A Phase Ib Trial of AVID200, a TGFß 1/3 Trap, in Patients with Myelofibrosis.

PURPOSE: Myelofibrosis (MF) is a clonal myeloproliferative neoplasm characterized by systemic symptoms, cytopenias, organomegaly, and bone marrow fibrosis. JAK2 inhibitors afford symptom and spleen burden reduction but do not alter the disease course and frequently lead to thrombocytopenia. TGFß, a pleiotropic cytokine elaborated by the MF clone, negatively regulates normal hematopoiesis, downregulates antitumor immunity, and promotes bone marrow fibrosis. Our group previously showed that AVID200, a potent and selective TGFß 1/3 trap, reduced TGFß1-induced proliferation of human mesenchymal stromal cells, phosphorylation of SMAD2, and collagen expression. Moreover, treatment of MF mononuclear cells with AVID200 led to increased numbers of progenitor cells (PC) with wild-type JAK2 rather than JAK2V617F. PATIENTS AND METHODS: We conducted an investigator-initiated, multicenter, phase Ib trial of AVID200 monotherapy in 21 patients with advanced MF. RESULTS: No dose-limiting toxicity was identified at the three dose levels tested, and grade 3/4 anemia and thrombocytopenia occurred in 28.6% and 19.0% of treated patients, respectively. After six cycles of therapy, two patients attained a clinical benefit by IWG-MRT criteria. Spleen and symptom benefits were observed across treatment cycles. Unlike other MF-directed therapies, increases in platelet counts were noted in 81% of treated patients with three patients achieving normalization. Treatment with AVID200 resulted in potent suppression of plasma TGFß1 levels and pSMAD2 in MF cells. CONCLUSIONS: AVID200 is a well-tolerated, rational, therapeutic agent for the treatment of patients with MF and should be evaluated further in patients with thrombocytopenic MF in combination with agents that target aberrant MF intracellular signaling pathways.

Patients with hypercortisolemic Cushing disease possess a distinct class of hematopoietic progenitor cells leading to erythrocytosis.

Although human cell cultures stimulated with dexamethasone suggest that the glucocorticoid receptor (GR) activates stress erythropoiesis, the effects of GR activation on erythropoiesis in vivo remain poorly understood. We characterized the phenotype of a large cohort of patients with Cushing disease, a rare condition associated with elevated cortisol levels. Results from hypercortisolemic patients with active Cushing disease were compared with those obtained from eucortisolemic patients after remission and from volunteers without the disease. Patients with active Cushing disease exhibited erythrocytosis associated with normal hemoglobin F levels. In addition, their blood contained elevated numbers of GR-induced CD163+ monocytes and a unique class of CD34+ cells expressing CD110, CD36, CD133 and the GR-target gene CXCR4. When cultured, these CD34+ cells generated similarly large numbers of immature erythroid cells in the presence and absence of dexamethasone, with raised expression of the GR-target gene GILZ. Of interest, blood from patients with Cushing disease in remission maintained high numbers of CD163+ monocytes and, although their CD34+ cells had a normal phenotype, these cells were unresponsive to added dexamethasone. Collectively, these results indicate that chronic exposure to excess glucocorticoids in vivo leads to erythrocytosis by generating erythroid progenitor cells with a constitutively active GR. Although remission rescues the erythrocytosis and the phenotype of the circulating CD34+ cells, a memory of other prior changes is maintained in remission.

Megakaryocytes Are Regulators of the Tumor Microenvironment and Malignant Hematopoietic Progenitor Cells in Myelofibrosis.

Megakaryocytes (MKs) are multifunctional hematopoietic cells that produce platelets, serve as components of bone marrow (BM) niches that support the development of hematopoietic stem and progenitor cell (HSPC) and provide inflammatory signals. MKs can dynamically change their activities during homeostasis and following stress, thereby regulating hematopoietic stem cell (HSC) function. Myelofibrosis (MF) is a progressive chronic myeloproliferative neoplasm (MPN) characterized by hyperactivation of JAK/STAT signaling and MK hyperplasia, which is associated with an aberrant inflammatory signature. Since JAK1/2 inhibitor alone is incapable of depleting the malignant HSC clones or reversing BM fibrosis, the identification of mechanisms that cooperate with MF JAK/STAT signaling to promote disease progression might help in developing combination therapies to modify disease outcomes. Chronic inflammation and MK hyperplasia result in an abnormal release of TGFß1, which plays a critical role in the pathobiology of MF by contributing to the development of BM fibrosis. Dysregulated TGFß signaling can also alter the hematopoietic microenvironment supporting the predominance of MF-HSCs and enhance the quiescence of the reservoir of wild-type HSCs. Upregulation of TGFß1 levels is a relatively late event in MF, while during the early pre-fibrotic stage of MF the alarmin S100A8/S100A9 heterocomplex promotes pro-inflammatory responses and sustains the progression of MF-HSCs. In this review, we will discuss the recent advances in our understanding of the roles of abnormal megakaryopoiesis, and the altered microenvironment in MF progression and the development of novel combined targeted therapies to disrupt the aberrant interplay between MKs, the BM microenvironment and malignant HSCs which would potentially limit the expansion of MF-HSC clones.

TGF-ß1 protein trap AVID200 beneficially affects hematopoiesis and bone marrow fibrosis in myelofibrosis.

Myelofibrosis (MF) is a progressive chronic myeloproliferative neoplasm characterized by hyperactivation of JAK/STAT signaling and dysregulation of the transcription factor GATA1 in megakaryocytes (MKs). TGF-ß plays a pivotal role in the pathobiology of MF by promoting BM fibrosis and collagen deposition and by enhancing the dormancy of normal hematopoietic stem cells (HSCs). In this study, we show that MF-MKs elaborated significantly greater levels of TGF-ß1 than TGF-ß2 and TGF-ß3 to a varying degree, and we evaluated the ability of AVID200, a potent TGF-ß1/TGF-ß3 protein trap, to block the excessive TGF-ß signaling. Treatment of human mesenchymal stromal cells with AVID200 significantly reduced their proliferation, decreased phosphorylation of SMAD2, and interfered with the ability of TGF-ß1 to induce collagen expression. Moreover, treatment of MF mononuclear cells with AVID200 led to increased numbers of progenitor cells (PCs) with WT JAK2 rather than mutated JAK2V617F. This effect of AVID200 on MF PCs was attributed to its ability to block TGF-ß1-induced p57Kip2 expression and SMAD2 activation, thereby allowing normal rather than MF PCs to preferentially proliferate and form hematopoietic colonies. To assess the in vivo effects of AVID200, Gata1lo mice, a murine model of MF, were treated with AVID200, resulting in the reduction in BM fibrosis and an increase in BM cellularity. AVID200 treatment also increased the frequency and numbers of murine progenitor cells as well as short-term and long-term HSCs. Collectively, these data provide the rationale for TGF-ß1 blockade, with AVID200 as a therapeutic strategy for patients with MF.

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

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

Shared and Tissue-Specific Expression Signatures between Bone Marrow from Primary Myelofibrosis and Essential Thrombocythemia.

Megakaryocytes have been implicated in the micro-environmental abnormalities associated with fibrosis and hematopoietic failure in the bone marrow (BM) of primary myelofibrosis (PMF) patients, the Philadelphia-negative myeloproliferative neoplasm (MPN) associated with the poorest prognosis. To identify possible therapeutic targets for restoring BM functions in PMF, we compared the expression profiling of PMF BM with that of BM from essential thrombocytopenia (ET), a fibrosis-free MPN also associated with BM megakaryocyte hyperplasia. The signature of PMF BM was also compared with published signatures associated with liver and lung fibrosis. Gene set enrichment analysis (GSEA) identified distinctive differences between the expression profiles of PMF and ET. Notch, K-Ras, IL-8, and apoptosis pathways were altered the most in PMF as compared with controls. By contrast, cholesterol homeostasis, unfolded protein response, and hypoxia were the pathways found altered to the greatest degree in ET compared with control specimens. BM from PMF expressed a noncanonical transforming growth factor ß (TGF-ß) signature, which included activation of ID1, JUN, GADD45b, and genes with binding motifs for the JUN transcriptional complex AP1. By contrast, the expression of ID1 and GADD45b was not altered and there was a modest signal for JUN activation in ET. The similarities among PMF, liver fibrosis, and lung fibrosis were modest and included activation of integrin-α9 and tropomyosin-α1 between PMF and liver fibrosis, and of ectoderm-neural cortex protein 1 and FRAS1-related extracellular matrix protein 1 between PMF and lung fibrosis, but not TGF-ß. These data identify TGF-ß as a potential target for micro-environmental therapy in PMF.

Calreticulin: Challenges Posed by the Intrinsically Disordered Nature of Calreticulin to the Study of Its Function.

Calreticulin is a Ca2+-binding chaperone protein, which resides mainly in the endoplasmic reticulum but also found in other cellular compartments including the plasma membrane. In addition to Ca2+, calreticulin binds and regulates almost all proteins and most of the mRNAs deciding their intracellular fate. The potential functions of calreticulin are so numerous that identification of all of them is becoming a nightmare. Still the recent discovery that patients affected by the Philadelphia-negative myeloproliferative disorders essential thrombocytemia or primary myelofibrosis not harboring JAK2 mutations carry instead calreticulin mutations disrupting its C-terminal domain has highlighted the clinical need to gain a deeper understanding of the biological activity of this protein. However, by contrast with other proteins, such as enzymes or transcription factors, the biological functions of which are strictly defined by a stable spatial structure imprinted by their amino acid sequence, calreticulin contains intrinsically disordered regions, the structure of which represents a highly dynamic conformational ensemble characterized by constant changes between several metastable conformations in response to a variety of environmental cues. This article will illustrate the Theory of calreticulin as an intrinsically disordered protein and discuss the Hypothesis that the dynamic conformational changes to which calreticulin may be subjected by environmental cues, by promoting or restricting the exposure of its active sites, may affect its function under normal and pathological conditions.

The Calreticulin control of human stress erythropoiesis is impaired by JAK2V617F in polycythemia vera.

Calreticulin (CALR) is a Ca2+-binding protein that shuttles among cellular compartments with proteins bound to its N/P domains. The knowledge that activation of the human erythropoietin receptor induces Ca2+ fluxes prompted us to investigate the role of CALR in human erythropoiesis. As shown by Western blot analysis, erythroblasts generated in vitro from normal sources and JAK2V617F polycythemia vera (PV) patients expressed robust levels of CALR. However, Ca2+ regulated CALR conformation only in normal cells. Normal erythroblasts expressed mostly the N-terminal domain of CALR (N-CALR) on their cell surface (as shown by flow cytometry) and C-terminal domain (C-CALR) in their cytoplasm (as shown by confocal microscopy) and expression of both epitopes decreased with maturation. In the proerythroblast (proEry) cytoplasm, C-CALR was associated with the glucocorticoid receptor (GR), which initiated the stress response. In these cells, Ca2+ deprivation and inhibition of nuclear export increased GR nuclear localization while decreasing cytoplasmic detection of C-CALR and C-CALR/GR association and proliferation in response to the GR agonist dexamethasone (Dex). C-CALR/GR association and Dex responsiveness were instead increased by Ca2+ and erythropoietin. In contrast, JAK2V617F proErys expressed normal cell-surface levels of N-CALR but barely detectable cytoplasmic levels of C-CALR. These cells contained GR mainly in the nucleus and were Dex unresponsive. Ruxolitinib rescued cytoplasmic detection of C-CALR, C-CALR/GR association, and Dex responsiveness in JAK2V617F proErys and its effects were antagonized by nuclear export and Ca2+ flux inhibitors. These results indicates that Ca2+-induced conformational changes of CALR regulate nuclear export of GR in normal erythroblasts and that JAK2V617F deregulates this function in PV.

2p15-p16.1 microdeletions encompassing and proximal to BCL11A are associated with elevated HbF in addition to neurologic impairment.

Elevated fetal hemoglobin (HbF) ameliorates the clinical severity of hemoglobinopathies such as ß-thalassemia and sickle cell anemia. Currently, the only curative approach for individuals under chronic transfusion/chelation support therapy is allogeneic stem cell transplantation. However, recent analyses of heritable variations in HbF levels have provided a new therapeutic target for HbF reactivation: the transcriptional repressor BCL11A. Erythroid-specific BCL11A abrogation is now actively being sought as a therapeutic avenue, but the specific impact of such disruption in humans remains to be determined. Although single nucleotide polymorphisms in BCL11A erythroid regulatory elements have been reported, coding mutations are scarcer. It is thus of great interest that patients have recently been described with microdeletions encompassing BCL11A. These patients display neurodevelopmental abnormalities, but whether they show increased HbF has not been reported. We have examined the hematological phenotype, HbF levels, and erythroid BCL11A expression in 3 such patients. Haploinsufficiency of BCL11A induces only partial developmental γ-globin silencing. Of greater interest is that a patient with a downstream deletion exhibits reduced BCL11A expression and increased HbF. Novel erythroid-specific regulatory elements in this region may be required for normal erythroid BCL11A expression, whereas loss of separate elements in the developing brain may explain the neurological phenotype.

Activation of non-canonical TGF-ß1 signaling indicates an autoimmune mechanism for bone marrow fibrosis in primary myelofibrosis.

Primary myelofibrosis (PMF) is characterized by megakaryocyte hyperplasia, dysplasia and death with progressive reticulin/collagen fibrosis in marrow and hematopoiesis in extramedullary sites. The mechanism of fibrosis was investigated by comparing TGF-ß1 signaling of marrow and spleen of patients with PMF and of non-diseased individuals. Expression of 39 (23 up-regulated and 16 down-regulated) and 38 (8 up-regulated and 30 down-regulated) TGF-ß1 signaling genes was altered in the marrow and spleen of PMF patients, respectively. Abnormalities included genes of TGF-ß1 signaling, cell cycling and abnormal in chronic myeloid leukemia (EVI1 and p21(CIP)) (both marrow and spleen) and Hedgehog (marrow only) and p53 (spleen only) signaling. Pathway analyses of these alterations predict an increased osteoblast differentiation, ineffective hematopoiesis and fibrosis driven by non-canonical TGF-ß1 signaling in marrow and increased proliferation and defective DNA repair in spleen. Since activation of non-canonical TGF-ß1 signaling is associated with fibrosis in autoimmune diseases, the hypothesis that fibrosis in PMF results from an autoimmune process triggered by dead megakaryocytes was tested by determining that PMF patients expressed plasma levels of mitochondrial DNA and anti-mitochondrial antibodies greater than normal controls. These data identify autoimmunity as a possible cause of marrow fibrosis in PMF.

Dexamethasone targeted directly to macrophages induces macrophage niches that promote erythroid expansion.

Cultures of human CD34(pos) cells stimulated with erythroid growth factors plus dexamethasone, a model for stress erythropoiesis, generate numerous erythroid cells plus a few macrophages (approx. 3%; 3:1 positive and negative for CD169). Interactions occurring between erythroblasts and macrophages in these cultures and the biological effects associated with these interactions were documented by live phase-contrast videomicroscopy. Macrophages expressed high motility interacting with hundreds/thousands of erythroblasts per hour. CD169(pos) macrophages established multiple rapid 'loose' interactions with proerythroblasts leading to formation of transient erythroblastic island-like structures. By contrast, CD169(neg) macrophages established 'tight' interactions with mature erythroblasts and phagocytosed these cells. 'Loose' interactions of CD169(pos) macrophages were associated with proerythroblast cytokinesis (the M phase of the cell cycle) suggesting that these interactions may promote proerythroblast duplication. This hypothesis was tested by experiments that showed that as few as 103 macrophages significantly increased levels of 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide incorporation frequency in S/G2/M and cytokinesis expressed by proerythroblasts over 24 h of culture. These effects were observed also when macrophages were co-cultured with dexamethasone directly conjugated to a macrophage-specific CD163 antibody. In conclusion, in addition to promoting proerythroblast proliferation directly, dexamethasone stimulates expansion of these cells indirectly by stimulating maturation and cytokinesis supporting activity of macrophages.

Identification of NuRSERY, a new functional HDAC complex composed by HDAC5, GATA1, EKLF and pERK present in human erythroid cells.

To clarify the role of HDACs in erythropoiesis, expression, activity and function of class I (HDAC1, HDAC2, HDAC3) and class IIa (HDAC4, HDAC5) HDACs during in vitro maturation of human erythroblasts were compared. During erythroid maturation, expression of HDAC1, HDAC2 and HDAC3 remained constant and activity and GATA1 association (its partner of the NuRD complex), of HDAC1 increased. By contrast, HDAC4 content drastically decreased and HDAC5 remained constant in content but decreased in activity. In erythroid cells, pull down experiments identified the presence of a novel complex formed by HDAC5, GATA1, EKLF and pERK which was instead undetectable in cells of the megakaryocytic lineage. With erythroid maturation, association among HDAC5, GATA1 and EKLF persisted but levels of pERK sharply decreased. Treatment of erythroleukemic cells with inhibitors of ERK phosphorylation reduced by >90% the total and nuclear content of HDAC5, GATA1 and EKLF, suggesting that ERK phosphorylation is required for the formation of this complex. Based on the function of class IIa HDACs as chaperones of other proteins to the nucleus and the erythroid-specificity of HDAC5 localization, this novel HDAC complex was named nuclear remodeling shuttle erythroid (NuRSERY). Exposure of erythroid cells to the class II-selective HDAC inhibitor (HDACi) APHA9 increased γ/(γ+ß) globin expression ratios (Mai et al., 2007), suggesting that NuRSERY may regulate globin gene expression. In agreement with this hypothesis, exposure of erythroid cells to APHA9 greatly reduced the association among HDAC5, GATA1 and EKLF. Since exposure to APHA9 did not affect survival rates or p21 activation, NuRSERY may represent a novel, possibly less toxic, target for epigenetic therapies of hemoglobinopaties and other disorders.

The role of glucocorticoid receptor (GR) polymorphisms in human erythropoiesis.

Glucocorticoids are endogenous steroid hormones that regulate several biological functions including proliferation, differentiation and apoptosis in numerous cell types in response to stress. Synthetic glucocorticoids, such as dexamethasone (Dex) are used to treat a variety of diseases ranging from allergy to depression. Glucocorticoids exert their effects by passively entering into cells and binding to a specific Glucocorticoid Receptor (GR) present in the cytoplasm. Once activated by its ligand, GR may elicit cytoplasmic (mainly suppression of p53), and nuclear (regulation of transcription of GR responsive genes), responses. Human GR is highly polymorphic and may encode > 260 different isoforms. This polymorphism is emerging as the leading cause for the variability of phenotype and response to glucocorticoid therapy observed in human populations. Studies in mice and clinical observations indicate that GR controls also the response to erythroid stress. This knowledge has been exploited in-vivo by using synthetic GR agonists for treatment of the erythropoietin-refractory congenic Diamond Blackfan Anemia and in-vitro to develop culture conditions that may theoretically generate red cells in numbers sufficient for transfusion. However, the effect exerted by GR polymorphism on the variability of the phenotype of genetic and acquired erythroid disorders observed in the human population is still poorly appreciated. This review will summarize current knowledge on the biological activity of GR and of its polymorphism in non-hematopoietic diseases and discuss the implications of these observations for erythropoiesis.

Transcriptomic and phospho-proteomic analyzes of erythroblasts expanded in vitro from normal donors and from patients with polycythemia vera.

Erythropoiesis is a tightly regulated process which becomes decoupled from its normal differentiation program in patients with polycythemia vera (PV). Somatic mutations in JAK2 are commonly associated with this myeloid proliferative disorder. To gain insight into the molecular events that are required for abnormally developing erythroid cells to escape dependence on normal growth signals, we performed in vitro expansion of mature erythroblasts (ERY) from seven normal healthy donors and from seven polycythemic patients in the presence of IL3, EPO, SCF for 10, 11, or 13 days. Normal ERYs required exposure to the glucocorticoid dexamethasone (Dex) for expansion, while PV-derived ERYs expanded in the absence of dexamethasone. RNA expression profiling revealed enrichment of two known oncogenes, GPR56 and RAB4a, in PV-derived ERYs along with reduced expression levels of transcription factor TAL1 (ANOVA FDR < 0.05). While both normal and polycythemic-derived ERYs integrated signaling cascades for growth, they did so via different signaling pathways which are represented by their differential phospho-profiles. Our results show that normal ERYs displayed greater levels of phosphorylation of EGFR, PDGFRß, TGFß, and cKit, while PV-derived ERYs were characterized by increased phosphorylation of cytoplasmic kinases in the JAK/STAT, PI3K, and GATA1 pathways. Together these data suggest that PV erythroblast expansion and maturation may be maintained and enriched in the absence of dexamethasone through reduced TAL1 expression and by accessing additional signaling cascades. Members of this acquired repertoire may provide important insight into the pathogenesis of aberrant erythropoiesis in myeloproliferative neoplasms such as polycythemia vera.

The expression of the glucocorticoid receptor in human erythroblasts is uniquely regulated by KIT ligand: implications for stress erythropoiesis.

Studies in mice indicated that activation of the erythroid stress pathway requires the presence of both soluble KIT ligand (KITL) and the glucocorticoid receptor (GR). To clarify the relative role of KITL and GR in stress erythropoiesis in humans, the biological activities of soluble full length- (fl-, 26-190 aa), carboxy-terminus truncated (tr-, 26-162 aa) human (hKITL) and murine (mKITL) KITL in cultures of cord blood (CB) mononuclear cells (MNCs) and CD34(pos) cells that mimic either steady state (growth factors alone) or stress (growth factors plus dexamethasone [DXM]) erythropoeisis were investigated. In steady state cultures, the KITLs investigated were equally potent in sustaining growth of hematopoietic colonies and expansion of megakaryocytes (MK) and erythroid precursors (EBs). By contrast, under stress erythropoiesis conditions, fl-hKITL generated greater numbers of EBs (fold increase [FI]=140) than tr-hKITL or mKITL (FI=20-40). Flow cytometric analyses indicated that only EBs generated with fl-hKITL remained immature (>70% CD36(pos)/CD235a(neg/low)), and therefore capable to proliferate, until day 8-12 in response to DXM. Signaling studies indicated that all KITLs investigated induced EBs to phosphorylate signal transducer and activator of transcription 5 (STAT5) but that extracellular-signaling-regulated-kinases (ERK) activation was observed mainly in the presence of fl-hKITL. EBs exposed to fl-hKITL also expressed higher levels of GRα than those exposed to mKITL (and tr-hKITL) which were reduced upon exposure to the ERK inhibitor U0126. These data reveal a unique requirement for fl-hKITL in the upregulation of GRα and optimal EB expansion in cultures that mimic stress erythropoiesis.

Under HEMA conditions, self-replication of human erythroblasts is limited by autophagic death.

The number of erythroblasts generated ex-vivo under human-erythroid massive-amplification conditions by mononuclear cells from one unit of adult blood (~10(10)) are insufficient for transfusion (~10(12) red cells), emphasizing the need for studies to characterize cellular interactions during culture to increase erythroblast production. To identify the cell populations which generate erythroblasts under human-erythroid-massive-amplification conditions and the factors that limit proliferation, day 10 non-erythroblasts and immature- and mature-erythroblasts were separated by sorting, labelled with carboxyfluorescein-diacetate-succinimidyl-ester and re-cultured either under these conditions (for proliferation, maturation and/or apoptosis/autophagy determinations) or in semisolid media (for progenitor cell determination). Non-erythroblasts contained 54% of the progenitor cells but did not grow under human-erythroid-massive-amplification conditions. Immature-erythroblasts contained 25% of the progenitor cells and generated erythroblasts under human-erythroid-massive-amplification conditions (FI at 48 h=2.57±1.15). Mature-erythroblasts did not generate colonies and died in human-erythroid-massive-amplification conditions. In sequential sorting/re-culture experiments, immature-erythroblasts retained the ability to generate erythroblasts for 6 days and generated 2-5-fold more cells than the corresponding unfractionated population, suggesting that mature-erythroblasts may limit erythroblast expansion. In co-cultures of carboxyfluorescein-diacetate-succinimidyl-ester-labelled-immature-erythroblasts with mature-erythroblasts at increasing ratios, cell numbers did not increase and proliferation, maturation and apoptotic rates were unchanged. However, Acridine Orange staining (a marker for autophagic death) increased from ~3.2% in cultures with immature-erythroblasts alone to 14-22% in cultures of mature-erythroblasts with and without immature-erythroblasts. In conclusion, these data identify immature-erythroblasts as the cells that generate additional erythroblasts in human-erythroid-massive-amplification cultures and autophagy as the leading cause of death limiting the final cellular output of these cultures.

The dominant negative ß isoform of the glucocorticoid receptor is uniquely expressed in erythroid cells expanded from polycythemia vera patients.

Glucocorticoid receptor (GR) agonists increase erythropoiesis in vivo and in vitro. To clarify the effect of the dominant negative GRß isoform (unable to bind STAT-5) on erythropoiesis, erythroblast (EB) expansion cultures of mononuclear cells from 18 healthy (nondiseased) donors (NDs) and 16 patients with polycythemia vera (PV) were studied. GRß was expressed in all PV EBs but only in EBs from 1 ND. The A3669G polymorphism, which stabilizes GRß mRNA, had greater frequency in PV (55%; n = 22; P = .0028) and myelofibrosis (35%; n = 20) patients than in NDs (9%; n = 22) or patients with essential thrombocythemia (6%; n = 15). Dexamethasone stimulation of ND cultures increased the number of immature EBs characterized by low GATA1 and ß-globin expression, but PV cultures generated great numbers of immature EBs with low levels of GATA1 and ß-globin irrespective of dexamethasone stimulation. In ND EBs, STAT-5 was not phosphorylated after dexamethasone and erythropoietin treatment and did not form transcriptionally active complexes with GRα, whereas in PV EBs, STAT-5 was constitutively phosphorylated, but the formation of GR/STAT-5 complexes was prevented by expression of GRß. These data indicate that GRß expression and the presence of A3669G likely contribute to development of erythrocytosis in PV and provide a potential target for identification of novel therapeutic agents.

Humanized culture medium for clinical expansion of human erythroblasts.

Ex vivo-generated erythroblasts represent alternative transfusion products. However, inclusion of bovine components in media used for their growth precludes clinical use, highlighting the importance of developing culture media based on pharmaceutical grade reagents. In addition, because adult blood generates ex vivo lower numbers of erythroblasts than cord blood, cord blood has been proposed as the source of choice for ex vivo erythroblast production. To clarify the potential of adult blood to generate erythroblasts ex vivo, experiments were designed to identify growth factors [stem cell factor (SCF), interleukin-3 (IL-3), erythropoietin (EPO), and/or thrombopoietin (TPO)] and the optimal concentration and addition schedule of hormones (dexamethasone and estradiol) sustaining maximal erythroid amplification from adult blood mononuclear cells (MNC) using media with serum previously defined as human erythroid massive amplification culture (HEMA(ser)). Adult MNC stimulated with SCF and IL-3 in combination with EPO generated a 6-12-fold increase in erythroid cells while TPO was ineffective. Dexamethasone and estradiol (both at 10(-6) M) exerted partially overlapping but nonredundant functions. Dexamethasone was indispensable in the first 10 days of culture while estradiol was required from day 10 on. The growth factor and hormone combinations identified in HEMA(ser) were then used to formulate a media composed of dialyzed pharmaceutical grade human albumin, human albumin-lipid liposomes, and iron-saturated recombinant human tranferrin (HEMA(def)). HEMA(def) sustained erythroid amplification as efficiently as HEMA(ser) for cord blood MNC and 10-fold higher than HEMA(ser) for adult blood MNC. In fact, the numbers of erythroblasts generated in HEMA(def) by adult MNC were similar to those generated by cord blood MNC. In conclusion, this study identifies growth factors, hormone combinations, and human protein-based media that allow similar levels of ex vivo erythroid expansion from adult and cord blood MNC, paving the way to evaluate adult blood as a source of ex vivo-expanded erythroblasts for transfusion.

Pathological interactions between hematopoietic stem cells and their niche revealed by mouse models of primary myelofibrosis.

Primary myelofibrosis (PMF) belongs to the Philadelphia-negative myeloproliferative neoplasms and is a hematological disorder caused by abnormal function of the hematopoietic stem cells. The disease manifests itself with a plethora of alterations, including anemia, splenomegaly and extramedullary hematopoiesis. Its hallmarks are progressive marrow fibrosis and atypical megakaryocytic hyperplasia, two distinctive features used to clinically monitor disease progression. In an attempt to investigate the role of abnormal megakaryocytopoiesis in the pathogenesis of PMF, several transgenic mouse models have been generated. These models are based either on mutations that interfere with the extrinsic (thrombopoietin and its receptor, MPL) and intrinsic (the GATA1 transcription factor) control of normal megakaryocytopoiesis, or on known genetic lesions associated with the human disease. Here we provide an up-to-date review on the insights into the pathobiology of human PMF achieved by studying these animal models, with particular emphasis on results obtained with Gata1(low) mice.

Inhibition of estradiol receptor/Src association and cell growth by an estradiol receptor alpha tyrosine-phosphorylated peptide.

This report offers direct evidence that association of the estradiol receptor (ER) with Src triggered by steroid agonists or growth factors controls breast and prostate cancer cell growth. This association is abolished in whole cells and in vitro by a six-amino-acid peptide that mimics the sequence around the phosphotyrosine residue in position 537 of the human ERalpha. The phosphorylated peptide, at nanomolar concentrations, is taken up by MCF-7 and LNCaP cells derived from human mammary and prostate cancers, respectively. In addition, to block the ER/Src interaction, the phosphopeptide inhibits Src/Erk pathway, cyclin D1 expression, and DNA synthesis induced by estradiol or androgen or triggered by epidermal growth factor. In contrast, no inhibition of the Src-mediated epidermal growth factor action on DNA synthesis is detectable in human mammary cancer cells that do not express ER (MDA-MB231), indicating that the peptide specifically targets the ER-associated Src. Remarkably, the peptide, in contrast with classic steroid antagonists, does not interfere in ER- or androgen receptor-dependent transcriptional activity. Nevertheless, it markedly inhibits the growth of MCF-7 cell xenografts induced in immunodepressed and estradiol-treated mice. The present report suggests that inhibition of association of steroid receptors with Src or other signaling effectors may have therapeutic applications for patients with ER-positive tumors.