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Multiple signaling pathways are involved in erythropoietin-independent differentiation of erythroid progenitors in polycythemia vera
Abstract
Polycythemia vera (PV) is a myeloproliferative disorder arising in a multipotent hematopoietic stem cell. The pathogenesis of PV remains poorly understood; however, the biologic hallmark of this disease is the presence of erythropoietin (Epo)-independent colony formation (endogenous erythroid colony [EEC]) and cytokine hypersensitivity. We have developed a simple liquid culture from CD34+ cells to study PV erythroid differentiation. PV erythroid differentiation was characterized in this culture system by two types of abnormalities: 1) an increased proliferation of progenitors in response to cytokines, associated with strict cytokine dependency for preventing apoptosis; and 2) Epo-independent terminal erythroid differentiation in the presence of stem cell factor and interleukin-3 as evidenced by the acquisition of glycophorin A. The level of Epo-independent terminal differentiation correlates in PV patients with the number of EEC. Epo-independent terminal differentiation as well as normal Epo-induced differentiation were repressed by inhibitors of JAK2 (AG490), PI3K (LY294002), and the Src family kinases (PP2). In contrast, an inhibitor of the ERK/MAP kinase pathway (PD98059) had no effect on Epo-independent terminal differentiation. These signaling abnormalities were not mediated by a decreased expression or activity of the membrane tyrosine phosphatase CD45, which dephosphorylates JAK2 and Src family kinases. This study demonstrates that early steps of PV erythroid differentiation are strictly cytokine dependent. In contrast, late erythroid differentiation is an Epo-independent phenomenon that is mediated by signaling pathways identical to those in Epo-induced differentiation.
... Later on were found evidences about the possible molecular mechanisms of Myeloproliferative Disorders, including the association between various chronic myeloid neoplasms and constitutive activation of specific proteins with tyrosine kinase function (Campbell PJ et al., 2006). The frequent presence of chromosomal abnormalities in the short arm of chromosome 9 (Kralovics R et al., 2002) and the evidence of the increased activity of some signal transduction pathways (Röder S et al., 2001, Ugo V et al., 2004 pointed the scientific research towards specific molecular targets. In April 2005 five independent research groups identified a single point mutation in exon 14 of the somatic gene Janus Kinase 2 (JAK2), present in most patients with MPNs Ph-negative (Levine RL et al., 2005;James C et al., 2005;Baxter EJ et al., 2005;Kralovics R et al., 2005;Zhao R et al., 2005). ...
... It has also been shown that transplantation of cells with a hyperactivated PI3K/mTOR pathway induces leukemia in mice (Feng Z et al., 2005;Dowling RJ et al., 2010). PI3K signaling is of key importance in normal erythropoietin-induced erythroid differentiation and in spontaneous PV erythroid differentiation (Ugo V et al., 2004); conceivably, several kinases which are part of this pathway such as Akt and mTOR have been reported constitutively phosphorylated in bone marrow (Grimwade LF et al., 2009), in megakaryocytes of MPN patients (Vicari L et al., 2012) and in JAK2 V617F mutated cell lines (James C et al., 2005). ...
... Substrates of mTORC2 include the FOXO1 and FOXO3a transcription factors (Su B et al., 2011). Therefore, the signaling network controlled by Akt and mTOR has a central role in a variety of cellular processes that include cell growth, metabolism and proliferation; the activity of this network is elevated in most human cancers, including MPN, as supported by findings that: erythroblasts of JAK2V617F conditional KI mice showed strong Akt activation, particularly in animals homozygous for the JAK2 mutation (Akada H et al., 2010), elevated levels of phosphorylated STAT5, Akt and mTOR were found in the bone marrow of MPN patients (Grimwade LF et al., 2009) and a strong inhibition of EEC formation and EPO-induced erythroid differentiation in PV progenitor cells was produced by a PI3K/Akt inhibitor (Ugo V et al., 2004). Therefore, the PI3K/Akt/mTOR pathway represents an attractive target for cancer therapy (Engelman JA et al., 2009). ...
A gain-of-function mutation in Janus kinase 2 (JAK2V617F) is at the basis of the majority of chronic myeloproliferative neoplasms (MPN). Enhanced activation of other downstream pathways including the PI3K/mTOR pathway has been documented as well. In this study we evaluated the effects of JAK1/2 inhibitors, alone and in combination with mTOR, with a dual mTOR/PI3K inhibitor and with a pan PI3K inhibitor in in-vitro and in-vivo MPN models. Our findings of strong synergy between the JAK2 inhibitors and mTOR/PI3K inhibitor suggested that we might be able to administer these drugs at lower concentrations than when the drugs are used individually. This provides a framework for combination trials using compounds in patients with myeloproliferative neoplasms
... The procedure was previously approved by the local Institutional Ethics Committee (FCM UNICAMP CAAE: 45878215.8.0000.5404). The quantification of gamma-globin (γ A + γ G ) chains induced by compound 10a and RVT was performed using the CD34 + cells [52,53]. The separation of the mononuclear cells from the peripheral blood samples was performed by centrifugation using a Ficoll-Hypaque gradient. ...
... The following cell surface-specific antibodies were used in the experiment: anti-transferrin receptor (FITC-conjugated) (CD 71), antiglycophorin A (PE-conjugated), and anti-fetal hemoglobin (FITC-conjugated). 52 These antibodies were commercially acquired from Caltag Laboratories (Burlingame, CA). In the flow cytometry experiments, the cellular content was 1 × 10 5 cells in a final volume of 300 µL in phosphate-buffered saline. ...
Resveratrol (RVT) derivatives (10a-i) were designed, synthesized, and evaluated for their potential as gamma-globin inducers in treating Sickle Cell Disease (SCD) symptoms. All compounds were able to release NO at different levels ranging from 0–26.3%, while RVT did not demonstrate this effect. In vivo, the antinociceptive effect was characterized using an acetic acid-induced abdominal contortion model. All compounds exhibited different levels of protection, ranging from 5.9–37.3%; the compound 10a was the most potent among the series. At concentrations between 3.13–12.5 µM, the derivative 10a resulted in a reduction of 41.1–64.3% in the TNF-α levels in the supernatants of macrophages that were previously LPS-stimulated. This inhibitory effect was higher than that of RVT used as the control. In addition, the compound 10a and RVT induced double the production of the gamma-globin chains (γG+γA), compared to the vehicle, using CD34+ cells. Compound 10a also did not induce membrane perturbation and it was not mutagenic in the in vivo assay. Thus, compound 10a emerged as a new prototype of the gamma-globin-inducer group with additional analgesic and anti-inflammatory activities and proving to be a useful alternative to treat SCD symptoms.
... Les EEC ainsi que les progéniteurs mégacaryocytaires (endogenous megakaryocyte colony « EMC »), sont également hypersensibles à de nombreux facteurs de croissance comme l'IL-3, l'insulin-like growth factor 1 (IGF-1), le SCF, le GM-CSF (Correa et al., 1994) (Correa, Blood 1994) (Dai et al., 1992) (Axelrad et al., 2000). Cette indépendance des EEC à l'EPO est altérée en utilisant des inhibiteurs de tyrosines kinases comme l'imatinib mesylate (inhibant ABL, KIT et PDGFR) (Oehler, 2003) et AG490 (inhibant JAK2) (Ugo et al., 2004), confirmant le rôle des tyrosines kinases dans ce phénotype. ...
... Les cibles de STATs sont globalement des gènes impliquées dans la prolifération cellulaire (cycline D1, c-myc ou pim-1), la survie (Dai et al., 2005). En plus, le LY294002, inhibiteur de PI3K, empêche la différenciation indépendante de l'EPO des progéniteurs de PV (Ugo et al., 2004). Par ailleurs, de nombreuses études réalisées in vitro ou in vivo sur des modèles de souris ont montrées que la mutation JAK2V617 est responsable de l'activation constitutive de la voie PI3K/AKT (James et al., 2005a) (Baker et al., 2007). ...
Les néoplasies myéloprolifératives (NMP) sont des maladies hématologiques acquises de la cellule souche hématopoïétique. Une mutation activatrice de la protéine de signalisation JAK2, JAK2V617F, a été identifiée chez la moitié des patients atteints de NMP Philadelphie négatives. Il a été rapporté que les patients avec des NMP avaient une augmentation du risque thrombotique et de la densité microvasculaire dans la rate et la moelle osseuse, sans explication physiopathologique claire. Des travaux récents ont mis en évidence la présence de la mutation JAK2V617F non seulement dans les cellules sanguines mais également dans les cellules endothéliales (CE) de ces patients. Nous faisons l’hypothèse que la présence de JAK2V617F dans les CE pourrait modifier leurs propriétés expliquant l’augmentation de l’angiogenèse dans les NMP. Pour répondre à cette hypothèse, nous avons voulu étudier le phénotype angiogénique des cellules endothéliales portant la mutation JAK2V617F. In vitro, nous disposons des particules lentivirales permettant d’obtenir des CE JAK2V617F par transduction lentivirale. In vivo, nous disposons des souris transgéniques exprimant la mutation JAK2V617F de manière conditionnelle (JAK2V617F/WT) grâce à la stratégie Cre-lox. Pour répondre à notre hypothèse, il été nécessaire de travailler avec des modèles murins exprimant la mutation JAK2V617F spécifiquement dans les CE sans atteinte concomitante de la lignée hématopoïétique. Dans un premier temps, nous avons voulu caractériser deux modèles endothéliaux inductibles couramment utilisés, Cdh5(PAC)-CreERT2 et Pdgfb-iCreERT2, en termes d’efficacité et de spécificité de recombinaison dans les cellules endothéliales vis-à-vis du compartiment hématopoïétique. Nous avons démontré que les souris adultes Cdh5(PAC)-CreERT2 pouvaient être utilisées comme modèles endothéliaux spécifiques, avec toutefois la mise en garde que la recombinaison est très variable entre les souris. Nous avons constaté que les souris PDGFB-iCreERT2 sont appropriées pour cibler les cellules endothéliales dans une large gamme d’organes à l'exception du foie, et devraient être utilisées dans les quatre premières semaines qui suivent l'induction, pour cibler un gène d’intérêt au niveau des cellules endothéliales, sans qu’il ait une atteinte concomitante dans la lignée hématopoïétique. Nous avons ensuite étudié les propriétés angiogéniques des cellules endothéliales JAK2V617F, in vitro en utilisant des HUVEC transduites avec un lentivirus permettant l’expression de JAK2V617F, et in vivo avec les souris Pdgfb-iCreERT2;JAK2V617F/WT. Nous avons démontré que les HUVEC JAK2V617F avaient un profil proangiogénique lié à une capacité proliférative élevée, résultant de l’activation de la voie JAK2/STAT3/PI3K. L’avantage hyperprolifératif que confère la mutation JAK2V617F aux cellules endothéliales a été confirmé in vivo avec le modèle de la vascularisation post-natale de la rétine, avec toutefois une diminution de la densité du réseau vasculaire due à une augmentation de la régression vasculaire au niveau de la rétine des souris Pdgfb-iCreERT2;JAK2V617F/WT.
... 1 The molecular characterization of PV came in 2005 with the discovery of the JAK2 V617F mutation in about 90% of PV patients. 2 JAK2 V617F mutation activates JAK2 kinase and therefore, the JAK kinase -signal transducer and activator of the transcription signaling (JAK/STAT) pathway. 3 The significance of JAK2 mutation in gastric cancer is not well established. ...
... However, the EPO receptor (EPOR) is weakly expressed on erythroid cells, and its expression quickly decreases with terminal maturation [192][193][194][195]. EPO binding leads to EPOR dimerisation and activation of JAK/STAT to induce the erythroid transcriptional programme. Other signalling pathways stimulated by EPO include the MAPK and PI3K pathways, which act to promote survival and/or proliferation [196][197][198][199]. There is evidence to suggest that EPO may prime erythroid commitment in HSPCs, and that EPOR may be more broadly expressed in HPCs, indicating that erythroid lineage commitment may occur early during haematopoiesis [195,[200][201][202]. EPO is produced in the liver and adult kidney, and acts to regulate the level of oxygen in the blood by modulating the number of circulating erythrocytes [203][204][205][206][207][208]. ...
The haematopoietic system plays an essential role in our health and survival. It is comprised of a range of mature blood and immune cell types, including oxygen-carrying erythrocytes, platelet-producing megakaryocytes and infection-fighting myeloid and lymphoid cells. Self-renewing multipotent haematopoietic stem cells (HSCs) and a range of intermediate haematopoietic progenitor cell types differentiate into these mature cell types to continuously support haematopoietic system homeostasis throughout life. This process of haematopoiesis is tightly regulated in vivo and primarily takes place in the bone marrow. Over the years, a range of in vitro culture systems have been developed, either to expand haematopoietic stem and progenitor cells or to differentiate them into the various haematopoietic lineages, based on the use of recombinant cytokines, co-culture systems and/or small molecules. These approaches provide important tractable models to study human haematopoiesis in vitro. Additionally, haematopoietic cell culture systems are being developed and clinical tested as a source of cell products for transplantation and transfusion medicine. This review discusses the in vitro culture protocols for human HSC expansion and differentiation, and summarises the key factors involved in these biological processes.
... Mononuclear BM cell samples were separated on Ficoll-Hypaque (Sigma-Aldrich). CD34-positive cells were enriched using the magnetically activated cell sorting isolation kit (Miltenyi Biotec), resulting in a purity of at least 90% [22]. CD34-positive cells were cultured in X-Vivo20 media (Lonza) with 20% FBS, 100 ng/mL Stem Cell Factor (SCF), 100 ng/mL Fmsrelated tyrosine kinase 3 ligand (FLT3L), 100 ng/mL Thrombopoietin (TPO), 60 ng/mL interleukin-3 (IL-3) and 10 ng/ mL interleukin-6 (IL-6) (PrepoTech), and were maintained at 37 °C and 5% CO 2 . ...
Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are characterized by risk of relapses, poor survival, unwanted side effects and high toxicity with the current therapies. In light of these facts, there are efforts to develop new drugs specific for deregulated molecules that participate in leukemia pathogenesis. Hematopoietic cell kinase (HCK), an Src kinase family member, is overexpressed on hematopoietic stem cells of MDS and de novo AML patients and involved in the oncogenic process. Thus, we investigated in vitro, ex vivo and in vivo effects of a novel chemical compound targeting HCK inhibition (iHCK-37), in combination with the most used drugs for the treatment of MDS and de novo AML, 5-Azacytidine and Cytarabine. Herein, the combination treatment with iHCK-37 and 5-Azacytidine or Cytarabine demonstrated additive effects against leukemia cells, compared to either drug alone. iHCK-37 plus 5-Azacytidine or Cytarabine treatment was able to reduce the activation of oncogenic pathways, MAPK/ERK and PI3K/AKT, leading to reduction of ERK and AKT phosphorylation, and increased BAX and decreased BCL-XL protein expression. Moreover, treatment with iHCK-37 reduced MDS and AML CD34-positive cell numbers inside a 3D-structure but did not affect normal CD34-positive cell numbers. In vivo analysis showed that leukemic mice treated with iHCK-37 had reduced ERK and AKT proteins phosphorylation levels and leukocyte numbers. In conclusion, the iHCK-37 inhibitor has anti-neoplastic activity in leukemia cells without altering apoptosis and survival rate of normal cells, suggesting on-target malignant cell killing activity as a single agent or in combination with 5-Azacytidine or Cytarabine.
... The JAK family of protein tyrosine kinases is involved in cytokine receptor signaling, transmits the activating signal in the Epo-EpoR signaling pathway, and also is a required subunit for surface expression of cytokine receptors, including EpoR [12]. Ugo et al. have shown that inhibition of JAK2 abrogated Epo independence of erythroid progenitors [13]; this observation was followed by a description of the principal defect of the molecular basis of Ph-negative MPD, that is, a loss-of-function somatic mutation of an autoinhibitory JAK2 domain resulting from a valine-to-phenylalanine mutation at amino acid JAK2V617F. JAK2V617F leads to ongoing phosphorylation activity, which then can bind to a receptor and promote STAT recruitment and increased expression of PRV-1 [14]. ...
... In addition to the JAK/STAT, other cellular signaling pathways are involved in myeloproliferative neoplasms including Phosphoinositide 3-kinase (PI3 K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway ( [50]). The PI3 K signaling is involved in erythropoietin-independent differentiation erythroid progenitors in PV ( [51]). In vitro studies of JAK2 V617F mutated cell lines, both human and murine, treated with mTOR inhibition demonstrated inhibition of proliferation ( [52]). ...
Introduction:
Current treatment for polycythemia vera (PV) is limited and primarily targets thrombosis risk. Agents targeting distinct mechanisms of action within myeloproliferation are undergoing clinical evaluation to optimize efficacy, improve tolerance and augment long term disease complications.
Area covered:
This article reviews the current data from completed early phase clinical trials in PV, either as monotherapy or in combination with the few currently approved agents.
Expert opinion:
There remains an opportunity in PV management to improve efficacy and decrease risk of disease progression. Evolving data from use of long acting interferons are serving to clarifying the potential front line role of this therapy. JAK2 inhibition has made a significant impact on decreasing morbidity in patients with hydroxyurea resistant/refractory disease. New approaches may soon expand options including histone deactylase inhibitors (HDACi), either as monotherapy or combination therapy, which showed promising activity and symptomatic control of pruritus. Drugs targeting new molecular pathways (mammalian target of rapamycin, insulin receptor substrates 1/2, MDM2 protein) or the iron metabolism pathway are in early phase trial. Further translational studies assessing efficacy, long term complications, survival, and constitutional symptom control could pave a way for future success in PV drug development either as monotherapy or in combination.
... 1 Proliferation, survival, and differentiation of the 3 myeloid lineages require these pathways in different proportions and at different stages of differentiation. The PI3K pathway was suggested to contribute to the Epo independence of erythroid progenitors in PV. 48 The MAPK-ERK pathway was suggested to induce megakaryocyte senescence at high Tpo signaling levels 39 and to be involved in the myelofibrosis induced by TpoR W515 mutants. 25 The p38 MAPK pathway activated by FLT3 was shown to contribute to megakaryocyte abnormal proliferation and differentiation in myelofibrosis. ...
The ATP-binding pocket of the kinase domain of JAK2 is the major target of the present treatment of myeloproliferative neoplasms. Several inhibitors of JAK2 that are ATP competitive have been developed, but they do not discriminate between wild-type and mutant JAK2. These inhibitors have been used in myelofibrosis and, for the first time, treatment induced a reduction in spleen size and in constitutional symptoms. However, no dramatic effects on BM fibrosis, allele burden, or peripheral blast numbers were observed. These data indicate that other avenues should be explored that would either target mutant molecules (JAKs or receptors) more specifically and spare wild-type JAK2 or that would address other pathways that contribute to the malignant proliferation. Future success in treating myeloproliferative neoplasms will depend on advances of the understanding of JAK-STAT signaling and also on a better understanding of the disease pathogenesis, especially the role that mutants in spliceosome factors and epigenetic regulators play in the phenotype of the disease and the precise mechanism of fibrosis development.
... Although ruxolitinib reduces to some extent the mutated allele burden after long-term treatment, only few patients eventually achieve complete molecular response [15]. The lack of a clear disease-modifying effect of ruxolitinib [16,17] might be ascribed to an underlying mutation complexity of clonal hematopoietic progenitors and/or to hyperactivated signaling pathways other than JAK/STAT, including in particular the PI3K/mTOR cascade [18][19][20][21]. ...
Inhibition of the constitutively activated JAK/STAT pathway in JAK2V617F mutated cells by the JAK1/JAK2 inhibitor ruxolitinib resulted in clinical benefits in patients with myeloproliferative neoplasms. However, evidence of disease-modifying effects remains scanty; furthermore, some patients do not respond adequately to ruxolitinib, or have transient responses, thus novel treatment strategies are needed. Here we demonstrate that ruxolitinib causes incomplete inhibition of STAT5 in JAK2V617F mutated cells due to persistence of phosphorylated serine residues of STAT5b, that conversely are targeted by PI3K and mTORC1 inhibitors. We found that PI3K/mTOR-dependent phosphorylation of STAT5b serine residues involves Protein Phosphatase 2A and its repressor CIP2A. The levels of CIP2A were found increased in cells harboring the JAK2V617F mutation, and we provide evidence of a correlation between clinical responses and the extent of CIP2A downregulation in myelofibrosis patients receiving the mTOR inhibitor RAD001 in a phase II clinical trial. To achieve maximal inhibition of STAT5 phosphorylation, we combined ruxolitinib with BKM120, a PI3K inhibitor, and RAD001, an mTOR inhibitor, obtaining improved efficacy in JAK2V617F mutated cell lines, primary patients' cells, and JAK2V617F knock-in mice. These findings contribute to understanding the effectiveness of PI3K/mTOR inhibitors in MPN and argue for the rationale to develop combination clinical trials.
... This cytokine hypersensitivity or independence induced by JAK2V617F in cell lines mimics the EPO hypersensitivity or independence previously described in PV erythroid progenitors. 4,44 In addition to this activation of the canonical pathway of cytokine receptors, 2 reports have described that JAK2V617F acts on chromatin by phosphorylating H3Y41, decreasing the binding of heterochromatin protein 1a and PRMT5, impairing its ability to methylate histone substrates. 45,46 It is unknown whether these effects on chromatin are really important in the pathogenesis of MPNs. ...
The genetic landscape of classical myeloproliferative neoplasm (MPN) is in large part elucidated. The MPN restricted driver mutations including those in JAK2, calreticulin (CALR) and MPL abnormally activate the cytokine receptor/JAK2 pathway and their downstream effectors, more particularly the STATs. The most frequent mutation, JAK2V617F, activates the three main myeloid cytokine receptors (EPOR, G-CSFR and MPL) whereas CALR or MPL mutants are restricted to MPL activation. This explains why JAK2V617F is associated with polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) whereas CALR and MPL mutants are found in ET and PMF. Other mutations in genes involved in epigenetic regulation, splicing and signaling cooperate with the three MPN drivers and play a key role in the PMF pathogenesis. Mutations in epigenetic regulators TET2 and DNMT3A are involved in disease initiation and may precede the acquisition of JAK2V617F. Other mutations in epigenetic regulators such as EZH2 and ASXL1 also play a role in disease initiation and disease progression. Mutations in the splicing machinery are predominantly found in PMF and implicated in the development of anemia or pancytopenia. Both heterogeneity of classical MPNs and prognosis are determined by a specific genomic landscape i.e. type of MPN driver mutations, association with other mutations and their order of acquisition. However, factors other than somatic mutations play an important role in disease initiation, as well as disease progression such as germline predisposition, inflammation and ageing. Delineation of these environmental factors will be important to better understand the precise pathogenesis of MPN.
... After lysing the red cells, primary human CD34 + bone marrow cells were purified by magnetic bead separation using the human CD34 MicroBead kit and the AutoMACS Pro separator (Miltenyi Biotec). Purity of the CD34 + fraction was assessed by flow cytometry using anti-CD34-phycoerythrin (PE; BD Biosciences), and only CD34 + fractions showing a purity of N90% were used [19]. ...
New drug development for neoplasm treatment is nowadays based on molecular targets that participate in the disease pathogenesis and tumor phenotype. Herein, we describe a new specific pharmacological hematopoietic cell kinase (HCK) inhibitor (iHCK-37) that was able to reduce PI3K/AKT and MAPK/ERK pathways activation after erythropoietin induction in cells with high HCK expression: iHCK-37 treatment increased leukemic cells death and, very importantly, did not affect normal hematopoietic stem cells. We also present evidence that HCK, one of Src kinase family (SFK) member, regulates early-stage erythroid cell differentiation by acting as an upstream target of a frequently deregulated pathway in hematologic neoplasms, PI3K/AKT and MAPK/ERK. Notably, HCK levels were highly increased in stem cells from patients with some diseases, as Myelodysplastic Syndromes and Acute Myeloid Leukemia, that are associated with ineffective erythropoiesis These discoveries support the exploration of the new pharmacological iHCK-37 in future preclinical and clinical studies.
... The MPNs comprise clonal hematologic diseases that are thought to arise from a transformation of a hematopoietic stem cell. A major characteristic of Ph-negative MPNs is an increased signaling through the Janus kinase (JAK) signal transducer and activator of transcription (STAT) pathway as well as through the phosphatidylinositol 3-kinase (PI3K)-AKT (also known as protein kinase B) pathway in erythroid and myeloid cells [14][15][16] . The most significant evidence of molecular pathology was reported in 2005 with the identification of the somatic mutation JAK2 -V617F [17][18][19][20] . ...
Primary myelofibrosis (PMF) is a rare chronic BCR-ABL1-negative myeloproliferative neoplasm characterized by progressive bone marrow fibrosis, inefficient hematopoiesis, and shortened survival. The clinical manifestations of PMF include splenomegaly, consequent to extramedullary hematopoiesis, pancytopenias, and an array of potentially debilitating constitutional symptoms. The diagnosis is based on bone marrow morphology and clinical criteria. Mutations in the JAK2 (V617F), MPL (W515), and CALR (exon 9 indel) genes are found in approximately 90% of patients whereas the remaining 10% are so-called triple negatives. Activation of the JAK/STAT pathway results in overproduction of abnormal megakaryocytes leading to bone marrow fibrosis. These mutations might be accompanied by other mutations, such as ASXL1. The commonly used prognostication scoring for PMF is based on the International Prognostic Scoring System. The subsequently developed Dynamic International Prognostic Scoring System-plus employs clinical as well as cytogenetic variables. In PMF, CALR mutation is associated with superior survival and ASXL1 with inferior outcome. Patients with triple-negative PMF have a higher incidence of leukemic transformation and lower overall survival compared with CALR- or JAK2-mutant patients. The impact of genetic lesions on survival is independent of current prognostic scoring systems. These observations indicate that driver and passenger mutations define distinct disease entities within PMF. Accounting for them is not only relevant to clinical decision-making, but should also be considered in designing clinical trials.
... Finally, inhibition of the PI3K-Akt-mTOR pathway has been identified as a potential therapeutic target. Inappropriate activation of this pathway has been demonstrated in multiple malignancies, including MPNs [45]. One study demonstrated increased expression of Akt in MPN bone marrow, specifically within megakaryocytes [46]. ...
Primary myelofibrosis is a unique entity among BCR-ABL-negative myeloproliferative diseases, manifesting as bone marrow fibrosis and pancytopenia. Considerable evidence indicates that genetic and epigenetic abnormalities can result in defective clonal hematopoietic stem cell proliferation in addition to bone marrow microenvironment alteration. The “bad seeds in bad soil” theory illustrates the orchestrating efforts of hematopoietic stem cells, stromal cells, and their surrounding signaling molecules in myelofibrosis progression and malignancy transformation, though the exact mechanism of myelofibrosis is still not clear. This study reviews current concepts and questions regarding the pathogenesis of primary myelofibrosis and discusses the emerging targeted therapy aimed at restoring normal bone marrow environment and halting bone marrow fibrotic deterioration.
... JAK/STAT is also a target for identifying the molecular abnormalities in PV. Furthermore, in erythropoietin-independent differentiation of erythroid progenitors in PV, where constitutive activation of STAT3 has been reported, STAT3 was found to be repressed by inhibitors of JAK2 [14,15]. Recently, several groups have identified a consistent, single somatic activating mutation in the JAK2 gene in the majority of patients with PV [16]. ...
Polycythemia vera (PV) is a chronic myeloproliferative disorder characterized by abnormal growth of erythroid precursors in the bone marrow. Almost all patients with PV, around 97%, have a mutation in Janus kinase 2 (JAK2). It is through the activation of JAK/Signal Transducers and Activators of Transcription (STAT) protein signaling pathway that the JAK2 mutation is thought to induce cellular proliferation, growth, hematopoiesis and immune response in PV patients. A summary of the current role of immunotherapy in the treatment of PV is provided. There are new JAK kinase inhibitors that are currently being evaluated and are at various stages of clinical trials and development, as well as the previously studied cytokines therapies. At the present time, ruxolitinib and interferon-α (IFN-α) are the only United States Food and Drug Administration (FDA) approved drugs for the management of advanced PV. JAK kinase inhibitors are better tolerated and less problematic than the interferons. Still, allogeneic stem cell transplantation is the only potentially curable method for end-stage PV. Additional genetic mutations have been implicated in PV pathogenesis. In this perspective, targeting different pathways might be required. Further investigations are needed to evaluate the promising role of immunotherapy in PV whether alone or in combination with other modalities.
... BCL-x L 25 and the JAK-STAT pathway 26 , and down regulation of thrombopoietin receptor (MPL) expression 27 . Many of these observations were subsequently explained by the identification of an activating mutation in the JAK2 tyrosine kinase. ...
The classical myeloproliferative disorders (MPD), comprising essential thrombocythaemia (ET), polycythaemia vera (PV) and idiopathic myelofibrosis (IMF), are clonal premalignant haematopoietic neoplasms associated with activating mutations in signalling pathway molecules and a variable tendency to develop acute myeloid leukaemia (AML). This thesis examined genotype-phenotype associations of JAK2 and MPL mutations, the presence of clonal diversity in the MPD and the genetic events associated with progressive disease.
Mutations in MPL were identified in 4% of ET and 7% of IMF but not in PV. Three different acquired MPL mutations were identified, one of which had been reported as an inherited allele. Although MPL mutations did not delineate a distinct clinical or histopathological subtype of ET, molecular testing provides an important new tool in the diagnostic armamentarium. Clones homozygous for the JAK2 V617F mutation were identified in female but not male patients with ET, suggesting that gender differences may be important in the determination of disease phenotype. In patients with two acquired genetic alterations, a signalling pathway mutation and a cytogenetic abnormality were usually present within the same clone. By contrast, coexistence of two signalling pathway mutations indicated the presence of biclonal disease that in two patients had arisen independently and not from a shared founder clone.
RAS mutations were identified as potential cooperating events in patients with JAK2 or MPL mutant IMF. In patients developing AML following a JAK2 V617F-positive MPD, those with V617F-positive leukaemia had progressed via an accelerated phase of disease and harboured acquired alterations of RUNX1 or EVI1. V617F-negative leukaemias tended to follow directly from ET or PV, and loss of the JAK2 mutation by reversion to wild-type due to mitotic recombination, gene deletion or gene conversion was excluded. The thesis concludes with a discussion of how clonal heterogeneity can be integrated into current models of MPD disease pathogenesis.
... Cytokines, such as IL-8, associated with acute respiratory distress syndrome (32), and IL-6, associated with severe cytokine-release syndrome, can occur in chronic lymphocytic leukemia patients after treatment with rituximab, an anti-CD20 monoclonal antibody (33). The underlying disease (polycythemia vera) in the second casepatient is associated with Janus kinase 2 mutation V617F, which leads to constitutive tyrosine phosphorylation activity that promotes cytokine hypersensitivity, including IL-3 and stem cell factor (34,35). The abnormal signaling pathway in cells of patients with polycythemia vera can also contribute to growth of fibroblasts and microvascular endothelial cells and induce the production of profibrogenic and angiogenic cytokines (36). ...
A nosocomial cluster induced by co-infections with avian influenza A(H7N9) and A(H1N1)pdm09 (pH1N1) viruses occurred in 2 patients at a hospital in Zhejiang Province, China, in January 2014. The index case-patient was a 57-year-old man with chronic lymphocytic leukemia who had been occupationally exposed to poultry. He had co-infection with H7N9 and pH1N1 viruses. A 71-year-old man with polycythemia vera who was in the same ward as the index case-patient for 6 days acquired infection with H7N9 and pH1N1 viruses. The incubation period for the second case-patient was estimated to be <4 days. Both case-patients died of multiple organ failure. Virus genetic sequences from the 2 case-patients were identical. Of 103 close contacts, none had acute respiratory symptoms; all were negative for H7N9 virus. Serum samples from both case-patients demonstrated strong proinflammatory cytokine secretion but incompetent protective immune responses. These findings strongly suggest limited nosocomial co-transmission of H7N9 and pH1N1 viruses from 1 immunocompromised patient to another.
... Описание митотической рекомбинации хромосомы 9p как наиболее распространенного цитогенетического дефекта при ИП [46] Гомозиготность мутации, вызываемая митотической рекомбинацией хромосомы 9p [32][33][34][35] 2001-2004 Эритропоэтиннезависимый рост при ИП зависит от JAK-STAT-пути передачи сигнала [47,48] STAT-белки конститутивно активируются при мутации [32,[34][35][36] 2005 ...
Current concepts of true polycythemia are reviewed. Results of numerous epidemiological studies on the prevalence of this form of myeloproliferative neoplasms are presented with special reference to recent findings concerning its pathogenesis and the role of JAK2V617F mutation that occurs in the majority of patients. The clinical picture and new diagnostic criteria are discussed The data on the incidence and prevalence of the disease and its complications are considered including venous and/or arterial thrombosis, transformation into post-polycythemic myelofibrosis and acute myeloleucosis. An algorithm for the treatment of patients with erythremia is proposed along with recommendations on the use of aspirin, hydroxyurea, alpha-interpheron, and imatinib. The prospects for clinical application of selective JAK2 inhibitors are discussed.
... et al. have confirmed that CD34+ cells from PV patients become more erythroid than healthy controls when cultured in liquid serum free medium without EPO (the medium contained IL-3 and SCF)193 . We were able to enhance the observation done by Ugo et al. in a culture medium containing Flt3, TPO, IL-6, SCF (modified expansion medium). ...
The molecular etiology of polycythemia vera (PV) remains incompletely understood. PV patients harbor increased numbers of hematopoietic stem cells (HSC), common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMP) and display Epo-independent erythroid maturation. However, the molecular mechanism underlying Epo-hypersensitivity and stem cell expansion is unclear. The transcription factor NF-E2 is overexpressed in the majority of PV patients. The work presented in this thesis demonstrates that elevation of NF-E2 expression in healthy CD34+ cells to levels observed in PV CD34+ cells causes Epo-independent erythroid maturation and expansion of the HSC, CMP and GMP cell number. Silencing NF-E2 in PV patients abrogates Epo-independent maturation, decreases the amount of HSC, CMP and GMP thus demonstrating that NF-E2 plays an important role in PV pathology.
... l'Epo350 . La différenciation érythroïde indépendante d'Epo des érythroblastes de patients peut être diminuée par les inhibiteurs de JAK2 mais aussi des Src kinases351 . D'autre part, au cours de l'érythroleucémie induite par le virus de Friend, les érythroblastes de souris déficientes pour Lyn ne développent pas de polyglobulie, suggérant un rôle de Lyn dans la phase tardive de la différenciation érythroïde terminale de la transformation352 . ...
Erythropoiesis is a process leading to red cells production. This differentiation program is mainly under control of the transcription factor GATA-1 that controls the expression of erythroid genes and the anti-apoptotic protein Bcl-xL. During apoptosis, GATA-1 is cleaved by activated caspase-3, leading to decreased Bcl-xL expression. During terminal erythroid differentiation, transient caspase-3 activation is required but GATA-1 remains uncleaved. In this study we demonstrated that during differentiation but not during apoptosis, the chaperone protein Hsp70 protects GATA-1 from caspase 3-mediated proteolysis. At the onset of caspase activation, Hsp70 translocates into nucleus and protects GATA-1, allowing Bcl-xL. In contrast, EPO starvation induces the nuclear export of Hsp70 and the cleavage of GATA-1 leading to apoptosis. On the other hand, GATA-1 overexpression induces a blocage of maturation, then GATA-1 expression must be tighly regulated for proper erythroid differentiation. Here, we showed that Hsp27 is accumulated into nucleus of differentiating erythroblasts through p38 MAPPK phosphorylation. Nuclear Hsp27 interacts with acetylated GATA-1 to favor it's ubiquitinylation and proteasomal degradation. Those results show a new role for Hsp70 and Hsp27 along terminal erythroid differentiation through the fine tuning of GATA-1 expression. Further, we determined the mechanisms of nuclear accumulation during terminal erythroid differentiation. Erythropoiesis is positively regulated by two factors necessary for proliferation and survival of erythroid progenitors, SCF from early stage until the stage of basophilic erythroblast and Epo since CFU-E until erythroblast. Before c-Kit (SCF receptor) down-modulation at basophilic stage, Hsp70 is mainly localized into cytoplasm. Indeed, SCF induced Hsp70 nuclear export via S400 Hsp70 AKT phosphorylation resulting in a weak nuclear Hsp70. At the onset of c-Kit down-modulation, SCF induced Hsp70 nuclear export was decreased. On the other hand, Epo activated Lyn induced Hsp70 nuclear accumulation. Then, we described here a new mechanism of c-Kit erythroid blocage since it's down modulation is necessary for caspase-3 GATA-1 protection by Hsp70. Moreover, we highlighted a new survival and differentiating role for Lyn kinase under Epo. We tested if our model could be applicated to low grade myelodysplastic syndrome (MDS), characterized by anemia, associated with excessive caspase activation leading to apoptosis and delayed expression of the glycophorin A marker of erythroid progenitors. Here, we demonstrated that a defect in nuclear localization of Hsp70 is partially responsible for the observed phenotype since expression of nuclear Hsp70 partially rescues phenotype observed in differentiating cells of MDS patients. These results confirm our physiologic model. Moreover, c-Kit could be a new therapeutic target in MDS.
... and colleagues demonstrate that the same signaling pathways necessary to obtain the erythroid differentiation of normal cells in the presence of Epo are involved in the spontaneous differentiation of PV erythroid cells. AG490 (JAK2 inhibitor), LY294002 (PI3K inhibitor) and PP2 (Src kinase inhibitor) were essential to the demonstration that JAK2, PI3K and Scr are involved in spontaneous erythroid differentiation of PV[74](A) Representation of the structure of JAKs. The similarity of the domains between the 4 different JAK proteins was used to define the 7 different domains (JH1-JH7). ...
Myeloproliferative neoplasms (MPNs) are a heterogeneous group of clonal malignant diseases, including polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (MF) and chronic myeloid leukemia (CML). The oncogenic event underlying CML is the 9-22 translocation, resulting in the fusion protein Bcr-Abl. PV, ET and MF are MPNs Bcr-Abl negative characterized by proliferation of one or more myeloid lineages with relatively normal and effective maturation. Excessive production of mature cells involves mainly the erythroid lineage in PV, megakaryocytic lineage in ET and granulocytic and megakaryocytic lineages in MF. Common findings in MPNs patients are hepatosplenomegaly, variable predisposition to thrombotic events, bleeding and transformation to acute leukemia or bone marrow fibrosis Standard treatment is the use of Hydroxyurea (HU), but acetylsalicylic acid can also be indicated, especially in ET patients. Bleeding can be the therapeutic treatment of choice for patients with PV. Recently, great progress has been achieved in understanding the molecular mechanisms of MPNs. A somatic mutation in the JAK2 gene (JAK2 V617F) was described in more than 95% of PV patients and 50-60% of ET or MF patients. JAK2V617F mutation is associated to the myeloproliferative phenotype and quickly turned into a useful clonal marker in the diagnosis and represents therapeutic target of great importance. Additional mechanisms, such as genetic alterations in genes such as ASXL1, CBL, IDH1, IDH2, RUNX1, TET2, EZH2, LNK, among others, can play a role in MPNs etiophatogenesis. Interestingly, some of these genes were shown to be directly or indirectly implicated in regulation of DNA methylation. Although this mechanism has not been fully elucidated in MPNs, it may have therapeutic potential, as hypomethylating agents are already used in the treatment of myeloid neoplasms.
... A major clue was the recognition of increased signaling through the JAK-signal transducer and activator of transcription (STAT) pathway, comprised of JAKs and STATs, as well as through the phosphatidylinositol 3-kinase (PI3K)-AKT (also known as protein kinase B) pathway in erythroid and myeloid cells. [22][23][24] The most significant clue to date came in 2005 with the identification of the somatic mutation JAK2 V617F . [25][26][27][28] This mutation in JAK2 exon 14, which occurs in at least 95% of patients with PV and about 60% of those with PMF and ET, results in a valine (V) to phenylalanine (F) substitution at codon 617. ...
Myelofibrosis (MF) is a rare chronic BCR-ABL1 (breakpoint cluster region-Abelson murine leukemia viral oncogene homologue 1)-negative myeloproliferative neoplasm characterized by progressive bone marrow fibrosis, inefficient hematopoiesis, and shortened survival. The clinical manifestations of MF include splenomegaly, consequent to extramedullary hematopoiesis, cytopenias, and an array of potentially debilitating abdominal and constitutional symptoms. Dysregulated Janus kinase (JAK)-signal transducer and activator of transcription signaling underlies secondary disease-associated effects in MF, such as myeloproliferation, bone marrow fibrosis, constitutional symptoms, and cachexia. Common fatal complications of MF include transformation to acute leukemia, thrombohemorrhagic events, organ failure, and infections. Potential complications from hepatosplenomegaly include portal hypertension and variceal bleeding, whereas extramedullary hematopoiesis outside the spleen and liver – depending on the affected organ – may result in intracranial hypertension, spinal cord compression, pulmonary hypertension, pleural effusions, lymphadenopathy, skin lesions, and/or exacerbation of abdominal symptoms. Although allogeneic stem cell transplantation is the only potentially curative therapy, it is suitable for few patients. The JAK1/JAK2 inhibitor ruxolitinib is effective in improving splenomegaly, MF-related symptoms, and quality-of-life measures. Emerging evidence that ruxolitinib may be associated with a survival benefit in intermediate- or high-risk MF suggests the possibility of a disease-modifying effect. Consequently, ruxolitinib could provide a treatment backbone to which other (conventional and novel) therapies may be added for the prevention and effective management of specific MF-associated complications.
... As an acquired mutation, JAK2 V617F plays a key role in the pathogenesis of MPNs [2,13,14,15]. The JAK2 protein is a cytoplasmic tyrosine kinase and takes part in signal transduction [16]. A single amino acid substitution (valine to phenylalanine) in the JAK2 tyrosine kinase encoding gene causes uncontrolled hematopoiesis. ...
Objective: Myeloproliferative neoplasms (MPNs) like essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF) are acquired clonal hematopoietic stem cell disorders and originate from a multipotent hematopoietic stem cell. The SOCS1 and SOCS3 genes are negative regulators of the JAK/STAT signal pathway. In this study we investigate the promoter methylation of these genes in the pathogenesis of MPNs and secondary erythrocytosis/thrombocythemia.
Materials and Methods: Promoter methylation of SOCS1 and SOCS3 genes was analyzed with methylation-specific PCR. PCR products were analyzed by agarose gel electrophoresis.
Results: No disease-specific CpG island methylation of SOCS1 was observed. Hypermethylation of the SOCS3 promoter was identified in 5 out of 19 (26.3%) PV cases, 2 out of 21 (9.5%) ET cases, 1 out of 5 (20%) PMF cases, and 9 out of 42 (21.4%) cases of secondary erythrocytosis/thrombocythemia.
Conclusion: The results revealed that promoter methylation of the SOCS3 gene suggests a possible role for SOCS3 methylation in the pathogenesis of MPNs and secondary erythrocytosis/thrombocythemia.
Conflict of interest:None declared.
... (MAPK), extracellular signal-regulated kinase (ERK) and Phosphatidylinositol 3-kinase (PI3)/Akt/Mammalian target of Rapamycin (mTOR) pathways [5][6][7][8]. Augmented 'genetic instability' and epigenetic modification may ensue (as described in Fig. 1). This genetic instability may subsequently facilitate disease progression. ...
The myeloproliferative neoplasms (MPN) are clonal myeloid disorders characterized by proliferation of mature myeloid cells, such that in polycythaemia vera (PV), the red cell proliferation dominates, platelets in essential thrombocythaemia (ET) and in myelofibrosis (MF), there may be cytopenia or proliferation, but the characteristic feature is the strikingly abnormal bone marrow stroma. These entities have a tendency to show phenotypic mimicry and may transform from one to another, for example, 20–30% of patients with PV are likely to develop MF. The significant event in this field was the recognition that Janus Kinase-2 (JAK2) activation was highly prevalent, followed by the description of the JAK2V617F mutation in 2005 (vide infra), which stimulated renewed interest in disease biology. Janus Kinase-2-targeted therapies have led to marked improvements for patients with this condition. However, it is obvious that the pathogenesis of these complex disorders reaches beyond this mutation; only 50–60% of patients with ET, for example, have the JAK2 mutation and several additional mutations have been described, which are of relevance in both the pathogenesis and clinical phenotype of these conditions.
Purpose:
Treatment options are limited beyond JAK inhibitors for patients with primary myelofibrosis (PMF), or secondary MF. Preclinical studies have revealed that PI3Kδ inhibition cooperates with ruxolitinib, a JAK1/2 inhibitor, to reduce proliferation and induce apoptosis of JAK2V617F mutant cell lines.
Patients and methods:
In a phase I dose-escalation and expansion study, we evaluated the safety and efficacy of a selective PI3Kδ inhibitor umbralisib in combination with ruxolitinib in MF patients who had a suboptimal response or lost response to ruxolitinib. Enrolled subjects were required to be on a stable dose of ruxolitinib for ≥8 weeks and continue that maximally tolerated dose at study enrollment. The recommended dose of umbralisib in combination with ruxolitinib was determined using a modified 3+3 dose escalation design. Safety, pharmacokinetics, and efficacy outcomes were evaluated, and spleen size was measured with a novel automated digital atlas.
Results:
Thirty-seven MF patients with prior exposure to ruxolitinib were enrolled. 2 patients treated with 800mg umbralisib experienced reversible Grade 3 asymptomatic pancreatic enzyme elevation, but no dose-limiting toxicities were seen at lower umbralisib doses. Two patients (5%) achieved complete response (CR), and 12 patients (32%) met the IWG-MRT response criteria of clinical improvement (CI). With a median follow-up of 50.3 months for censored patients, overall survival was greater than 70% after 3 years of follow-up.
Conclusions:
Adding umbralisib to ruxolitinib in patients was well-tolerated and may re-sensitize MF patients to ruxolitinib without unacceptable rates of adverse events seen with earlier generation PI3Kδ inhibitors.
Our knowledge about the biology of myeloproliferative neoplasms (MPNs) has exploded in the last 20 years, and this increased knowledge has led to advances in therapy. Introduced by Associate Editor Mario Cazzola, this Review Series brings readers up to date on our understanding of the natural history of the classical MPNs—polycythemia vera, essential thrombocythemia, and myelofibrosis—and the approaches to diagnosis, prognostication, and treatment for patients with these conditions.
To enable effective oxygen transport, approximately 200 billion red blood cells (RBCs) need to be produced every day in the bone marrow through the fine-tuned process of erythropoiesis. Erythropoiesis is regulated at multiple levels to ensure that defective RBC maturation or overproduction can be avoided. Here, we provide an overview of different layers of this control, ranging from cytokine signaling mechanisms that enable extrinsic regulation of RBC production to intrinsic transcriptional pathways necessary for effective erythropoiesis. Recent studies have also elucidated the importance of post-transcriptional regulation and highlighted additional gatekeeping mechanisms necessary for effective erythropoiesis. We additionally discuss the insights gained by studying human genetic variation impacting erythropoiesis and highlight the discovery of BCL11A as a regulator of hemoglobin switching through genetic studies. Finally, we provide an outlook of how our ability to measure multiple facets of this process at single-cell resolution, while accounting for the impact of human variation, will continue to refine our knowledge of erythropoiesis and how this process is perturbed in disease. As we learn more about this intricate and important process, additional opportunities to modulate erythropoiesis for therapeutic purposes will undoubtedly emerge.
Introduction: Polycythemia vera (PV) is the most common myeloproliferative neoplasm (MPN). PV is characterized by erythrocytosis, leukocytosis, thrombocytosis, increased hematocrit and hemoglobin in the peripheral blood. Splenomegaly and myelofibrosis often occur in PV patients. Almost all PV patients harbor a mutation in the JAK2 gene, mainly represented by the JAK2V617F point mutation.
Areas covered: This article examines the recent in vitro and in vivo available models of PV and moreover, it offers insights on emerging biomarkers and therapeutic targets. The evidence from mouse models, resembling a PV-like phenotype generated by different technical approaches is discussed. The authors searched PubMed, books and clinicaltrials.gov for original and review articles and drugs development status including the terms: Myeloproliferative Neoplasms, Polycythemia Vera, erythrocytosis, hematocrit, splenomegaly, bone marrow fibrosis, JAK2V617F, Hematopoietic Stem Cells, MPN cytoreductive therapy, JAK2 inhibitor, histone deacetylase inhibitor, PV-like phenotype, JAK2V617F BMT, transgenic JAK2V617F mouse, JAK2 physiologic promoter.
Expert opinion: Preclinical models of PV are valuable tools for enabling an understanding of the pathophysiology and the molecular mechanisms of the disease. These models provide new biological insights on the contribution of concomitant mutations and the efficacy of novel drugs in a “more faithful” setting. This may facilitate an enhanced understanding of pathogenetic mechanisms and targeted therapy.
Des mutations dans le gène de la calréticuline (CALR), codant pour une protéine résidente du réticulum endoplasmique (RE), ont été découvertes récemment dans les syndromes myéloprolifératifs (SMP). Elles sont associées à augmentation de prolifération cellulaire portant spécifiquement sur la lignée mégacaryocytaire. Ceci est le résultat d’une activation constitutive de la signalisation des voies JAK-STAT et MAP Kinases, consécutive à l’interaction des protéines mutantes CALR avec le récepteur à la thrombopoïétine. Plusieurs études ont montré la faible expression de ces protéines mutées dans les cellules, mais aucune n’a déterminé l’impact de leur expression sur l’homéostasie du RE ni les acteurs mis en jeu dans leur élimination. Dans ce travail, nous avons montré que l’expression des protéines CALR mutées ne perturbe pas sensiblement l’équilibre du RE et ne modifie pas la sensibilité des cellules à l’apoptose induite par un stress du RE. Nous avons ensuite démontré dans différents modèles, y compris des cellules engagées dans la différenciation mégacaryocytaire, que les faibles niveaux intracellulaires de variants protéiques CALR n’étaient pas liés à une sécrétion accrue dans le milieu extracellulaire ni à un défaut transcriptionnel. Cette faible expression est en fait la conséquence d’une dégradation mettant en jeu principalement la voie ERAD-protéasome. Dans ce processus, la reconnaissance de motifs glycans n’est pas impliquée, mais EDEM3 semble avoir un rôle majeur puisque son extinction augmente l’expression des formes mutées de CALR. La modulation de cette dégradation pourrait constituer une approche thérapeutique innovante dans les SMP.
Polycythemia vera (PV) is a chronic myeloproliferative neoplasm (MPN) characterized by erythrocytosis and clonal Janus kinase 2 (JAK2) V617F or JAK2 exon 12 mutations. The 2016 World Health Organization update has incorporated clinical and laboratory findings, molecular testing, and bone marrow pathology in its new classification, thereby improving the sensitivity to detect patients with PV and distinguishing the disease from other MPNs. Crystal structures and modeling of the JAK2 molecule have offered insight into possible therapeutic strategies with novel small molecules. These small molecules provide precision by affecting the mutated protein over the activity of wild type protein. Newer molecular techniques, especially next-generation sequencing, are revolutionizing molecular biology with the identification of somatic mutations modifying the course and prognosis of the disease. These mutations are found to have a potential role in responsiveness to therapy and may guide therapy decisions as well. Here we review recent advances in the clinical features, the diagnosis, the approaches, and the treatment of PV.
The recent acceleration in sequencing and related technologies has revealed the myeloproliferative neoplasms (MPNs) as genetically complex disorders. Constitutional alleles have also been identified that alter the risk of acquiring a clonal MPN or modulate its clinical phenotype. In addition, a group of rare inherited conditions lead to polyclonal overproduction of mature blood cells and represent important differential diagnoses for the clonal MPNs. This chapter presents an overview of these genetic events divided into five broad categories. The first three categories outline the genetic events that are central to MPN pathogenesis, comprising mutations in cytokine signalling pathways mutations in pathways controlling transcriptional regulation and events associated with transformation to advanced-phase disease. The fourth category comprises constitutional alleles that are associated with an increased risk of developing a clonal MPN. The fifth category comprises inherited conditions characterized by polyclonal blood cell overproduction that phenocopy the clonal MPN.
Advances in defining the mutational landscape of myeloproliferative neoplasms (MPNs) over the past decade have revolutionized the molecular diagnosis of these entities. Several methods have been developed for molecular analysis of MPN-related mutations, each with different analytical and diagnostic sensitivities. Molecular testing has become standard of care for the diagnostic workup of any suspected MPN, with JAK2 V617F being the most useful first test for polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Well-established second-order tests include: JAK2 exon 12 mutation analysis (suspected PV) and CALR and MPL mutation screening (suspected PMF or ET). This chapter reviews clinical and technical aspects of molecular testing in MPN, with focus on the most commonly mutated genes involved in these disorders. An overview of the molecular pathogenesis of MPN and emerging testing approaches is also provided.
There has been a major revolution in the management of patients with myeloproliferative neoplasms (MPN), in particular those with myelofibrosis and extensive splenomegaly and symptomatic burden, following the introduction of the JAK1 and JAK2 inhibitor ruxolitinib. The drug has been later approved also as second line therapy for polycythemia vera (PV). However, the therapeutic armamentarium for MPN is still largely inadequate to cope with the major unmet patients' needs, that include normalization of life span (MF and some PV patients), reduction of cardiovascular complications (mainly PV and essential thrombocythemia (ET)), prevention of hematological progression and improved quality of life (all MPN). In fact, none of available drugs has shown clear evidence of a disease-modifying activity, even if some patients treated with interferon and ruxolitinib showed reduction of mutated allele burden, and ruxolitinib might extend survival of patients with higher-risk MF. Raised awareness of the molecular abnormalities and cellular pathways involved in the pathogenesis of MPN is facilitating the development of clinical trials with novel target drugs, either alone or in combination with ruxolitinib. Although for most of these molecules a convincing preclinical rationale was provided, the results of early phase I and II clinical trials have been quite disappointing to date, and toxicities sometimes limiting. In this review, we will critically illustrate the current landscape of novel therapies that are under evaluation for patients with MPN on the basis of current guidelines, patient risk stratification criteria and previous experience, looking ahead to the chance of a cure for these disorders.
Background: Chronic myeloproliferative disorders (MPDs) are a heterogeneous group of diseases in which a clonal disorder of hematopoietic stem cells leads to an increase in the level of production in one or more of the blood cell. JAK2V617F recently acquired mutations in many patients with chronic myeloproliferative disorders (MPDs) is described by changing G to T mutation at nucleotide 1849 in exon 12 of the JAK2 gene is located on chromosome 9 identified, which leads to substitution of the amino acid phenylalanine instead of valine at position 617 of the protein JAK2 is the purpose of this study to evaluate the frequency of these mutations in patients with MPDs was. Materials and Methods: In this study, 117 patients were evaluated MPD. In patients identify mutations by ARMS-PCR method.
This method is a fast and easy test. Moreover, it is possible to distinguish between homozygous and heterozygous individuals with Mutation provides JAK2V617F. Patient information was obtained by questionnaire, records and sampling were carried out with the consent of the patient. Three patients were also sequencing. Results: Mutations in 6/86% (30/26) of patients with polycythemia vera, 6/46% (15/07) of patients with essential thrombocythemia, 5/61% (08/13) of patients with primary myelofibrosis and 14% (34/4 ) patients with chronic myeloid leukemia were identified. Polycythemia vera patients have a mutation, the white blood cells were significantly higher (p = 0.03). In addition, 16 of 26 patients with polycythemia vera JAK2-positive women with mutations associated with sex Showed.in other groups, no significant differences were found. The mutation was confirmed by sequencing. Conclusion: Our results are compatible with other studies and the highest prevalence of the mutation in polycythemia vera and lowest in chronic myeloid leukemia is identify. Discovery this mutation in the differential diagnosis, prognosis and prediction of response to treatment is useful opportunity to amend the criteria for diagnosis and provides a classification of disease.
The elucidation of the molecular pathogenesis of a variety of serious or life-threatening conditions has led to the expectation that the development of safe and effective therapies will soon follow. This principle has previously been demonstrated in malignant hematology by the effectiveness of ABL kinase inhibitors in chronic myelogenous leukemia (CML). A new opportunity to deliver on this premise is underway in the closely related non-CML myeloproliferative neoplasms (MPNs).
Myelofibrosis (MF) is a myeloproliferative neoplasm that presents either as a primary disease or evolves secondarily from polycythemia vera or essential thrombocythemia to post-polycythemia vera MF or post-essential thrombocythemia MF, respectively. Myelofibrosis is characterized by stem cell-derived clonal myeloproliferation, abnormal cytokine expression, bone marrow fibrosis, anemia, splenomegaly, extramedullary hematopoiesis, constitutional symptoms, cachexia, leukemic progression, and shortened survival. Therapeutic options for patients with MF have been limited to the use of cytoreductive agents, predominantly hydroxyurea; splenectomy and splenic irradiation for treatment of splenomegaly; and management of anemia with transfusions, erythropoiesis-stimulating agents, androgens, and immunomodulatory agents along with steroids. The only curative option is allogeneic stem cell transplantation (ASCT), which is associated with high morbidity and mortality risks. Recently, JAK (Janus kinase) inhibitor therapies have become available and proven to be palliative in primary MF patients with hydroxyurea-refractory splenomegaly and severe constitutional symptoms. The purpose of this article is to review the clinical features of MF; discuss different treatment strategies, including ASCT; and discuss the potential danger and benefit of using JAK inhibitors prior to ASCT.
Polycythemia vera (PV) is a myeloproliferative neoplasm characterized by increased red blood cell mass and usually overproduction of granulocytes and platelets and increased spleen size. JAK2V617F mutation is present in more than 95% of PV patients. Bone marrow examination reveals excessive proliferation of erythroid, myeloid, and megakaryocytic elements. The prognosis of PV depends on the severity of the complications occurring during the clinical course. Thrombotic complications are the main cause of morbidity and mortality in PV. Survival is affected whether appropriate therapy is applied during the erythrocytotic phase of the disease. Uncontrolled erythrocytosis poses very high risk for development of thrombosis. Some studies suggest that PV patients have a normal or near-normal lifeexpectancy. Most studies, however, report excess mortality caused by thrombotic complications and acute leukemia transformation during course of PV. This review aims to highlight the pathogenesis, diagnosis and current management in PV
Introduction:
Myelofibrosis (MF) is a myeloproliferative neoplasm associated with significant disease burden composed of splenomegaly, constitutional symptoms and a reduced life expectancy. The advent of targeted treatments has provided new means by which to improve MF associated splenomegaly, symptoms, health-related quality of life and even mortality. Areas covered: We discuss the spectrum of targeted treatments currently under investigation for MF. We furthermore compare their effects on improving anemia, reducing fibrosis and splenomegaly and enhancing symptom control. Expert opinion: MF is a complex disorder, partly attributable to its heterogeneity. Although the severity of patient symptoms correlates with risk category, high symptom burden may also be observed in low-risk patients. Serial use of PRO tools allows clinicians to objectively evaluate the MF symptom burden, compare efficacy of therapies and adjust medications to improve symptom control. Novel targeted agents have proven superior to historic treatment regimens for symptom management. Promising treatment categories include JAK2 inhibitors, histone deacetylase inhibitors, hypomethylating agents, heat shock protein-90 inhibitors, hedgehog inhibitors, PI3-AKT-mTOR inhibitors, antifibrosing agents and telomerase inhibitors. The majority of therapies remain under investigation, either alone or in combination with other treatments. It is anticipated that these agents will be increasingly integrated into standard treatment algorithms for MF symptom management.
Durch Überexpression von NF-E2 konnten wir in transgenen Tieren ein neues Mausmodell zur Entwicklung myeloproliferativer Erkrankungen (MPE) etablieren. Unsere Mausstämme simulieren viele der bereits vom Patienten bekannten Aspekte der MPE. Transgene Tiere zeigen eine trilineare Hyperplasie im Knochenmark mit den typischen Veränderungen der Megakaryozyten. In den Kolonie-Assays konnte ein deutlich stärkeres Proliferationspotential im Vergleich zum Wildtyp festgestellt werden. Darüber hinaus fand sich im peripheren Blut eine Leukozytose und Thrombozytose. Zusätzlich konnten transgene Tiere die für Polyzythämia vera charakteristischen EPO-unabhängigen erythroiden Kolonien ausbilden.
Außergewöhnlich an unserem Mausmodell sind drei Beobachtungen, die weder durch das JAK2V617F-Modell noch bei dem MPLW515L-Modell beobachtet werden konnten:
1. Transgene Tiere mit NF-E2-Überexpression sterben an massiven gastrointestinalen Blutungen.
2. Es tritt eine massive, möglicherweise durch Milzinfarkte hervorgerufene Einschränkung der Milzfunktion auf.
3. Die NF-E2-Überexpression von NF-E2 ermöglicht den Übergang in eine akute myeloische Leukämie.
Interessanterweise sind Thrombose, Blutungen und die maligne Transformation klinisch die bedeutendsten Komplikationen myeloproliferativer Erkrankungen. In dieser Arbeit kann somit ein neuer molekularer Marker als möglicher Ursprung dieser Erkrankungen vorgestellt werden. Insbesondere die unterschiedlichen Überlebenskurven der Tiere sowie die unterschiedlich ausgeprägten Phänotypen hinsichtlich der peripheren Blutparameter legen einen Dosiseffekt von NF-E2 nahe. Ob die Höhe des NF-E2-Spiegels mit den Komplikationsraten und folglich mit dem Überleben der Patienten korreliert, muss in ausführlichen klinischen Studien geklärt werden. Da bis dato noch keine molekularen Marker in die Risikostratifizierung der MPE aufgenommen wurden, erscheint die Untersuchung des Transkriptionsfaktors NF-E2 und seine Rolle in der Pathogenese myeloproliferativer Erkrankungen somit als lohnenswertes Ziel für weiterführende klinische Forschung.
Polycythaemia vera (PV) was first described in 1892 by Vazquez [1] in a case report describing a patient with ruddy cyanosis, splenomegaly, and an increased red cell count not associated with a congenital form of heart disease. A decade later in 1903 it was defined more clearly by Osler [2], and the disease then became known as Vasquez–Osler disease until this eponymous term was superseded by PV. PV together with essential thrombocythaemia (ET) and primary myelofibrosis (PMF) (Chap. 12) represent an overlapping spectrum of clonal haematological disorders called the human myeloproliferative neoplasms (MPN). The MPNs were first grouped together, along with chronic myeloid leukaemia (CML), by Dameshek in his seminal paper of 1951 [3]. CML is now generally considered as a distinct entity, but shares several features with the other MPNs. All of these disorders result from acquired genetic changes in the haematopoietic stem cell compartment and are characterized by proliferation of various cells of the myeloid lineages. They also all share the propensity to develop into acute myeloid leukaemia (AML), albeit with varying incidence, and, as will be described next, the majority demonstrate abnormalities of intracellular signalling. Taking all of these characteristics together, the MPN and CML therefore provide in vivo model systems to study the multistep development of AML. In addition, as MPNs are associated with full terminal differentiation of myeloid lineages, they allow the study of the effects of oncogenic mutations on normal myeloid homeostasis before this is complicated by cooperating mutations which block differentiation.
Introduction: Myelofibrosis (MF), classified as a myeloproliferative neoplasm (MPN) by the WHO, is an orphan disorder in which therapeutic strategies have been slow to develop, likely due to a lack of understanding of the disease pathogenesis. Areas covered: In this article, progress dating from the seminal descriptions of a highly prevalent mutation in the JAK2 gene, JAK2 V617F in 2005 is reviewed. This article describes, in brief, the current status of our scientific understanding in this field and reflects on how this is now stimulating a change in therapeutic strategies for patients. Information was gathered from a review of the medical literature and conference abstracts, in particular the most recent American Society of Haematology meeting. Each novel therapy is described, in turn, and then future strategies for evaluating this plethora of potential agents are discussed. Concerns are highlighted regarding the difficulty in comparing some data and the lack of a clear surrogate marker such as BCR/ABL transcript levels as used in cell-mediated lympholysis. Expert opinion: Overall, for the field of MF and MPNs as a whole, this is a time of unprecedented interest, activity, and a very real opportunity to make significant inroads into this difficult disorder. The approval of ruxolitinib demonstrates already the great potential for benefit in this field. However, economic constraint also impinges upon its reimbursement and collection of proper data with regard to both disease burden and health benefit is necessary to ensure patient access after clinical efficacy and safety approval.
Myeloproliferative disorders are clonal hematopoietic diseases that are characterized by the amplification of one or more myeloid lineages. Polycythemia vera, essential thrombocythemia, idiopathic myelofibrosis and chronic myeloid leukemia are considered classic myeloproliferative disorders and share common clinical and biological features. While the genetic basis of chronic myeloid leukemia is shown to be the constitutive active protein BCR-ABL, the main molecular lesions in polycythemia vera, essential thrombocythemia and idiopathic myelofibrosis remain unknown. This review focuses on the recent discovery of the JAK2 V617F mutation, its relationship to the myeloproliferative phenotype and implications in the clinical approach of patients.
In the last decade, genomic studies have identified multiple recurrent somatic mutations in myeloproliferative neoplasms (MPNs). Beginning with the discovery of the JAK2 V617F mutation, multiple additional mutations have been found that constitutively activate cell-signaling pathways, including MPL, CBL, and LNK. Furthermore, several classes of epigenetic modifiers have also been identified, in patients with MPN, revealing a requirement for mutations in other pathways to cooperate with JAK-STAT pathway mutations in MPN pathogenesis. Mutations in the de novo DNA methylation protein, DNMT3A, demethylation machinery, TET2 and related IDH1/2 production of oncometabolite 2-hydroxygluterate, and polycomb complex proteins EZH2 and ASXL1 have opened new pathophysiologic clues into these diseases. The prognostic relevance of these novel disease alleles remains an important area of investigation, and clinical trials are currently underway to determine if these findings represent tractable therapeutic targets, either alone, or in combination with JAK2 inhibition.
JAK2-V617F is central to the pathogenesis of myeloproliferative neoplasms. We examined whether lestaurtinib decreased JAK2-V617F allele burden and evaluated its clinical benefits and tolerability in patients with polycythaemia vera (PV) and essential thrombocythaemia (ET). This phase 2, open-label, multicentre study was designed to detect ≥15% reduction in JAK2-V617F allele burden in 15% of patients. Eligible patients received lestaurtinib 80 mg twice daily for 18 weeks and could participate in a 1-year extension phase of treatment. Of 39 enrolled patients, 27 (69%) had PV; 12 (31%) had ET. While the pre-specified responder rate of 15% was not met, lestaurtinib modestly reduced JAK2-V617F allele burden and reduced spleen size in a subset of patients. Of 37 patients in the full efficacy analysis, 5 (14%) responded clinically. Every patient had ≥1 adverse event, most commonly gastrointestinal (95%). Fifteen patients (38%) experienced serious adverse events; 23 (59%) withdrew due to adverse events. This is the first reported study of JAK2-inhibitor treatment in patients with PV/ET and highlights both the need for further studies to assess the role of JAK2 inhibition in treatment of PV/ET and the use of JAK2-V617F as a biomarker for response. This trial was registered at www.clinicaltrials.gov as NCT00586651.
The chronic myeloproliferative neoplasms (MPNs), are characterized by a Janus Kinase (JAK)-2 V617F point mutation but this molecular abnormality does not explain by itself the pathogenesis of these disorders, or the phenotypic diversity associated with essential thrombocythemia, polycythemia vera (PV), and myelofibrosis. Beyond the JAK/signal transducer and activator of transcription network, a wide number of molecular alterations were described in MPN including the fosfatidilinositolo-3-chinasi (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway constitutive activation. Several pathway inhibitors were developed, including everolimus, up to the latest class of catalytic inhibitors such as BKM120 and BEZ235. In this review, we present some clinical and experimental evidence showing that the PI3K/Akt/mTOR pathway could represent a therapeutic target in MPNs. In in vitro studies, everolimus has been shown to inhibit cell proliferation and clonogenic potential in human and murine JAK2 V617F mutated cell lines. Patients with PV and primary myelofibrosis hematopoietic progenitors were significantly more sensitive to everolimus compared with healthy control subjects. Of much interest, a combination of everolimus and the JAK1/2 inhibitor, ruxolitinib, showed strong synergism in inducing cell cycle arrest and blockade of cell proliferation. Similar data were obtained using a dual PI3K/mTOR inhibitor, BEZ235, with activity that was also shown in preclinical murine models. A multicenter phase I/II trial with everolimus in myelofibrosis documented a well tolerated clinical efficiency in terms of spleen size reduction and resolution of systemic symptoms and pruritus. These observations indicate that the PI3K/Akt/mTOR pathway might represent a novel target for treatment in MPN. The synergism demonstrated in vitro with JAK2 inhibitors could open additional therapeutic possibilities based on concurrent targeting of different pathways that might optimize efficacy and reduce toxicity in patients.
Bone marrow cells from two glucose-6-phosphate dehydrogenase (G-6-PD) heterozygotes with polycythemia vera were cultured to determine whether progenitors which wre not of the polycythemia vera clone were present, and, if present, which cell lines contributed to the increase in erythroid colonies observed in response to added erythropoietin (ESF). To accomplish this, the G-6-PD isoenzyme activity of individual erythroid colonies was determined. All of the erythroid colonies analyzed in cultures without added ESF, contained the G-6-PD isoenzyme type characteristic of the abnormal clone. With higher ESF concentrations in the culture, however, there was an increase in the colonies that were not of the polycythemia vera clone. Analysis of the ratio of the various types of colonies indicated that normal and polycythemia vera cells are capable of responding to ESF in vitro. In selected patients, this technique permits analysis of the ratios of normal to abnormal cells during the course of the disease, in response to therapy and during late complications, such as myelofibrosis or leukemic transformation.
Three families with polycythemia inherited through apparently different modes are described. Secondary causes of polycythemia were ruled out. Erythropoietin (EPO) levels were normal or low, even after phlebotomy. In vitro erythroid colony growth in standard assay cultures containing EPO was normal; however, in the absence of added EPO, a few progenitors from most of the affected individuals were able to generate recognizable colonies of mature erythroblasts, although these were smaller and proportionately less numerous than seen in polycythemia vera (PV). To search for EPO-receptor changes as a possible pathophysiologic mechanism, we examined, by Southern blot analysis, genomic DNA samples from affected and nonaffected family members, as well as three patients with PV. Two different probes, derived from the human EPO-receptor, were used. We found no evidence for chromosomal rearrangements or gene amplification in hereditary polycythemia or PV patients. Further, no nucleotide sequences were found that were homologous to the Friend spleen focus-forming virus glycoprotein gp55, which has been shown to bind to and activate the murine EPO-receptor. Functional studies examining number and binding affinity of the EPO-receptor on erythroid progenitors from three hereditary polycythemia patients demonstrated no abnormalities. We conclude that the mechanism(s) for the erythrocytosis in familial and congenital polycythemia and in PV may not involve the EPO-receptor and, therefore, may result from alterations of postreceptor responses.
In order to demonstrate whether 'spontaneous' erythroid colonies observed in vitro in polycythemia vera (PV) using standard colony assays were independent from erythropoietin (epo) or exquisitely sensitive to the hormone, we used methyl cellulose serum-free cultures, in which serum was completely replaced by iron-saturated transferrin, α-thioglycerol albumin, and low density lipoproteins. Connaught Step III epo was used. In 6 cases of PV, no epo-independent colony (CFU-E or BFU-E derived) was observed in serum-free conditions, while spontaneous colonies were present after plating the same PV bone marrow in culture with serum. The epo dose-response curves showed a tenfold increase in the sensitivity to epo of PV erythroid progenitors compared to normal controls. In PV, the first CFU-E and BFU-E colonies were observed after addition of 0.001-0.01 IU/ml of epo, while in controls they appeared at an epo concentration between 0.01 and 0.1 IU/ml. Numbers of spontaneous colonies in cultures with serum compared with the epo dose-response curve in the same patient in serum-free cultures are much higher than expected from the small amount of epo present in the serum. These results confirm that PV erythroid progenitors able to differentiate spontaneously in standard culture conditions are in fact dependent of epo and hypersensitive to the hormone. They show that these abnormal progenitors do not represent a homogeneous population, but exhibit different degrees in their hypersensitivity to epo. Since the small amount of epo present in the normal serum cannot explain the growth of spontaneous colonies by itself, a hypersensitivity to other serum factors cannot be excluded.
Erythroid progenitor cells isolated from patients with polycythemia vera (PV) proliferate and differentiate in methylcellulose in the absence of exogenous erythropoietin (EPO). To investigate the potential role of the erythropoietin receptor (EPO-R) in the pathogenesis of PV, we cultured bone marrow-derived or peripheral blood-derived erythroid progenitors in the presence of neutralizing monoclonal antibodies (MoAbs) specific for EPO or EPO-R. Mononuclear cells were obtained from 9 healthy adults and 9 PV patients by Ficoll-Hypaque gradients and cultured with or without EPO in methylcellulose for 12 days under standard or serum-free conditions. Neutralizing anti-EPO and anti-EPO-R MoAbs, added to cultures at day 0, caused dose-dependent growth inhibition of all normal burst-forming units-erythroid (BFU-E) derived from health adult controls. The MoAbs had no effect on the growth of nonerythroid progenitor cells under the same culture conditions. In contrast, neutralizing antibodies distinguished two classes of BFU-E derived from PV patients. Class I BFU-E from PV patients were EPO-dependent. These progenitors, like those derived from healthy adults, had normal EPO dose-dependent growth characteristics and showed a normal period of EPO requirement in vitro that extended 6 days after the initiation of culture. These results indicate that EPO exerts its critical effect early during erythroid differentiation; the addition of neutralizing antibodies to normal progenitors after 6 days had no effect on the subsequent size or maturation of the colonies. Class II BFU-E from PV patients were EPO-independent. They proliferated and differentiated even in the presence of high concentrations of neutralizing anti-EPO or anti-EPO-R MoAbs. We conclude that the class II BFU-E from PV patients are independent of free EPO.
Erythropoietin (EPO) regulates the proliferation and differentiation of erythroid cells through interaction with its receptor (EPOR). Although EPOR is a member of the cytokine receptor superfamily and lacks a kinase domain, EPO induces tyrosine phosphorylation, which is correlated with gene transcription and mitogenesis. Here we demonstrate that EPO induces tyrosine phosphorylation of JAK2 kinase and activates its in vitro autophosphorylation. Using EPOR mutants, phosphorylation and activation of kinase activity correlate with the induction of mitogenesis. Furthermore, JAK2 physically associates with a membrane-proximal region of the EPOR cytoplasmic domain that is required for biological activity. The results support the hypothesis that JAK2 is the kinase that couples EPO binding to tyrosine phosphorylation and mitogenesis.
Human erythroid malignancies (polycythemia vera [PV] and erythroleukemia) are associated with erythropoietin (Epo)-independent growth and differentiation. Missense or nonsense mutations in the Epo receptor (Epo-R) have been recently described in experimental erythroleukemia in mice and in cases of erythrocytosis in humans. To search for a similar genetic alteration in erythroleukemia and PV, we entirely sequenced the exons of the Epo-R gene as well as the intron-exon junctions in these disorders using polymerase chain reaction. In 1 of 10 cases of erythroleukemia, a single allele mutation was found in the 8th Epo-R gene exon that changed asparagine 487 into a serine. No Epo-r gene mutation was found in 12 PV cases studied, but the same mutation (N487S) was found in 1 patient with polycythemia that did not fulfill the criteria of PV (polycythemia of unknown origin). We did not detect this mutation after sequencing part of the 8th exon of the Epo-R gene from 21 other patients with polycythemia of unknown origin and 51 normal controls. The Epo-R mutation was also found in Epstein-Barr virus-derived cell lines from both cases, suggesting that it is not related to the malignant clone. Therefore, this mutation does not appear to be somatic, although no familial cases were found. The biologic effect of this mutation was subsequently studied. Erythroid progenitors from the polycythemic patient normally responded to Epo, whereas those from the erythroleukemic patient were Epo-independent due to autocrine stimulation by Epo. The normal and the mutated Epo-R were transfected into the murine Ba/F3 cell line. Both types of cells displayed the same response to Epo for proliferation, differentiation, and inhibition of apoptosis. Although this mutation may destroy a consensus binding site for Grb2, no obvious differences either in the pattern of Epo-induced tyrosine phosphorylated proteins or in the binding of Grb2 to the Epo-R were observed. In conclusion, a somatic Epo-R missense mutation does not appear to be a molecular mechanism involved in the abnormal growth of human erythroleukemia and PV. However, the Epo-R mutation (N487S) that we describe is located in the same tyrosine sequence beginning at AA 485 as the one previously observed (P488S) in as case of polycythemia (Sokol et al, Exp Hematol 22:447, 1994). These results suggest that this phosphopeptide sequence may play an important role in Epo signalling.
Deregulating the expression of Bcl-xL, an inhibitor of apoptosis, in an erythropoietin-dependent erythroblast cell line averts apoptosis induced by the withdrawal of erythropoietin. Since in polycythemia vera an abnormal clone of erythroid progenitors is independent of erythropoietin, we investigated whether the endogenous expression of Bcl-xL was deregulated in these cells.
Erythroid colonies from patients with polycythemia vera and normal subjects were cultured in the presence and absence of erythropoietin and assessed by immunocytochemical and flow-cytometric analysis with anti-Bcl-x antibodies that recognize the two species of Bcl-x (Bcl-xL and Bcl-xS). Reverse-transcriptase-polymerase-chain-reaction analysis was used to determine which one of the two species was responsible for anti-Bcl-x staining. Bone marrow mononuclear cells from 8 healthy bone marrow donors, 14 patients with polycythemia vera, 19 patients with other myeloproliferative syndromes, and 12 patients with secondary erythrocytosis were analyzed by flow cytometry with antibodies against Bcl-x and glycophorin A, an erythroid marker.
Erythroid cells from patients with polycythemia vera survived in vitro without erythropoietin, and this finding correlated with the expression of Bcl-x protein (Bcl-xL messenger RNA was the main species of Bcl-x found), even in mature erythroblasts that normally do not express Bcl-x. The mean (+/-SD) percentage of cells positive for both glycophorin A and Bcl-x in the 14 patients with polycythemia vera (21.8+/-3.6 percent) was significantly higher than that in 8 normal donors (6.62+/-1.58 percent), 12 patients with secondary erythrocytosis (6.87+/-1.95 percent), 9 patients with essential thrombocythemia (3.81+/-0.97 percent), and 10 patients with chronic myeloid leukemia (2.7+/-0.41 percent).
Deregulated expression of Bcl-x may contribute to the erythropoietin-independent survival of erythroid-lineage cells in polycythemia vera and thereby contribute to the pathogenesis of this disease.
To evaluate the functional conservation of signal transduction mechanisms between haematopoietic receptors and to characterize the molecules activated in this phenomenon, we introduced granulocyte colony-stimulating factor receptor (G-CSFR) cDNA into mouse fetal liver cells using a retroviral vector. In semi-solid medium assays, G-CSFR-infected cells gave rise to all types of colonies [granulocyte-macrophage (GM), megakaryocyte (MK) and mixed lineage (GEMM) colony-forming units (CFU) and erythroid burst-forming units (BFU-E)] in the presence of G-CSF alone. The direct effect of G-CSF on erythroid differentiation of G-CSFR-transduced erythroid progenitors was demonstrated by the development of erythroid colonies using G-CSFR-expressing Lin- cells cloned at one cell per well in liquid culture in the presence of G-CSF. Interestingly, while Stat5, but not Stat3, was activated in erythroid cells in response to erythropoietin (EPO), both were activated in erythroid and granulocytic cells stimulated by G-CSF. Furthermore, G-CSF induced the growth of erythroid colonies from G-CSFR-expressing fetal liver cells from EPO receptor-/- (EPO-R-/-) or Stat5a-/- Stat5b-/- mice, demonstrating that erythroid differentiation can occur in the absence of EPO-R or Stat5. These data show that forced expression of G-CSFR allows G-CSF-dependent multilineage proliferation and differentiation of haematopoietic progenitors and rescues EPO-R-/- erythroid cells. While G-CSF induces Stat5 activation in G-CSFR-expressing erythroid cells, this activation is not necessary for the terminal erythroid differentiation induced by G-CSF.
The regulation of tyrosine phosphorylation and associated signalling through antigen, growth-factor and cytokine receptors is mediated by the reciprocal activities of protein tyrosine kinases and protein tyrosine phosphatases (PTPases). The transmembrane PTPase CD45 is a key regulator of antigen receptor signalling in T and B cells. Src-family kinases have been identified as primary molecular targets for CD45 (ref. 4). However, CD45 is highly expressed in all haematopoietic lineages at all stages of development, indicating that CD45 could regulate other cell types and might act on additional substrates. Here we show that CD45 suppresses JAK (Janus kinase) kinases and negatively regulates cytokine receptor signalling. Targeted disruption of the cd45 gene leads to enhanced cytokine and interferon-receptor-mediated activation of JAKs and STAT (signal transducer and activators of transcription) proteins. In vitro, CD45 directly dephosphorylates and binds to JAKs. Functionally, CD45 negatively regulates interleukin-3-mediated cellular proliferation, erythropoietin-dependent haematopoieisis and antiviral responses in vitro and in vivo. Our data identify an unexpected and novel function for CD45 as a haematopoietic JAK phosphatase that negatively regulates cytokine receptor signalling.
The cysteine proteases known as caspases play a central role in most apoptotic pathways. Here, we show that caspase inhibitors arrest the maturation of human erythroid progenitors at early stages of differentiation, before nucleus and chromatin condensation. Effector caspases such as caspase-3 are transiently activated through the mitochondrial pathway during erythroblast differentiation and cleave proteins involved in nucleus integrity (lamin B) and chromatin condensation (acinus)without inducing cell death and cleavage of GATA-1. These observations indicate a new function for caspases as key proteases in the process of erythroid differentiation.
Erythropoiesis results from the proliferation and differentiation of pluripotent stem cells into immature erythroid progenitors (ie, erythroid burst-forming units (BFU-Es), whose growth, survival, and terminal differentiation depends on erythropoietin (Epo). Ineffective erythropoiesis is a common feature of myelodysplastic syndromes (MDS). We used a 2-step liquid-culture procedure to study erythropoiesis in MDS. CD34(+) cells from the marrow of patients with MDS were cultured for 10 days in serum-containing medium with Epo, stem cell factor, insulin-like growth factor 1, and steroid hormones until they reached the proerythroblast stage. The cells were then placed in medium containing Epo and insulin for terminal erythroid differentiation. Numbers of both MDS and normal control cells increased 10(3) fold by day 15. However, in semisolid culture, cells from patients with refractory anemia (RA) with ringed sideroblasts and RA or RA with excess of blasts produced significantly fewer BFU-Es than cells from controls. Fluorescence in situ hybridization analysis of interphase nuclei from patients with chromosomal defects indicated that abnormal clones were expanded in vitro. Epo-signaling pathways (STAT5, Akt, and ERK 1/2) were normally activated in MDS erythroid progenitors. In contrast, apoptosis was significantly increased in MDS cells once they differentiated, whereas it remained low in normal cells. Fas was overexpressed on freshly isolated MDS CD34(+) cells and on MDS erythroid cells throughout the culture. Apoptosis coincided with overproduction of Fas ligand during the differentiation stage and was inhibited by Fas-Fc chimeric protein. Thus, MDS CD34(+)-derived erythroid progenitors proliferated normally in our 2-step liquid culture with Epo but underwent abnormal Fas-dependent apoptosis during differentiation that could be responsible for the impaired erythropoiesis.
In this study, we examined the in vitro and in vivo effects of forced expression of Mpl-R (the thrombopoietin receptor) on the progeny of murine hematopoietic stem cells. Bone marrow cells from 5-FU–treated mice were transduced with retroviral vectors containing the human Mpl-R cDNA, or the neomycine gene as a control. After 7 days cocultivation on virus-producer cells, GpE86-Mpl-R or Gp86-Neo, the types of hematopoietic progenitor cells responding to thrombopoietin (TPO) were studied by clonogenic assays. Mpl-R–infected cells gave rise to CFU-GEMM, BFU-E, CFU-MK, but not CFU-GM while Neo-infected cells produced only megakaryocytic colonies. In addition, when nonadherent cells from GpE86-Mpl-R cocultures were grown with TPO as the only stimulus for 7 days, a marked expansion of CFU-GEMM, BFU-E, and CFU-MK was observed, while no change in CFU-GM number was seen. Erythroid and megakaryocytic maturation occurred in the presence of TPO while a block in granulocytic differentiation was observed at the myeloblast stage. The direct effects of TPO on Mpl-R–transduced progenitor cells were demonstrated by single cell cloning experiments. To analyze the effects of the constitutive expression of Mpl-R on the determination of multipotent progenitors (CFU-S) and long-term repopulating stem cells, Mpl-R– or Neo-infected cells were injected into lethally irradiated recipient mice. No difference was seen in (1) the number of committed progenitor cells contained in individual CFU-S12 whether colonies arose from noninfected or Mpl-R–infected CFU-S; (2) the mean numbers of progenitor cells per leg or spleen of mice reconstituted with Mpl-R– or Neo-infected cells, 1 or 7 months after the graft; and (3) the blood parameters of the two groups of animals, with the exception of a 50% reduction in circulating platelet counts after 7 months in mice repopulated with Mpl-R–infected bone marrow cells. These results indicate that retrovirus-mediated expression of Mpl-R in murine stem cells does not modify their ability to reconstitute all myeloid lineages of differentiation and does not result in a preferential commitment toward the megakaryocytic lineage.
Essential thrombocythemia (ET) and idiopathic myelofibrosis (PMF) are two myeloproliferative diseases characterized by a marked megakaryocytic (MK) involvement. The pathogenesis of these two diseases is unknown. Recently it has been shown that overexpression of Mpl-ligand (Mpl-L) in mice induces thrombocytosis and myelofibrosis. In this study, we investigated whether Mpl-L was responsible for the pathogenesis of ET and PMF. Using in vitro cultures of blood or marrow CD34+ cells, we investigated whether MK growth was abnormal in these two diseases. Spontaneous MK growth involving only a fraction (20%) of the MK progenitors, as compared with growth in the presence of pegylated recombinant human megakaryocyte growth and development factor (PEG-rhuMGDF), was found in both diseases (21ET and 14PMF) using serum-free semisolid and liquid cultures, including cultures at one cell per well. We first searched for ac-mpl mutation/deletion by sequencing the entire coding region of the gene by polymerase chain reaction (PCR) in nine ET patients and five PMF patients, but no mutation was found. We subsequently investigated whether an autocrine stimulation by Mpl-L could explain the autonomous MK growth. Addition of different preparations of soluble Mpl receptor (sMpl) containing a Fc domain of IgG1 (sMpl-Fc) markedly inhibited MK spontaneous growth in both ET and PMF patients. This effect was specific for sMpl because a control soluble receptor (s4-1BB-Fc) had no inhibitory effect and an sMpl devoid of the Fc fragment had the same inhibitory efficacy as the sMpl-Fc. This inhibition was reversed by addition of PEG-rhuMGDF or a combination of cytokines. The sMpl-Fc markedly altered the entry into cell cycle of the CD34+ cells and increased the apoptosis that occurs in most patient CD34+ cells in the absence of exogenous cytokine, suggesting an autocrine stimulation. In contrast, a neutralizing antibody against Mpl-L did not alter the spontaneous MK growth, whereas it totally abolished the effects of 10 ng/mL PEG-rhuMGDF on patient or normal CD34+ cells. Mpl-L transcripts were detected at a very low level in the patient CD34+cells and MK and only when a highly sensitive fluorescent PCR technique was used. By quantitative reverse-transcription (RT)-PCR, the number of Mpl-L transcripts per actin transcripts was lower than detected in human Mpl-L–dependent cell lines, suggesting that this synthesis of Mpl-L was not biologically significant. In favor of this hypothesis, the Mpl-L protein was not detected in culture supernatants using either an enzyme-linked immunosorbent assay (ELISA) or a biological (Ba/F3huc-mpl) assay, except in one PMF patient. Investigation of Mpl-L signaling showed an absence of constitutive activation of STATs in spontaneously growing patient MKs. Addition of PEG-rhuMGDF to these MKs activated STATs 3 and 5. This result further suggests that spontaneous growth is neither related to a stimulation by Mpl-L nor to ac-mpl mutation. In conclusion, our results show that Mpl-L or Mpl are not directly implicated in the abnormal proliferation of MK cells from ET and PMF. The mechanisms by which the sMpl mediates a growth inhibition will require further experiments.
Erythroid progenitor cells isolated from patients with polycythemia vera (PV) proliferate and differentiate in methylcellulose in the absence of exogenous erythropoietin (EPO). To investigate the potential role of the erythropoietin receptor (EPO-R) in the pathogenesis of PV, we cultured bone marrow-derived or peripheral blood-derived erythroid progenitors in the presence of neutralizing monoclonal antibodies (MoAbs) specific for EPO or EPO-R. Mononuclear cells were obtained from 9 healthy adults and 9 PV patients by Ficoll-Hypaque gradients and cultured with or without EPO in methylcellulose for 12 days under standard or serum-free conditions. Neutralizing anti-EPO and anti-EPO-R MoAbs, added to cultures at day 0, caused dose-dependent growth inhibition of all normal burst-forming units-erythroid (BFU-E) derived from health adult controls. The MoAbs had no effect on the growth of nonerythroid progenitor cells under the same culture conditions. In contrast, neutralizing antibodies distinguished two classes of BFU-E derived from PV patients. Class I BFU-E from PV patients were EPO- dependent. These progenitors, like those derived from healthy adults, had normal EPO dose-dependent growth characteristics and showed a normal period of EPO requirement in vitro that extended 6 days after the initiation of culture. These results indicate that EPO exerts its critical effect early during erythroid differentiation; the addition of neutralizing antibodies to normal progenitors after 6 days had no effect on the subsequent size or maturation of the colonies. Class II BFU-E from PV patients were EPO-independent. They proliferated and differentiated even in the presence of high concentrations of neutralizing anti-EPO or anti-EPO-R MoAbs. We conclude that the class II BFU-E from PV patients are independent of free EPO.
The mechanisms of the chronic myeloid leukemia (CML) clones proliferative advantage over normal clones are currently unknown. They may involve an insensitivity to a negative regulation of a growth factor-independent proliferation. Clonogenic progenitors from CML patient blood or marrow in chronic phase were grown either in the presence or absence of recombinant growth factors. No erythroid colonies were observed in the absence of any cytokine. In contrast, erythroid colonies composed of fully mature hemoglobinized erythroblasts (day 12 burst-forming units-erythroid) were obtained in the presence of Steel factor (SF) alone. Addition of erythropoietin (Epo) to SF either had no effect on the cloning efficiency or increased up to 50% the number of erythroid colonies. No erythroid growth was observed when cultures were stimulated by interleukin-3 or granulocyte- macrophage colony-stimulating factor alone. Similar erythroid growth in the presence of SF but without Epo was obtained in “serum-free” cultures when purified blood CML CD34+ cells were grown. This growth of erythroid colonies in the absence of Epo was not accounted for by an autocrine stimulation loop by Epo, because neutralizing antibodies against Epo did not inhibit it. This abnormal response to growth factor was specifically observed in the CML clone, as shown by the presence of the BCR-ABL transcript in all of these erythroid colonies. The direct implication of BCR-ABL was further documented (1) by studies of alpha- interferon-treated patients with a chimerism in which the abnormal growth correlates with the presence of the malignant clone and (2) by the use of antisense oligonucleotide against BCR-ABL transcript, which abrogated this abnormal growth. Finally, erythroid growth in the SF presence was greatly diminished by herbimycin A, whereas, at the same concentration, this tyrosine kinase inhibitor had no marked effect on erythroid colony formation in the presence of SF plus Epo on CML or normal marrow cells. This result suggests that the BCR-ABL kinase activity leads directly to this Epo-independent terminal differentiation requiring, however, the presence of SF.
In polycythemia vera (PV) erythroid colonies that grow in vitro in the absence of exogenous erythropoietin (EPO) arise from the abnormal clone that is responsible for overproduction of red blood cells. Although the mechanism of autonomous formation of burst-forming units-erythroid (BFU-E) is not fully understood, a spontaneous release of growth regulatory molecules by PV cells and/or by accessory cells is likely to be involved. Because of its cytokine synthesis inhibiting action, interleukin-10 (IL-10) could be a potentially useful molecule to modulate abnormal erythropoiesis in PV. We studied the effect of recombinant human IL-10 on the EPO-independent growth of erythroid bursts derived from peripheral blood mononuclear cells (PBMNCs) of patients with PV. IL-10 showed a profound, dose-dependent, and specific inhibitory effect on autonomous BFU-E formation. Ten nanograms per milliliter of IL-10 significantly suppressed spontaneous growth of erythroid colonies in methylcellulose in five of five PV patients tested with a mean inhibition by 81% (range, 72-94). To elucidate the possible mechanism of the inhibitory action of IL-10 we further studied the effect of anticytokine antibodies on autonomous BFU-E growth and the ability of exogenous cytokines to restore IL-10–induced suppression of erythroid colony growth. Among a panel of growth regulatory factors tested (granulocyte-macrophage colony-stimulating factor [GM-CSF], IL-3, granulocyte colony-stimulating factor, stem cell factor, and insulin-like growth factor-1) GM-CSF was the only molecule for which both an inhibition of spontaneous BFU-E formation by its respective antibody as well as a significant restimulation of erythroid colonies in IL-10-treated cultures by exogenous addition was found. Moreover, inhibition of GM-CSF production by IL-10 was shown in PV PBMNCs at the mRNA level. Our data indicate that autonomous BFU-E growth in PV can be profoundly inhibited by IL-10 and that this inhibitory effect seems to be at least in part secondary to suppression of endogenous GM-CSF production.
© 1998 by The American Society of Hematology.
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.
Three families with polycythemia inherited through apparently different modes are described. Secondary causes of polycythemia were ruled out. Erythropoietin (EPO) levels were normal or low, even after phlebotomy. In vitro erythroid colony growth in standard assay cultures containing EPO was normal; however, in the absence of added EPO, a few progenitors from most of the affected individuals were able to generate recognizable colonies of mature erythroblasts, although these were smaller and proportionately less numerous than seen in polycythemia vera (PV). To search for EPO-receptor changes as a possible pathophysiologic mechanism, we examined, by Southern blot analysis, genomic DNA samples from affected and nonaffected family members, as well as three patients with PV. Two different probes, derived from the human EPO-receptor, were used. We found no evidence for chromosomal rearrangements or gene amplification in hereditary polycythemia or PV patients. Further, no nucleotide sequences were found that were homologous to the Friend spleen focus-forming virus glycoprotein gp55, which has been shown to bind to and activate the murine EPO-receptor. Functional studies examining number and binding affinity of the EPO- receptor on erythroid progenitors from three hereditary polycythemia patients demonstrated no abnormalities. We conclude that the mechanism(s) for the erythrocytosis in familial and congenital polycythemia and in PV may not involve the EPO-receptor and, therefore, may result from alterations of postreceptor responses.
Three classes of normal human erythropoietic progenitor cells were investigated with respect to their erythropoietin (Ep) sensitivity under various culture conditions. Dose-response curves extending from 0.001 to 10 units Ep/ml indicated that under optimal conditions the formation of very large erythroid colonies or 'bursts' (from primitive BFU-E) required about three times the Ep concentration needed for small bursts (from mature BGU-E) and about five times that needed for the production of small erythroid clusters (from CFU-E). These findings suggest that by the primitive BFU-E stage erythropoietic cells in man have the capacity to respond to EP and that this responsiveness then progressively increases. Removal of the the adherent marrow cell fraction and/or omission of leukocyte-conditioned medium from the cultures resulted in failure to achieve maximum burst formation by primitive BFU-E even in the presence of 10 units Ep/ml. Elimination of these sources of burst-enhancing factors had less effect on the Ep dose-response curve for mature BFU-E, and the effect on CFU-E detection was minimal. These data show that red cell precursors at sequential stages of differentiation also show progressive changes in their response to factors other than Ep that affect burst formation in vitro. Ep dose-response data were also obtained for primitive and mature BFU-E and CFU-E present in specimens from five patients with polycythemia vera (PV). Simple analysis of the curves obtained indicated two coexisting population in this disease: (1) a population of abnormally (hyper) Ep-responsive cells (sensitive to <0.001 units/ml) and (2) a population of cells whose Ep sensitivity in vitro fell within the appropriate normal range. Quantitation of the ratio of phenotypically normal and abnormal progenitors defined in this way showed a progressive increase in favor of the abnormal cells in the later, mature BFU-E and CFU-E compartments. This could reflect a growth advantage in PV of the abnormally Ep-sensitive line proliferating in a low Ep environment. Such an interpretation would also be consistent with the concept that Ep can influence the production of early red cell precursors in man.
Polycythemia vera (PV) is a clonal disease of the hematopoietic stem cell characterized by a hyperplasia of marrow erythropoiesis, granulocytopoiesis, and megakaryocytopoiesis. We previously reported that highly purified PV blood burst-forming units-erythroid (BFU-E) are hypersensitive to recombinant human interleukin-3 (rIL-3). Because these cells may be only a subset, and not representative of marrow progenitors, we have now studied partially purified marrow hematopoietic progenitor cells. Dose-response experiments with PV marrow BFU-E showed a 38-fold increase in sensitivity to rIL-3 and a 4.3-fold increase in sensitivity to recombinant human erythropoietin (rEpo) compared with normal marrow BFU-E. In addition, PV marrow colony-forming units-granulocyte-macrophage (CFU-GM) and CFU-megakaryocyte (CFU-MK) also showed a marked hypersensitivity to rIL-3 and to human recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF). Dose-response curves with rGM-CSF and blood BFU-E showed a 48-fold increase in sensitivity. No effect of rIL-4, rIL-6, human recombinant granulocyte-CSF (rG-CSF), or macrophage-CSF (rM-CSF) was evident, nor was there any effect of PV cell-conditioned medium on normal BFU-E, when compared with normal cell-conditioned medium. Autoradiography with 125I-rEpo showed an increase in Epo receptors after maturation of PV BFU-E to CFU-E similar to that shown with normal BFU-E, but no increase of specific binding of 125I-rIL-3 by PV CD34+ cells was seen compared with normal CD34+ cells. These studies show that PV marrow hematopoietic progenitor cells are hypersensitive to rIL-3 and rGM-CSF, similar to PV blood BFU-E. While the mechanism does not appear to be due to enhanced binding of rIL-3, the hypersensitivity of PV progenitor cells to IL-3 and GM-CSF may be a key factor in the pathogenesis of PV.
Because polycythemia vera (PV) is a clonal hematopoietic stem cell disease with a trilineage hyperplasia, and interleukin-3 (IL-3) stimulates trilineage hematopoiesis, we have studied the response of highly purified PV blood burst-forming units-erythroid (BFU-E) to recombinant human IL-3 (rIL-3). Whereas the growth of normal blood BFU-E in vitro rapidly declined by 40 and 60% after 24 and 48 h of incubation without 50 U/ml of rIL-3, the growth of PV BFU-E declined by only 10 and 30% under the same conditions, demonstrating a reduced dependence on rIL-3. A reduced dependence of PV BFU-E on recombinant human erythropoietin (rEP) was also present. Dose-response experiments showed a 117-fold increase in PV BFU-E sensitivity to rIL-3, and a 6.5-fold increase in sensitivity to rEP, compared to normal BFU-E, whereas blood BFU-E from patients with secondary polycythemia responded like normal BFU-E. Endogenous erythroid colony (EEC) formation, which is independent of the addition of rEP, was reduced by 50% after erythroid colony-forming cells were generated from PV BFU-E in vitro without rIL-3 for 3 d, whereas rEP-stimulated erythroid colonies were unaffected. These studies demonstrate a striking hypersensitivity of PV blood BFU-E to rIL-3, which may be the major factor in the pathogenesis of increased erythropoiesis without increased EP concentrations.
In vitro cultures of erythroid progenitors and radioimmunoassay of erythropoietin (Epo) are 2 recently available techniques. It is possible to assess their relevance in various hematological disorders. Erythroid cultures can be performed in the investigation of polycythemias, pure red cell aplasias (PRCA) and refractory anemias. In primary polycythemias "spontaneous" colonies appear in vitro whereas this phenomenon is never observed in secondary polycythemias. These so called "spontaneous" colonies have been demonstrated with a lower incidence in all myeloproliferative disorders. Therefore, if the absence of spontaneous colonies does not permit us to eliminate the presence of a myeloproliferative syndrome aside from polycythemia vera, their presence does seem pathognomonic of a myeloproliferative disorder. In acquired chronic pure red cell aplasia in adults, a strong correlation is found between the in vitro growth of erythroid colonies and the results of immuno-suppressive treatment. In refractory anemias erythroid cultures do not have either diagnostic, or prognostic interest. Serum epo level does not have a high discriminatory value in distinguishing between primary and secondary erythrocytosis. Indeed in PV, the Epo level is generally low or normal, in secondary polycythemias Epo level is high or normal. There is an important overlap between the two groups. Epo level determination can have a therapeutic incidence. Administration of recombinant Epo seems justified only in patients both sufficiently anemic to warrant transfusions and in whom Epo level is low in comparison with the degree of anemia.
The role of erythropoietin (EP) in polycythemia vera (PV) is controversial, with some experiments suggesting that erythroid progenitors in PV are exquisitely sensitive to EP and EP dependent, and others suggesting that PV progenitors are EP independent. We have examined the characteristics of the EP receptor (EP-R) on erythroid colony-forming cells (ECFC) from patients with PV. In contrast to normal ECFC, which have two classes of EP-R, with 20% showing high affinity (Kd = 0.13 nM; range, 0.04-0.20 nM) and the remainder lower affinity (Kd = 0.37 nM; range, 0.28-0.57 nM), PV ECFC show a single class of 851 low affinity EP-R with Kd = 0.72 nM (range, 0.36-0.85 nM). ECFC from patients with secondary (EP driven) polycythemia or anemia show two classes of EP-R (Kd = 0.18 and 1.10 nM, respectively). Attempts to remove tightly bound EP from putative high affinity EP-R in PV did not reveal any higher affinity receptors. Determination of molecular size by crosslinking showed two proteins of 90 and 100 kD similar to those seen with normal EP-R. These studies indicate the PV ECFC have EP-R that are structurally similar to normal EP-R but lack the higher binding affinity for EP.
To further define the growth factors required for the in vitro proliferation of erythroid progenitors in polycythemia vera (PV), we have compared the ability of interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) to support the growth of erythropoietin (Epo)-dependent and -independent erythroid colony formation. By using nonadherent mononuclear cells from peripheral blood, Epo-dependent colony formation was enhanced by IL-3 and GM-CSF in PV patients. Comparable results were obtained with normal erythroid progenitors. Augmenting effects of IL-3 and GM-CSF were observed on spontaneous erythroid colony formation, i.e., erythroid colony formation in the absence of exogenous supplied Epo. This was not due to a small amount of Epo in the culture media because an anti-Epo antibody did not prevent endogenous colony formation, nor did it prevent the enhancing effects of IL-3. Finally it was observed that in contrast to IL-3, monocyte depletion was required for the enhancing effects of GM-CSF on erythroid colony formation. These results provide evidence that endogenous colony formation in PV is independent of Epo but can be augmented by IL-3 or GM-CSF.
S ummary
Peripheral blood mononuclear cells from healthy subjects, patients with secondary polycythaemia and patients with polycythaemia vera (PV) were cultured using the plasma clot technique, and large erythroid colonies or bursts were scored after 14 d. Erythroid bursts appeared without addition of erythropoietin in all 20 patients with PV but never in normal subjects ( n =10) nor in patients with secondary polycythaemia ( n =6). The erythropoietin dose‐response curves in six PV patients were characterized by an initial plateau followed by a nearly normal response. This suggests the coexistence of two populations of erythroid progenitors in PV, one abnormally sensitive, the other normally responsive to erythropoietin. In nine patients the ratio of spontaneous bursts to bursts induced by high doses of erythropoietin from the blood on day 14 was identical with the ratio of spontaneous colonies to colonies induced by high doses of erythropoietin from the bone marrow of the same patients on day 7. This argues against the hypothesis that spontaneous erythroid bursts are derived from erythroid progenitors (BFU E ) abnormally sensitive to erythropoietin. Moreover blood mononuclear cells from healthy subjects were found to give rise to erythroid bursts when erythropoietin addition was delayed until 6 d in culture. This is consistent with the concept that erythropoietin is not normally required in vivo for the differentiation of early (BFU E ) to late (CFU E ) erythroid progenitors. These findings suggest that in PV all BFU E are normally responsive to the physiological factor(s) different from erythropoietin and responsible for their differentiation into CFU E , and that the abnormal response to erythropoietin, distinctive of a population of erythroid progenitors, is expressed only at the most mature (CFU E ) level.
We previously demonstrated that highly purified normal human blood burst-forming units-erythroid (BFU-E) need the direct action of recombinant human stem cell factor (rSCF) in the presence of recombinant human erythropoietin (rEP) and recombinant human interleukin-3 (rIL-3) for further development in a serum-free medium. To study the response of polycythaemia vera (PV) BFU-E to rSCF, we performed dose-response experiments in a serum-free medium using highly purified BFU-E from PV patients. A marked increase in the number of PV bursts occurred with increasing concentrations of rSCF, compared to normal burst formation, when the cells were cultured in the presence of rIL-3 at 1 U/ml. The percentage of maximum growth for normal BFU-E was 31 +/- 11% while for PV it was 64 +/- 9% at the highest concentration of rSCF (P < 0.01). Without rIL-3, only 11% of maximum normal BFU-E growth occurred as the rSCF concentration was increased and the size of the colonies was very small, but PV BFU-E still expressed 48% of the maximum number of large erythroid bursts (P < 0.001). This demonstrated an enhanced sensitivity of PV BFU-E to rSCF, compared to normal BFU-E. The pattern of 59Fe incorporation into haem after 8 d of cell culture indicated that PV BFU-E had a time course of maturation and a degree of cellular maturity similar to normal BFU-E. The percentage positivity and intensity of c-kit receptors on PV erythroid cells were examined using immunofluorescence flow cytometry. When BFU-E, CFU-E, or erythroblasts were incubated with phycoerythrin-conjugated SR-1 anti-c-kit receptor monoclonal antibody, 90% of the PV and normal BFU-E displayed c-kit receptor at comparable intensities, as well as 80% of the PV and normal CFU-E. A distinct loss of c-kit expression occurred with erythroid differentiation beyond the CFU-E stage, but at all stages no difference of c-kit receptor expression was evident for PV erythroid precursors compared to normal precursors. These results indicate that the hypersensitivity to rSCF did not appear to be related to the number of c-kit receptors. Since we have previously shown that highly purified PV BFU-E are hypersensitive to rIL-3 and rGM-CSF, as well as rEP, it is now evident that PV BFU-E are hypersensitive to each of the cytokines that have a prominent role in guiding their normal proliferation and differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)
The mechanisms of the chronic myeloid leukemia (CML) clones proliferative advantage over normal clones are currently unknown. They may involve an insensitivity to a negative regulation of a growth factor-independent proliferation. Clonogenic progenitors from CML patient blood or marrow in chronic phase were grown either in the presence or absence of recombinant growth factors. No erythroid colonies were observed in the absence of any cytokine. In contrast, erythroid colonies composed of fully mature hemoglobinized erythroblasts (day 12 burst-forming units-erythroid) were obtained in the presence of Steel factor (SF) alone. Addition of erythropoietin (Epo) to SF either had no effect on the cloning efficiency or increased up to 50% the number of erythroid colonies. No erythroid growth was observed when cultures were stimulated by interleukin-3 or granulocyte-macrophage colony-stimulating factor alone. Similar erythroid growth in the presence of SF but without Epo was obtained in "serum-free" cultures when purified blood CML CD34+ cells were grown. This growth of erythroid colonies in the absence of Epo was not accounted for by an autocrine stimulation loop by Epo, because neutralizing antibodies against Epo did not inhibit it. This abnormal response to growth factor was specifically observed in the CML clone, as shown by the presence of the BCR-ABL transcript in all of these erythroid colonies. The direct implication of BCR-ABL was further documented (1) by studies of alpha-interferon-treated patients with a chimerism in which the abnormal growth correlates with the presence of the malignant clone and (2) by the use of antisense oligonucleotide against BCR-ABL transcript, which abrogated this abnormal growth. Finally, erythroid growth in the SF presence was greatly diminished by herbimycin A, whereas, at the same concentration, this tyrosine kinase inhibitor had no marked effect on erythroid colony formation in the presence of SF plus Epo on CML or normal marrow cells. This result suggests that the BCR-ABL kinase activity leads directly to this Epo-independent terminal differentiation requiring, however, the presence of SF.
Polycythaemia vera (PV) patients' blood burst-forming units-erythroid (BFU-E) have an enhanced sensitivity to stem cell factor (SCF) compared to normal BFU-E. To characterize SCF receptors on erythroid progenitors from normal individuals and PV patients, we performed binding experiments using radioiodinated recombinant SCF (rSCF), day 1 BFU-E and day 8 erythroid colony-forming cells (ECFC), which are mostly colony-forming units-erythroid (CFU-E). 125I-rSCF binds to a single class of cell surface receptors (23,000/ECFC) at 0 degrees C with a high-binding affinity (Kd = 17 pM). Saturation occurred at 0.5 nM (10 ng/ml) which produces a nearly maximum biological effect. One half of the radiolabelled rSCF was internalized by the cells after 30 min at 37 degrees C. No significant differences in the receptor number, dissociation constant, or internalization rate were found between normal and PV ECFC. Autoradiographic analysis of 125I-rSCF binding to normal BFU-E and ECFC showed that no differences were present in either the percentage of positive cells or the number of radioactive grains/cell between the normal and PV erythroid progenitors. The enhanced sensitivity of PV BFU-E and CFU-E to SCF does not appear to be related to changes in SCF receptor number, binding affinity or internalization and the hypersensitivity of PV erythroid progenitors to SCF must reside in a further internal cellular abnormality.
The effect of mast cell growth factor (MGF) was studied on erythropoietin (Epo)-dependent and Epo-independent ("spontaneous") erythroid colony formation in patients with polycythemia vera (PV). MGF stimulated both Epo-dependent and Epo-independent erythroid colony formation from PV peripheral blood progenitor cells in vitro at a dose similar to normal erythroid progenitor. In addition, evidence was obtained that the stimulating effect of MGF was a direct effect on the erythroid progenitor and independent of serum. Antibodies against interleukin-1 (IL-1), IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and Epo could not abolish the enhancing effect of MGF. This was also supported by the finding that sorted CD34+ cells could be stimulated by MGF in the presence and absence of Epo. Finally, it was demonstrated that the spontaneous erythroid colony formation could not be ascribed to spontaneous release of MGF in the culture medium since anti-MGF did not affect the colony numbers. In conclusion, MGF has a direct stimulatory effect, independent of serum, on both Epo-dependent and Epo-independent erythroid colony formation in PV.
Erythropoietin (EPO) is a potent regulator of the viability, proliferation and differentiation of erythroid progenitor cells. Its effect is mediated by binding to the erythropoietin receptor (EPO-R), a member of a new cytokine receptor family. Alterations of the EPO/EPO-R system have recently been shown to be involved in the pathogenesis of familial erythrocytosis and polycythaemia vera (PV). In order to define whether genetic changes in the EPO-R gene and its ligand play a role in the development of PV, the structure and expression levels of the EPO-R and EPO genes were examined in samples from bone marrow and/or peripheral blood mononuclear cells of 24 patients with PV. As expected, EPO serum levels were low and no detectable level of EPO mRNA was found by reverse polymerase chain reaction (RT-PCR) in the peripheral blood mononuclear cells of our PV patients. To search for structural alterations of the EPO-R, cDNA samples were subjected to PCR and SSCP analysis as well as sequencing. Heterogenous expression of EPO-R mRNA was observed without any structural changes, as revealed by RT-SSCP analysis using overlapping primers spanning the whole coding region of the EPO-R gene. Structural integrity of the EPO-R was further confirmed by sequencing of cloned PCR products. These data suggest that the mechanisms for the development of PV do not involve structural changes of the EPO-R gene.
We have investigated the question of erythropoietin (Epo) hypersensitivity versus Epo independence as the basis for the endogenous erythroid bursts (EEBs) that develop in cultures without added Epo from hematopoietic cells of polycythemia vera (PV) patients. Using an improved serum-free (SF) medium containing interleukin (IL)-3, but no insulin-like growth factor-1 (IGF-1), and devoid of contaminants that influence erythropoiesis, we compared circulating normal and PV early erythroid progenitors (BFU-E) with respect to their responses in vitro to recombinant human (rHu) Epo. Cultures were seeded with Ficoll-Hypaque density-separated peripheral blood (PB) mononuclear cells (MNCs), and erythroid bursts, together with their component colonies of > or = 50 cells, were scored in situ at 13 to 16 days of culture. The Epo dose-response curve of BFU-E from PV patients was found to be statistically indistinguishable from that of normal subjects. This observation provides compelling evidence against the Epo-hypersensitivity hypothesis. In the complete SF medium minus Epo, the sensitivity of BFU-E to IGF-1 was much greater in PV than in normals, the dose-response curve being shifted to the left by at least 2 orders of magnitude. These data show that the erythroid progenitor cell response in PV is hypersensitive to IGF-1, and independent of Epo. The data also emphasize the importance of truly SF medium conditions for assessment of progenitor cell sensitivities to recombinant growth factors. Depletion of adherent cells totally prevented erythroid burst formation by normal circulating progenitors, but did not prevent the hypersensitive response to IGF-1 of such cells from PV patients. Hence, again unlike its normal counterpart, the progenitor cell response in PV appears to be independent of adherent cell control.
Mice with the recessive motheaten (me) or the allelic viable motheaten (mev) mutations express a severe autoimmune and immunodeficiency syndrome. We have shown that the basic defect in these mice involves lesions in the gene which encodes haematopoietic cell phosphatase (HCP). These mice thus provide excellent models for investigating the roles of phosphatases in haematopoiesis and the nature of the genetic and cellular events linking impaired haematopoiesis to severe immunodeficiency and expression of systemic autoimmunity.
Polycythemia vera (PV) is an acquired clonal disorder characterized by increased production of mature red cells and growth of erythroid colonies in the absence of erythropoietin. Mutation of the erythropoietin receptor has been demonstrated to cause familial polycythemia, but no mutations have been found in PV. Moreover, both erythroid and myeloid progenitors from patients with PV have been reported to be hypersensitive to a number of different growth factors. Attention has therefore focused on post-receptor signal transduction pathways. The SHP-1 gene is an especially attractive candidate gene. Firstly, SHP-1 binds to and negatively regulates signalling from the erythropoietin receptor and is likely to regulate other cytokine receptors in a similar manner. Secondly, absence of SHP-1 protein in the motheaten mouse is accompanied by increased sensitivity of hematopoietic progenitors to a number of cytokines including erythropoietin. Thirdly, familial or sporadic polycythemia in man may result from mutations of the SHP-1 binding domain of the erythropoietin receptor. We have therefore searched for mutations of the SHP-1 gene in genomic DNA from patients with PV. In this disease the majority of peripheral blood lymphocytes are not part of the malignant clone and a variable proportion of myeloid cells may arise from normal progenitors. We have therefore chosen to study DNA from purified peripheral blood granulocytes obtained from nine women in whom the granulocytes were clonally derived. Southern analysis was used to show that the gene was not rearranged and densitometry confirmed the presence of two copies of the gene in each DNA sample. Sequencing of the entire coding region and all splice junctions revealed no mutations. Hematopoietic transcription factor binding sites in the SHP-1 promoter region were intact and the methylation status of the two SHP-1 promoters in PV patients was identical to that in three normal controls. Finally, we showed that levels of SHP-1 protein in granulocytes from patients was similar to those from normal controls. These results demonstrate that the SHP-1 gene is structurally and transcriptionally intact in patients with PV.
Erythropoietin (EP) and stem cell factor (SCF) are essential growth factors for erythroid progenitor cell proliferation and differentiation in serum-free culture. It has been previously shown that burst-forming units-erythroid and colony-forming units-erythroid from patients with polycythemia vera (PV) have enhanced sensitivity to EP and SCF compared with normal erythroid progenitors, but little is known about the mechanism for this difference. In the present investigation, the effect of EP and SCF on protein tyrosine phosphorylation in day-8 normal and PV erythroid colony-forming cells, which give rise to colonies of 2-49 hemoglobinized cells, was studied. EP rapidly induced tyrosine phosphorylation of the EP receptor, whereas the most prominent phosphorylated protein induced by SCF was identified as the SCF receptor. No additional phosphorylated proteins were evident when PV cells were compared with normal cells. Culture of normal erythroid progenitors with orthovanadate, an inhibitor of protein tyrosine phosphatases, resulted in an increased number of erythroid colonies and enhanced protein tyrosine phosphorylation. However, in contrast, little enhancement was evident with PV cells. These results indicate that, although vanadate may be acting in normal erythroid progenitors as a phosphatase inhibitor that potentiates the kinase activity induced by SCF and EP, this function is diminished in PV cells. Because erythropoiesis is regulated by a balance between protein tyrosine kinase activity and protein tyrosine phosphatase activity, PV patients may have an abnormal phosphatase activity allowing increased cell proliferation.
In this study, we examined the in vitro and in vivo effects of forced expression of Mpl-R (the thrombopoietin receptor) on the progeny of murine hematopoietic stem cells. Bone marrow cells from 5-FU-treated mice were transduced with retroviral vectors containing the human Mpl-R cDNA, or the neomycine gene as a control. After 7 days cocultivation on virus-producer cells, GpE86-Mpl-R or Gp86-Neo, the types of hematopoietic progenitor cells responding to thrombopoietin (TPO) were studied by clonogenic assays. Mpl-R-infected cells gave rise to CFU-GEMM, BFU-E, CFU-MK, but not CFU-GM while Neo-infected cells produced only megakaryocytic colonies. In addition, when nonadherent cells from GpE86-Mpl-R cocultures were grown with TPO as the only stimulus for 7 days, a marked expansion of CFU-GEMM, BFU-E, and CFU-MK was observed, while no change in CFU-GM number was seen. Erythroid and megakaryocytic maturation occurred in the presence of TPO while a block in granulocytic differentiation was observed at the myeloblast stage. The direct effects of TPO on Mpl-R-transduced progenitor cells were demonstrated by single cell cloning experiments. To analyze the effects of the constitutive expression of Mpl-R on the determination of multipotent progenitors (CFU-S) and long-term repopulating stem cells, Mpl-R- or Neo-infected cells were injected into lethally irradiated recipient mice. No difference was seen in (1) the number of committed progenitor cells contained in individual CFU-S12 whether colonies arose from noninfected or Mpl-R-infected CFU-S; (2) the mean numbers of progenitor cells per leg or spleen of mice reconstituted with Mpl-R- or Neo-infected cells, 1 or 7 months after the graft; and (3) the blood parameters of the two groups of animals, with the exception of a 50% reduction in circulating platelet counts after 7 months in mice repopulated with Mpl-R-infected bone marrow cells. These results indicate that retrovirus-mediated expression of Mpl-R in murine stem cells does not modify their ability to reconstitute all myeloid lineages of differentiation and does not result in a preferential commitment toward the megakaryocytic lineage.
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.
Polycythaemia vera (PV) is a myeloproliferative disorder characterized by haematopoietic progenitor cells being hypersensitive to cytokines such as erythropoietin, interleukin-3, stem cell factor and insulin-like growth factor 1, which results in an increased production of mature blood cells. The pathogenetic cellular mechanism(s) behind this hypersensitivity to cytokines is unknown, but the number of cytokine receptors and the interaction between ligand and receptor are normal in PV. Interest has therefore focused on post-receptor mechanism(s). Haematopoietic cell phosphatase (HCP) is an intracellular tyrosine phosphatase that has been demonstrated to regulate proliferative signals negatively induced by the cytokines mentioned above. Moreover, motheaten mice that genetically lack HCP have an increased amount of erythroid progenitors that are hypersensitive to Epo, and patients with familial polycythaemia have been shown to exhibit a mutation of the Epo receptor gene that includes the docking site for HCP. We therefore studied mRNA expression of HCP in pure populations of CD34+ cells, granulocytes, platelets and lymphocytes from patients with PV, chronic myeloid leukaemia (CML) or essential thrombocythemia (ET), as well as healthy controls. Using a polymerase chain reaction analysis employing specific primers for HCP, we failed to detect any abnormalities of HCP expression in PV in any of the cell populations that were examined. Moreover, HCP mRNA expression was similar in ET and CML compared to controls. Finally, Western blot analysis revealed a normal HCP protein content in PV granulocytes and platelets. We therefore conclude that neither an impaired expression of the HCP gene nor a defect in HCP protein synthesis is present in PV, and does not seem to play a role in the aetiology of this disorder.
In polycythemia vera (PV) erythroid colonies that grow in vitro in the absence of exogenous erythropoietin (EPO) arise from the abnormal clone that is responsible for overproduction of red blood cells. Although the mechanism of autonomous formation of burst-forming units-erythroid (BFU-E) is not fully understood, a spontaneous release of growth regulatory molecules by PV cells and/or by accessory cells is likely to be involved. Because of its cytokine synthesis inhibiting action, interleukin-10 (IL-10) could be a potentially useful molecule to modulate abnormal erythropoiesis in PV. We studied the effect of recombinant human IL-10 on the EPO-independent growth of erythroid bursts derived from peripheral blood mononuclear cells (PBMNCs) of patients with PV. IL-10 showed a profound, dose-dependent, and specific inhibitory effect on autonomous BFU-E formation. Ten nanograms per milliliter of IL-10 significantly suppressed spontaneous growth of erythroid colonies in methylcellulose in five of five PV patients tested with a mean inhibition by 81% (range, 72-94). To elucidate the possible mechanism of the inhibitory action of IL-10 we further studied the effect of anticytokine antibodies on autonomous BFU-E growth and the ability of exogenous cytokines to restore IL-10-induced suppression of erythroid colony growth. Among a panel of growth regulatory factors tested (granulocyte-macrophage colony-stimulating factor [GM-CSF], IL-3, granulocyte colony-stimulating factor, stem cell factor, and insulin-like growth factor-1) GM-CSF was the only molecule for which both an inhibition of spontaneous BFU-E formation by its respective antibody as well as a significant restimulation of erythroid colonies in IL-10-treated cultures by exogenous addition was found. Moreover, inhibition of GM-CSF production by IL-10 was shown in PV PBMNCs at the mRNA level. Our data indicate that autonomous BFU-E growth in PV can be profoundly inhibited by IL-10 and that this inhibitory effect seems to be at least in part secondary to suppression of endogenous GM-CSF production.
The SHP-1 phosphatase associates with the receptors for erythropoietin, stem cell factor, and interleukin-3, and negatively regulates the mitogenic signals generated during engagement by their respective ligands. The erythroid progenitors of patients with polycythemia vera are hypersensitive to the mitogenic effects of these growth factors despite the fact that the numbers and binding affinities for their receptors are not increased. To determine whether post-receptor signaling defects may account for growth factor-hypersensitivity in polycythemia vera, we determined the expression of SHP-1 in highly purified erythroid progenitors from polycythemia vera patients. Our data demonstrate that in approximately 60% of the patients, expression of SHP-1 in the colony forming unit-erythroid population is diminished. The decreased expression of the protein may result from a transcriptional defect, as suggested by the diminished SHP-1 mRNA expression in the erythroid progenitors of these patients. Studies to determine the level of maturation of polycythemia vera and normal cells indicated that there was no difference between the two at early colony forming unit-erythroid stage of differentiation although polycythemia vera cells showed retarded differentiation kinetics at late colony forming unit-erythroid stage of differentiation. Furthermore, SHP-1 expression in normal colony forming unit-erythroid demonstrated downregulation of mRNA and protein levels during terminal differentiation, suggesting that its function is required for growth control during the early stages of erythropoiesis. These results indicate an important role for SHP-1 in the regulation of normal human erythroid progenitors and suggest that defective expression of the protein may contribute to the pathogenesis of polycythemia vera.
The erythropoietin receptor (EpoR) is essential for production of red blood cells; a principal function of EpoR is to rescue committed erythroid progenitors from apoptosis. Stat5 is rapidly activated following EpoR stimulation, but its function in erythropoiesis has been unclear since adult Stat5a-/-5b-/- mice have normal steady-state hematocrit. Here we show that Stat5 is essential for the high erythropoietic rate during fetal development. Stat5a-/-5b-/- embryos are severely anemic; erythroid progenitors are present in low numbers, show higher levels of apoptosis, and are less responsive to Epo. These findings are explained by a crucial role for Stat5 in EpoR's antiapoptotic signaling: it mediates the immediate-early induction of Bcl-X(L) in erythroid cells through direct binding to the Bcl-X promoter.
Erythropoietin (Epo)-independent differentiation of erythroid progenitors is a major characteristic of myeloproliferative disorders, including chronic myeloid leukemia. Epo receptor (EpoR) signaling is crucial for normal erythroid development, as evidenced by the properties of Epo(-/-) and EpoR(-/-) mice, which contain a normal number of fetal liver erythroid progenitors but die in utero from a severe anemia attributable to the absence of red cell maturation. Here we show that two constitutively active cytoplasmic protein tyrosine kinases, P210(BCR-ABL) and v-SRC, can functionally replace the EpoR and support full proliferation, differentiation, and maturation of fetal liver erythroid progenitors from EpoR(-/-) mice. These protein tyrosine kinases can also partially complement the myeloid growth factors IL-3, IL-6, and Steel factor, which are normally required in addition to Epo for erythroid development. Additionally, BCR-ABL mutants that lack residues necessary for transformation of fibroblasts or bone marrow cells can fully support normal erythroid development. These results demonstrate that activated tyrosine kinase oncoproteins implicated in tumorigenesis and human leukemia can functionally complement for cytokine receptor signaling pathways to support normal erythropoiesis in EpoR-deficient cells. Moreover, terminal differentiation of erythroid cells requires generic signals provided by activated protein tyrosine kinases and does not require a specific signal unique to a cytokine receptor.
Polycythemia rubra vera (PV) is one of four diseases collectively called the myeloproliferative disorders (MPDs). Each disorder leads to an increased production of one or several hematopoietic cell lineages. MPDs arise from acquired mutations in a pluripotent hematopoietic stem cell. However, the molecular mechanisms leading to the development of these diseases are poorly understood. This review will summarize and evaluate recent advances in our understanding of one particular MPD, PV.
The absolute polycythemias--those with increased red blood cell mass--can be divided into two groups: primary, caused by acquired or inherited mutations leading to a "gain-of-function" abnormalities expressed within the erythroid progenitors; and secondary, due to circulating serum factors, typically erythropoietin, stimulating erythropoiesis. This overview concentrates on the molecular biology of polycythemia vera (PV) discussed in the context of other polycythemic disorders. Recent advances in the regulation of erythropoiesis, as they may relate to polycythemic states, are discussed as a background for those well-defined polycythemic states wherein the molecular defect has not yet been elucidated. A number of cellular abnormalities associated with PV, including the hyperresponsiveness of PV progenitors to many cytokines as well as decreased expression of the thrombopoietin receptor on platelets and increased expression of Bcl-xL, suggest that the PV defect alters a number of cellular functions and is not restricted to cytokine receptor signal transduction. The increasing number of recognized instances of familial incidence of PV suggests that in these families the predisposition for PV is inherited as a dominant trait, and that PV is acquired as a new mutation that leads to a clonal hematopoiesis and may be due to loss of heterozygosity. The existence of these families provides a unique opportunity for isolation of the mutations in the gene leading to PV. Semin Hemaol 38(suppl 2):10-20.
In this study, we examined the molecular mechanism of erythropoietin-initiated signal transduction of erythroid differentiation through Src and phosphatidylinositol 3-kinase (PI3-kinase). Antisense oligonucleotides against src but not lyn inhibited the formation of erythropoietin-dependent colonies derived from human bone marrow cells and erythropoietin-induced differentiation of K562 human erythroleukaemia cells. Antisense p85alpha oligonucleotide or LY294002, a selective inhibitor of PI3-kinase, independently inhibited the formation of erythropoietin-dependent colonies. In K562 cells, Src associated with PI3-kinase in response to erythropoietin. Antisense src RNA expression in K562 cells inhibited the erythropoietin-induced activation of PI3-kinase and its association with erythropoietin receptor. PP1, a selective inhibitor of the Src family, reduced erythropoietin-induced tyrosine phosphorylation of erythropoietin receptor and its association with PI3-kinase in F-36P human erythroleukaemia cells. The coexpression experiments and in vitro kinase assay further demonstrated that Src directly tyrosine-phosphorylated erythropoietin receptor, and associated with PI3-kinase. In vitro binding experiments proved that glutathione S-transferase-p85alpha N- or C-terminal SH2 domains independently bound to erythropoietin receptor, which was tyrosine-phosphorylated by Src. Taken together, Src transduces the erythropoietin-induced erythroid differentiation signals by regulating PI3-kinase activity.
Signal transducers and activators of transcription (STAT) proteins are transcription factors activated by phosphorylation on tyrosine residues after cytokine stimulation. In erythropoietin receptor (EPOR)-mediated signaling, STAT5 is tyrosine-phosphorylated by EPO stimulation. Although Janus Kinase 2 (JAK2) is reported to play a crucial role in EPO-induced activation of STAT5, it is unclear whether JAK2 alone can tyrosine-phosphorylate STAT5 after EPO stimulation. Several studies indicate that STAT activation is caused by members of other families of protein tyrosine kinases such as the Src family. We previously reported that reduction of Src by induction of antisense src RNA expression suppressed EPO-promoted erythroid differentiation in K562 cells. In the present study, we explored the function of Src downstream of the EPOR-initiated signaling. Reduction of Src diminished tyrosine phosphorylation of STAT5 in K562 cells regardless of EPO treatment. The tyrosine phosphorylation level of STAT5 induced by EPO in F-36P cells was reduced in the presence of PP1 or PP2 selective Src inhibitor. In addition, the expression of dominant negative Src in F-36P cells reduced the tyrosine phosphorylation of STAT5. When Src and STAT5 were co-expressed in COS7 cells, tyrosine phosphorylation of STAT5 was observed, and tyrosine residue 694 (Tyr 694) of STAT5A was identified as the major phosphorylation site by Src. In vitro kinase assay revealed that GST-STAT5 fusion protein with the conserved C-terminal, but not the C-terminal-truncated mutant which lacks Tyr 694, was tyrosine-phosphorylated by Src. Src can thus directly tyrosine-phosphorylate the activation site of STAT5 (Tyr 694 in STAT5A), and Src may contribute to EPO-induced signal transduction via STAT5.