Amittha Wickrema

Distinct roles of erythropoietin, insulin-like growth factor I, and stem cell factor in the development of erythroid progenitor cells.

Erythropoietin (EP), insulin-like growth factor I (IGF-I) and stem cell factor (SCF) each reduce apoptosis of human erythroid progenitor cells. To determine if these growth factors have additional roles in stimulating erythropoiesis, the proliferation, maturation, and survival of highly purified human erythroid colony-forming cells (ECFCs) were studied during the application of different combinations of these growth factors in a serum-free liquid culture. EP maintained cell viability and supported heme synthesis during erythroid maturation, with little increase in viable cell number or stimulation of DNA synthesis. The addition of SCF with EP resulted in a substantial increase in DNA synthesis, which was greater than that seen with the addition of EP and was associated with a large expansion in the number of ECFCs. Thus EP, by itself, produces little increase in cell proliferation, and expansion of the number of erythroid cells depends upon the presence of SCF with EP. The addition of IGF-I with EP led to enhanced heme synthesis and moderate cellular proliferation, but also greatly enhanced nuclear condensation and enucleation in the late erythroblasts. Thus EP, by itself, is not sufficient for complete end-terminal nuclear condensation/enucleation and the presence of IGF-I is necessary for this complete process. While EP greatly reduced apoptosis during 16 h of incubation at 37 degrees C, the addition of SCF and IGF-I with EP had little additional effect, but these additions enhanced DNA synthesis > 3.4-fold. Thus SCF may have an additional role in directly stimulating proliferation through a process that is distinct from apoptosis. Our observations indicate that EP prevents apoptosis and maintains erythroid cell viability and development. IGF-I enhances erythroid maturation and proliferation, but the proliferation of erythroid progenitors is mainly controlled by the addition of SCF with EP, independent of an effect on apoptosis.

Fludarabine, Melphalan, and Alemtuzumab Conditioning in Adults With Standard-Risk Advanced Acute Myeloid Leukemia and Myelodysplastic Syndrome

PURPOSE This prospective phase II study evaluated toxicity, relapse rate, progression-free survival, and overall survival after allogeneic transplantation and conditioning with fludarabine, melphalan, and alemtuzumab in patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). PATIENTS AND METHODS Fifty-two consecutive adults with AML and MDS were enrolled onto the study. Median age was 52 years (range, 17 to 71 years) and the majority of patients had high-risk disease, comorbidities, and/or modest reduction in performance status. Fifty-six percent of patients had unrelated or mismatched related donors. RESULTS After a median follow-up of 18 months (range, 2 to 34 months), 1-year survival was 48% (95% CI, 34% to 61%), progression-free survival was 38% (95% CI, 25% to 52%), relapse rate was 27% (95% CI, 15% to 40%), and treatment-related mortality was 33% (95% CI, 20% to 46%). The cumulative probability of extensive chronic graft-versus-host disease (GVHD) was only 18% (95% CI, 8% to 40%); extensive chronic GVHD was only observed in recipients of unrelated donor transplants. Performance score and disease status were the major predictors of outcome. High-risk disease (ie, active AML or MDS with > 5% blasts) or even modest decreases in performance status were associated with poor outcomes. Patients with standard-risk leukemia (first or second complete remission) or MDS (< 5% blasts) had excellent outcomes despite unfavorable disease characteristics. CONCLUSION Fludarabine and melphalan combined with in vivo alemtuzumab is a promising transplantation regimen for patients with AML or MDS and low tumor burden. For patients with active disease, this regimen provides at best modest palliation. Despite a low incidence of GVHD, transplantation is still associated with considerable nonrelapse mortality in patients with decreased performance status.

Differentiation stage-specific activation of p38 mitogen-activated protein kinase isoforms in primary human erythroid cells

p38α, p38β, p38γ, and p38δ are four isoforms of p38 mitogen-activated protein (MAP) kinase (MAPK) involved in multiple cellular functions such as cell proliferation, differentiation, apoptosis, and inflammation response. In the present study, we examined the mRNA expression pattern of each of the four isoforms during erythroid differentiation of primary erythroid progenitors. We show that p38α and p38γ transcripts are expressed in early hematopoietic progenitors as well as in late differentiating erythroblasts, whereas p38δ mRNA is only expressed and active during the terminal phase of erythroid differentiation. On the other hand, p38β is minimally expressed in early CD34+ hematopoietic progenitors but not expressed in lineage-committed erythroid progenitors. We also determined the phosphorylation/activation of p38α, MAPK kinase 3/6, and MAPKAP-2 in response to erythropoietin and stem cell factor. We found that phosphorylation of p38α, MAPK kinase kinase 3/6 and MAPKAP-2 occurs only upon growth factor withdrawal in primary erythroid progenitors. Moreover, our data indicate that activation of p38α does not induce apoptosis or promote proliferation of erythroid progenitors. On the other hand, under steady-state culture conditions, both p38α and p38δ isoforms are increasingly phosphorylated activated in the terminal phase of differentiation. This increased phosphorylation/activity was accompanied by up-regulation of heat shock protein 27 phosphorylation. Finally, we demonstrate that tumor necrosis factor α, an inflammatory cytokine that is modulated by p38α, is expressed by differentiating erythroblasts and inhibition of p38α or tumor necrosis factor α results in reduction in differentiation. Taken together, our data demonstrate that both p38α and δ isoforms function to promote the late-stage differentiation of primary erythroid progenitors and are likely to be involved in functions related to erythrocyte membrane remodeling and enucleation.

Reduced-intensity conditioning with combined haploidentical and cord blood transplantation results in rapid engraftment, low GVHD, and durable remissions.

We conducted a 45 patient prospective study of reduced-intensity conditioning (RIC) and transplantation of unrelated umbilical cord blood (UCB) and CD34(+) stem cells from a haploidentical family member. Median age was 50 years; weight was 80 kg. Fifty-eight percent had active disease. Neutrophil engraftment occurred at 11 days (interquartile range [IQR], 9-15) and platelet engraftment at 19 days (IQR, 15-33). In the majority of patients, early haploidentical engraftment was replaced by durable engraftment of UCB by 100 days, with regular persistence of minor host and/or haplo-hematopoiesis. Percentage of haplochimerism at day 100 correlated with the haplo-CD34 dose (P = .003). Cumulative incidence of acute GVHD (aGVHD) was 25% and chronic GVHD (cGVHD) was 5%. Actuarial survival at 1 year was 55%, progression-free survival (PFS) was 42%, nonrelapse mortality (NRM) was 28%, and relapse was 30%. RIC and haplo-cord transplantation results in fast engraftment of neutrophils and platelets, low incidences of aGVHD and cGVHD, low frequency of delayed opportunistic infections, reduced transfusion requirements, shortened length of hospital stay, and promising long-term outcomes. UCB cell dose had no impact on time to hematopoietic recovery. Therefore, UCB selection can prioritize matching, and better matched donors can be identified rapidly for most patients. This study is registered at http://clinicaltrials.gov as NCI clinical trial no. NCT00943800.

The hSK4 (KCNN4) isoform is the Ca2+-activated K+ channel (Gardos channel) in human red blood cells

The question is, does the isoform hSK4, also designated KCNN4, represent the small conductance, Ca2+-activated K+ channel (Gardos channel) in human red blood cells? We have analyzed human reticulocyte RNA by RT-PCR, and, of the four isoforms of SK channels known, only SK4 was found. Northern blot analysis of purified and synchronously growing human erythroid progenitor cells, differentiating from erythroblasts to reticulocytes, again showed only the presence of SK4. Western blot analysis, with an anti-SK4 antibody, showed that human erythroid progenitor cells and, importantly, mature human red blood cell ghost membranes, both expressed the SK4 protein. The Gardos channel is known to turn on, given inside Ca2+, in the presence but not the absence of external \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{K}}_{{\mathrm{o}}}^{+}\end{equation*}\end{document} and remains refractory to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{K}}_{{\mathrm{o}}}^{+}\end{equation*}\end{document} added after exposure to inside Ca2+. Heterologously expressed SK4, but not SK3, also shows this behavior. In inside–out patches of red cell membranes, the open probability (Po) of the Gardos channel is markedly reduced when the temperature is raised from 27 to 37°C. Net K+ efflux of intact red cells is also reduced by increasing temperature, as are the Po values of inside–out patches of Chinese hamster ovary cells expressing SK4 (but not SK3). Thus the envelope of evidence indicates that SK4 is the gene that codes for the Gardos channel in human red blood cells. This channel is important pathophysiologically, because it represents the major pathway for cell shrinkage via KCl and water loss that occurs in sickle cell disease.

Engagement of Gab1 and Gab2 in Erythropoietin Signaling

Several signaling cascades are activated during engagement of the erythropoietin receptor to mediate the biological effects of erythropoietin. The members of the insulin receptor substrate (IRS) family of proteins play a central role in signaling for various growth factor receptors and cytokines by acting as docking proteins for the SH2 domains of signaling elements, linking cytokine receptors to diverse downstream pathways. In the present study we provide evidence that the recently cloned IRS-related proteins, Gab1 and Gab2, of the Gab family of proteins, are rapidly phosphorylated on tyrosine during erythropoietin treatment of erythropoietin-responsive cells and provide docking sites for the engagement of the SHP2 phosphatase and the p85 subunit of the phosphatidylinositol 3′-kinase. Furthermore, our data show that Gab1 is the primary IRS-related protein activated by erythropoietin in primary erythroid progenitor cells. In studies to identify the erythropoietin receptor domains required for activation of Gab proteins, we found that tyrosines 425 and 367 in the cytoplasmic domain of the erythropoietin receptor are required for the phosphorylation of Gab2. Taken together, our data demonstrate that Gab proteins are engaged in erythropoietin signaling to mediate downstream activation of the SHP2 and phosphatidylinositol 3′-kinase pathways and possibly participate in the generation of the erythropoietin-induced mitogenic responses.

Inhibition of the TGF-β receptor I kinase promotes hematopoiesis in MDS

MDS is characterized by ineffective hematopoiesis that leads to peripheral cytopenias. Development of effective treatments has been impeded by limited insight into pathogenic pathways governing dysplastic growth of hematopoietic progenitors. We demonstrate that smad2, a downstream mediator of transforming growth factor-beta (TGF-beta) receptor I kinase (TBRI) activation, is constitutively activated in MDS bone marrow (BM) precursors and is overexpressed in gene expression profiles of MDS CD34(+) cells, providing direct evidence of overactivation of TGF-beta pathway in this disease. Suppression of the TGF-beta signaling by lentiviral shRNA-mediated down-regulation of TBRI leads to in vitro enhancement of hematopoiesis in MDS progenitors. Pharmacologic inhibition of TBRI (alk5) kinase by a small molecule inhibitor, SD-208, inhibits smad2 activation in hematopoietic progenitors, suppresses TGF-beta-mediated gene activation in BM stromal cells, and reverses TGF-beta-mediated cell-cycle arrest in BM CD34(+) cells. Furthermore, SD-208 treatment alleviates anemia and stimulates hematopoiesis in vivo in a novel murine model of bone marrow failure generated by constitutive hepatic expression of TGF-beta1. Moreover, in vitro pharmacologic inhibition of TBRI kinase leads to enhancement of hematopoiesis in varied morphologic MDS subtypes. These data directly implicate TGF-beta signaling in the pathobiology of ineffective hematopoiesis and identify TBRI as a potential therapeutic target in low-risk MDS.

Phosphorylation of forkhead transcription factors by erythropoietin and stem cell factor prevents acetylation and their interaction with coactivator p300 in erythroid progenitor cells.

The mammalian forkhead transcription factors, FOXO3a (FKHRL1), FOXO1a (FKHR) and FOXO4 (AFX) are negatively regulated by PKB/Akt kinase. In the present study we examined the engagement of forkhead family of transcription factors in erythropoietin (Epo)- and stem cell factor (SCF)-mediated signal transduction. Our data show that all three forkhead family members, FOXO3a, FOXO1a and FOXO4 are phosphorylated in human primary erythroid progenitors. Experiments performed to determine various upstream signaling pathways contributing to phosphorylation of forkhead family members show that only PI-3-kinase pathway is required for inactivation of FOXO3a. Our data also demonstrate that during Epo deprivation FOXO3a interacts with the transcriptional coactivator p300 and such interaction is disrupted by stimulation of cells with Epo. To determine the domains in FOXO3a, mediating its interaction with p300, we performed GST pull-down assays and found that the N-terminus region containing the first 52 amino acids was sufficient for binding p300. Finally, our data demonstrate that FOXO3a and FOXO1a are acetylated during growth factor deprivation and such acetylation is reversed by stimulation with Epo. Thus mammalian forkhead transcription factors are involved in Epo and SCF signaling in primary erythroid progenitors and may play a role in the induction of apoptotic and mitogenic signals.

Cutting Edge: Activation of the p38 Mitogen-Activated Protein Kinase Signaling Pathway Mediates Cytokine-Induced Hemopoietic Suppression in Aplastic Anemia

Myelosuppressive cytokines, in particular IFN-γ and TNF-α, play an important role in the pathogenesis of idiopathic aplastic anemia in humans. It is unknown whether these negative regulators of hemopoiesis suppress stem cells by activating a common signaling cascade or via distinct nonoverlapping pathways. In this study, we provide evidence that a common element in signaling for IFN-γ and TNF-α in human hemopoietic progenitors is the p38/MapKapK-2 signaling cascade. Our studies indicate that pharmacological inhibition of p38 reverses the suppressive effects of IFN-γ and TNF-α on normal human bone marrow-derived erythroid and myeloid progenitors. Most importantly, inhibition of p38 strongly enhances hemopoietic progenitor colony formation from aplastic anemia bone marrows in vitro. Thus, p38 appears to play a critical role in the pathogenesis of aplastic anemia, suggesting that selective pharmacological inhibitors of this kinase may prove useful in the treatment of aplastic anemia and other cytokine-mediated bone marrow failure syndromes.

AP1 Regulation of Proliferation and Initiation of Apoptosis in Erythropoietin-Dependent Erythroid Cells

ABSTRACT The transcription factor AP1 has been implicated in the induction of apoptosis in cells in response to stress factors and growth factor withdrawal. We report here that AP1 is necessary for the induction of apoptosis following hormone withdrawal in the erythropoietin (EPO)-dependent erythroid cell line HCD57. AP1 DNA binding activity increased upon withdrawal of HCD57 cells from EPO. A dominant negative AP1 mutant rendered these cells resistant to apoptosis induced by EPO withdrawal and blocked the downregulation of Bcl-XL. JunB is a major binding protein in the AP1 complex observed upon EPO withdrawal; JunB but not c-Jun was present in the AP1 complex 3 h after EPO withdrawal in HCD57 cells, with a concurrent increase injunB message and protein. Furthermore, analysis of AP1 DNA binding activity in an apoptosis-resistant subclone of HCD57 revealed a lack of induction in AP1 DNA binding activity and no change injunB mRNA levels upon EPO withdrawal. In addition, we determined that c-Jun and AP1 activities correlated with EPO-induced proliferation and/or protection from apoptosis. AP1 DNA binding activity increased over the first 3 h following EPO stimulation of HCD57 cells, and suppression of AP1 activity partially inhibited EPO-induced proliferation. c-Jun but not JunB was present in the AP1 complex 3 h after EPO addition. These results implicate AP1 in the regulation of proliferation and survival of erythroid cells and suggest that different AP1 factors may play distinct roles in both triggering apoptosis (JunB) and protecting erythroid cells from apoptosis (c-Jun).