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Polycythemia Vera. II. Hypersensitivity of bone marrow erythroid, granulocyte-macrophage, and megakaryocyte progenitor cells to interleukin-3 and granulocyte-macrophage colony-stimulating factor
Abstract
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.
... As such, Several authors have described that both peripheral blood mononuclear cells and purified BFU-E progenitors obtained from patients with PV are hypersensitive to human IL-3 and to GM-CSF. [28][29][30] In addition, PV colony-forming unit-granulocyte-macrophage (CFU-GM) and CFU-megakaryocyte also show a marked hypersensitivity to IL-3 and to GM-CSF. 30 However, another group has found that the spontaneous megakaryocyte colonies derived from blood and bone marrow of patients with myeloproliferative disorders, including PV, cannot be neutralized by antibodies against IL-3 and GM-CSF either alone or in combination. ...
... [28][29][30] In addition, PV colony-forming unit-granulocyte-macrophage (CFU-GM) and CFU-megakaryocyte also show a marked hypersensitivity to IL-3 and to GM-CSF. 30 However, another group has found that the spontaneous megakaryocyte colonies derived from blood and bone marrow of patients with myeloproliferative disorders, including PV, cannot be neutralized by antibodies against IL-3 and GM-CSF either alone or in combination. 31 This suggests that the megakaryocyte progenitor growth in these disorders under in vitro conditions is independent of IL-3 and GM-CSF. ...
Polycythemia vera (PV) is an acquired clonal myeloproliferative disorder characterized by increased production of mature red cells. We still lack a molecular target responsible for this disorder; however, recent investigations have focused on a number of molecules involved in signal transduction pathways mediated by erythropoietin (Epo) and other growth factors. Here we review the implication of these molecules in the pathogenesis of PV.
The material reviewed in this work includes articles published in journals covered by Medline. We also include data obtained in our laboratory regarding to the significance of apoptosis inhibitory proteins in erythroid development.
Overproduction of erythroid cells in PV is particular in that it occurs in the absence of a recognizable physiologic stimulus, since circulating serum levels of Epo are normal or lower than normal. Genetic analysis as well as in vitro studies, have established an essential role for Epo in the survival and maturation of committed erythroid progenitors. Epo initiates its cellular response by binding to the Epo receptor (EpoR) expressed on the surface of immature erythroblasts. Following ligand binding, EpoR is known to activate a cytoplasmic protein tyrosine kinase, Jak2 which triggers a signal transduction cascade that leads to the development of early erythroid progenitors into mature erythroblast cells. Although the mechanism underlying the increased erythroid production in PV is not well understood, a number of causes have recently came for which may provide insights not only for the pathogenesis of PV but also for a fundamental biological process: the mechanism whereby a multipotential stem cell gives rise to a particular cell lineage.
... This mutation renders JAK-STAT signaling pathway to be active constitutively which results in unchecked proliferation of hematopoietic stem cells. Mutations in the JAK-2 gene appear to account for hyperresponsiveness of the hematopoietic progenitor cells to various cellular growth factors and other cytokines [25]. However, the presence of this mutation alone cannot explain all the other findings associated with myelofibrosis, including cytopenia and the tendency to transform into acute myeloid leukemia. ...
... Erythroid and megakaryocyte progenitors from MPN patients can form colonies without erythropoietin and thrombopoietin, respectively. Moreover, hematopoietic progenitors are hypersensitive to IGF-1, IL-3, GM-CSF, Epo, and Tpo (Axelrad et al., 2000;Correa et al., 1994;Dai et al., 1991Dai et al., , 1992. In-vitro, co-expression of JAK2V617F and cytokine receptors like EpoR, MPL, and GCSF-R in cell lines was sufficient to induce cytokine-independent growth in these experimental systems Lu et al., 2005;Ugo et al., 2005). ...
Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell (HSC) disorders with overproduction of mature myeloid blood cells, including essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). In 2005, several groups identified a single gain-of-function point mutation JAK2V617F in the majority of MPN patients. The JAK2V617F mutation confers cytokine independent proliferation to hematopoietic progenitor cells by constitutively activating canonical and non-canonical downstream pathways. In this chapter, we focus on (1) the regulation of JAK2, (2) the molecular mechanisms used by JAK2V617F to induce MPNs, (3) the factors that are involved in the phenotypic diversity in MPNs, and (4) the effects of JAK2V617F on hematopoietic stem cells (HSCs). The discovery of the JAK2V617F mutation led to a comprehensive understanding of MPN; however, the question still remains about how one mutation can give rise to three distinct disease entities. Various mechanisms have been proposed, including JAK2V617F allele burden, differential STAT signaling, and host genetic modifiers. In vivo modeling of JAK2V617F has dramatically enhanced the understanding of the pathophysiology of the disease and provided the pre-clinical platform. Interestingly, most of these models do not show an increased hematopoietic stem cell self-renewal and function compared to wildtype controls, raising the question of whether JAK2V617F alone is sufficient to give a clonal advantage in MPN patients. In addition, the advent of modern sequencing technologies has led to a broader understanding of the mutational landscape and detailed JAK2V617F clonal architecture in MPN patients.
... Erythroid and megakaryocyte progenitors from MPN patients can form colonies without erythropoietin and thrombopoietin, respectively. Moreover, hematopoietic progenitors are hypersensitive to IGF-1, IL-3, GM-CSF, Epo, and Tpo (Axelrad et al., 2000;Correa et al., 1994;Dai et al., 1991Dai et al., , 1992. In-vitro, co-expression of JAK2V617F and cytokine receptors like EpoR, MPL, and GCSF-R in cell lines was sufficient to induce cytokine-independent growth in these experimental systems Lu et al., 2005;Ugo et al., 2005). ...
Classical Philadelphia-negative myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell-derived disorders characterized by uncontrolled proliferation of differentiated myeloid cells and close pathobiologic and clinical features. According to the 2016 World Health Organization (WHO) classification, MPNs include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The 2016 revision aimed in particular at strengthening the distinction between masked PV and JAK2-mutated ET, and between prefibrotic/early (pre-PMF) and overt PMF. Clinical manifestations in MPNs include constitutional symptoms, microvascular disorders, thrombosis and bleeding, splenomegaly secondary to extramedullary hematopoiesis, cytopenia-related symptoms, and progression to overt MF and acute leukemia. A dysregulation of the JAK/STAT pathway is the unifying mechanistic hallmark of MPNs, and is guided by somatic mutations in driver genes including JAK2, CALR and MPL. Additional mutations in myeloid neoplasm-associated genes have been also identified, with established prognostic relevance, particularly in PMF. Prognostication of MPN patients relies on disease-specific clinical models. The increasing knowledge of MPN biology led to the development of integrated clinical and molecular prognostic scores that allow a more refined stratification. Recently, the therapeutic landscape of MPNs has been revolutionized by the introduction of potent, selective JAK inhibitors (ruxolitinib, fedratinib), that proved effective in controlling disease-related symptoms and splenomegaly, yet leaving unmet critical needs, owing the lack of disease-modifying activity. In this review, we will deal with molecular, clinical, and therapeutic aspects of the three classical MPNs aiming at highlighting either shared characteristics, that overall define a continuum within a single disease family, and uniqueness, at the same time.
... IL-3 could induce the differentiation of hematopoietic stem cells into myeloid progenitor cells [29]. It is also essential for early hematopoiesis, especially in erythrocytosis [30][31][32], might play a role in a new mechanism in the development of HAPC [23]. TNF-alpha, a major mediator of inflammation, is involved in the pathophysiological processes of tissue damage, inflammation, and shock [24], and it has also been reported to be involved in CMS [33]. ...
High-altitude polycythemia (HAPC) is a common aspect of chronic mountain sickness (CMS) caused by hypoxia and is the main cause of other symptoms associated with CMS. However, its pathogenesis and the mechanisms of high-altitude acclimation have not been fully elucidated. Exposure to high altitude is associated with elevated inflammatory mediators. In this study, the subjects were recruited and placed into a plain control (PC) group, plateau control (PUC) group, early HAPC (eHAPC) group, or a confirmed HAPC (cHAPC) group. Serum samples were collected, and inflammatory factors were measured by a novel antibody array methodology. The serum levels of interleukin-2 (IL-2), interleukin-3 (IL-3), and macrophage chemoattractant protein-1 (MCP-1) in the eHAPC group and the levels of interleukin-1 beta (IL-1 beta), IL-2, IL-3, tumor necrosis factor-alpha (TNF-alpha), MCP-1, and interleukin-16 (IL-16) in the cHAPC group were higher than those in the PUC group. More interestingly, the expression of IL-1 beta, IL-2, IL-3, TNF-alpha, MCP-1, and IL-16 in the PUC group showed a remarkable lower value than that in the PC group. These results suggest that these six factors might be involved in the pathogenesis of HAPC as well as acclimation to high altitudes. Altered inflammatory factors might be new biomarkers for HAPC and for high-altitude acclimation.
... Dans la PV, TE et MFP certaines cellules érythroïdes sont capables de proliférer et de se différencier sans EPO, et sont appelées colonies érythroïdes spontanées (endogenous erythroid colony « EEC ») (Prchal and Axelrad, 1974) (Kralovics et al., 2003), suggérant un mécanisme physiopathologique commun entre ces trois NMP. 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 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.
... 7 This phenomenon represented one of the minor diagnostic criteria in the 2008 World Health Organization definition of PV but does not figure any longer in the 2016 revised revision. 5,34 During the 1980s, works of several groups pointed out the EPO hypersensitivity of PV erythropoietic progenitor cells from peripheral blood and in some cases even their EPO independency to form colonies. 8,9 In the 1990s, this phenomenon was complemented by the discovery of increased reactivity to other cytokines, such as interleukin 1, interleukin 3, granulocyte macrophage colony-stimulating factor, and insulin-like growth factor 1. 35,36 Biochemical background of V617F point mutation in JAK2 is a substitution of phenylalanine for valine at amino acid position 617 (V617F) of the JH2 domain. This gain of function mutation results in a permanent activation of JAK2. ...
The discovery of JAK2 V617F mutation in the mid-2000s started to fill the gap between clinical presentation of polycythemia vera (PV), first described by Vaquez at the end of the 19th century, and spontaneous erythroid colony formation, reported by Prchal and Axelrad in the mid-1970s. The knowledge on this mutation brought an important insight to our understanding of PV pathogenesis and led to a revision of the World Health Organization diagnostic criteria in 2008. JAK–STAT is a major signaling pathway implicated in survival and proliferation of hematopoietic precursors. High prevalence of JAK2 V617F mutation among myeloproliferative neoplasms (>95% in PV and ~50% in primary myelofibrosis and essential thrombocythemia) together with its role in constitutively activating JAK–STAT made JAK2 a privileged therapeutic target. Ruxolitinib, a JAK 1 and 2 inhibitor, has already proven to be efficient in relieving symptoms in primary myelofibrosis and PV. In the latter, it also appears to improve microvascular involvement. However, evidence regarding its potential role in altering the natural course of PV and its use as an adjunct to current standard therapies is sparse. Therapeutic advances are needed in PV as phlebotomy, low-dose aspirin, cytoreductive agents, and interferon alpha are the only therapeutic tools available at the moment to influence outcome. Even though several questions are still unanswered, this review aims to serve as an overview article of the potential role of ruxolitinib in PV according to current literature and expert opinion. It should help hematologists to visualize the place of this tyrosine kinase inhibitor in the field of current practice and offer criteria for a careful patient selection.
... 25 Although EECs were also observed in some patients with ET, 26 their presence is a hallmark of PV and was used as a clinical diagnostic tool. 4 The observed increased proliferative responsiveness of PV progenitor cells to EPO, IGF-1, and other growth factors (eg, interleukin [IL]-3, granulocyte-macrophage colonystimulating factor [GM-CSF], thrombopoietin, and stem cell factor) implicated abnormal cytokine signaling pathways in the molecular underpinnings of PV. 4,5,26,27 However, studies that examined mutations related to cytokine signaling targets (eg, the EPO receptor, IGF-1 receptor, IGF-1-binding proteins, and tyrosine phosphatases) were unsuccessful in elucidating the pathogenesis of PV. 4 It was also hypothesized that abnormal cytokine signaling was not necessarily related to a limited number of specific mutations but rather to more general defects in transcriptional regulation that could affect a variety of metabolic pathways that play a role in the pathogenesis of PV. 4 The transcriptional dysregulation hypothesis was supported by studies of cells from patients with PV, which reported decreased levels of the thrombopoietin receptor c-MPL in platelets 28 and an increased proportion of erythroid progenitors expressing BCL-x (an antiapoptotic protein). 29 Downstream signal transduction molecules important to cytokine receptor signaling, including EPO-mediated pathways, were further studied to identify the potential PV candidate genes. ...
Kris Vaddi,1 Srdan Verstovsek,2 Jean-Jacques Kiladjian3 1Drug Discovery, Incyte Corporation, Wilmington, DE, 2Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 3Clinical Investigations Center, Hôpital Saint-Louis et Université Paris Diderot, Paris, France Abstract: Polycythemia vera (PV) is a chronic myeloproliferative neoplasm characterized by erythrocytosis and the presence of Janus kinase (JAK) 2V617F or similar mutations. This review summarizes the pathophysiology of PV, the challenges associated with traditional treatment options, and the scientific rationale and supportive clinical evidence for targeted therapy with ruxolitinib. Accumulating evidence indicates that activating mutations in JAK2 drive the PV disease state. Traditional PV treatment strategies, including aspirin, phlebotomy, and cytoreductive agents such as hydroxyurea, provide clinical benefits for some but not all patients and may not adequately treat PV-related symptoms. Furthermore, traditional treatment approaches are associated with potential side effects that may limit their usage and lead some patients to discontinue the treatment. Ruxolitinib is an orally available small-molecule tyrosine kinase inhibitor that is a potent and selective inhibitor of JAK1/JAK2. Ruxolitinib is approved in the US for patients with PV with an inadequate response or intolerance to hydroxyurea and in Europe for adults with PV who are resistant to or intolerant of hydroxyurea. In the Phase III RESPONSE registration trial, ruxolitinib was superior to the best available therapy in patients with PV who were resistant to or intolerant of hydroxyurea in controlling hematocrit levels, reducing spleen volume, and improving PV-related symptoms and quality-of-life measures. The most common nonhematologic adverse events in ruxolitinib-treated patients were headache, diarrhea, pruritus, and fatigue in the RESPONSE trial; hematologic adverse events were primarily grade 1 or 2. In the Phase IIIb nonregistration RELIEF trial, there were nonsignificant trends toward an improved symptom control in patients with PV on a stable hydroxyurea dose who were generally well controlled but reported disease-associated symptoms and switched to ruxolitinib vs those who continued hydroxyurea therapy. Updated treatment guidelines will be important for educating physicians about the role of ruxolitinib in the treatment of patients with PV. Keywords: myeloproliferative disorder, polycythemia vera, Janus kinase inhibitor
... Cytokine independent or hypersensitive growth of hematopoietic progenitors is a cardinal feature of ET, PV and PMF. Endogenous erythroid colonies (EECs) 56,57 and endogenous megakaryocyte colonies (EMCs) 58 are evidenced in these patients along with the hypersensitivity to IGF-1, IL-3, GM-CSF, Epo and Tpo [59][60][61][62] . The mutation JAK2 V617F renders the tyrosine kinase JAK2 an authority to constitutively auto phosphorylate41 with increased kinase activity 43 . ...
... Since NF-E2 has an important role in HSC and progenitor physiology, as well as in terminal differentiation processes its regulation must be under a tight control of cytokine signals. Also, PV cells are known to be hypersensitive to certain cytokines [179][180][181][182] . The rationale for performing the experiments described bellow was: to see if promoting Epo -independent erythroid differentiation by increasing the numbers of cells with erythroid phenotype around 2 fold (Fig.4.5.1 a). ...
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.
... Les EEC peuvent être présentes aussi dans des cas de thrombocytémie essentielle (TE) et de myélofibrose idiopathique (MFI)380 .Bien que la croissance soit indépendante de l'Epo, la séquence, l'expression et l'affinité de liaison de l'Epo au REpo sont normales chez les patients de PV381,382,383,384 . De plus, les progéniteurs de patients sont hypersensibles à l'Epo mais aussi à l'IGF-1, l'IL-3, le GM-CSF et au SCF385,386,387,388 . Ces résultats suggèrent qu'un événement en aval du récepteur soit responsable de la formation d'EEC. ...
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.
... The biologic hallmark of NMPs is cytokine hypersensitivity, including erythropoietin (EPO), interleukin 3 (IL-3), stem cell factor (SCF), insulin like growth factor (IGF-1), granulocyte-macrophage colony stimulating factor (GM-CSF) and thrombopoietin (TPO) [69][70][71][72]. In PV, hematopoietic progenitors have the ability to generate EPO-independent erythroid colonies in vitro, known as endogenous erythroid colonies (EEC) [72]. ...
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.
... Important manifestations of these disorders are bone marrow hypercellularity and pan-myeloid myeloproliferation , leading to overproduction of one or more hematopoietic lineages (Spivak et al., 2003). In addition, constant hallmarks of MPD bone marrow cells are their hypersensitivity to several cytokines (Prchal and Axelrad, 1974; Dai et al., 1992; Correa et al., 1994; Dai et al., 1994) and their ability to generate EPO-independent erythroid colonies in vitro (Prchal and Axelrad, 1974), commonly referred to as endogenous erythroid colonies (EECs). Recently, gene JAK2, which encodes a tyrosine kinase required for effective signaling in response to several cytokines (Parganas et al. 1998), was found mutated in these conditions. ...
Polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF) are myeloproliferative disorders (MPD) that arise from the clonal proliferation of a pluripotent hematopoietic progenitor, leading to the overproduction of one or more myeloid lineages. Recently, a specific mutation in the JAK2 gene, which encodes a tyrosine kinase, has been shown to be associated with the myeloproliferative phenotype observed in PV, ET and IMF. In this study of Brazilian patients, the JAK2 V617F mutation [c.1887G > T) was detected in four out of 49 patients with PV (96%), 14 out of 25 patients with IMF (56%), and in eight out of 29 patients with ET, which is in accordance with previous screenings of this mutation in other populations.
... (Berlin, 1975; Landolfi, 1998; Streiff et al., 2002). A constant hallmark of PV as well as other MPD bone marrow cells is their hypersensitivity to several cytokines (Prchal and Axelrad, 1974; Dai et al., 1992; Correa et al., 1994; Dai et al., 1994 ) and their ability to generate EPO-independent erythroid colonies in vitro (Prchal and Axelrad, 1974), commonly referred to as endogenous erythroid colonies (EECs). The gene JAK2, codes for a tyrosine kinase and is essential for effective signaling in response to several cytokines (Parganas et al., 1998). ...
... The number of receptors for the growth factors and their dissociation constants are normal. These observations suggest that signal transduction pathways may be altered in these cells [4,[11][12][13] . Imatinib mesylate is a potent inhibitor of Bcr-Abl tyrosine kinase activity. ...
It has been shown that imatinib mesylate, a drug used in the treatment of chronic myelogenous leukemia, inhibits the effect of stem cell factor, which has a central role in erythropoiesis. In some polycythemia vera (PV) patients, it has inhibited autonomous erythroid colony growth in vitro and decreased the need for phlebotomy. In this study we have investigated the effect of insulin like growth factor (IGF)-I, stem cell factor (SCF) and erythropoietin (Epo) with interleukin (IL)-3, granulocyte macrophage-colony stimulating factor (GM-CSF) and granulocyte-colony stimulating factor (G-CSF) in the presence of imatinib mesylate on the erythroid progenitors derived from peripheral blood mononuclear cells of three patients with PV and four healthy controls in semisolid medium. Erythroid colony formation from hematopoietic progenitors obtained from healthy controls was observed only in the presence of all cytokines. However, the number of erythroid colonies could not reach that of patients with PV. Inhibition of imatinib mesylate on erythroid colony growth was evident. Hematopoietic progenitors of patients with PV displayed two types of colony formation: the first type was exogenous cytokine-independent and was hypersensitive to current cytokines, and the second displayed hypersensitivity to current exogenous cytokines, but was exogenous cytokine-dependent. For both types, the inhibitory effect of imatinib mesylate was striking in the presence of all cytokines including IL-3, GM-CSF and Epo. There is no direct evidence yet that imatinib mesylate could inhibit the effect of IL-3, G-CSF, GM-CSF, Epo and IGF-I on erythropoiesis. Considering former studies together with results of this study, it can be argued that imatinib mesylate is effective in PV on the intersecting signal transduction mechanisms in which stem cell factor and its receptor may have a part.
... In retrospect it is not surprising that the EpoR has not been shown to be involved in the pathogenesis of PV. Erythroid progenitors from PV patients have been shown to be hypersensitive not only to Epo but also to a range of growth factors, and in fact, myeloid and megakaryocyte progenitors from these patients are also hypersensitive to growth factors [48,49]. It is likely that PV results from a somatic mutation of a multi-potent progenitor cell leading to dysregulated growth of a clone of abnormal cells; this is reviewed by Hinshelwood et al. [50]. ...
This review will discuss evidence for the role of the erythropoietin (Epo) receptor in the development of erythrocytosis and other hematological disorders. The possible causative role of mutations of other genes in the pathogenesis of idiopathic erythrocytosis will be considered. Polycythemia vera (PV) is a myeloproliferative disorder that is caused by an undefined stem cell abnormality, characterized by a significant erythrocytosis, leukocytosis, and thrombocytosis. However, erythrocytosis may arise from apparent (or relative) polycythemia in which the hematocrit is raised due to a low plasma volume. In such cases the red cell mass is normal. A group of disorders with increased red cell mass caused by stimulation of erythrocyte production is known as secondary polycythemia. Investigation of such patients may reveal a congenital abnormality such as high affinity hemoglobin or an acquired abnormality caused, for example, by smoking, renal vascular impairment, or an Epo-producing tumor. Even after thorough examination there remains a cohort of patients for whom no definite cause for the erythrocytosis can be established. A careful clinical history may reveal whether this idiopathic erythrocytosis is likely to be congenital and/or familial, in which case the term “primary familial and congenital polycythemia” is sometimes applied. Access to a range of laboratory investigations may define the molecular pathophysiology. We will now discuss how this process can be investigated. Am. J. Hematol. 60:55–60, 1999. © 1999 Wiley-Liss, Inc.
... Eine Hyperreaktivität auf "Granulocyte Macrophage colony-stimulating factor" (GM-CSF), "Stem cell factor" (SCF) und "Interleukin-3" (IL-3) wurde für die PV ebenfalls gezeigt (42,43). Hyperreaktivität bedeutet, dass bei einer bestimmten Konzentration eines Zytokins, Zellen von PV-Patienten mehr Kolonien ausbilden als Zellen gesunder Spender. ...
Die Polyzythämia Vera (PV) ist eine von drei malignen hämatologischen Erkrankungen, die unter der Bezeichnung „myeloproliferative Erkrankungen“ zusammengefasst werden. Im Knochenmark dieser Patienten kommt es zu einer verstärkten Bildung erythoider, myeloischer und megakaryozytärer Zellen, woraus ein erhöhter Hämatokrit sowie erhöhte Thrombozyten- und Granulozytenzahlen resultieren. PV-Patienten leiden häufig an Thrombosen und anderen vaskulären Komplikationen. Genexpressionsanalysen in unserem Labor zeigten, dass in PV-Patienten der Transkriptionsfaktor NF-E2 überexprimiert ist. NF-E2 wird in den hämatopoetischen Stamm- und Progenitorzellen, sowie in erythroiden und megakaryozytären Zellen exprimiert. Dies entspricht den Zellreihen, die in der Polyzythämia vera beeinträchtigt sind.
Ziel dieser Arbeit war die Untersuchung des Einflusses des Transkriptionsfaktors NF-E2 auf die Erythropoese. Es konnte gezeigt werden, dass eine Modulation der Expression von NF-E2 in hämatopoetischen Stammzellen signifikante Auswirkungen auf deren Fähigkeit hat, erythroide Kolonien in Methylzellulose zu bilden. So führte in humanen CD34+-Zellen, wie auch in murinen KSL- und KL-Zellen, eine retro- bzw. lentiviral induzierte NF-E2-Überexpression zu einer signifikanten Reduktion des erythroiden Koloniebildungspotenzials dieser Zellen. Im Kontrollexperiment führte eine Reduktion der NF-E2-Expression mittels siRNA zu einer Zunahme der erythroiden Kolonien. Darüber hinaus konnte nachgewiesen werden, dass die Überexpression von NF-E2 ebenfalls die Morphologie der Kolonien humaner CD34+-Zellen verändert. NF-E2-überexprimierende Zellen bildeten fast ausschließlich BFU-Es, wohingegen die mit einem Kontrollvektor transduzierten Zellen zu 70% CFU-Es ausbildeten.
Die Ergebnisse dieser Arbeit zeigen in Zusammenhang mit aktuellen Ergebnissen unserer Arbeitsgruppe, dass eine NF-E2-Überexpression zur Verzögerung der frühen erythroiden Differenzierung führt, welches sich in einer erhöhten Produktion erythroider Zellen je Vorläuferzelle niederschlägt. Die NF-E2-Überexpression liefert somit ein mögliches Modell für die Entstehung der Erythrozytose in PV-Patienten. Weitere Untersuchungen deuten an, dass NF-E2 ebenfalls einen großen Einfluss auf die Megakaryopoese hat. Weitere Untersuchungen müssen nun zeigen, ob NF-E2 einen möglichen therapeutischen Ansatzpunkt darstellen könnte.
This review focuses on polycythemia vera (PV)—its diagnosis, cellular and genetic pathology, and management. In Section I, Dr. Pearson, with Drs. Messinezy and Westwood, reviews the diagnostic challenge of the investigation of patients with a raised hematocrit. The suggested approach divides patients on their red cell mass (RCM) results into those with absolute (raised RCM) and apparent (normal RCM) erythrocytosis. A standardized series of investigations is proposed for those with an absolute erythrocytosis to confirm the presence of a primary (PV) or secondary erythrocytosis, with abnormal and normal erythropoietic compartments respectively, leaving a heterogenous group, idiopathic erythrocytosis, where the cause cannot be established. Since there is no single diagnostic test for PV, its presence is confirmed following the use of updated diagnostic criteria and confirmatory marrow histology.
In Section II, Dr. Green with Drs. Bench, Huntly, and Nacheva reviews the evidence from studies of X chromosome inactivation patterns that support the concept that PV results from clonal expansion of a transformed hemopoietic stem cell. Analyses of the pattern of erythroid and myeloid colony growth have demonstrated abnormal responses to several cytokines, raising the possibility of a defect in a signal transduction pathway shared by several growth factors. A number of cytogenetic and molecular approaches are now focused on defining the molecular lesion(s).
In the last section, Dr. Barbui with Dr. Finazzi addresses the complications of PV, notably thrombosis, myelofibrosis and acute leukemia. Following an evaluation of published data, a management approach is proposed. All patients should undergo phlebotomy to keep the hematocrit (Hct) below 0.45, which may be all that is required in those at low thrombotic risk and with stable disease. In those at high thrombotic risk or with progressive thrombocytosis or splenomegaly, a myelosuppressive agent should be used. Hydroxyurea has a role at all ages, but 32P or busulfan may be used in the elderly. In younger patients, interferon-α or anagrelide should be considered. Low-dose aspirin should be used in those with thrombotic or ischemic complications.
This review focuses on polycythemia vera (PV)—its diagnosis, cellular and genetic pathology, and management. In Section I, Dr. Pearson, with Drs. Messinezy and Westwood, reviews the diagnostic challenge of the investigation of patients with a raised hematocrit. The suggested approach divides patients on their red cell mass (RCM) results into those with absolute (raised RCM) and apparent (normal RCM) erythrocytosis. A standardized series of investigations is proposed for those with an absolute erythrocytosis to confirm the presence of a primary (PV) or secondary erythrocytosis, with abnormal and normal erythropoietic compartments respectively, leaving a heterogenous group, idiopathic erythrocytosis, where the cause cannot be established. Since there is no single diagnostic test for PV, its presence is confirmed following the use of updated diagnostic criteria and confirmatory marrow histology.
In Section II, Dr. Green with Drs. Bench, Huntly, and Nacheva reviews the evidence from studies of X chromosome inactivation patterns that support the concept that PV results from clonal expansion of a transformed hemopoietic stem cell. Analyses of the pattern of erythroid and myeloid colony growth have demonstrated abnormal responses to several cytokines, raising the possibility of a defect in a signal transduction pathway shared by several growth factors. A number of cytogenetic and molecular approaches are now focused on defining the molecular lesion(s).
In the last section, Dr. Barbui with Dr. Finazzi addresses the complications of PV, notably thrombosis, myelofibrosis and acute leukemia. Following an evaluation of published data, a management approach is proposed. All patients should undergo phlebotomy to keep the hematocrit (Hct) below 0.45, which may be all that is required in those at low thrombotic risk and with stable disease. In those at high thrombotic risk or with progressive thrombocytosis or splenomegaly, a myelosuppressive agent should be used. Hydroxyurea has a role at all ages, but 32P or busulfan may be used in the elderly. In younger patients, interferon-α or anagrelide should be considered. Low-dose aspirin should be used in those with thrombotic or ischemic complications.
Myeloproliferative neoplasms (MPNs) are clonal stem cell disorders characterized by the presence of JAK2V617F mutation. Thrombohemorrhagic as well as autoimmune or inflammatory phenomena are common clinical outcomes of these disorders. Recent studies have shown that abnormality in frequency and function of blood cells manifested by an alteration in CD markers’ expression patterns play a key role in these complications. So, there may be a relationship between CD markers’ expressions and prognosis of JAK2V617F positive MPNs. Therefore, in this review, we have focused on these abnormalities from the perspective of changing expressions of CD markers and assessment of the relationship between these changes with prognosis of JAK2V617F positive MPNs. It can be stated that the abnormal expression of a large number of CD markers can be used as a prognostic biomarker for clinical outcomes including thrombohememorrhagic events, as well as autoimmune and leukemic transformation in JAK2V617F positive MPNs. Considering the possible role of CD markers’ expressions in JAK2V617F MPNs prognosis, further studies are needed to confirm the relationship between the expression of CD markers with prognosis to be able to find an appropriate therapeutic approach via targeting CD markers.
Essential thrombocythemia (ET) is a chronic myeloproliferative neoplasm that is associated with diminished quality of life, thrombohemorrhagic complications, and transformation to myelofibrosis (MF) and acute leukemia (AML). The important recent discoveries of driver mutations, including the calreticulin gene in addition to JAK2 and MPL, have led to a greater understanding of disease pathogenesis and set the stage for the advent of more sophisticated prognostic, diagnostic, and therapeutic strategies. In this paper we summarize recent studies describing the molecular basis of ET. We review the prognostic importance of establishing a ‘true’ ET diagnosis, as well as risk factors for the development of adverse outcomes including thrombosis, AML (2% risk at 15 years), and MF (9% risk at 15 years). Finally, we discuss the decision to initiate treatment and assess the quality of evidence supporting the use of established, available therapies as well as novel treatments. Special situations, such as pregnancy, familial ET, and extreme thrombocytosis will also be discussed.
Each member of the family of chronic myeloproliferative disorders (cMPDs) is believed to originate as a regulatory defect in a single pluripotential bone marrow stem cell [1–5]. These disorders are characterized by trilineal marrow hyperplasia with the major clinicopathological emphasis usually on a single cell lineage only [6–8]. The basis for this selective emphasis is at present unknown. In polycythemia vera (PV), it has long been known that erythroid progenitor cells grown in cultures containing serum produce colonies in the absence of added erythropoietin (EPO) [9]. The debate that followed as to whether the cells were independent of, or hypersensitive to, the small quantities of EPO in serum, could not be resolved as long as the cells were grown in media containing serum [10]. In 1994, Paulo Correa and I developed a strictly serum-free medium [11] and applied it to this question. We were able to show that the PV erythroid progenitor cells were not hypersensitive to EPO [10]. In the same year, Fisher and associates found that among the erythroid progenitor cells in PV there were those that proliferated and differentiated in the presence of even high concentrations of EPO- and EPO receptor-neutralizing monoclonal antibodies; the cells were clearly independent of EPO [12]. Then we found that the PV progenitor cells were in fact hypersensitive to another growth factor – insulin-like growth factor-I (IGF-I) [10]. IGF-I, like EPO, we now know stimulates erythropoeisis [11,13].
Hematopoietic progenitor cell survival is mediated via the interaction of stimulatory and inhibitory hematopoietins (i.e., cytokines, growth factors) and their receptors. Among these, erythropoietin (EPO) is the principal growth factor that promotes the survival of erythroid progenitors. Analysis of mutant mice has clearly established an essential role for EPO and its receptor (EPOR) in erythroid development. In embryos of mice deficient in EPO or the EPOR, definitive erythropoiesis is completely impaired and liver tissue contains increased numbers of nucleated erythroid cells undergoing apoptosis[2]. In addition, in vitro studies show that in the absence of EPO, erythroid progenitors die and their genomic DNA is degraded into oligonucleosomal fragments, a feature of apoptotic cell death [2]. Based on these data, a model has been proposed whereby the concentration of circulating EPO controls the number of erythroid progenitors that survive by inhibiting apoptotic cell death [3]. Although the precise mechanisms by which EPO suppresses apoptosis of erythroid cells remains to be elucidated, several recent publications have thrown some interesting light on this issue.
Hematologic diseases often share clinical features within the choroid and retina because of the limited number of cell line or lines affected. No one chorioretinal change is specific for a particular hematologic anomaly; rather, it is the context and pattern that help guide clinical diagnoses. Retinal and choroidal manifestations of hematologic illness may be critical not only in identifying new or recurrent illness but also in ultimately reducing patient morbidity and mortality. Therefore, it is paramount that an open and regular dialogue be maintained between a patients hematologist and ophthalmologist.
The Janus family tyrosine kinases are indispensible for cytokine signaling and play a crucial role in blood cell production. However, their excessive activity causes various hematological phenotypes associated with overproduction of terminally differentiated cells and/or blastic transformation. Here we review the somatic mutations in the Janus family kinases and the associated hematological phenotypes.
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.
Síndromes mieloproliferativas (SMPs) são doenças hematopoéticas de origem clonal que apresentam amplificação de uma ou mais linhagens mielóides. Policitemia vera (PV), trombocitemia essencial (TE), mielofibrose idiopática (MF) e leucemia mielóide crônica (LMC) são consideradas SMPs clássicas e apresentam características clínicas e biológicas comuns. Ao contrário de LMC, cuja etiologia está relacionada à proteína constitutivamente ativa Bcr-Abl, o mecanismo molecular de PV, TE e MF permaneceu por muito tempo desconhecido. Esta revisão se foca na recente descoberta da mutação JAK2 V617F em pacientes com PV, TE e MF, sua relação com o fenótipo mieloproliferativo e implicações na abordagem clínica de pacientes.
: The evolution of myelofibrosis accompanying chronic myeloproliferative disorders (CMPDs) is often linked with megakaryopoiesis. However, it is not known whether or to what extent megakaryocytes of normal human bone marrow are capable of stimulating fibroblast growth. For this reason, an in vitro study was performed to elucidate possible cytokine-dependent interactions between megakaryocytes and fibroblasts derived from healthy volunteers. Fibroblast growth was significantly promoted by the presence of megakaryocytes and modulated by additional application of various cytokines. While recombinant human (rh) interleukin (IL)-1α had no obvious effect on fibroblast proliferation, a slight increase was detected on adding granulocyte-macrophage colony stimulating factor (rhGM-CSF). Application of rhIL-3 caused a significant increase in the number of fibroblasts. In contrast, administration of rhIL-11 suppressed the megakaryocyte-dependent growth-promoting effect and co-stimulation with rhIL-3 led to a significant decrease of fibroblast number in comparison to rhIL-3-stimulated co-cultures. Inhibition of cell-cell contact in unstimulated, as well as in rhIL-3-stimulated co-cultures led to a conspicuous impairment of fibroblast growth. A similar effect was observed when neutralizing antibodies directed against platelet-derived growth factor (PDGF) and transforming growth factor (TGF)β1 were added to rhIL-3-stimulated cultures. Our findings are in keeping with the assumption that interactions between megakaryocytes and fibroblasts involve in cytokine-mediated functional network regulated by factors such as spatial relationship, cytokine stimulation, and low concentrations of mediators, particularly PDGF and TGFβ. In this complex system rhIL-3 seems to play a crucial role in the promotion of these various interrelationships.
The understanding of myeloproliferative neoplasms has changed dramatically since Dameshek proposed his classification over 50 years ago. Our knowledge of the types of cells which constitute the hematopoietic system and of how they are regulated has also appreciated significantly over this time. This review relates what is currently known about the acquired genetic mutations associated with adult myeloproliferative neoplasms to how they lead to the hematopoietic perturbations of myeloproliferative disease. There is a particular focus on how stem and progenitor cell compartments are affected by BCR-ABL1 and JAK2V617F mutations, and the particular issue of resistance of leukemic stem cells to conventional and targeted therapies.
The human myeloproliferative neoplasms (MPN) have long been associated with abnormal responses to cytokines and activation of signaling pathways, although the exact molecular mechanisms underlying these observations were unknown. This situation altered with the discovery of the JAK2 V617F, which presaged the ongoing description of further mutations predicted to activate canonical signaling pathways in MPN. This article covers the nature of these mutations and summarizes functional experiments in model systems and in human MPN cells to define the signaling pathways altered and how these drive and determine the MPN cellular phenotype. Also discussed are recently described, novel noncanonical signaling pathways to chromatin predicted to alter gene transcription more directly and to also contribute to the MPN phenotype.
Essential thrombocythemia (ET) is a myeloproliferative neoplasm essentially characterized by excessive production of platelets. Molecular pathogenesis of ET is linked in approximately half of the patients to intracellular cytokine signaling dysregulation as a result of thrombopoietin receptor or Janus kinase 2 (JAK2) mutations. However, genetic defects underlying cytokine transcription have not been associated with ET. Using molecular cytogenetics and whole-genome array analyses, we uncovered a submicroscopic deletion at 20q13.2 in a JAK2V617F-positive ET patient with an acquired complex chromosome translocation. The deletion encompassed the nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 2 (NFATC2) gene that encodes a transcription factor involved in the regulation of hematopoietic cytokines. RNA interference-mediated suppression of NFATC2 mRNA or pharmacological inhibition of NFATC2 protein with 11R-VIVIT in cultured JAK2V617F-positive SET-2 megakaryocytes increased colony stimulating factor 2 (granulocyte-macrophage) (CSF2) mRNA and promoted cell proliferation. Moreover, impairment of NFATC2-calcineurin interaction with 11R-VIVIT further reduced the transcription of the NFATC2 gene. Antibody-mediated neutralization of CSF2 cytokine in inhibitor-treated cells prevented 11R-VIVIT-induced cell proliferation, indicating that impairment of NFATC2-calcineurin interaction promotes megakaryocyte proliferation through up-regulation of CSF2 transcription. Our results suggest a model in which haplo-insufficiency of NFATC2 cooperates with activation of the JAK-STAT signaling pathway in the pathogenesis of JAK2V617F-positive ET with del(20q). These results further indicate that pathogenesis of ET may be linked to genetic defects of other transcription factor genes involved in the regulation of cytokine expression. © 2012 Wiley Periodicals, Inc.
The pathogenesis of polycythemia vera (PV), a disease involving a multipotent hematopoietic progenitor cell, is unknown. Thrombopoietin (TPO) is a newly characterized hematopoietic growth factor which regulates the production of multipotent hematopoietic progenitor cells as well as platelets. To evaluate the possibility that an abnormality in TPO-mediated signal transduction might be involved in the pathogenesis of PV, we examined TPO-induced protein tyrosine phosphorylation using platelets as a surrogate model system. Platelets were isolated from the blood of patients with PV as well as from patients with other chronic myeloproliferative disorders and control subjects. Impaired TPO-mediated platelet protein tyrosine phosphorylation was a consistent observation in patients with PV as well as those with idiopathic myelofibrosis (IMF), in contrast to patients with essential thrombocytosis, chronic myelogenous leukemia, secondary erythrocytosis, iron deficiency anemia, hemochromatosis, or normal volunteers. Thrombin-mediated platelet protein tyrosine phosphorylation was intact in PV platelets as was expression of the appropriate tyrosine kinases and their cognate substrates. However, expression of the platelet TPO receptor, Mpl, as determined by immunoblotting, chemical crosslinking or flow cytometry was markedly reduced or absent in 34 of 34 PV patients and also in 13 of 14 IMF patients. Impaired TPO-induced protein tyrosine phosphorylation in PV and IMF platelets was uniformly associated with markedly reduced or absent expression of Mpl. We conclude that reduced expression of Mpl is a phenotypic characteristic of platelets from patients with PV and IMF. The abnormality appears to distinguish PV from other forms of erythrocytosis and may be involved in the platelet function defect associated with PV.
Megakaryocytes are difficult to isolate because of their fragility, their tendency to aggregate, and their varying sizes. For purification of cells at different stages of maturation and of different sizes (ploidy classes) we developed an immunomagnetic cell sorting method (MACS) to enrich the whole spectrum of the megakaryocytic cell lineage. The use of small magnetic beads coupled to various antibodies and labelling with fluorescent antibodies allowed direct analysis of enrichment and evaluation of the isolated fraction without further staining or detachment procedures. CD 61 (Y2/51), a monoclonal antibody directed against platelet glycoprotein IIIa, was employed to perform the separation procedure. An enrichment up to 47% of CD 61-positive cells with an average of 37% and a recovery rate of 37% was obtained by using the MACS technique. Pre-enrichment by Percoll density centrifugation, followed by MACS separation, resulted in an enrichment of 65% and a recovery rate of 67%. The relative amount of small megakaryocytic cells in only MACS-enriched cell populations, however, was higher than in Percoll/MACS fractions. As a parameter of vitality we tested cytokine secretion of the enriched megakaryocytes in reverse haemolytic plaque assays. Secretion of IL-1, IL-6, GM-CSF, and PDGF with and without stimulation by phorbol myristate acetate was demonstrable at the single cell level.
The classic chronic myeloproliferative disorders (MPDs) polycythemia vera (PV), essential thrombocythemia (ET), and idiopathic
myelofibrosis (MF) have been historically grouped together because of their overlapping clinical phenotypes. The recent discovery
of a single acquired point mutation (V617F) in the Janus kinase 2 (JAK2) gene in most patients with PV and about half of ET and MF patients provides a new starting point for exploring the genetic
and biologic mechanisms of disease pathogenesis in these related disorders. This chapter highlights the different roads of
discovery to the JAK2 V617F mutation, both in vitro and in vivo experimental data relevant to the mutant JAK2 tyrosine kinase, and new questions raised by the discovery.
To examine the possible involvement of protein kinase C (PKC) in the regulation of aberrant erythropoiesis of polycythemia vera (PV), we investigated the effects of PKC inhibitors on in vitro burst-forming unit of erythroid (BFU-E)-derived colony formation by bone marrow (BM) and peripheral blood (PB) cells obtained from five PV patients. 1-(Isoquinoline-sulfonyl)-2-methylpiperazine dihydrochloride (H-7), an inhibitor of PKC, suppressed the colony formation by BM and PB cells of PV patients in a dose-dependent manner, similar to those in the normal individuals. However, the 50% inhibitory concentrations (IC50) of H-7 in PV BM and PB cells were significantly higher than those in normal BM and PB cells, respectively. The BFU-E-derived colony formation by PV BM and PB cells was also less affected by Staurosporine, another PKC inhibitor, than those in a normal subject. Furthermore, in the study of PV, the IC50 of endogenous colonies formed in the absence of erythropoietin was much higher than that of colonies formed by the stimulation of erythropoietin. By contrast, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide dihydrochloride (HA1004), a cyclic AMP-dependent kinase inhibitor, did not have such inhibitory effects. These findings suggest that PKC, as a second messenger, is involved in the regulation of aberrant erythropoiesis of PV.
ANCA-Associated Systemic Vasculitis (AASV) is characterized by leukocytoclasis, accumulation of unscavenged apoptotic and necrotic neutrophils in perivascular tissues. Dysregulation of neutrophil cell death may contribute directly to the pathogenesis of AASV.
Neutrophils from Healthy Blood Donors (HBD), patients with AASV most in complete remission, Polycythemia Vera (PV), Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA) and renal transplant recipients (TP) were incubated in vitro, and the rate of spontaneous apoptosis was measured by FACS. Plasma levels of cytokines and sFAS were measured with cytometric bead array and ELISA. Expression of pro/anti-apoptotic factors, transcription factors C/EBP-α, C/EBP-β and PU.1 and inhibitors of survival/JAK2-pathway were measured by real-time-PCR.
AASV, PV and RA neutrophils had a significantly lower rate of apoptosis compared to HBD neutrophils (AASV 50 ± 14% vs. HBD 64 ± 11%, p<0.0001). In RA but not in AASV and PV, low apoptosis rate correlated with increased plasma levels of GM-CSF and high mRNA levels of anti-apoptotic factors Bcl-2A1 and Mcl-1. AASV patients had normal levels of G-CSF, GM-CSF and IL-3. Both C/EBP-α, C/EBP-β were significantly higher in neutrophils from AASV patients than HBD. Levels of sFAS were significantly higher in AASV compared to HBD.
Neutrophil apoptosis rates in vitro are decreased in AASV, RA and PV but mechanisms seem to differ. Increased mRNA levels of granulopoiesis-associated transcription factors and increased levels of sFAS in plasma were observed in AASV. Additional studies are required to define the mechanisms behind the decreased apoptosis rates, and possible connections with accumulation of dying neutrophils in regions of vascular lesions in AASV patients.
Acute myeloid leukemia (AML) is one of the most common leukemias with a 20% 5-year event-free survival in adults and 50% overall survival in children, despite aggressive chemotherapy treatment and bone marrow transplantation. The incidence and mortality rates for acute leukemia have only slightly decreased over the last 20 years, and therefore greater understanding of the molecular mechanisms associated with leukemic progression is needed. To this end, a number of transcription factors that appear to play a central role in leukemogenesis are being investigated; among them is the cAMP response element binding protein (CREB). CREB is a transcription factor that can regulate downstream targets involving in various cellular functions including cell proliferation, survival, and differentiation. In several studies, the majority of bone marrow samples from patients with acute lymphoid and myeloid leukemia demonstrate CREB overexpression. Moreover, CREB overexpression is associated with a poor outcome in AML patients. This review summarizes the role of CREB in leukemogenesis.
Myelofibrosis (MF; primary or post-polycythemia vera/essential thrombocythemia) carries the worst prognosis among BCR-ABL-negative myeloproliferative neoplasms (MPNs). Stem cell transplantation is the only curative approach but is hampered by significant nonrelapse mortality. Thus, effective, targeted therapies are needed. A mutated Janus kinase 2 (JAK2) gene (JAK2(V617F)), found in a significant portion of patients with MPN, results in increased JAK2 tyrosine kinase activity, leading to clonal proliferation; several small molecules inhibit the growth of hematopoietic colonies harboring JAK2(V617). Several JAK2 inhibitors have reached the clinical trial stage and are reviewed here. The most developed among them is INCB018424, which has demonstrated noteworthy clinical activity, with a rapid and profound reduction in splenomegaly and associated improvement in constitutional symptoms in MF patients receiving 10-25 mg orally twice daily, continuously. Thrombocytopenia (reversible) was the most common adverse event, seen in 30% of patients treated with 25 mg twice daily but not with 10 mg twice daily. Interestingly, INCB018424 was equally active in patients with and without JAK2 mutation. Other JAK2 inhibitors are less developed but show a similar type of clinical benefit. Conclusively, JAK2 inhibitors, particularly INCB018424, are clinically active in MF and are well tolerated. Whether they have an effect on the natural course of MF in treated patients remains to be elucidated.
The discovery of the JAK2V617F mutation followed by the discovery of JAK2 exon 12 and MPLW515 mutations has completely modified the understanding, diagnosis, and management of the classic myeloproliferative disorders (MPDs), which include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Nonetheless, genetic defects have not yet been identified in about 40% of ET and PMF. There is now strong evidence that these mutations are the oncogenic events that drive these disorders and are responsible for most biologic and clinical abnormalities. In addition, there are convincing data indicating that the number of JAK2V617F copies (homozygosity vs. heterozygosity) is important in explaining how a single mutation can be associated with several disorders. However, it is still uncertain whether these mutations are sufficient to explain the full development, heterogeneity, and progression of MPD, or if other genetic or epigenetic events are also necessary. In this review, we discuss different hypothetical models of MPD pathogenesis supported by recent findings. Further characterization of the molecular events operating in these disorders will be essential in fully understanding their pathogenesis and in developing new therapeutic approaches.
Polycythemia vera and essential thrombocythemia are the most common chronic myeloproliferative neoplasms; their molecular basis has been appreciated only recently and is briefly discussed in this article. Major causes of morbidity and mortality are represented by arterial and venous thrombosis, as well as by evolution to myelofibrosis or transformation to acute leukemia. Therapy is currently aimed at reducing the rate of thrombosis without increasing the risk of hematologic transformation that might be caused by exposure to cytotoxic drugs. A risk-oriented approach is employed for stratifying patients to the most appropriate therapeutic options. However, results of clinical trials with interferon, and the expected effects of novel drugs selectively targeting the abnormal pathways that are involved in the clonal myeloproliferation, are pushing therapeutic goals from disease control only to cure. These different issues, and current recommendations for treatment, will be discussed in the review.
Dysregulated signaling is a hallmark of chronic myeloproliferative neoplasms (MPNs), as evidenced by the identification of the activating JAK2 V617F somatic mutation in almost all patients with polycythemia vera (PV) and 50-60% of essential thrombocythemia and primary myelofibrosis patients. These disorders are clinically distinct, raising the question of how a single mutation can result in such phenotypic diversity. Mouse models have demonstrated that the level of JAK2 V617F expression can modulate the phenotype, and clinical studies of JAK2 V617F allele burden have reported similar findings. It has also been hypothesized that one or more pre-JAK2 V617F events may modify the MPN phenotype. However, the molecular basis of JAK2 V617F-negative essential thrombocythemia and primary myelofibrosis remains largely unexplained. Mutations in the TET2 gene have been identified in both JAK2 V617F-positive and -negative MPNs and other myeloid neoplasms, but their functional and clinical significance have yet to be clarified. In addition, recent reports have identified a specific germline haplotype that increases the predisposition to MPNs. The role of inhibitory pathways (e.g., SOCS and LNK) in regulating JAK-STAT signaling in MPNs is being increasingly recognized. The implications of these findings and their clinical relevance are the focus of this article.
The discovery of a common Janus kinase 2 (JAK2) point mutation, JAK2V617F, in myeloproliferative neoplasms has generated enormous interest in the development and therapeutic use of small molecule JAK2 inhibitor-targeted therapy in these diseases. A handful of compounds are currently in clinical development in primary myelofibrosis or post-polycythemia vera (PV)/essential thrombocythemia (ET) myelofibrosis. To date, clinical benefit has been demonstrated in terms of reduction of splenomegaly, improvement in constitutional symptoms, and control of leukocytosis. Some of the drugs have also been evaluated in PV and ET, with demonstrated activity against erythrocytosis, thrombocytosis, pruritus, and splenomegaly. However, drug effect on bone marrow fibrosis or JAK2 allele burden has been modest so far. Regardless, it is important to keep in mind that current anti-JAK2 treatment trials constitute only the beginning of many upcoming similar clinical trials, and that it is premature to make generalizations or any form of comparative conclusions regarding drug activity or toxicity.
Analyse der Transkritionsfaktor Bindung an den Promotor von prv-1, das in Granulozyten von Patienten mit Polycythaemia rubra vera überexprimiert wird. Characterization of the transcription factor binding sites of the promotor of prv-1. Prv-1 is overexpressed in neurophil granulocytes of pationts with polycythaemia rubra vera.
Auf der Suche nach der molekularbiologischen Ursache der Erkrankung "Polycythaemia vera" wurden mehrere Kandidatengene in einem Screening-Verfahren (Subtraktive Hybridisierung) gefunden. In dieser Arbeit wurden zwei dieser Kandidatengene, PRV-1 (Projekt I) und AB3 (Projekt II), näher untersucht.
Um Aussagen über die Funktion von PRV-1 treffen zu können, sollten mittels eines Verfahrens zur Detektion von Protein-Protein-Interaktionen potentielle Bindungspartner von PRV-1 aufgespürt werden. Aufgrund der verhältnismäßig physiologischen Bedingungen wurde hierzu eine Yeast-two-hybrid-Screen verwendet. Dabei wurde PRV-1 ohne die natürliche Leadersequenz in-frame in den Bait-Vektor pGBKT7 kloniert. Anschließend wurde das Bait-Konstrukt in den Hefestamm Sacharomyces cerevisiae AH109 transformiert. Die korrekte Protein-Expression von PRV-1 in S. cerevisiae wurde durch Western Blots mit Antikörpern gegen PRV-1 und das im Bait-Vektor kodierte c-myc Tag überprüft. Nach Phänotypentestung des bait-tragenden Hefestammes wurde das Screening einer "Pretransformed cDNA Human Bone Marrow Library" durch Mating durchgeführt. Insgesamt wurden dabei 307, auf Quadrupel Dropout Medium wachstumsfähige, Kolonien gefunden. Aus drei exemplarischen Kolonien wurden dann die Prey-Plasmide in einem zweischrittigen Prozess aufgereinigt und die funktionelle Prey-Bait-Interaktion ihres Proteinproduktes nach Retransfomation bestätigt. Nach Sequenzierung konnten die Prey-Plasmide schließlich durch Datenbankabgleich, soweit bekannt, identifiziert werden.
Um mehr über das Gen AB3 heraus zu finden, sollte mittels einer 5' RACE mehr Sequenzinformation von AB3 aufgeklärt werden. Durch einen, mit einer 32P-gelabelten AB3-Sonde hybridisierten, Northern Territory RNA Blot gelang es, Lungengewebe als ein Gewebe zu identifizieren, in dem AB3 deutlich exprimiert wird. In vier Runden 5-RACE mit der "Marathon Ready cDNA Library: Human Lung" gelang es insgesamt 4230bp neue 5'cDNA-Sequenz aufzuklären und die bekannte AB3 cDNA-Sequenz damit von 3441bp auf 7671bp zu verlängern. Durch Datenbankvergleiche mit anderen Genen konnten zwei Domänen postuliert werden. Zusammenfassend handelt es sich bei AB3 wahrscheinlich um eine Tyrosinkinase mit C-terminaler katalytischer Tyrosinkinasedomäne und etwas N-terminalerer leucin-rich repeat Domäne. Da innerhalb der bekannten Sequenz keine Transmembran-Domäne gefunden wurde, könnte AB3 somit als intrazelluläre Tyrosinkinase an der Signaltransduktion beteiligt sein.
Die Polycythaemia Rubra Vera (=PV) ist eine klonale Stammzellerkrankung des hämatologischen Systems. PRV-1 (=Polyzythämia Rubra Vera-1) ist ein hämatologischer Oberflächenrezeptor, dessen mRNA in den Granulozyten von nahezu allen PV Patienten, sowie bei einigen Patienten mit Osteomyelofibrose, mit Essentieller Thrombozythämie, bei entzündlichen Erkrankungen und nach G-CSF-/ GM-CSF-Gabe überexprimiert wird. Da bei sekundären Erythrozytosen und Leukämien diese Überexpression nicht besteht, kann PRV-1 als molekularer Marker zur Diagnose der PV beitragen.
In der vorliegenden Doktorarbeit wurde nach Interaktionspartnern für PRV-1 gesucht, um darüber Hinweise auf dessen bisher unbekannte Funktion zu erhalten. Dazu wurden 153 aus einem mit einer Knochenmark cDNA Bank durchgeführten Yeast Two Hybrid Assay hervorgegangene positive cDNA-Klone isoliert. Nach Sequenzierung wurden die Klone durch Abgleich mit der NCBI Datenbank identifiziert. Davon wurden acht cDNAs, die potentiell für eine in vivo Interaktion mit PRV-1 in Frage kommen, zur weiteren Analyse ausgewählt. Da ein mit eukaryontisch exprimiertem Protein durchgeführter GST Pull Down Assay keine konsistenten Ergebnisse lieferte, wurde ein bakterieller GST Pull Down Assay etabliert. Darüber konnte für die Proteine Decorin, C1qB und Defensin in vitro eine physikalische Interaktion mit PRV-1 nachgewiesen werden. Während C1qB und Defensin eine schwächere Interaktion zeigten, ließ sich für Decorin eine starke Bindung an PRV-1 nachweisen. Da die cDNA Bank oft nur ein Fragment der Proteine enthielt, kann die für die Bindung an PRV-1 verantwortliche Region näher eingegrenzt werden.
Decorin ist durch Interaktionen mit verschiedenen Wachstumsfaktoren an der Regulation der Proliferation, Apoptose, Aktivierung und Adhäsion von Zellen im Rahmen von entzündlichen, tumorösen und fibrotischen Prozessen beteiligt. C1q als erste Komponente des Komplementsystems aktiviert einerseits über eine Bindung an die Fc-Domäne von Antikörpern die Komplementkaskade und sorgt andererseits über direkte Bindung an Makrophagen und Granulozyten für deren Aktivierung. Das von Granulozyten synthetisierte Defensin-α3 wirkt chemotaktisch auf Makrophagen und moduliert die Zytokinproduktion.
Die hier nachgewiesenen Interaktionen legen daher einen Einfluss von PRV-1 auf die Kontrolle von Wachstum, Überleben und Funktion von myeloischen Zellen, sowohl bei entzündlichen Prozessen als auch im Rahmen der Pathogenese der PV nahe.
Juvenile chronic myelogenous leukemia (JCML) is a good model for the study of myeloproliferation because JCML hematopoietic progenitor cells grow in vitro at very low cell densities without the addition of exogenous stimulus. Previous studies have demonstrated that this proliferation is dependent on granulocyte-macrophage colony-stimulating factor (GM-CSF), and that removal of monocytes from the cell population before culture eliminates this "spontaneous" myeloproliferation, suggesting a paracrine role of monocyte stimulation. However, subsequent studies have shown that increased GM-CSF production from the JCML monocytes is not a consistent finding and therefore not a plausible sole mechanism. In examining hematopoietic growth factor dose-response curves, both JCML GM and erythroid nonadherent progenitor cell populations displayed a marked and selective hypersensitivity to GM-CSF. Responses to interleukin-3 and G-CSF were identical to control dose-response curves. This is the first demonstration of a myeloid leukemia in which hypersensitivity to a specific growth factor appears to be involved in the pathogenesis of the disease.
We investigated the effect of interleukin-6 (IL-6) on murine megakaryocytopoiesis in a serum-free culture system. The addition of IL-6 to a culture containing interleukin-3 (IL-3) resulted in a significant increase in the number of megakaryocyte colonies by bone marrow cells of normal mice. The megakaryocytic progenitors that survive exposure to 5-fluorouracil (5-FU) exhibited a more significant response to IL-6 and IL-3. Polyclonal anti-IL-6 antibody neutralized the stimulatory effect of IL-6 on megakaryocyte colony growth supported by IL-3. Delayed addition experiments and replating experiments of blast cell colonies showed that megakaryocytic progenitors are supported by IL-3 in the early stage of the development but require IL-6 for their subsequent proliferation and differentiation. In addition, IL-6 increased the size of megakaryocytes in granulocyte-macrophage-megakaryocyte colonies. The combination of granulocyte colony-stimulating factor or granulocyte-macrophage colony stimulating factor with IL-3 resulted in an increase in the granulocyte-macrophage colony growth of bone marrow cells of 5-FU-treated mice or normal mice, respectively, but had little effect on the enhancement of pure and mixed megakaryocyte colony growth. These results suggest that IL-6 plays an important role in murine megakaryocytopoiesis.
The growth-promoting activities of interleukin-6 (IL-6) in combination with different factors were assessed in bone marrow (BM) cultures prepared from normal mice and from mice treated with 5-fluorouracil (5-FU). Effects on hematopoietic colony formation with respect to number, size, and cellular composition were evaluated. In agreement with previous reports, IL-6 acts synergistically with IL-3 to stimulate increased numbers of granulocyte/macrophage (GM) and multilineage colonies in day-2 and day-4 post-5-FU BM cultures. Furthermore, day 4 but not day 2 post-5-FU BM showed enhanced GM colony formation when stimulated with IL-6 plus interleukin-4 (IL-4) or granulocyte colony-stimulating factor (G-CSF). In contrast, IL-6 did not increase the number of colonies supported by M-CSF or GM-CSF. Nevertheless IL-6 interacted with all factors, including M-CSF and GM-CSF, to stimulate an increase in colony size. Many of these myeloid colonies attained a diameter of greater than or equal to 0.5 mm, suggesting they derive from high proliferative potential cells (HPP-CFC). The response of normal and day-8 post-5-FU BM containing high numbers of more mature progenitors was also assessed. We found IL-6 enhanced colony formation by lineage-restricted megakaryocytic and erythroid progenitors in the presence of IL-3 and IL-4 plus erythropoietin (Epo), respectively. The sum of these results shows that IL-6 interacts with a variety of factors to regulate the growth of progenitor cells at different stages of lineage commitment and maturation.
Interleukin-6 (IL-6, also known as B-cell stimulatory factor 2/interferon beta 2) was previously shown to support the proliferation of granulocyte/macrophage progenitors and indirectly support the formation of multilineage and blast cell colonies in cultures of spleen cells from normal mice. We report here that IL-3 and IL-6 act synergistically in support of the proliferation of murine multipotential progenitors in culture. The time course of total colony formation by spleen cells isolated from mice 4 days after injection of 5-fluorouracil (150 mg/kg) was significantly shortened in cultures containing both lymphokines relative to cultures supported by either of the two factors. Serial observations (mapping) of individual blast cell colonies in culture revealed that blast cell colonies emerged after random time intervals in the presence of IL-3. The average time of appearance in IL-6 alone was somewhat delayed, and in cultures containing both factors the appearance of multilineage blast cell colonies was significantly hastened relative to cultures grown in the presence of the individual lymphokines. In cultures of day-2 post-5-fluorouracil bone marrow cells, IL-6 failed to support colony formation; IL-3 alone supported the formation of a few granulocyte/macrophage colonies, but the combination of factors acted synergistically to yield multilineage and a variety of other types of colonies. In this system, IL-1 alpha also acted synergistically with IL-3, but the effect was smaller, and no multilineage colonies were seen. Together these results indicate that IL-3 and IL-6 act synergistically to support the proliferation of hemopoietic progenitors and that at least part of the effect results from a decrease in the G0 period of the individual stem cells.
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.
We have found that the peripheral blood of normal humans contains a significant number of committed erythroid stem cells of high proliferative capacity. These erythroid stem cells closely resemble the murine erythroid burst-forming unit (BFU-E) with respect to proliferative capacity, colony morphology, and erythropoietin requirement. BFU-E were isolated from the peripheral blood of normal individuals by Ficoll-Hypaque density gradient centrifugation and cultured in vitro using the plasma culture technique. Macroscopic erythroid colonies of between 100 and 1000 cells were observed after 10-14 days of culture in the presence of either sheep or human erythropoietin at 0.5-4 units/ml. Individual colonies contained between 3 and 20 subcolonies and reached a maximum mean size of approximately 500 cells. Colony number was linearly related to the cell input, suggesting that a single cellular entity was precursor to each colony. The frequency of cells capable of giving rise to an erythroid colony was at least 100 per ml of blood in a number of individuals tested. The ability to assay significant numbers of erythroid precursor cells of high proliferative capacity from normal peripheral blood should facilitate the study of both normal erythropoiesis and of disease states affecting erythropoiesis in which marrow samples are not available on a routine basis.
We performed in vitro culture studies examining the interaction of erythropoietin with red cell progenitors in polycythemia vera. Bone marrow was obtained from five patients with typical disease and from five healthy volunteers, and assayed for erythroid colony formation (CFU-E) by the methylcellulose technique. In cultures without added erythropoietin, a mean eightfold greater cloning efficiency was noted with the polycythemia vera marrows, as compared to normal. There was prominent stimulation of colony formation by erythropoietin, and the shape of the erythropoietin dose-response cruves appeared to be similar in both patients and controls. Anti-erythropoietin antibody reduced the number of CFU-E in cultures not containing added erythropoietin, but did not eliminate them. Dexamethasone (10(-9) M) caused a consistent increase in CFU-E in the patients' cultures. These studies provide evidence for functional erythropoietin and glucocorticosteroid receptor mechanisms on erythroid precursors in polycythemia vera. The observations are consistent with a concept of this disease as a disorder of hematopoietic stem cells in which peripheral erythrocytosis is caused by an expanded erythroid progenitor compartment which maintains responsiveness to hormonal modulation.
Two women with polycythemia vera and heterozygosity (GdB/GdA) at the X-chromosome-linked locus for glucose-6-phosphate dehydrogenase were studied to determine the nature of the cellular origin of their polycythemia. In contrast to unaffected tissue, such as skin fibroblasts, which consisted of both B and A types, the glucose-6-phosphate dehydrogenase of the patients' erythrocytes, granulocytes and platelets was only of Type A. These results provide direct evidence for the stem-cell nature of polycythemia vera and strongly imply a clonal origin for this disease. The fact that no descendants of the presumed normal stem cells were found in circulation suggests that bone-marrow proliferation in this disorder is influenced by local (intramarrow) regulatory factors.
To understand the factors that regulate the early growth and development of immature erythroid progenitor cells, the burst-forming units-erythroid (BFU-E), it is necessary to have both highly purified target cells and a medium free of serum. When highly purified human blood BFU-E were cultured in a serum-free medium adequate for the growth of later erythroid progenitors, BFU-E would not grow even with the addition of recombinant human interleukin-3 (rIL-3), known to be essential for these cells. However, the addition of recombinant human stem cell factor (rSCF), which supports germ cell and pluripotential stem cell growth, stimulated BFU-E to grow equally well in serum-free as in serum-containing medium. Limiting dilution studies showed that rSCF acts directly on the BFU-E that do not require accessory cells for growth. Furthermore, rSCF was necessary for BFU-E development during the initial 7 days of culture, until these cells reached the stage of the late progenitors, the colony-forming units-erythroid (CFU-E). These studies indicate that early erythropoiesis is dependent on the direct action of SCF that not only affects early stem cells but is continually necessary for the further development of committed erythroid progenitor cells until the CFU-E stage of maturation.
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.
The presence of heterogeneous erythroid progenitor cells, contaminant cells, or serum may alter erythroid colony development in vitro. To obtain highly purified colony-forming units-erythroid (CFU-E), we cultured partially purified human blood burst-forming units-erythroid (BFU-E) in methylcellulose with recombinant human erythropoietin (rHuEPO) for 7 d and generated cells that consisted of 30-60% CFU-E, but no BFU-E. A serum-free medium was used that allowed development of the same number of erythroid colonies as serum containing medium, but with a greater percentage of larger colonies. This medium consisted of delipidated crystalline bovine serum albumin, iron saturated transferrin, lipid suspension, fibrinogen, thrombin, Iscove's modified Dulbecco's medium/F-12[HAM], and insulin plus rHuEPO. When CFU-E were cultured in a limiting dilution assay and the percentage of nonresponder wells was plotted against cell concentration, both serum-free cultures and serum-containing cultures yielded overlapping straight lines through the origin indicating that CFU-E development did not depend on accessory cells and that insulin acted directly on the CFU-E. Human recombinant interleukin 3 (IL-3) and/or granulocyte-macrophage colony-stimulating factor had no effect on CFU-E growth, while they markedly enhanced BFU-E growth. Physiological concentrations of recombinant human insulin-like growth factor I (IGF-I) enhanced CFU-E growth in the absence of insulin and, together with rHuEPO in serum-free medium, provided a plating efficiency equal to that of serum-containing medium. Limiting dilution analysis in serum-free medium with IGF-I showed a straight line through the origin indicating that IGF-I also acted directly on the CFU-E and not through an effect on accessory cells. These data demonstrate that CFU-E do not require accessory cells, but do require IGF-I and/or insulin which act directly on the CFU-E.
The antigenic characteristics of erythropoietin (Ep) dependent and independent ("endogenous") erythroid progenitor cells (BFU-E and CFU-E) from patients with polycythaemia vera (PV) and idiopathic myelofibrosis (MF) were studied using 9 selected murine monoclonal antibodies (McAbs) in a complement dependent cytotoxicity assay followed by culture in methyl cellulose. McAbs L343 (HLA-DR antigen) S3-13 and S17-25 reacted with about 50% of BFU-E in MF and PV. In contrast HLA-DR antigens were not presented on CFU-E. McAbs R1.B19, WHGS29.1, PMN29 and PM81 recognized the antigens on less than 25% of BFU-E and CFU-E. The reactivity of Ep-dependent and Ep-independent BFU-E and CFU-E was similar with the majority of McAbs. However, McAbs S4-7 and VIM-2 reacted with a higher proportion of Ep-independent BFU-E than of Ep-dependent BFU-E. These results indicate that Ep independent, neoplastic BFU-E in PV and MF are more mature than their Ep dependent counterparts. The antigens expressed on erythroid progenitor cells from patients with PV and MF were earlier found on BFU-E and CFU-E from normal bone marrow and peripheral blood, usually on a higher proportion of cells, than in myeloproliferative disorders.
Marrow cells from seven untreated patients with polycythemia vera (PV) were used to initiate standard single inoculum long-term marrow cultures. The numbers, erythropoietin independence, and cycling behavior of all detectable classes of erythroid, granulopoietic, and multilineage progenitors were then evaluated and the results obtained compared with preculture values. Time course studies showed that the long-term marrow culture system supports the continuous proliferation of primitive neoplastic progenitor cells from PV patients for many weeks. However, these progenitors fail to respond to signals from the adherent layer that return their counterparts in normal long-term marrow cultures to a quiescent state 5-7 d after each medium change. This abnormal cycling behavior of PV cells in the long-term culture system appears to mimic that operative in vivo, where primitive hemopoietic progenitors are also in a continuous state of turnover, in contrast to similar primitive progenitor compartments in normal individuals, which are quiescent. The long-term marrow culture system thus offers new possibilities for the further analysis of abnormal cellular and molecular mechanisms underlying clonal expansion at the stem cell level in PV.
The My-10 glycoprotein is an hematopoietic cell surface antigen expressed specifically by undifferentiated (blast) cells, constituting 1%-4% of normal adult bone marrow leukocytes. We used several immunological and in vitro culture methods to analyze the expression of this unique antigen on a variety of lymphohematopoietic progenitor cells. Colony-forming cells (CFC) for granulocyte-monocyte colonies (CFC-GM) and erythroid colonies (BFU-E) were predominantly My-10 positive. CFC with higher proliferative potential were more strongly My-10 positive than CFC with lower proliferative potential, and those for mixed-lineage and blast cell colonies were even more uniformly My-10 positive. Cells maintaining CFC-GM number in short-term marrow culture (pre-CFC) were found to be My-10 positive, as were lymphoid precursors defined by their content of intranuclear terminal deoxynucleotidyl transferase. More mature erythroid precursors (CFU-E) were heterogeneous for antigen expression and lost My-10 antigen progressively, in parallel with advancing maturational stage. The My-10 antigen permits rapid identification and purification of hematopoietic progenitor cells for further study or potential clinical application. The disappearance of the My-10 antigen, moreover, may be a probe for differentiation-linked cellular events.
An improved plasma culture system is described for the production of erythrocytic colonies by mammalian adult hemopoietic cells in vitro under the influence of erythropoietin. The concentration of fetal calf serum in the medium used for dilution of the cells was critical for erythrocytic colony formation when low numbers of cells were plated. Optimal concentrations were found for plasma, fetal calf serum, bovine serum albumin, and L-asparagine in the culture medium, and the colony-forming efficiency was shown to depend on the concentration of erythropoietin. With erythropoietin at plateau concentration, the number of erythrocytic colonies produced was directly proportional to the number of bone marrow or spleen cells plated, over a wide range of cell concentrations. Colony numbers per culture conformed to a Poisson distribution. Thus, the improved plasma culture system may be used for the quantitative assay of CFU-E. The method is rapid (2 days), reliable, convenient, and inexpensive. Since the improved plasma culture system also supports granulocytic colony formation by bone marrow cells in the presence of conditioned medium (CSA), and the number of granulocytic colonies produced is proportional to the number of cells plated, the same hemopoietic cell suspensions can be simultaneously assayed for CFU-E and CFU-C under virtually identical conditions.
The anti-My-10 mouse monoclonal antibody was raised against the immature human myeloid cell line KG-1a and was selected for nonreactivity with mature human granulocytes. Anti-My-10 immunoprecipitated a KG-1a cell surface protein with an apparent Mr of approximately 115 kD. We describe the binding of this antibody to human hematopoietic cell types and show that My-10 is expressed specifically on immature normal human marrow cells, including hematopoietic progenitor cells. My-10 is also expressed by leukemic marrow cells from a subpopulation of patients. Thus, this antibody allows the identification and purification of hematopoietic progenitor cells from normal human marrow and the subclassification of leukemia.
We measured serum concentrations of erythropoietin in 59 patients with polycythemia using a sensitive and specific radioimmunoassay. The mean concentration was 17.5 +/- 8.4 mU/mL (+/- SD) in 26 patients with polycythemia vera and 14.9 +/- 4.2 mU/mL in 26 normal persons. In contrast, the average concentration was 94.3 +/- 101.2 mU/mL in 33 patients with secondary polycythemia, representing a highly significant elevation (p < 0.0001) compared to both normal and polycythemia vera groups. The average hematocrit value did not differ between the polycythemia vera and the secondary polycythemia patients, and both groups had higher values (median, 55%) than the normal donors (median, 41%). Erythropoietin concentrations ascertained by radioimmunoassay helped discriminate between polycythemia vera and secondary polycythemia. Ninety-two percent of polycythemia vera patients had concentrations less than 30 mU/mL (the concentration used as a cut off point), and 94% of secondary polycythemia patients had concentrations greater than 30 mU/mL. This represents an overall correct classification of 93% of the patients. Serum erythropoietin levels as ascertained by radioimmunoassay can distinguish between most polycythemia vera and secondary polycythemia patients and should prove useful in the differential diagnosis of polycythemia.
Polycythemia vera: Spontaneous growth of hemoglobinized colonies is mediated by adherent cells
- C Nissen
- E Hasler
- K Moor
- Y Moser
- Speck
Nissen C, Hasler E, Moor K, Moser Y, Speck B: Polycythemia vera: Spontaneous growth of hemoglobinized colonies is mediated by adherent cells. Exp Hematol 14549,1986 (abstr)
Intracellular growth factors in polycythemia vera and other myeloproliferative disorders Cashman JD, Eaves CJ, Eaves AC: Unregulated prolifera-tion of primitive neoplastic progenitor cells in long-term polycythemia vera marrow cultures
- J Eid
- Rf Ebert
- Ms Gesell
- J Spivak
Eid J, Ebert RF, Gesell MS, Spivak J L Intracellular growth factors in polycythemia vera and other myeloproliferative disorders. Proc Natl Acad Sci USA 84532,1987 32. Cashman JD, Eaves CJ, Eaves AC: Unregulated prolifera-tion of primitive neoplastic progenitor cells in long-term polycythemia vera marrow cultures. 3 Clin Invest 81537, 1988
An antagonist to IL-3 produced by mouse cells (abstr, suppl 1) For personal use only
- Fg Pluthero
- M Shreeve
- D Eskinazi
- Axelrad
Pluthero FG, Shreeve M, Eskinazi D, Axelrad AA: An antagonist to IL-3 produced by mouse cells. Blood 7474a, 1989 (abstr, suppl 1) For personal use only. by guest on July 24, 2011. bloodjournal.hematologylibrary.org From
colonies in a serum-free system: Results in primary proliferative polycythemia and thrombocythemia
colonies in a serum-free system: Results in primary proliferative polycythemia and thrombocythemia. Br J Hematol 67:387,
1987
Polycythemia vera: In vitro analysis of regulatory defects Regulation of Erythropoiesis
- Ac Eaves
- G Krystal
- Jd Cashman
- Cj Eaves
Eaves AC, Krystal G, Cashman JD, Eaves CJ: Polycythemia
vera: In vitro analysis of regulatory defects, in Zanjani E, Tavassoli
M, Ascensao JL (eds): Regulation of Erythropoiesis. New York,
NY, PMA, 1988, p 523
Intracellular growth factors in polycythemia vera and other myeloproliferative disorders Unregulated proliferation of primitive neoplastic progenitor cells in long-term polycythemia vera marrow cultures. 3 Clin Invest 81537 An antagonist to IL-3 produced by mouse cells
- J Eid
- Rf Ebert
- Ms Gesell
- J Spivak
- Jd Cashman
- Cj Eaves
- Ac Eaves
- Fg Pluthero
- M Shreeve
- D Eskinazi
- Aa Axelrad
Eid J, Ebert RF, Gesell MS, Spivak J L Intracellular growth
factors in polycythemia vera and other myeloproliferative disorders. Proc Natl Acad Sci USA 84532,1987
32. Cashman JD, Eaves CJ, Eaves AC: Unregulated proliferation of primitive neoplastic progenitor cells in long-term polycythemia vera marrow cultures. 3 Clin Invest 81537, 1988
33. Pluthero FG, Shreeve M, Eskinazi D, Axelrad AA: An
antagonist to IL-3 produced by mouse cells. Blood 7474a, 1989
(abstr, suppl 1)
Koury MJ, Bondurant M C Erythropoietin retards DNA breakdown and prevents programmed death in erythroid progenitor cells
- Rosner Boston
Rosner B: Fundamentals of Biostatistics (ed 2). Boston,
MA, PWS, 1986, p 472
22. Koury MJ, Bondurant M C Erythropoietin retards DNA
breakdown and prevents programmed death in erythroid progenitor cells. Science 248378, 1990
Akabane T Interleukin-6 enhances murine megakaryocytopoiesis in serumfree culture Polycythemia vera: Spontaneous growth of hemoglobinized colonies is mediated by adherent cells
- K Koike
- T Nakahata
- T Kubo
- T Kikuchi
- M Takagi
- A Ishiguro
- K Tsuji
- K Naganuma
- A Okano
- Y Akiyama
- C Nissen
- E Hasler
- K Moor
- Y Moser
- B Speck
Koike K, Nakahata T, Kubo T, Kikuchi T, Takagi M,
Ishiguro A, Tsuji K, Naganuma K, Okano A, Akiyama Y, Akabane
T Interleukin-6 enhances murine megakaryocytopoiesis in serumfree culture. Blood 75:2286,1990
30. Nissen C, Hasler E, Moor K, Moser Y, Speck B: Polycythemia vera: Spontaneous growth of hemoglobinized colonies is
mediated by adherent cells. Exp Hematol 14549,1986 (abstr)