Burst-forming Units Research Articles

Regulation of hematopoietic stem cells using protein transduction domain–fused Polycomb

The Polycomb-group complex is a chromatin regulatory factor that is classified into two different complexes: Polycomb repressive complex 1 and 2. Components of Polycomb repressive complex 1 are involved in the self-renewal of hematopoietic stem cells. Bmi1, one of these components, maintains the immaturity of neural and cancer stem cells as well as that of hematopoietic stem cells. We constructed recombinant protein transduction domain (PTD)-Polycomb proteins and transduced them into murine bone marrow (BM) cells. We designed and fused the PTD-protein transduction domain to three proteins (i.e., green fluorescent protein, Bmi1, and Mel18). Murine BM cells were incubated for 48 h and each PTD-Polycomb protein was added. Then, we analyzed the function of hematopoiesis using the colony assay and transplantation. BM cells exposed to PTD-Bmi1 showed an increased number of colonies. In contrast, BM cells exposed to PTD-Mel18 or to both proteins showed a decreased number of colonies. Hematopoietic cells derived from PTD-Bmi1-transduced BM cells were significantly increased in the peripheral blood at 6 weeks after transplantation. Moreover, 80% of mice transplanted with PTD-Bmi1-transduced BM cells died at 8 to 24 weeks after transplantation. However, only a few early deaths were observed in the mice transplanted with BM cells exposed to both PTD-Bmi1 and PTD-Mel18. We expect that hematopoietic stem cells could proliferate after transduction with PTD-Bmi1, but this may generate undesirable effects, e.g., tumorigenesis. Thus, Bmi1 and Mel18 have opposing functions and are present in distinct complexes.

Methylation of AR locus does not always reflect X chromosome inactivation state

AbstractClonality can be established by a lack of mosaicism in a female because of random inactivation of either the maternal or paternal X chromosome early in embryogenesis. The methylation status of CpG sites close to the trinucleotide repeats in exon 1 of the human androgen receptor (AR) X chromosome gene assay (HUMARA) has been used to determine clonality. This HUMARA at times indicated clonal hematopoiesis in healthy elderly women, thus precluding its applicability. We used a clonality assay based on quantitative expression of polymorphic X chromosome genes (qTCA) and found no evidence of clonal hematopoiesis in healthy nonanemic elderly persons. We found instances of discordance between HUMARA results and those obtained by pyrosequencing and qTCA methods, as well as by directly quantifying AR gene expression. To determine the basis of this discrepancy we examined the methylation pattern of the AR locus subject to HUMARA. Notably, we found the extent of DNA methylation to be highly variable at the AR gene in granulocytes of persons with discordant results and also in erythroid burst-forming unit colonies but not in those with clonal hematopoiesis. These data provide the molecular basis of incomplete correlation with the pattern of DNA methylation of this X chromosome AR gene locus.

Deletion of the Scl +19 enhancer increases the blood stem cell compartment without affecting the formation of mature blood lineages

The stem cell leukemia (Scl)/Tal1 gene is essential for normal blood and endothelial development, and is expressed in hematopoietic stem cells (HSCs), progenitors, erythroid, megakaryocytic, and mast cells. The Scl +19 enhancer is active in HSCs and progenitor cells, megakaryocytes, and mast cells, but not mature erythroid cells. Here we demonstrate that in vivo deletion of the Scl +19 enhancer (SclΔ19/Δ19) results in viable mice with normal Scl expression in mature hematopoietic lineages. By contrast, Scl expression is reduced in the stem/progenitor compartment and flow cytometry analysis revealed that the HSC and megakaryocyte-erythroid progenitor populations are enlarged in SclΔ19/Δ19 mice. The increase in HSC numbers contributed to enhanced expansion in bone marrow transplantation assays, but did not affect multilineage repopulation or stress responses. These results affirm that the Scl +19 enhancer plays a key role in the development of hematopoietic stem/progenitor cells, but is not necessary for mature hematopoietic lineages. Moreover, active histone marks across the Scl locus were significantly reduced in SclΔ19/Δ19 fetal liver cells without major changes in steady-state messenger RNA levels, suggesting post-transcriptional compensation for loss of a regulatory element, a result that might be widely relevant given the frequent observation of mild phenotypes after deletion of regulatory elements.

Ex vivo fucosylation improves human cord blood engraftment in NOD-SCID IL-2Rγnull mice

Delayed engraftment remains a major hurdle after cord blood (CB) transplantation. It may be due, at least in part, to low fucosylation of cell surface molecules important for homing to the bone marrow microenvironment. Because fucosylation of specific cell surface ligands is required before effective interaction with selectins expressed by the bone marrow microvasculature can occur, a simple 30-minute ex vivo incubation of CB hematopoietic progenitor cells with fucosyltransferase-VI and its substrate (GDP-fucose) was performed to increase levels of fucosylation. The physiologic impact of CB hematopoietic progenitor cell hypofucosylation was investigated in vivo in NOD-SCID interleukin (IL)-2Rγ(null) (NSG) mice. By isolating fucosylated and nonfucosylated CD34(+) cells from CB, we showed that only fucosylated CD34(+) cells are responsible for engraftment in NSG mice. In addition, because the proportion of CD34(+) cells that are fucosylated in CB is significantly less than in bone marrow and peripheral blood, we hypothesize that these combined observations might explain, at least in part, the delayed engraftment observed after CB transplantation. Because engraftment appears to be correlated with the fucosylation of CD34(+) cells, we hypothesized that increasing the proportion of CD34(+) cells that are fucosylated would improve CB engraftment. Ex vivo treatment with fucosyltransferase-VI significantly increases the levels of CD34(+) fucosylation and, as hypothesized, this was associated with improved engraftment. Ex vivo fucosylation did not alter the biodistribution of engrafting cells or pattern of long-term, multilineage, multi-tissue engraftment. We propose that ex vivo fucosylation will similarly improve the rate and magnitude of engraftment for CB transplant recipients in a clinical setting.

The effect of iron overload and chelation on erythroid differentiation

We investigated the mechanisms of hematopoietic disorders caused by iron overload and chelation, in particular, the inhibition of erythroblast differentiation. Murine c-kit(+) progenitor cells or human CD34(+) peripheral blood hematopoietic progenitors were differentiated in vitro to the erythroid lineage with free iron and/or an iron chelator. Under iron overload, formation of erythroid burst-forming unit colonies and differentiation to mature erythroblasts were significantly suppressed; these effects were canceled by iron chelation with deferoxamine (DFO). Moreover, excessive iron burden promoted apoptosis in immature erythroblasts by elevating intracellular reactive oxygen species (ROS). Interestingly, both DFO and a potent anti-oxidant agent reduced intracellular ROS levels and suppressed apoptosis, thus restoring differentiation to mature erythroblasts. Accordingly, intracellular ROS may represent a new therapeutic target in the treatment of iron overload.

P190-B RhoGAP Is Critical for Hematopoietic Stem Cell Niche Regulation

Abstract 221Hematopoiesis is regulated by components of the stromal microenvironment, so-called niche. Although the concept of hematopoietic stem and progenitor cell (HSC/P) niche is well known, its molecular regulation remains ill-defined. Here, we provide evidence that p190-B GTPase Activating Protein (p190-B), a negative regulator of Rho activity, is a regulator of mesenchymal/stromal cell functions necessary for normal hematopoiesis during fetal development. Mice lacking p190-B die before birth. At day 14.5 post coitum, p190-B−/− embryos are paler than WT embryos with a 40% lower hematocrit. Cellularity and numbers of burst forming-unit (BFU) erythroid (E), colony forming-unit (CFU)-granulo-monocytic and CFU-erythroid per p190-B−/− fetal livers (FL) were 50% lower than WT. In addition, the frequency of LongTerm-HSC (LinnegScaposKitposCD150posCD48neg), ShortTerm-HSC (LSK-CD150posCD48pos), and progenitors (LK) appeared significantly reduced in the bone marrow of e17.5/18 embryos. These data suggest a defect in fetal hematopoiesis. However, p190-B−/− FL cells were able to fully reconstitute hematopoiesis of adult irradiated recipients (Xu, Blood 2009). Mice reconstituted with p190-B−/− FL cells exhibited bone marrow content, white blood count, red blood count and hematocrit similar to those reconstituted with WT cells. Furthermore, hematopoietic recovery of p190-B−/− reconstituted animals following 5-Fluorouracil or phenylhydrazine-induced stress was comparable to that of WT. Therefore, FL HSC/P retain hematopoietic potential, which suggests a non-cell autonomous defect in p190-B−/− embryos. In the present study, we examined in more detail this possibility. The numbers of CFU-fibroblast (CFU-F) were 2-fold lower in p190-B−/− FL compared to WT FL. We next examined the hematopoietic supportive capacity of p190-B−/− FL microenvironment using stromal cell cultures derived from e14.5 FL. No noticeable differences were observed during the establishment of the stromal cell cultures. p190-B−/− stromal cells exhibited a spindle-like shape and expressed CD90, CD44 and Sca-1 but not CD45, CD31 or CD11b similarly to WT stromal cells. Cobblestone Area Forming Cell assays (CAFC) was performed in co-culture between various number of WT BM cells and p190-B−/− and WT stromal cells. We examined 5 independent stromal cell cultures from both genotypes. Frequency of CAFC was 75% lower on p190-B−/− stroma than WT stroma at both 1 week (1 in 5 × 10 3 versus 1 in 21 × 10 3, n = 5 p < 0.01) and 2 weeks (1 in 21 × 10 3 versus 1 in 87 × 10 3, n = 4 p < 0.01) in culture. CFU with cells recovered from one week coculture was also performed. Cocultures with p190-B−/− stroma gave rise to 2-fold less CFU than with WT stroma (538+90 versus 255+81, p < 0.01, one representative experiment of 4). Using competitive repopulation assay, we then assessed the in vivo repopulation ability of CD45.2+ hematopoietic cells co-cultured for one week on each stroma. Fifteen weeks following transplantation, the frequency of CD45.2+ cells in the peripheral blood of mice that received cells cocultured on p190-B−/− stroma was dramatically reduced compared to mice that received cells cocultured on WT stroma (0.43% + 0.37 versus 32.4% + 7.5, n = 6, p < 0.01), equivalent to a 100-fold difference in calculated competitive repopulation unit. Therefore, p190-B−/− stroma exhibited defective hematopoietic supportive activity. Interestingly, p190-B−/− stromal cells, like WT, showed characteristic of mesenchymal stem cells (MSC), i.e. ability to give rise to CFU-F and to differentiate to adipocytes and osteoblasts. They express relatively high level of nestin and osteopontin. At a mechanistic level, gene expression analysis of molecules known to play a critical role in HSC maintenance in the niche indicated 200-fold downregulation of Kit-ligand and Wnt3a in p190-B−/− stroma compared to WT stroma (p<0.05). Surprisingly, BMP4, angiopoietin and CXCL12 were upregulated in p190-B−/− stroma, 35-fold, 14-fold and 16-fold, respectively (p<0.05). Hence, p190-B expression in MSC/stromal cell microenvironment fine-tunes niche regulatory pathways to maintain normal hematopoiesis. Therefore, p190-B may be a critical regulator of the mesenchymal stem/hematopoietic stem and progenitor niche. Disclosures:No relevant conflicts of interest to declare.

Erythropoietin-Driven Signaling Complements the Survival Defect of DMT1-Mutant Erythroid Cells

Abstract 2104Mutations inactivating the divalent metal transporter 1 (DMT1) cause impaired erythroid iron utilization and lead to the development of hypochromic microcytic anemia associated with ineffective erythropoiesis (IE). The anemia can be ameliorated with high-dose erythropoietin (EPO) therapy (Pospisilova D, et al. Blood. 2006. 108:404–5). In contrast to β-thalassemia mouse model with dramatically elevated EPO levels which were proposed to protect erythroid cells from apoptosis (Libani IV, et al. Blood. 2008. 112:875–885), DMT1-mutant mice (mk/mk) have only 2.8-fold higher EPO levels when compared to the wild-type littermates. This corresponds to 2-fold elevation of serum EPO above the normal range for DMT-1-mutant patient before initiation of EPO therapy. Different mechanisms may therefore drive IE in anemia due to DMT1 mutation. In this study we analyzed the bases for the clinical success of high-dose EPO supplementation in a DMT1-mutant patient and mk/mk mice. EPO administration significantly increased hemoglobin levels (7.4 g/dL to 9.1 g/dL for the patient and 7.5±0.6 to 9.5±0.4 g/dL for mk/mk mice) and partially ameliorated IE. Colony forming assay using patient’s cells showed significantly improved in vitro growth of post-treatment DMT1-mutant burst-forming unit erythroid (BFU-E) progenitors when compared to pre-treatment BFU-Es. In addition, the reduced plating efficiency and colony-forming capacity of pre-treatment DMT1-mutant BFU-Es can be corrected by the addition of the broad spectrum caspase inhibitor z-VAD-fmk to the cultures. This indicates involvement of caspase-dependent apoptosis in the defective survival of pre-treatment BFU-E progenitors and in their impaired capacity to form erythroid colonies. TUNEL assay on patient’s bone marrow smears showed markedly decreased rate of apoptosis (from 4% to 1.5% of TUNEL-positive erythroblasts) after EPO supplementation. No profound changes in erythroblast maturation were noted in post-treatment bone marrow with the exception of additional expansion of polychromatophilic pool suggesting that inhibition of apoptosis rather than increased differentiation of DMT1-mutant erythroid cells predominantly accounts for amelioration of anemia and IE. In accordance with the patient’s results, EPO administration to mk/mk mice did not alter the distribution of erythroblasts of different maturation stages. On the other hand, augmented STAT5 activation and enhanced expression of anti-apoptotic proteins BCL-XL and MCL-1 was detected in EPO treated mice. This correlated with decreased number of erythroid Ter119+ precursors undergoing apoptosis in EPO treated mk/mk bone marrow (12.4±2.3% to 5.4±0.9%) and spleen (7.3±0.7% to 3.1±0.9%). EPO supplementation also significantly reduced susceptibility of mk/mk erythrocytes to undergo stress-induced death that could reflect increased eryptosis (apoptosis of DMT1-mutant erythrocytes) in vivo and protective effect of EPO. Low to undetectable expression of hepcidin in mk/mk liver could be attributed to 16-fold increase in GDF15 expression in the bone marrow; the expression of TWSG1 was comparable to wild-type littermates. Also patient’s urinary hepcidin is low (55.3 ng/mg creatinine; normal range 71–1762), however, in contrast to mk/mk mice and β-thalassemia patients the suppression of hepcidin seems to be only partly mediated by GDF15 as patient’s GDF15 plasma levels are only 1.9-fold higher (548.4 pg/mL) in comparison to gender- and age-matched controls (288.4±56.9 pg/mL). These results indicate that mouse models may not fully mimic the human disease and suggest existence of additional bone marrow-derived regulator of hepcidin expression. In summary we present the bases for the clinically approved success of EPO treatment under condition of iron-deprived erythropoiesis. We conclude that EPO-driven signaling rescues the survival defect of DMT1-mutant erythroid cells.Grant support: Czech Grant Agency, grants No. P305/10/P210 and P305/11/1745; Internal Grant of Palacky University Olomouc (LF_2011_011), and Ministry of Health Czech Republic Grant NS10281-3/2009. Disclosures:No relevant conflicts of interest to declare.

1, 25-Dihydroxyvitamin D Suppresses TNFα-Mediated NF-κB Activation in K562 and Human CD34+ Cells and Promotes Recovery of Erythroid Colony Formation in Human CD34+ Cells

Abstract 2097Anemia of inflammation (AI) has been widely associated with chronic rheumatologic, infectious, and cardiovascular disorders and comprises one-third of cases of anemia in the elderly. Although the antimicrobial peptide hepcidin is felt to be the prime regulator of AI, emerging evidence supports the premise that subsets of AI may be mediated via hepcidin-independent inflammatory pathways, such as those modulated by tumor necrosis factor alpha (TNFα), that directly suppress erythropoiesis. A better understanding of the pathophysiology of anemia subtypes is necessary if we are to develop more effective targeted, oral therapies for patients with inflammatory anemia. Our recent analysis of elderly participants in the Third National Health and Nutrition Examination Survey (NHANES III) revealed that vitamin D deficiency was strongly associated with AI in this population, with this subgroup exhibiting a rate of vitamin D deficiency nearly twice that of similarly aged non-anemic individuals. In the current study we aimed to evaluate whether vitamin D treatment may ameliorate TNFα-mediated erythroid colony suppression in humans. Human bone marrow-derived CD34+ cells from 3 healthy adults were first cultured in methylcellulose medium containing IL-3 (10ng/ml), EPO (1U/ml), SCF (50ng/ml) in the presence or absence of TNFα (25 ng/ml) and increasing doses of 1, 25-dihydroxyvitamin D3 [calcitriol, (0.01-1nM)], and erythroid burst-forming units (BFU-E) measured on day 14. The percentage of BFU-E colonies was reduced by 50% at day 14 in the presence of 25ng/ml TNFα (p < 0.01, t-test). TNFα-mediated suppression of erythropoiesis was reversed by the addition of vitamin D3 in a dose-dependent manner, with 1nM vitamin D3 resulting in a 50% recovery of BFU-E colony numbers at day 14 (p < 0.05, t-test). Vitamin D3 alone exhibited no significant effect on BFU-E formation at all concentrations tested. Based on studies showing that vitamin D receptor may serve as a negative regulator of NF-κB transcriptional activity, we next investigated whether vitamin D3 may alter TNFα-induced NF-κB activation in both K562 and human CD34+ cells. K562 cells pre-incubated with 1nM vitamin D3 for 3, 24, 48 and 72 hours were treated with 5 ng/ml TNFα for 30 minutes, and NF-κB activity in nuclear fraction was determined by an ELISA-format oligonucleotide binding assay. TNFα treatment alone markedly increased the oligonucleotide binding activity of the NF-κB p65 subunit, which was completely blocked by co-incubation with competitor oligonucleotide, confirming the assay specificity. Pre-incubation with vitamin D3 for 3 – 48 hours showed little effect on TNFα-mediated NF-κB activation, but pre-incubation for 72 hours resulted in suppression of TNFα-induced NF-κB activity by 46% (p < 0.02, t-test). Under similar conditions, human CD34+ cells pre-incubated with 1 nM vitamin D3 for 48 hours and 72 hours exhibited 38% and 84% suppression of TNFα-induced NF-κB activity, respectively (p < 0.01 for both conditions, t-test). Reduced NF-κB activity was correlated with the decreased nuclear translocation of NF-κB in K562 cells with no significant changes in IκB degradation, suggesting that vitamin D3 regulates the NF-κB nuclear translocation independent of IκB. Western analysis of whole cell lysates from both K562 and human CD34+ cells using antibodies recognizing known TNFα-associated signaling pathways revealed robust increases in phospho-p38, phospho-JNK, and phospho-ERK1/2 in response to TNFα stimulation compared with control, with no inhibition of these signaling pathways noted in response to vitamin D3 treatment at all doses tested, suggesting that vitamin D3 does not inhibit these TNFα-induced signaling cascades. Our study indicates that 1, 25-dihydroxyvitamin D3 significantly ameliorates TNFα-mediated suppression of erythroid colony development in human CD34+ cells via mechanisms that involve modulation of NF-κB signaling pathways and supports the design of future clinical trials examining whether vitamin D3 supplementation may prove to be an effective therapy for subgroups of patients with TNFα-mediated inflammatory anemia. Disclosures:No relevant conflicts of interest to declare.

EPO-Dependent Recovery of Late-Stage Erythroid Progenitors in the Marrow Precedes Splenic Expansion: Insights From a Sublethal Radiation Model

Abstract 180Erythropoiesis is a robust process of cellular expansion and maturation that occurs in the bone marrow and spleen of mice. Following clastogenic injury such as total body irradiation (TBI), erythroblasts are severely depleted in these organs, resulting in loss of reticulocyte output and the development of a mild anemia (Peslak et al., Exp. Hematol. 2011). However, the mechanistic and microenvironmental factors underlying erythroid recovery following sublethal TBI are poorly understood. To this end, we utilized colony assays to quantify erythroid progenitors, which consist of immature d7 erythroid burst-forming units (BFU-E) and more mature d3 BFU-E and erythroid colony forming units (CFU-E). Imaging flow cytometry was used to quantify erythroblast precursors. We found that d7 BFU-E undergo a slow, incomplete recovery during the first 10 days post-4 Gy TBI of C57Bl/6 mice. In contrast, d3 BFU-E exhibit a robust recovery beginning at 4 days post-TBI that is immediately followed by a rapid increase in CFU-E numbers to over 200 percent of steady-state levels. This initial erythroid progenitor recovery is followed by a wave of erythroid precursor maturation and red cell formation that occurs in close association with macrophages in the bone marrow. These erythroblast islands undergo a rapid synchronous expansion that peaks at 6 days post-TBI, suggesting that the bone marrow microenvironment plays a role in the recovery of the erythron from sublethal TBI. We hypothesized that erythropoietin (EPO), the primary regulator of erythroid survival and proliferation, mediates the rapid, specific expansion of late-stage erythroid progenitors following radiation injury. We found that plasma EPO levels increase 13-fold 4 days after 4 Gy TBI, temporally correlated with expansion of d3 BFU-E. Furthermore, maintenance of steady-state hematocrit levels following TBI prevented EPO induction and blocked expansion of late-stage erythroid progenitors, while exogenous EPO administered at 1 hour post-radiation specifically advanced recovery of late-stage progenitors. These data indicate that EPO is required for expansion of d3 BFU-E and CFU-E following radiation-induced marrow depletion.During times of acute hypoxia, such as the severe anemia induced by bleeding or phenylhydrazine exposure, EPO production is rapidly upregulated and splenic stress erythropoiesis is induced. Surprisingly, splenic erythropoiesis is absent during the rapid initial recovery of erythropoiesis in the bone marrow at 4–6 days post-TBI. However, a massive expansion of CFU-E begins at 7–8 days post-4 Gy TBI in spleen. EPO administration at 4 days following 4 Gy TBI significantly enhances late-stage progenitor recovery exclusively in the marrow, indicating that erythroid progenitors are not present in spleen at the time of rapid bone marrow expansion and that late-stage erythroid progenitor recovery initiates in the marrow and subsequently proceeds to the spleen. Furthermore, we found that erythroid progenitors transiently emerge in the bloodstream at 6–8 days post-TBI, following marrow recovery and prior to initiation of splenic erythropoiesis. These data are consistent with endogenous migration of the erythron from the bone marrow to the spleen during recovery from radiation-induced erythroid injury. Taken together, our data indicate that recovery from sublethal irradiation injury is regulated primarily by the EPO-induced expansion of late-stage erythroid progenitors in the bone marrow. This form of clastogenic injury is critically different from bleeding or hemolysis, which preserve bone marrow and splenic erythroblasts and induce expansion of splenic erythroid stress progenitors. Sublethal irradiation injury thus provides a unique model for the in vivo study of endogenous erythroid recovery. This model may be clinically useful for the functional evaluation of therapeutic factors that regulate or modulate erythroid cell maturation. Disclosures:Bemis:Litron Laboratories: Employment, Patents & Royalties.

CD34(+) stem cells from umbilical cord blood.

We describe the relation between umbilical cord clamping time and two different enrichment system of CD34+ stem cells from umbilical cord blood with the proliferative ability and bone marrow reconstitution of the stem cells obtained. After an obstetrician performed the cord blood collection, the purification of stem cells was performed either with a combination of monoclonal antibodies (negative selections) using the Stem Sep method, or with a positive cells selection based on their surface CD34 antigens using the Mini Macs system. An excellent recovery of haematopoietic progenitors [Burst Forming Unit Erythroids (BFUE); Colony Forming Unit Granulocytes and Macrophages (CFU-GM); and Colony Forming Unit Granulocytes, Erythroids, Monocytes and Macrophages (CFU-GME)], inversely related to the increase in clamping time, was performed with the Mini Macs system (54% of colonies, with 90% purity). With Stem Sep method, haematopoietic progenitor's recovery was 35% (with 80% purity). By applying early clamping of umbilical cord blood we obtained a greater number of CD34+ cells and their clonogenic activity was increased with enrichment. This is a useful technique considering that the number of CD34+ stem cells usually contained from a unit of placental blood is enough for the transplant to a child, but not for an adult. Thus, using these methods, we can get a larger number of CD34+ stem cells which reduces the risk of Graft versus Host Disease also in adult patients, producing survival rates similar to those obtained with transplantation of bone marrow from unrelated donors.

Chemosensitivity of nonleukemic clonogenic precursors in AML patients in complete remission: Association with CD34 + mobilization and with disease-free survival

A high number of CD34(+) cells in the peripheral blood during mobilization in patients with acute myeloid leukemia (AML) in complete remission (CR) is associated with a high relapse rate. The variability in chemoresistance of normal bone marrow precursors has been hypothesized as explanation for the variable CD34 mobilization in AML. In 37 patients with AML in CR, we determined the chemosensitivity of bone marrow clonogenic precursors to maphosphamide and etoposide, which was then correlated with the degree of CD34(+) mobilization. In an enlarged set of 49 patients, we also studied the importance of chemosensitivity of marrow precursors for disease-free survival and relapse incidence. Significant correlations were demonstrated between the peak number of CD34(+) cells and residual growth of colony-forming unit granulocyte-macrophage (CFU-GM) after maphosphamide (R = 0.550; p = 0.0003) and after etoposide (R = 0.793; p = 0.0003). It was possible to identify three groups of AML patients based on chemosensitivity. The mean CD34(+) peak was 33 × 10(6)/L in the hyperchemosensitive group, 141×10(6)/L in the normochemosensitive (p = 0.03), and 379 × 10(6)/L in the chemoresistant group (p = 0.002). Failed CD34(+) mobilization was observed in 72% of the hyperchemosensitive group, 23% of the normochemosensitive group, and 0% of the chemoresistant group (p = 0.001). Hyperchemosensitivity of CFU-GM, together with a low platelet count, were independent factors important in the failure of CD34(+) cell mobilization. A disease-free survival significantly inferior to that of all other patients was associated with chemoresistance of CFU-GM (log rank, p=0.030) and with chemoresistance of burst-forming unit erythroid (BFU-E) (log rank, p=0.033). Chemoresistance of CFU-GM (p = 0.048) and BFU-E (p = 0.017) was also associated with increase relapse incidence. Nonleukemic nature of these precursors was demonstrated studying minimal residual disease from single colony cells. In conclusion, we found that hyperchemosensitivity of normal nonleukemic CFU-GM is associated with a high risk of CD34(+) cell mobilization failure, while a chemoresistant pattern in CFU-GM and BFU-E is associated with poor disease-free survival and increased cumulative incidence of relapse.

SWAP-70 regulates erythropoiesis by controlling 4 integrin

Background The regulation of normal and stress-induced erythropoiesis is incompletely understood. Integrin-dependent adhesion plays important roles in erythropoiesis, but how integrins are regulated during erythropoiesis remains largely unknown. To obtain novel insights into the regulation of erythropoiesis, we used cellular and molecular approaches to analyze the role of SWAP-70 and the control of integrins through SWAP-70. In addition, mice deficient for this protein were investigated under normal and erythropoietic stress conditions. We show that SWAP-70, a protein involved in cytoskeletal F-actin rearrangements and integrin regulation in mast cells, is expressed in hematopoietic stem cells and myeloid-erythroid precursors. Although Swap-70(-/-) mice are not anemic, erythroblastic differentiation is perturbed, and SWAP-70 is required for an efficient erythropoietic stress response to acute anemia and for erythropoietic recovery after bone marrow transplantation in irradiated mice. SWAP-70 deficiency impairs colony-forming unit erythroid development, while burst-forming unit erythroid development is normal, and significantly affects development of late erythroblasts in the spleen and bone marrow. The α(4) integrin is constitutively hyper-activated in Swap-70(-/-) colony-forming unit erythroid cells, which hyper-adhere to fibronectin. Blocking α(4) and β(1) integrin chains in vivo restored erythroblastic differentiation and the erythropoietic stress response in Swap-70(-/-) mice. Conclusions Our study reveals that SWAP-70 is a novel regulator of integrin-mediated red blood cell development and stress-induced erythropoiesis.

Aberrant Epigenetic and Genetic Marks Are Seen in Myelodysplastic Leukocytes and Reveal Dock4 as a Candidate Pathogenic Gene on Chromosome 7q

Myelodysplastic syndromes (MDS) are characterized by abnormal and dysplastic maturation of all blood lineages. Even though epigenetic alterations have been seen in MDS marrow progenitors, very little is known about the molecular alterations in dysplastic peripheral blood cells. We analyzed the methylome of MDS leukocytes by the HELP assay and determined that it was globally distinct from age-matched controls and was characterized by numerous novel, aberrant hypermethylated marks that were located mainly outside of CpG islands and preferentially affected GTPase regulators and other cancer-related pathways. Additionally, array comparative genomic hybridization revealed that novel as well as previously characterized deletions and amplifications could also be visualized in peripheral blood leukocytes, thus potentially reducing the need for bone marrow samples for future studies. Using integrative analysis, potentially pathogenic genes silenced by genetic deletions and aberrant hypermethylation in different patients were identified. DOCK4, a GTPase regulator located in the commonly deleted 7q31 region, was identified by this unbiased approach. Significant hypermethylation and reduced expression of DOCK4 in MDS bone marrow stem cells was observed in two large independent datasets, providing further validation of our findings. Finally, DOCK4 knockdown in primary marrow CD34(+) stem cells led to decreased erythroid colony formation and increased apoptosis, thus recapitulating the bone marrow failure seen in MDS. These findings reveal widespread novel epigenetic alterations in myelodysplastic leukocytes and implicate DOCK4 as a pathogenic gene located on the 7q chromosomal region.

HIF1α synergizes with glucocorticoids to promote BFU-E progenitor self-renewal

AbstractWith the aim of finding small molecules that stimulate erythropoiesis earlier than erythropoietin and that enhance erythroid colony-forming unit (CFU-E) production, we studied the mechanism by which glucocorticoids increase CFU-E formation. Using erythroid burst-forming unit (BFU-E) and CFU-E progenitors purified by a new technique, we demonstrate that glucocorticoids stimulate the earliest (BFU-E) progenitors to undergo limited self-renewal, which increases formation of CFU-E cells > 20-fold. Interestingly, glucocorticoids induce expression of genes in BFU-E cells that contain promoter regions highly enriched for hypoxia-induced factor 1α (HIF1α) binding sites. This suggests activation of HIF1α may enhance or replace the effect of glucocorticoids on BFU-E self-renewal. Indeed, HIF1α activation by a prolyl hydroxylase inhibitor (PHI) synergizes with glucocorticoids and enhances production of CFU-Es 170-fold. Because PHIs are able to increase erythroblast production at very low concentrations of glucocorticoids, PHI-induced stimulation of BFU-E progenitors thus represents a conceptually new therapeutic window for treating erythropoietin-resistant anemia.

Split chimerism between nucleated and red blood cells after bone marrow transplantation for haemoglobinopathies

Previous studies have shown that a stable presence of both donor and recipient haematopoietic derived cells after allogeneic haematopoietic stem cell transplantation (HSCT) occurs in approximately ten percent of the patients affected by Beta-Thalassemia. Once achieved this condition, defined as persistent mixed chimerism (PMC), the patients do not require additional red blood cells (RBCs) support and, regardless of the presence in some cases of an extremely low percentage of donor-derived nucleated cells, they are clinically cured by an incomplete, but functional graft. Most of the published papers have however investigated the impact of donor engraftment in the nucleated cells, rather than in the mature erythrocytes. We have recently published a paper showing that in four long-term transplanted patients affected by haemoglobinopathies, characterized by the presence of few donor engrafted nucleated cells - both in the peripheral blood and in the bone marrow – the majority of the erythrocytes were of donor origin. Moreover we showed that the proportion of donor-derived erythroid precursors, determined by analyzing singularly picked-up burst-forming unit erythroid colonies, was equivalent to that observed in the mature nucleated cells, rather than in the red blood cells. These results suggest that in patients characterized by the presence of PMC after HSCT a selective advantage of the donor erythroid precursors maturation might successfully contrast the problems bound to the recipient ineffective erythropoiesis. When genetically modified HSCT will be a possible option for treating Thalassemia Major, the co-existence of the repaired cells with those still expressing the genetic defect will be an expected scenario, not in an allogeneic, but in an autologous, environment.

The Risk of Secondary Myelodysplastic Syndrome/Acute Leukemia Following High-Dose Yttrium-90 Ibritumomab Tiuxetan Is Analogous to That Observed Following High-Dose Chemotherapy: a Matched-Pair Analysis In Non-Hodgkin Lymphoma Patients.

AbstractAbstract 1289 Introduction:Myeloablative doses of Yttrium-90 (90Y)-Ibritumomab Tiuxetan combined with tandem autologous peripheral blood stem cell (PBSC) support can be safely administered to patients aged up to 76 years (Devizzi L et al, J Clin Oncol 2008). Whether marrow exposure to myeloablative doses of radiolabelled antibodies (hd-radio-immunotherapy) could increase the risk of secondary myelodysplastic syndrome and acute leukemia (sMDS/AL) remains a matter of concern. Therefore, we prospectively investigated by means of a longitudinal study the incidence of sMDS/AL in a cohort of high-risk non-Hodgkin lymphoma (NHL) patients receiving hd-radio-immunotherapy. The cumulative incidence of sMDS/AL in the study group was compared with that observed in a historical group of patients autografted following chemotherapy-based myeloablative conditioning. Additionally, to further investigate marrow damage induced by hd-radio-immunotherapy, hematopoietic and stromal progenitors, as well as telomere length (TL) were assessed. Patients and Methods:From July 2004 through December 2007, 53 relapsed/refractory (n= 36) or de novo high-risk (n= 17) NHL patients (median age, 64 years; range, 26 – 76 years) received a high-dose sequential (HDS) chemotherapy program, including a myeloablative phase with 90Y-Ibritumomab Tiuxetan (1.2 mCi/kg body weight). Patients were monitored for frequency of colony-forming cells (CFCs), cytogenetic and TL analysis. Bone marrow (BM) samples were collected prior to hd-radio-immunotherapy, every six months for two years and annually thereafter. The study was approved by the Institutional Ethical Committee and written informed consent was obtained from each patient. The study group included 29 patients with aggressive lymphoma (diffuse large B-cell lymphoma, n = 19; mantle cell lymphoma, n = 8; Richter syndrome, n = 2) and 24 with indolent NHL subtypes (follicular lymphoma, n = 20; marginal zone lymphoma, n = 3; lymphocytic lymphoma, n = 1). Thirty patients were relapsed after one (n=15) or more (n=15) previous chemotherapy regimens, 6 patients were refractory and 17 patients were previously untreated with high-risk prognostic index. Pre-exposure to alkylating agent involved 68% of the patients. A historical group of 55 NHL patients who received an analogous HDS program with final chemotherapy-based autografting was used as control group. Study and control groups were pair-matched for age, gender, histological subtype, disease status at transplant and type of grafted PBSC. Results:At a median follow-up of 49 months (range, 32 – 74), 4 out of 53 patients of the study group developed sMDS/AL at 6, 12, 27 and 36 months from transplant. According to WHO criteria, refractory anemia with excess of blasts 1 (RAEB-1) was diagnosed in one patient, whereas refractory cytopenia with unilineage dysplasia (RCUD) was diagnosed in the other three cases, with acute leukemia transformation being observed in two patients at 12 and 18 months after initial detection of abnormal cytogenetics, respectively. The cumulative incidence of sMDS/AL resulted of 8.29% at 5-years. An analogous 5-year cumulative incidence of 8.05% was observed in the historical control group autografted following a chemotherapy-based conditioning. Radioimmunotherapy-treated patients showed a significant but transient decrease in granulocyte-macrophage colony forming unit (CFU-GM) at 6 months after transplant, with recovery to baseline values at 12 months. Multilineage colony-forming units (CFU-Mix), erythroid burst-forming units (BFU-E) and fibroblast CFU (CFU-F) were not show significantly affected over time. In contrast, a progressive and significant decrease in BM TL was evident from 12 months after transplant. Conclusion:In our series of elderly NHL patients receiving hd-radio-immunotherapy, the incidence of sMDS/AL was found increased compared to recently reported series of younger lymphoma patients receiving chemotherapy-based conditioning. However, the increased risk of sMDS/AL was not associated with the type of myeloablative conditioning, i.e. chemotherapy vs radio-immunotherapy. Other factors may have favored the development of sMDS/AL, including advanced age at transplant, extensive pre-transplant therapy and type of reinfused PBSC. The observed progressive TL loss gives support to the concept that TL loss might be involved in sMDS/AL development. Disclosures:No relevant conflicts of interest to declare.

Inhibition of BCL11B Expression Leads to Apoptosis of Malignant T Cells but Not CD34+ Cells.

AbstractAbstract 3755Altered expression, mutation, disruption, or rearrangement of the B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11B gene (BCL11B) has been associated with T-cell malignancies. BCL11B overexpression occurs primarily in T-cell malignancies, suggesting that it may be a target gene for cancer therapy. Our previous study showed that the inhibition of BCL11B expression by siRNA led to apoptosis in human T-cell acute lymphoblastic leukemia cell lines (Jurkat, huT78, and Molt-4), but not in normal, mature T cells. To confirm the potential of BCL11B siRNA as a therapeutic agent and to determine its safety, the present study further analyzed the effects of BCL11B suppression on hemopoietic stem/progenitor cells, CD34+ cells were collected from three samples of umbilical cord blood using magnetic-activated cell sorting. BCL11B expressions in mRNA level of umbilical cord blood CD34+ cells were analyzed by the real-time quantitative PCR with TaqMan technique. For nucleofection of CD34+ cells, BCL11B-siRNA935 and the U-001 program were used of a Nucleofection Device II. Mock-transfected cells nucleofected without siRNA were used as a negative control. To evaluate the role of differentiation and proliferation in CD34+ cells after BCL11B-siRNA935 transfection, erythroid burst-forming units (BFU-E), granulocyte/macrophage colony-forming units (CFU-GM), and megakaryocyte colony-forming units (CFU-Meg) from BCL11B siRNA935-, mock-CD34+ cells were performed using methylcellulose assays. Colonies were assessed 14 days after plating. The BCL11B mRNA expression level in the CD34+ cells was significantly lower than that in Molt-4 cells and in peripheral blood mononuclear cells from eight healthy individuals. There was no significant difference between BCL11B siRNA935-transfected and mock-transfected CD34+ cells with respect to the formation of BFU-E, CFU-GM, or CFU-Meg (P > 0.05). We speculate that the BCL11B gene, because of its very low expression level, may not play a major role in proliferation and differentiation of hematopoietic stem/progenitor cells, but we cannot rule out another anti-apoptotic mechanism. Nevertheless, BCL11B gene silencing alone does not affect the proliferation and differentiation of hematopoietic stem/progenitor cells in vitro. In conclusion, our findings provide evidence for an anti-apoptotic function of BCL11B in T-cell malignancies, but not in hemopoietic stem/progenitor cells, suggesting that BCL11B siRNA is safe and may be considered a new targeted therapeutic strategy for T-cell malignancies.* National Natural Science Foundation(30771980)& Guangdong Science and Technology Project (2007B030703008) Grants Disclosures:Li:National Natural Science Foundation (30771980) and Guangdong Science and Technology Project (2007B030703008, 2009B050700029) Grants: Research Funding.

Stress Erythropoiesis: HIF-1 Alpha Synergizes with Glucocorticoids to Induce BFU-E Progenitor Self-Renewal

AbstractAbstract 814The goal of this study was to find small molecules that stimulate erythropoiesis at an earlier stage than erythropoietin (Epo). We therefore studied the mechanisms by which glucocorticoids (GCs) promote erythroblast formation during stress erythropoiesis (SE). Since the target cell of GCs in SE was not known we first established a FACS-based method to purify burst-forming unit erythroid (BFU-E) and colony-forming unit of erythroid lineage (CFU-E) cells from mouse fetal liver. BFU-E and CFU-E cells were resolved by differences in CD71 or CD24a expression (BFU-E = CD71/CD24a10% low; CFU-E = CD71/CD24a20% high) from a population of cells positive for Kit, and negative for the cell surface markers Ter119, B220, Mac-1, CD3, Gr-1, Sca-1, FcGR, CD41 and CD34. Purified “BFU-E” cells formed 94% BFU-E colonies and “CFU-E” cells formed 95% CFU-E colonies at cloning efficiencies of 55–70%. Using pure CFU-E and BFU-E progenitors we demonstrated that while 100nM Dexamethasone (Dex) does not increase proliferation or self-renewal of CFU-E cells, Dex stimulates the earlier BFU-E progenitors to undergo limited self-renewal, which increases formation of CFU-E cells and erythroblasts >20-fold. After identifying BFU-E progenitors as the target cells of GCs during SE, we next determined how DEX changes miRNA and mRNA expression in BFU-Es by next generation sequencing. No miRNAs changed the level of expression more than 50%. Of the 10,000 most expressed mRNAs 83 were up-regulated and 112 mRNAs down-regulated more than 2-fold in response to Dex. Since previous studies show that dimerization (trans activation) of the GC receptor is necessary for GCs to induce erythroblast production, the most important GC target genes are likely up- rather than down-regulated. We hypothesized that other molecules that are able to enhance the expression of the 83 up-regulated genes could enhance or replace the stimulatory effect of Dex. We therefore computationally analyzed the promoter regions of the up-regulated genes using motif enrichment analysis. We found that Dex induces expression of genes in BFU-E cells that contain promoter regions highly enriched for hypoxia-induced factor 1 alpha (HIF1a) binding sites (p-value = 1e–27). Analysis of six published gene expression datasets, where the effect of GCs on gene expression had been determined in cell types other than BFU-E cells, showed very low or no enrichment of HIF1a targets among genes up-regulated by Dex. We confirmed the suggested overlapping effects of HIF1a and GCs on gene expression in BFU-E cells by comparing the effect of Dex with that of pharmacological HIF1a activation by the prolyl hydroxylase inhibitor (PHI) Dimethyloxalylglycine (DMOG). As determined by next generation mRNA sequencing, DMOG increased expression of 98 genes more than 50%, of which 28 were among 109 genes up-regulated >50% by Dex (p-value for overlap = 1e–26). Importantly the overlap in effects on gene expression also resulted in overlapping biological effects. Similar to Dex, DMOG had little effect on CFU-E cells (Figure A). In contrast DMOG synergized with Dexamethasone to promote BFU-E self-renewal and prevent BFU-E cell exhaustion, enhancing the production of CFU-E cells 170-fold and maximum erythroblast production 300-fold (Figure B). DMOG thus enhanced the stimulatory effect of 100nM Dex on erythroblast production 7-fold. In similar experiments where BFU-E cells were cultured in GCs levels corresponding to physiological cortisol levels (1 nM Dex), DMOG enhanced erythroblast production 10-fold. We further showed that DMOG and Dex also synergistically enhance proliferation of adult mouse and human erythroid progenitors. Our results support a physiological model of SE where increased systemic levels of free cortisol, in combination with local anoxia, induce BFU-E self-renewal and increase erythroblast production. This suggests that the therapeutic potential for PHIs in treatment for anemia goes beyond replacing recombinant Epo. Here we show that in addition to enhancing Epo production from the kidneys, PHIs have an intrinsic stimulatory effect on BFU-E progenitors, leading to increased production of Epo-responsive CFU-E cells. Thus PHIs provide a new window for treating Epo-resistant anemia. The synergistic effect of GCs and PHIs further suggests that PHIs may enhance or replace the effect of Prednisone in the treatment of Diamond-Blackfan Anemia and other bone marrow failure syndromes. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Erythropoietin Induction by Anemia Is Required for CFU-E Expansion During Erythroid Recovery From Sublethal Radiation Injury

AbstractAbstract 3218The massive steady-state output of the erythron makes the erythroid lineage exquisitely sensitive to clastogenic injury. While the rapid loss of reticulocytes is well-described, the response of bone marrow progenitors and precursors to sublethal irradiation and the mechanisms underlying recovery of the erythron remain poorly defined. Following 4 Gy total body irradiation (TBI) in C57Bl/6 mice, functional colony assays were utilized to study the erythroid progenitor compartment, consisting of immature day 7 erythroid burst-forming units (BFU-E) and more mature day 3 BFU-E and erythroid colony-forming units (CFU-E). Multispectral imaging flow cytometry was used to quantify the erythroid precursor compartment, consisting of proerythroblasts and progressively more mature basophilic, polychromatophilic, and orthochromatic erythroblasts. At 2 days after 4 Gy TBI, greater than 95% of erythroid progenitors and precursors in the marrow were lost. A significant decrease in peripheral reticulocyte output and a gradual 9% drop in hematocrit accompanied this marrow loss over the first 3–4 days after radiation exposure. Following this initial injury and mild radiation-induced anemia, a robust recovery of the erythron began with a significant increase in day 3 BFU-E at 5 days post-radiation immediately followed by a rapid expansion of CFU-E at 5–6 days post-radiation to 200% of unirradiated control marrow. In contrast, day 7 BFU-E only partially recovered in a gradual linear fashion. Subsequent maturation of CFU-E resulted in progressive waves of erythroid precursors, reticulocytes, and mature red cells, creating a “ripple effect” following sublethal radiation injury. These results indicate that erythroid repopulation following radiation damage is centered on specific expansion and maturation of later erythroid progenitors (day 3 BFU-E and CFU-E). Erythropoietin (EPO) is known to be the primary regulator of the erythroid lineage, and day 3 BFU-E and CFU-E form the EPO-responsive compartment of the erythron. Therefore, we hypothesized that EPO may be the primary driving force underlying day 3 BFU-E/CFU-E expansion and subsequent erythroid recovery from radiation injury. Endogenous plasma EPO levels increased 13-fold above steady-state levels at 4 days post-radiation. This spike in EPO levels preceded the CFU-E expansion seen at 5–6 days post-radiation. To specifically determine both the etiology of the endogenous EPO spike and the necessity of supra-physiologic levels of EPO for erythroid recovery from radiation injury, we performed loss-of-function studies in which mice were transfused with packed red cells post-radiation to maintain a normal hematocrit. Prevention of the mild radiation-induced anemia by transfusion also prevented the increase in endogenous EPO and significantly abrogated day 3 BFU-E/CFU-E recovery. These findings directly link radiation-induced anemia with EPO induction and erythroid lineage reconstitution. Gain-of-function studies were performed to determine whether EPO is sufficient to drive erythroid expansion after radiation injury. Administration of exogenous EPO at 1 hour post-radiation significantly advanced the timing of CFU-E expansion and subsequent recovery of the erythron. In addition, the accelerated synchronous wave of recovery following exogenous EPO very closely mirrored the physiological wave of recovery during the endogenous EPO response, indicating that the previously observed “ripple effect” is an inherent component of EPO-induced erythroid recovery from radiation injury. Finally, administration of EPO at 4 days post-radiation, at the peak of the endogenous EPO response, further enhanced CFU-E recovery to over 330% of unirradiated control levels, providing additional evidence that EPO drives expansion of irradiated CFU-E. These studies, taken together, indicate that the anemia-induced EPO response following radiation injury is both necessary and sufficient for CFU-E expansion that leads to recovery of the erythron. A better understanding of the response of the erythroid lineage to clastogenic injury will ultimately lead to improved therapies to protect and mitigate the hematopoietic system from radiation and chemotherapy damage. Disclosures:No relevant conflicts of interest to declare.

Mdm2 Is Required for Survival of Hematopoietic Stem Cells/Progenitors via Dampening of ROS-Induced p53 Activity

Mdm2 is an E3 ubiquitin ligase that targets p53 for degradation. p53(515C) (encoding p53R172P) is a hypomorphic allele of p53 that rescues the embryonic lethality of Mdm2(-/-) mice. Mdm2(-/-) p53(515C/515C) mice, however, die by postnatal day 13 resulting from hematopoietic failure. Hematopoietic stem cells and progenitors of Mdm2(-/-) p53(515C/515C) mice were normal in fetal livers but were depleted in postnatal bone marrows. After birth, these mice had elevated reactive oxygen species (ROS) thus activating p53R172P. In the absence of Mdm2, stable p53R172P induced ROS and cell cycle arrest, senescence, and cell death in the hematopoietic compartment. This phenotype was partially rescued with antioxidant treatment and upon culturing of hematopoietic cells in methycellulose at 3% oxygen. p16 was also stabilized because of ROS, and its loss increased cell cycling and partially rescued hematopoiesis and survival. Thus, Mdm2 is required to control ROS-induced p53 levels for sustainable hematopoiesis.