+) erythroid progenitors. Clonality analysis demonstrated that although granulocytes of these PV patients were clonal, expanded erythroid cells were polyclonal. However, in vitro-expanded PV erythroid progenitors still had approximately a twofold increased proliferative capacity in comparison with erythroid progenitors from healthy individuals. Erythropoietin favors the cells without JAK2(V617F) allele. Dendritic cells in one out of three patients remained clonal. JAK2(V617F) mutation does not provide a proliferative/survival advantage to the PV clone during in vitro expansion. These data suggest that the JAK2(V617F) mutation plays an important role in the biology of PV, yet it may not be the PV-initiating event.

Dysregulated Expression of miRNAs in Polycythemia Vera Erythroid Progenitors.

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.

JAK1 and Tyk2 Activation by the Homologous Polycythemia Vera JAK2 V617F Mutation: CROSS-TALK WITH IGF1 RECEPTOR

The majority of polycythemia vera (PV) patients harbor a unique somatic mutation (V617F) in the pseudokinase domain of JAK2, which leads to constitutive signaling. Here we show that the homologous mutations in JAK1 (V658F) and in Tyk2 (V678F) lead to constitutive activation of these kinases. Their expression induces autonomous growth of cytokine-dependent cells and constitutive activation of STAT5, STAT3, mitogen-activated protein kinase, and Akt signaling in Ba/F3 cells. The mutant JAKs exhibit constitutive signaling also when expressed in fibrosarcoma cells deficient in JAK proteins. Expression of the JAK2 V617F mutant renders Ba/F3 cells hypersensitive to insulin-like growth factor 1 (IGF1), which is a hallmark of PV erythroid progenitors. Upon selection of Ba/F3 cells for autonomous growth induced by the JAK2 V617F mutant, cells respond to IGF1 by activating STAT5, STAT3, Erk1/2, and Akt on top of the constitutive activation characteristic of autonomous cells. The synergic effect on proliferation and STAT activation appears specific to the JAK2 V617F mutant. Our results show that the homologous V617F mutation induces activation of JAK1 and Tyk2, suggesting a common mechanism of activation for the JAK1, JAK2, and Tyk2 mutants. JAK3 is not activated by the homologous mutation M592F, despite the presence of the conserved GVC preceding sequence. We suggest that mutations in the JAK1 and Tyk2 genes may be identified as initial molecular defects in human cancers and autoimmune diseases.

Erythropoietin-independent erythroid colony formation by bone marrow progenitors exposed to interleukin-11 and interleukin-8

Endogenous erythroid colonies (EECs), formed in vitro without erythropoietin (EPo) or other exogenous cytokines, are characteristic of Polycythemia vera (PV). Our aim was to identify specific conditions of culture of bone marrow (BM) progenitors allowing formation of erythroid colonies without EPo. BM mononuclear cells (BMMCs), purified CD34+ cells, and purified CD36+ erythroid progenitors were cultured in serum-free media without and with cytokines: EPo, stem cell factor (SCF), and interleukin (IL)-11 and IL-8, produced by BM stromal cells and found elevated in PV. EECs were formed in PV cultures of either BMMCs or CD34+ cells, which include cytokine-secreting cells, but not in cultures of purified CD36+ erythroid progenitors (EP). Despite expression of V617F JAK-2, no constitutive activation of JAK-2, Stat-5, or Erk-1/2 was detected in erythroblasts issued from PV CD36+ progenitors. However, when SCF was provided, PV CD36+ progenitors formed erythroid colonies without EPo. The ability to form erythroid colonies with SCF alone was conferred to BM progenitors of healthy donors and secondary erythrocytosis by exposure to IL-11 and IL-8. Both IL-11 and IL-8 enhanced formation of erythroid colonies in response to EPo and interfered with the activation of Erk-1/2 and Stat-5 induced, respectively, by SCF and EPo in erythroblasts. Anti-IL-11 antibody inhibited formation of erythroid colonies by PV BMMCs and CD34+ cells. The data indicate that PV erythroid progenitors remain cytokine-dependent and that normal BM progenitors exposed to IL-11 and IL-8 can acquire the ability to form erythroid colonies without EPo.

Increased erythropoiesis in polycythemia vera is associated with increased erythroid progenitor proliferation and increased phosphorylation of Akt/PKB

The aim of this study was to explore the mechanism by which increased erythropoiesis occurs in polycythemia vera (PV). CD34(+) and erythroid colony-forming cells (ECFC) were purified from normal or PV peripheral blood and then incubated in the presence of erythropoietin (EPO) to generate erythroid progenitor cells. Measurement of proliferation by Ki-67 staining, TUNEL assays to measure apoptosis, and Western blots for detection of Akt/PKB and glycogen synthase kinase 3 (GSK3) phosphorylation were performed in both normal and PV erythroid progenitors. Polycythemia vera erythroid progenitor cells generated 60% more cells compared to normal cells in liquid medium cell cultures. TUNEL assays revealed no difference between PV and normal erythroid progenitors, but Ki-67 staining for cell proliferation showed many more positive cells in the PV samples. A marked increase of phosphorylation of Akt/PKB occurred in the day-8 erythroid progenitors of 4/5 PV patients, compared to normal cells, after incubation with either stem cell factor (SCF) or EPO. PV cells also had much greater glycogen synthase kinase 3 (GSK3) alpha,beta phosphorylation compared to normal cells after incubation with SCF or EPO. These results are parallel to the cellular hypersensitivity of PV cells to SCF and EPO previously reported. Increased erythropoiesis in PV is associated with increased cellular proliferation and increased phosphorylation of Akt/PKB and GSK3. This study provides additional insight into the pathogenesis of PV and the regulation of normal erythropoiesis, even though a specific molecular defect of the disease is still not apparent.

PTP-MEG2 is activated in polycythemia vera erythroid progenitor cells and is required for growth and expansion of erythroid cells

AbstractPolycythemia vera (PV) is a human clonal hematologic disorder. Previously we demonstrated that erythroid colony-forming cells (ECFCs) from PV patients contained a hyperactive membrane-associated tyrosine phosphatase. We now show that this phosphatase corresponded to protein tyrosine phosphatase (PTP)-MEG2, an intracellular enzyme with a putative lipid-binding domain. The increased activity of PTP-MEG2 in PV cells is due to its elevated distribution in the membrane fraction. With the development of ECFCs to mature red cells, the protein level of PTP-MEG2 decreased gradually, but membrane-associated PTP-MEG2 was sustained for a longer period of time in PV cells, which correlated with an enhanced colony-forming capability of the cells. Importantly, expression of dominant-negative mutant forms of PTP-MEG2 suppressed in vitro growth and expansion of both normal and PV ECFCs. The data indicate that PTP-MEG2 has an important role in the development of erythroid cells. (Blood. 2003;102:4354-4360)

Two New EPO Receptor Mutations: Truncated EPO Receptors Are Most Frequently Associated With Primary Familial and Congenital Polycythemias

AbstractPrimary polycythemias are caused by an acquired or inborn mutation affecting hematopoietic/erythroid progenitors that results in an abnormal response to hematopoietic cytokines. Primary familial and congenital polycythemia (PFCP; also known as familial erythrocytosis) is characterized by elevated red blood cell mass, low serum erythropoietin (EPO) level, normal oxygen affinity of hemoglobin, and typically autosomal dominant inheritance. In this study we screened for mutations in the cytoplasmic domain of the EPO receptor (EPOR; exons 7 and 8 of the EPOR gene) in 27 unrelated subjects with primary or unidentified polycythemia. Two new EPOR mutations were found, which lead to truncation of the EPOR similarly to previously described mutations in PFCP subjects. The first is a 7-bp deletion (del59855991) found in a Caucasian family from Ohio. The second mutation (5967insT) was found in a Caucasian family from the Czech Republic. In both cases the EPO dose responses of the erythroid progenitors of the affected subjects were examined to confirm the diagnosis of PFCP. In one of these families, the in vitro behavior of erythroid progenitors in serum-containing cultures without the addition of EPO mimicked the behavior of polycythemia vera progenitors; however, we show that antibodies against either EPO or the EPOR distinguish the in vitro growth abnormality of polycythemia vera erythroid progenitors from that seen in this particular PFCP family. We conclude that PFCP is a disorder that appears to be associated in some families with EPOR mutations. So far, most of the described EPOR mutations (6 out of 8) associated with PFCP result in an absence of the C-terminal negative regulatory domain of the receptor.

Identification of Increased Protein Tyrosine Phosphatase Activity in Polycythemia Vera Erythroid Progenitor Cells

Polycythemia vera (PV) is a clonal hematologic disease characterized by hyperplasia of the three major bone marrow lineages. PV erythroid progenitor cells display hypersensitivity to several growth factors, which might be caused by an abnormality of tyrosine phosphorylation. In the present study, we have investigated protein tyrosine phosphatase (PTP) activity in highly purified erythroid progenitor cells and found that the total PTP activity in the PV cells was twofold to threefold higher than that in normal cells. Protein separation on anion-exchange and gel-filtration columns showed that the increased activity was due to a major PTP eluted at approximately 170 kD. This enzyme was sensitive to PTP inhibitors and it did not cross-react with antibodies to SHP-1, SHP-2, or CD45. Subcellular fractionation showed that the PTP localized with the membrane fraction, where its activity was increased by threefold in PV erythroid progenitors when compared with normal cells. As the erythroid progenitors progressively matured, activity of the PTP declined rapidly in the normal cells but at a much slower rate in the PV cells. These studies suggest that a potentially novel membrane or membrane-associated PTP, representing a major PTP activity, may have an important role in proliferation and/or survival of human erythroid progenitors and that its hyperactivation in PV erythroid progenitors might be responsible for the increased erythropoiesis in PV patients.

Identification of Increased Protein Tyrosine Phosphatase Activity in Polycythemia Vera Erythroid Progenitor Cells

AbstractPolycythemia vera (PV) is a clonal hematologic disease characterized by hyperplasia of the three major bone marrow lineages. PV erythroid progenitor cells display hypersensitivity to several growth factors, which might be caused by an abnormality of tyrosine phosphorylation. In the present study, we have investigated protein tyrosine phosphatase (PTP) activity in highly purified erythroid progenitor cells and found that the total PTP activity in the PV cells was twofold to threefold higher than that in normal cells. Protein separation on anion-exchange and gel-filtration columns showed that the increased activity was due to a major PTP eluted at approximately 170 kD. This enzyme was sensitive to PTP inhibitors and it did not cross-react with antibodies to SHP-1, SHP-2, or CD45. Subcellular fractionation showed that the PTP localized with the membrane fraction, where its activity was increased by threefold in PV erythroid progenitors when compared with normal cells. As the erythroid progenitors progressively matured, activity of the PTP declined rapidly in the normal cells but at a much slower rate in the PV cells. These studies suggest that a potentially novel membrane or membrane-associated PTP, representing a major PTP activity, may have an important role in proliferation and/or survival of human erythroid progenitors and that its hyperactivation in PV erythroid progenitors might be responsible for the increased erythropoiesis in PV patients.

Endogenous BFU‐E in peripheral blood in diagnosis of polycythemia vera

Erythroid progenitors (CFU-E and BFU-E) growth in vitro from bone marrow and peripheral blood of patients with polycythemia vera (PV) was studied using a methylcellulose culture technique. The aim of the study was to find out whether the in vitro colony formation of peripheral blood could be used in the differential diagnosis of PV. In all 25 patients studied, endogenous colonies were found in the bone marrow and peripheral blood. The parallel study of both bone marrow and peripheral blood erythroid progenitors indicates that the presence of endogenous BFU-E in peripheral blood is a dependable test for PV. The results presented here showed that the abnormalities in PV erythroid progenitors are expressed at the level of both CFU-E and BFU-E, suggesting multiple changes in the erythroid progenitors. Our finding indicate that peripheral blood BFU-E differ from bone marrow BFU-E with regard to their dependence for further differentiation on BPA, the activity present in PHA-LCM.