Erythropoiesis In Myelodysplastic Syndromes Research Articles

Iron chelation improves ineffective erythropoiesis and iron overload in myelodysplastic syndrome mice.

Myelodysplastic syndrome (MDS) is a heterogeneous group of bone marrow stem cell disorders characterized by ineffective hematopoiesis and cytopenias, most commonly anemia. Red cell transfusion therapy for anemia in MDS results in iron overload, correlating with reduced overall survival. Whether the treatment of iron overload benefits MDS patients remains controversial. We evaluate underlying iron-related pathophysiology and the effect of iron chelation using deferiprone on erythropoiesis in NUP98-HOXD13 transgenic mice, a highly penetrant well-established MDS mouse model. Our results characterize an iron overload phenotype with aberrant erythropoiesis in these mice which was reversed by deferiprone-treatment. Serum erythropoietin levels decreased while erythroblast erythropoietin receptor expression increased in deferiprone-treated MDS mice. We demonstrate, for the first time, normalized expression of the iron chaperones Pcbp1 and Ncoa4 and increased ferritin stores in late-stage erythroblasts from deferiprone-treated MDS mice, evidence of aberrant iron trafficking in MDS erythroblasts. Importantly, erythroblast ferritin is increased in response to deferiprone, correlating with decreased erythroblast ROS. Finally, we confirmed increased expression of genes involved in iron uptake, sensing, and trafficking in stem and progenitor cells from MDS patients. Taken together, our findings provide evidence that erythroblast-specific iron metabolism is a novel potential therapeutic target to reverse ineffective erythropoiesis in MDS.

AG-946, an Activator of Pyruvate Kinase, Improves Ineffective Erythropoiesis in the Bone Marrow of Mouse Models of Myelodysplastic Syndromes

Background: Myelodysplastic syndromes (MDS) are a heterogeneous group of hematologic malignancies characterized by ineffective erythropoiesis. Anemia is the most common cytopenia and major clinical problem in MDS. Acquired pyruvate kinase (PK) deficiency has been observed in MDS, and a direct impact on the glycolytic pathway in the pathogenesis of anemia associated with MDS has been demonstrated. AG-946 is an investigational, small-molecule, allosteric activator of PK that has the potential to enhance red blood cell (RBC) functionality and survival by increasing glycolysis and adenosine triphosphate (ATP) production, and improve differentiation of erythroid cells in bone marrow, potentially improving anemia caused by ineffective erythropoiesis in MDS. Previous findings showed that RBCs from patients with MDS treated ex-vivo with AG-946, led to an increase in PK activity. Here, we used two MDS-related mouse models to evaluate the effects of the PK activator AG-946 in this mechanism of anemia. Aim: Evaluate the effect of AG-946 treatment on impaired erythropoiesis in the bone marrow of two mouse models of MDS. Methods:NUP98-HOXD13 (NHD13) is a fusion gene mutation observed in some patients with MDS. Mouse models with this mutation develop key characteristics of MDS such as impaired erythropoiesis and progressive anemia leading to malignant leukemia around 12 months of age. Male and female NHD13 and C57BL/6J mice were placed on 0.006% AG-946 (approximate dose 10 mg/kg/day) formulated diet starting at 10 months given ad libitum and continuing for 3 months. Numbers of animals for wild type (WT) Control, WT AG-946, NHD13 Control, NHD13 AG-946 were 10, 10, 13, and 11, respectively.Polg D257A (Polg) mouse model, while not a mutation observed in patients with MDS, does display many key features of MDS, such as impaired erythropoiesis and progressive anemia. Male and female Polg mice were placed on 0.006% AG-946 (approximate dose 10 mg/kg/day) formulated diet starting at 4 months given ad libitum and continuing for 8 months. Numbers of animals for WT Control, WT AG-946, Polg Control, Polg AG-946 were 10, 10, 19, and 18, respectively. Bone marrow from 1 femur per mouse was collected and analyzed by flow cytometry. Bone marrow cell pellets were washed and treated with ACK lysing buffer prior to staining. Samples with fewer than 1000 Ter119+ events were removed from analysis due to insufficient cell count. Bone marrow erythroblast populations were gated using Single Cells/Live/B220-/Ter119+/CD44 vs FSC, which were then gated into three populations: basophilic erythroblasts (BasoE), polychromatic erythroblasts (PolyE), and orthochromatic erythroblasts and reticulocytes (OrthoE + Retic). Results:No differences in any erythroblast populations were observed between untreated WT and NHD13 mice. No changes in the BasoE population were observed in WT or NHD13 mice. The PolyE population in NHD13 mice treated with AG-946 was significantly decreased compared with both the NHD13 and WT untreated mice (p=0.0021 and 0.0003, respectively). NHD13 mice treated with AG-946 showed significant increases in the OrthoE + Retic populations over the untreated NHD13 mice (p=0.0275).No differences in BasoE or OrthoE + Retic populations were observed between untreated WT and Polg mice while the PolyE population was significantly decreased in Polg mice compared with WT (p<0.0001). The BasoE population was significantly decreased in Polg mice treated with AG-946 compared with untreated Polg (p=0.0002). No difference in PolyE population was observed in Polg mice with AG-946. OrthoE + Retic population was significantly increased in Polg mice treated with AG-946 compared with WT and Polg untreated mice (p=0.0001 and 0.0304, respectively). Data graphed as average +/- SEM. Conclusions: Our data suggest AG-946 treatment improves erythroblast maturation in two MDS mouse models as demonstrated by a reduction in early-stage erythroblasts (BasoE, PolyE) coupled with an increase in late-stage erythroblast populations (OrthoE + Retic). These data are the first to suggest that PK activation by AG-946 could improve ineffective erythropoiesis in MDS.

As4S4 nanoparticles promote effective terminal erythropoiesis in bone marrow mononuclear cells from patients with myelodysplastic syndromes

Therapeutic choice for refractory anemia arising from ineffective erythropoiesis in myelodysplastic syndromes (MDS) has long remained an unmet demand. In this work, we revealed a novel function of As4S4 nanoparticles with a hydrophilic polymeric coating (ee-As4S4) to promote terminal erythropoiesis in the bone marrow mononuclear cells (BMMNCs) of MDS patients with different subtypes. We showed that ee-As4S4 effectively boosted the mRNA and protein levels of globins, increased the expression of the erythroid differentiation marker CD235a and the proportion of the CD235a+ subpopulation, and reduced the cellular volume of CD235a+ cells. Mechanistic studies demonstrated that ee-As4S4 was able to trigger the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), consequently leading to the increase of globins in mRNA and protein levels, as well as inducing the formation of stress granules (SGs) that caused the segregation and inactivation of eIF4E and inhibited the synthesis of general protein in the BMMNCs. Besides, ee-As4S4 mitigated the oxidative stress in BMMNCs by eliminating intracellular reactive oxygen species. All mechanisms together contributed to the effective differentiation of erythroblasts of early and terminal stages. Therefore, ee-As4S4 hold significant potentials for the treatment of refractory anemia that arises from ineffective erythropoiesis.

Analysis of Immunophenotypic Changes during Ex Vivo Human Erythropoiesis and Its Application in the Study of Normal and Defective Erythropoiesis.

Erythropoiesis is a highly regulated process and undergoes several genotypic and phenotypic changes during differentiation. The phenotypic changes can be evaluated using a combination of cell surface markers expressed at different cellular stages of erythropoiesis using FACS. However, limited studies are available on the in-depth phenotypic characterization of progenitors from human adult hematopoietic stem and progenitor cells (HSPCs) to red blood cells. Therefore, using a set of designed marker panels, in the current study we have kinetically characterized the hematopoietic, erythroid progenitors, and terminally differentiated erythroblasts ex vivo. Furthermore, the progenitor stages were explored for expression of CD117, CD31, CD41a, CD133, and CD45, along with known key markers CD36, CD71, CD105, and GPA. Additionally, we used these marker panels to study the stage-specific phenotypic changes regulated by the epigenetic regulator; Nuclear receptor binding SET Domain protein 1 (NSD1) during erythropoiesis and to study ineffective erythropoiesis in myelodysplastic syndrome (MDS) and pure red cell aplasia (PRCA) patients. Our immunophenotyping strategy can be used to sort and study erythroid-primed hematopoietic and erythroid precursors at specified time points and to study diseases resulting from erythroid dyspoiesis. Overall, the current study explores the in-depth kinetics of phenotypic changes occurring during human erythropoiesis and applies this strategy to study normal and defective erythropoiesis.

Iron Chelation Improves Ineffective Erythropoiesis and Iron Overload in a Mouse Model of Myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) is a heterogeneous group of bone marrow stem cell disorders characterized by ineffective hematopoiesis and cytopenias, most commonly anemia. Patients are treated with periodic red cell (RBC) transfusions, resulting in iron overload. Iron overload in MDS patients correlates with decreased survival, yet despite the availability of effective, safe, and orally administered iron chelation therapy, the treatment of iron overload in MDS remains controversial. The TELESTO trial, using deferasirox in low risk MDS patients, revealed an increase in event-free survival without impacting overall survival, hemoglobin, or RBC transfusion requirements [Angelucci Ann Int Med 2020]. However, iron chelators have different affinities for iron relative to transferrin; deferiprone has the lowest affinity of available iron chelators [Sohn Blood 2008], consistent with its ability to increase hepcidin expression in mice [Schmidt Am J Hematol 2015; Casu haematol 2016]. We hypothesize that deferiprone (DFP) impacts systemic and cellular iron distribution to enhance erythropoiesis in MDS. To test this hypothesis, we employed a highly penetrant well-established mouse model of MDS, NUP98:HOXD13 fusion transgenic mice. These MDS mice exhibit hypercellular marrow, increased apoptosis in the bone marrow compartment, and impaired survival relative to control; a proportion of mice progress to acute leukemic transformation after 7 months of age [Lin Blood 2005]. Using MDS mice, we further characterize parameters of iron trafficking in erythroblasts, evaluate the effects of DFP on erythropoiesis and iron distribution, and provide correlatory evidence in stem and progenitor cells from MDS patients. MDS mice were treated at 5 months of age with 1.25 mg/mL DFP in the drinking water for 4 weeks and compared with untreated MDS mice. Our results characterize an iron overload phenotype with aberrant erythropoiesis in MDS mice, reversed in DFP-treated MDS mice (Figure 1). Specifically, DFP leads to a redistribution of iron from liver and spleen to increase serum iron concentration and transferrin saturation, increased hemoglobin and RBC concentration in circulation, and normalized hepcidin responsiveness to iron. While MDS mice exhibit relatively decreased WBC count, no changes in WBC count are observed in DFP-treated MDS mice. Importantly, serum erythropoietin (Epo) concentration, splenomegaly, expanded erythropoiesis and erythroblast differentiation in the bone marrow are normalized in DFP-treated MDS mice. As expected when erythropoiesis is reversed, erythroblast expression of Fam132b (erythroferrone) is suppressed but no changes in STAT5 or AKT signaling, pathways downstream of Epo, are evident in DFP-treated MDS mice. These findings suggest that the expected changes in signaling downstream of Epo are unaffected by DFP administration, implicating possible changes in erythroblast Epor expression in DFP-treated MDS mice. We used sorted bone marrow to evaluate Epor expression in progressive stages of terminal erythropoiesis and demonstrate decreased Epor expression in later stage bone marrow erythroblasts from MDS mice, normalized in DFP-treated MDS mice. These findings provide evidence that increased Epor expression is a potential mechanism by which DFP leads to enhanced Epo-responsiveness and enhanced erythroblast differentiation despite decreased serum Epo concentration in MDS mice. In addition, we demonstrate for the first time increased ferritin concentration and normalized Tfr1, Pcbp1 and Ncoa4 expression in late stage erythroblasts from DFP-treated MDS mice, evidence of aberrant iron trafficking in MDS erythroblasts and their normalization with DFP treatment (Figure 2). Importantly, this effect is not observed in DFP-treated WT mice. Our data also provides evidence that reversal of ineffective erythropoiesis in DFP-treated MDS mice occurs independently of effects on erythroblast apoptosis. Finally, we confirm increased expression of genes involved in Epo-mediation as well as iron uptake and trafficking-Epor, Fam132b, Tfr1, Pcbp1 and Ncoa4-in stem and progenitor cells from MDS patients. Taken together, our findings provide evidence that erythroblast-specific iron metabolism is a novel potential therapeutic target to reverse ineffective erythropoiesis in MDS and provide a rationale for exploring the effect of DFP in patients with low risk MDS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Arsenic Dispensing Powder Promotes Erythropoiesis in Myelodysplastic Syndromes via Downregulation of HIF1A and Upregulation of GATA Factors.

Traditional Chinese Medicine (TCM) is a practical medicine based on thousands of years of medical practice in China. Arsenic dispensing powder (ADP) has been used as a treatment for MDS patients with a superior efficacy on anemia at Xiyuan Hospital of China Academy of Chinese Medical Sciences. In this study, we retrospectively analyzed MDS patients that received ADP treatment in the past 9 years and confirmed that ADP improves patients' anemia and prolongs overall survival in intermediate-risk MDS patients. Then, we used the MDS transgenic mice model and cell line to explore the drug mechanism. In normal and MDS cells, ADP does not show cellular toxicity but promotes differentiation. In mouse MDS models, we observed that ADP showed significant efficacy on promoting erythropoiesis. In the BFU-E and CFU-E assays, ADP could promote erythropoiesis not only in normal clones but also in MDS clones. Mechanistically, we found that ADP could downregulate HIF1A in MDS clones through upregulation of VHL, P53 and MDM2, which is involved in two parallel pathways to downregulate HIF1A. We also confirmed that ADP upregulates GATA factors in normal clones. Thus, our clinical and experimental studies indicate that ADP is a promising drug to promote erythropoiesis in both MDS and normal clones with a superior outcome than current regular therapies. ADP promotes erythropoiesis in myelodysplastic syndromes via downregulation of HIF1A and upregulation of GATA factors.

Increased FGF-23 levels are linked to ineffective erythropoiesis and impaired bone mineralization in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are clonal malignant hematopoietic disorders in the elderly characterized by ineffective hematopoiesis. This is accompanied by an altered bone microenvironment, which contributes to MDS progression and higher bone fragility. The underlying mechanisms remain largely unexplored. Here, we show that myelodysplastic NUP98‑HOXD13 (NHD13) transgenic mice display an abnormally high number of osteoblasts, yet a higher fraction of nonmineralized bone, indicating delayed bone mineralization. This was accompanied by high fibroblast growth factor-23 (FGF-23) serum levels, a phosphaturic hormone that inhibits bone mineralization and erythropoiesis. While Fgf23 mRNA expression was low in bone, brain, and kidney of NHD13 mice, its expression was increased in erythroid precursors. Coculturing these precursors with WT osteoblasts induced osteoblast marker gene expression, which was inhibited by blocking FGF-23. Finally, antibody-based neutralization of FGF-23 in myelodysplastic NHD13 mice improved bone mineralization and bone microarchitecture, and it ameliorated anemia. Importantly, higher serum levels of FGF‑23 and an elevated amount of nonmineralized bone in patients with MDS validated the findings. C‑terminal FGF‑23 correlated negatively with hemoglobin levels and positively with the amount of nonmineralized bone. Thus, our study identifies FGF-23 as a link between altered bone structure and ineffective erythropoiesis in MDS with the prospects of a targeted therapeutic intervention.

Severe ineffective erythropoiesis discriminates prognosis in myelodysplastic syndromes: analysis based on 776 patients from a single centre

The underlying mechanisms and clinical significance of ineffective erythropoiesis in myelodysplastic syndromes (MDS) remain to be fully defined. We conducted the ex vivo erythroid differentiation of megakaryocytic-erythroid progenitors (MEPs) from MDS patients and discovered that patient-derived erythroblasts exhibit precocity and premature aging phenotypes, partially by inducing the pro-aging genes, like ERCC1. Absolute reticulocyte count (ARC) was chosen as a biomarker to evaluate the severity of ineffective erythropoiesis in 776 MDS patients. We found that patients with severe ineffective erythropoiesis displaying lower ARC (<20 × 109/L), were more likely to harbor complex karyotypes and high-risk somatic mutations (p < 0.05). Lower ARCs are associated with shorter overall survival (OS) in univariate analysis (p < 0.001) and remain significant in multivariable analysis. Regardless of patients of lower-risk who received immunosuppressive therapy or higher-risk who received decitabine treatment, patients with lower ARC had shorter OS (p < 0.001). Whereas no difference in OS was found between patients receiving allo-hematopoietic stem cell transplantations (Allo-HSCT) (p = 0.525). Our study revealed that ineffective erythropoiesis in MDS may be partially caused by premature aging and apoptosis during erythroid differentiation. MDS patients with severe ineffective erythropoiesis have significant shorter OS treated with immunosuppressive or hypo-methylating agents, but may benefit from Allo-HSCT.

Transforming growth factor (TGF)-β pathway as a therapeutic target in lower risk myelodysplastic syndromes.

The transforming growth factor (TGF)-β superfamily comprises more than 30 soluble growth factors that play a central role in erythropoiesis and are part of a tightly regulated myelosuppressive negative feedback loop under physiologic conditions. TGF-β receptor activation and phosphorylation trigger a regulatory circuit of activating and inhibitory SMAD proteins and increased activation of the TGF-β signaling pathway either by a loss of negative feedback or constitutive activation has been associated with the myelosuppression and ineffective erythropoiesis in myelodysplastic syndromes (MDS). Anemia is the predominant cause of morbidity and quality of life impairment in patients with lower-risk (LR)-MDS, and there are very limited therapy options for these patients after failure of erythropoiesis stimulating agents (ESAs). Targeting the aberrant TGF-ß signaling pathway has therefore been investigated as a promising therapeutic approach to resolve the ineffective erythropoiesis in LR-MDS. In this article, we provide a brief overview of the TGF-β signaling cascade in hematopoiesis under physiologic conditions and its role in MDS pathogenesis. We also review preclinical and clinical data for the activin receptor type IIA ligand traps sotatercept and luspatercept that have recently shown promising results in overcoming the myelosuppressive effects of TGF-β signaling alterations to improve hematopoiesis in transfusion-dependent, non-del(5q) LR-MDS patients. Additional potential targets within the TGF-β pathway have also been identified in preclinical experiments and may provide further therapeutic options. Finally, combining different TGF-β pathway inhibitors or using them in combination with ESAs or the immunomodulator lenalidomide might have synergistic effects as well.

Ineffective erythropoiesis in myelodysplastic syndrome

Ineffective hematopoiesis is one of the hallmarks of myelodysplastic syndrome (MDS). Recently, several signaling pathways responsible for inefficient erythropoiesis in MDS have been uncovered. The p53-S100a8/S100a9-TLR4 pathway is involved in ineffective erythropoiesis in 5q minus (5q-) syndrome. Somatic mutations target multiple components of the messenger RNA (mRNA) splicing machinery including Splicing Factor 3 Subunit b1 (SF3b1) and Serine Arginine Rich Splicing Factor 2 (SRSF2) in patients with MDS. SF3b1 is the most frequently mutated spliceosome component in MDS and is mutated approximately 85% of the time in MDS with ring sideroblasts (MDS-RS). SF3b1 mutations are not simple loss-of-function or inactivating mutations but result in a change of function and cause aberrant splicing of genes that may be involved in the pathogenesis of MDS-RS. Recurrent mutations are also found in epigenetic regulator genes in MDS, including polycomb repressive complex 2 (PRC2) genes. The loss of enhancer of zeste homolog 2 (EZH2), an enzymatic component of PRC2 in mice, markedly accelerates the development of MDS in combination with representative driver mutations. The loss of EZH2 causes impaired erythropoiesis and increases ineffective hematopoiesis. Of interest, EZH2 is one of the targets of SRSF2 mutants in MDS, and mis-spliced EZH2 mRNA undergoes nonsense-mediated decay (NMD) -dependent degradation. All these data suggest that EZH2 insufficiency causes ineffective erythropoiesis.

Dual Deficiency of mDia1 and Mir-146a in an Age-Related Inflammatory Bone Marrow Microenvironment Induces Ineffective Erythropoiesis in Del(5q) MDS

Myelodysplastic syndromes (MDS) are a group of age-related clonal hematologic diseases characterized by anemia, neutropenia, and thromobocytopenia. The mechanisms of anemia in MDS are unclear, which is partially due to the heterogeneity of MDS involving many cytogenetic and molecular abnormalities. Using a mouse genetic approach, here we show that dual deficiency of mDia1 and miR-146a, two genes located at chromosome 5q that is commonly deleted in MDS, causes an aged-related anemia and ineffective erythropoiesis that closely mimics human MDS. Bone marrow erythropoiesis at various stages was dramatically affected in mDia1/miR-146a double knockout mice, which induced a massive splenomegaly with potent extramedullary erythropoiesis. Old (> 1 year of age), but not young (2-4 months of age), wild type recipient mice that were transplanted with bone marrow cells from mDia1/miR-146a double knockout mice, exhibited severe anemia and rapid lethality, which indicates that the aged microenvironment is important for the development of ineffective erythropoiesis. Consistent with the roles of mDia1 and miR-146a in the innate immune response, the serum levels of TNFα and IL-6 were significantly elevated in mDia1/miR-146a double knockout mice. Pathogen-associated molecular pattern proteins (PAMPs), or damage-associated molecular pattern proteins (DAMPs), whose levels increase in aged microenvironment, both induced TNFα and IL-6 upregulation in mDia1/miR-146a double knockout granulocytes and T cells. Mechanistically, we demonstrated that the anemia and ineffective erythropoiesis was independent of hepcidin expression in mDia1/miR-146a double knockout mice. Instead, pathologic levels of TNFα and IL-6 inhibit erythroid colony formation and differentially affect terminal erythropoiesis through reactive oxygen species-induced caspase-3 activation and cell apoptosis. Our study highlights the dual roles of age-related microenvironment and cytogenetic abnormalities in the pathogenesis of ineffective erythropoiesis in MDS. DisclosuresNo relevant conflicts of interest to declare.

Erythroferrone (ERFE) Is Selectively Expressed in Human CD71+ Erythroprogenitor Cells and Deregulated Overexpression Is Associated with a Favorable Outcome in Low Risk Myelodysplastic Syndrome (MDS)

IntroductionRecently, Erythroferrone (ERFE) was discovered as a new regulator of hepcidin in the context of hematopoietic stress and erythropoietin (EPO) stimulation (Kautz et al., Nature Genetics 2014). ERFEhas been shown to be expressed by erythroprogenitor cells of the bone marrow in response to increased erythroid activity induced by phlebotomy, EPO treatment or simulation of infectious situations in mice. It induces increased iron availability by downregulation of hepcidin in the liver and therefore represents an important new factor in iron homeostasis to be explored as a potential diagnostic or therapeutic target in the context of anemia and iron overload. Myelodysplastic Syndromes (MDS) are a group of heterogeneous malignant hematologic diseases characterized by inefficient hematopoiesis, severe anemia and deregulated iron homeostasis. In order to determine the specific role of ERFE in MDS, we analyzed the gene expression of ERFE in different hematopoietic compartments of MDS patients and healthy controls and correlated the differential expression data with clinical parameters and survival.MethodsCD71+ erythroprogenitor cells (n=198 samples) were immunomagnetically purified from mononuclear bone marrow (BM) cells of a total of n=148 MDS and n=18 sAML patients. Chronological samples were available in n=21 cases. For controls, CD71+ BM cells were analyzed from n=35 healthy donors. In addition to CD71+ cells, CD61+, CD15+ , CD34+, selected from BM, as well as CD3+ selected peripheral blood (PB) cells were immunomagnetically collected from three MDS patients as well as two healthy young and two healthy old volunteers. After total RNA extraction using the AllPrep DNA/RNA Mini kit (Qiagen), cDNA was transcribed from RNA via Quantitect cDNA synthesis kit (Qiagen). Subsequently, ERFE expression was quantified from cDNA by quantitative PCR.ResultsIn comparative expression analyses of different hematopoietic BM progenitor fractions (CD34+, CD15+, CD61+ and CD71+), ERFE was almost exclusively expressed in the erythropoietic CD71+ compartment. ERFE expression profiles in the CD71+ subset revealed a highly significant overexpression of this gene in MDS IPSS-low/int-1-risk (fold change (FC)=4.3, p<0.0001), IPSS-int-2/high-risk (FC=6.23, p<0.0001) and sAML (FC=6.69, p<0.0001) relative to healthy controls. ERFE expression profiles in MDS and sAML did not correlate with clinical laboratory parameters such as hemoglobin, EPO levels, ferritin, cobalamine, folic acid, transferrin, transferrin saturation, soluble transferrin receptor, reticulocytes, zinc protoporphyrin and lactate dehydrogenase. A negative correlation was observable for c-reactive protein levels (p=0.0053, Spearman r=-0.29) suggesting a possible link between an inflammatory environment and ERFE regulation. In exemplary chronological time course samples, ERFE expression was upregulated subsequent to clinical therapies such as 5-Azacytidine or Lenalidomide. Interestingly, in the total cohort of MDS patients with survival data follow up (n=90), low ERFE expression was associated with a significantly worse survival than high ERFE expression (median survival 2.1 years versus not reached, HR: 4.4, p=0.0007). This observation was even more pronounced in the subgroup analysis of MDS IPSS low/int-1 risk patients (n=54, median survival 2.1 years versus not reached, HR: 22, p<0.0001).ConclusionThe observed highly aberrant overexpression of ERFE in CD71+ erythropoietic progenitor cells suggests an important role for this gene in the dysfunctional erythropoiesis of MDS. The observation of a correlation between ERFE expression and survival, especially in low risk MDS patients with no apparent coherence to other established clinical markers warrants further pursuit of ERFE expression profiles in CD71+ BM cells of MDS patients as a possible independent prognostic marker. Moreover, aberrant levels of ERFE could provide a promising target for novel therapeutic avenues that mechanistically address dysfunctional erythropoiesis in MDS. DisclosuresNo relevant conflicts of interest to declare.

The Proinflammatory Protein S100A9 Suppresses Erythropoietin Elaboration in Patients with Myelodysplastic Syndromes

Introduction: Accumulating evidence implicates innate immune activation in the pathobiology of myelodysplastic syndromes (MDS) and its inflammatory bone marrow microenvironment. Excess elaboration of S100A9 accompanied by secondary induction of TNFα and IL-1β, characterize the inflammatory milieu found in lower risk MDS. Erythroid stimulating agents (ESA) and lenalidomide (LEN) are erythropoietic promoters with known anti-inflammatory properties. To date, the role of such inflammation parameters in endogenous erythropoietin (Epo) regulation and response to such treatments has not been investigated. Herein, we investigated the role of these inflammatory cytokines on Epo elaboration and response to ESA and LEN in MDS.Materials and Methods: The HepG2 hepatoma cell line was used to investigate Epo elaboration in vitro. Serum collected from 311 MDS patients from 3 centers (AOU Careggi, University of Firenze; Saint Louis hospital in Paris; H. Lee Moffitt Cancer Center in Tampa) were investigated. 125 were obtained prior to ESA treatment and 186 prior to LEN or LEN+EPO treatment. ELISAs for S100A9, S100A8, TNFα, IL1β and EPO were performed. Clinical data were analyzed from all patients. Spearman's correlation, Mann-Withney and Jonckheere-Terpstra tests were used for analysis of continuous variables. Chi-square test was used for analysis of non-continuous variables.Results: Median age of the patients was 76 years [41-94]. IPSS was low, intermediate-1, intermediate-2 and high risk in 47%, 49%, 3% and 1% of patients, respectively; whereas 28%, 42%, 22%, 7% and 1% of patients were very low, low, intermediate, high and very high risk according to IPSS-R. Serum S100A9 concentration was significantly higher in patients with lower risk versus higher risk MDS (17,798 pg/ml vs 15,291 pg/ml, p=0.01). No significant difference was observed for TNFα and IL1 β according to IPSS or IPSS-R. ELISA quantitation following 24h in vitro stimulation of HepG2 cells with S100A9 (10 to 20 µg/ml), TNFα (10 to 100 ng/ml) or IL1β (10 to 100ng/ml) showed that each cytokine significantly suppressed Epo elaboration in a concentration-dependent manner. To validate these findings, we assessed the relationship between serum concentration of these inflammatory cytokines and Epo level in MDS patients. We observed a significant negative correlation between TNFα and Epo concentration (r=-0.158, p=0.01), and a corresponding significant negative correlation between S100A9 and Epo (r=-0.143, p=0.01). We also found a significant positive correlation between S100A9 and S100A8 (r=0.757, p<0.001), S100A9 and TNFα (r=0.222, p<0001), S100A9 and IL1β (r=175, p=0.004), and IL1β with TNFα concentration (r=257, p<0.001). Moreover, we found that ESA responders had a significant higher serum TNFα concentration prior to treatment than non-responders (12 pg/ml vs 6 pg/ml, p=0.02) with a corresponding significantly lower Epo concentration in ESA responders. There was no significant correlation between S100A9 serum concentration and EPO response. Finally, in patients treated with LEN + ESA, we observed a significant positive correlation between S100A9 and duration of LEN or LEN + ESA response (r=-0.418, p=0.03). In vitro, 1µM LEN significantly reduced suppression of Epo elaboration by S100A9 or TNFα in HepG2 cells. Magnitude of LEN's improvement in EPO elaboration was greater for S100A9 vs TNF (43% vs 17%, p=0.08).Moreover, in peripheral blood mononuclear cells (PBMC) from MDS patients, 1µM LEN significantly decreased steady state S100A9 generation, and LPS-induced TNFα elaboration.Conclusion: These findings demonstrate that S100A9 and its NFκ-B transcriptional target, TNFα, directly suppress Epo elaboration and endocrine response to anemia in MDS. These inflammatory proteins may serve as rational biomarkers for response to lenalidomide and/or ESA treatment and merit prospective validation. Therapeutic strategies that either neutralize S100A9 or suppress elaboration of this alarmin may improve erythropoiesis in MDS by restoring Epo response and suppressing innate immune effectors. DisclosuresFenaux:Janssen: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Santini:celgene, Janssen, Novartis, Onconova: Honoraria, Research Funding. List:Celgene Corporation: Honoraria, Research Funding.

O-015 Inhibition of Smad2/3 signaling by ACE-536 corrects anemia and ineffective erythropoiesis in myelodysplastic syndromes

O-015 Inhibition of Smad2/3 signaling by ACE-536 corrects anemia and ineffective erythropoiesis in myelodysplastic syndromes

Reduction of Erythropoiesis in MDS with 5q Deletion Is Associated with Aberrant SPARC and TNF-Alpha Gene Expression and Predicts the Efficacy of Lenalidomide Therapy.

AbstractAbstract 2826Background of anemia in myelodysplastic syndromes (MDS) with 5q deletion (del(5q)) is a haplo-insufficiency of the RPS14 gene. However, degree of anemia and quantity of erythropoiesis vary markedly between MDS patients with del(5q), even with a similar degree of RPS14 insufficiency, so that the question arises whether other influences may play a further relevant role in erythroid failure in this disease.From 35 patients with low or intermediate-1 risk MDS with del(5q) who developed a transfusion-dependent anemia and were recruited and treated with lenalidomide (10 mg / d) within the MDS-003 study 38 +/− 33 months after diagnosis of MDS, bone marrow biopsies taken at 6 to 12-month time intervals after diagnosis of disease were evaluated for changes of erythropoiesis having occurred prior to lenalidomide therapy by morphometric, immunohistochemical, and molecular methods. A second group of patients with MDS treated with best supportive care (n = 307) served as a control. From each bone marrow biopsy, 20 000 nucleated cells were evaluated marking (1) nucleated erythroid cells (NEC) immunohistochemically using anti-HbA, glycophorin-C (GPC), CD71, and CD34 antibodies and (2) marrow macrophages by anti-CD68 antibody. By analogy to burst and colony forming units from in-vitro models, the numbers of erythroid precursor cells (EPC) were estimated by the numbers of erythroblastic islands (IslE) within marrow. Morphometrically assessed erythropoiesis was related to the mRNA expression of RPS14, SPARC, and TNF-alpha genes, nuclear p53 protein expression of NEC and their relevance for transfusion dependence and AML-free survival time of patients.5q deletion predicted erythroid hypoplasia independently of the type of MDS, evidence of marrow fibrosis and AML transformation (P < 0.0005). During early-phase MDS with del(5q), anemia correlated with p53 overexpression of NEC and decreased numbers of HbA+ NEC (P < 0.00005). After the first year of disease, two alterations of erythropoiesis became more relevant which did not correlate to p53 overexpression of NEC: (1) a decline in the numbers of GPC+HbA- NEC indicating impaired differentiation of EPC to proerythroblasts which correlated with RPS14 insufficiency, and (2) a progressive decrease in the number of IslE indicating a block of differentiation at an earlier stage, the IslE forming EPC, which did not correlate to RPS14 gene expression, but to two other independent factors, a reduced SPARC and an increased TNF-alpha gene expression (P < 0.00005). The degree of TNF-alpha expression correlated to the number of a central component of the IslE, the (iron-presenting) macrophages, the potential source of TNF-alpha overexpression. Both alterations contributed significantly to erythroid hypoplasia and progressive transfusion dependence (P < 0.00005). Response to lenalidomide therapy continued for > 4 years (100% AML-free survival) when started during a phase with high numbers of p53 overexpressing NEC, but was short or poor with < 20 % AML-free survival when started during a phase when differentiation of erythropoiesis was impaired at a less mature stage (P = 0.0001). On the basis of these observations, a score predicting the efficacy of lenalidomide in individual patients could be constructed. Conclusions:In MDS with del(5q), erythropoiesis significantly changes over time. In the early stage of disease, maturation defect of erythropoiesis mainly affects HbA+ NEC and correlates to p53 overexpression of NEC. In the course of disease, erythropoiesis becomes hypoplastic due to differentiation defects at the level of more immature erythroid (precursor) cells relating to a p53-independent mechanism of RPS14 insufficiency and altered SPARC as well as TNF-alpha gene expression. Efficacy of lenalidomide therapy is significantly impaired in the case of vanishing IslE, reduced SPARC and increased TNF-alpha gene expression. Disclosures:Giagounidis:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Knight:Celgene Corp.: Employment.

Impact of Decrease of Serum Haptoglobin in Patients with Acquired Bone Marrow Failure Syndromes

Acquired bone marrow failure (BMF) syndromes include aplastic anemia (AA), paroxysmal nocturnal hemoglobinuria (PNH), and myelodysplastic syndromes (MDS). In this study, to clarify what the frequencies of borderline PNH patients with lower proportions of glycosylphosphatidylinositol (GPI)-protein-deficient erythrocytes and no apparent clinical symptoms of PNH, such as visible hemoglobinuria and thrombotic events, are and what the significance of serum haptoglobin (Hpl) in patients with BMF is, we examined flow cytometry of erythrocytes with expression of CD59 and quantified the concentrations of serum Hpl in 142 Japanese patients with BMF, including 54 AA, 36 PNH, and 52 MDS patients. The diagnosis and grading of the severity of AA were based on the criteria of the International Agranulocytosis and Aplastic Anemia Study Group (Blood, 1987) and that of Frickhoten et al (N Engl J Med, 1991), respectively. A patient with over 1% of CD59− erythrocytes, determined by flow cytometry, was judged to have PNH erythrocytes. The diagnosis and phenotypes of MDS were determined according to the French-American-British criteria (Br J Haematol, 1982). The concentrations of serum Hpl were measured by the nephelometric procedure, developed by Van Lente et al (Clin Chem, 1979), with some modifications. The IgG bound to erythrocytes on the cell surfaces was measured by immunoradiometoric assay, developed by Jeje et al (Transfusion, 1984), with some modifications. The proportions of CD59− erythrocytes from 32 healthy individuals, AA patients, MDS patients, and PNH patients were 0.047 ± 0.052% (mean ± standard deviation), 0.143 ± 0.183%, 0.103 ± 0.126%, and 37.529 ± 33.442%, respectively. Thirteen (36.1%) of 36 PNH patients had lower proportions (range, 1–10%) of CD59− erythrocytes and no apparent clinical symptoms. Subsequently, the concentrations of serum Hpl in 436 Japanese healthy individuals, AA patients, MDS patients, and PNH patients were 152.4 ± 72.7 mg/dl (range, 42.6–309 mg/dl), 127.6 ± 130.4 mg/dl (range, 7–551 mg/dl), 73.2 ± 74.3 mg/dl (range, 3–430 mg/dl), and 13.2 ± 24.6 mg/dl (range, 2–130 mg/dl), respectively. There were significant differences in the values between AA and MDS (p<0.01), MDS and PNH (p<0.01), and AA and PNH patients (p<0.001). The frequencies of AA, MDS, and PNH patients with below 40 mg/dl of serum Hpl were 27.8%, 38.5%, and 94.4%, respectively. The frequency of PNH patients was significantly higher than that of AA or MDS patients (p<0.001 or p<0.001, respectively). Surprisingly, the white blood cell counts, absolute neutrophil counts, and platelet counts in the group (n=15) with low concentrations of serum Hpl in AA patients or the concentrations of hemoglobin and serum iron values in the group (n=20) with low concentrations of serum Hpl in MDS patients significantly decreased compared with those in the group with normal concentrations of serum Hpl in AA patients (n=39) or in MDS patients (n=32), respectively (p<0.005, p<0.01, and p<0.02; or p<0.05 and p<0.02, respectively). These findings suggest the possibility that decrease of serum Hpl might be due to the destruction of hematopoietic precursor cells via immunological mechanisms in AA or due to ineffective erythropoiesis in MDS, although it is unclear how the concentrations of serum Hpl decrease in AA patients. Recently, Theilgaard-Mönch K et al (Blood, 2006) reported that the production of Hpl by immature granulocytes in addition to hepatocytes may partially contribute to concentrations of serum Hpl. Then, we also considered another reason of decrease of concentrations of serum Hpl in patients with AA and MDS. The mean erythrocyte-associated IgG in 100 healthy individuals or 15 AA and 17 MDS patients with low concentrations of serum Hpl was 33 ± 13 molecules/one erythrocyte or 76.5 ± 64.5 molecules/one erythrocyte and 93.5 ± 60.8 molecules/one erythrocyte, respectively. The values of erythrocyte-associated IgG of 3 (20%) of 15 AA patients and 5 (29.4%) of 17 MDS patients were clinically significant, indicating that autoimmune hemolysis was associated with the decrease of concentrations of serum Hpl in these patients. In conclusion, our findings suggest that the concentrations of serum Hpl can be used as a hallmark knowing existence of over 1% of GPI-protein-negative erythrocytes in PNH, and that some mechanisms, including autoimmune hemolysis, contribute to decrease of concentrations of serum Hpl in AA and MDS.

Aberrant promotor methylation in MDS hematopoietic cells during in vitro lineage specific differentiation is differently associated with DNMT isoforms

Aberrant promoter methylation may contribute to the hematopoietic disturbances in myelodysplastic syndromes (MDS). To explore a possible mechanism, we therefore analyzed expression of DNA methyltransferase ( DNMT) subtypes kinetics and aberrant promoter methylation of key regulatory genes during MDS hematopoiesis. An in vitro model of MDS lineage-specific hematopoiesis was generated by culturing CD34+ cells from healthy donors ( n = 7) and MDS patients (low-risk: RA/ n = 6, RARS/ n = 3; high-risk: RAEB/ n = 4, RAEB-T/ n = 2) with EPO, TPO and GCSF. Promoter methylation analysis of key genes involved in the control of apoptosis ( p73, survivin, DAPK), DNA-repair ( hMLH1), differentiation ( RARb, WT1) and cell cycle control ( p14, p15, p16, CHK2) was performed by methylation specific PCR of bisulfite-treated genomic DNA. Expression of DNMT1, DNMT3a and DNMT3b was analyzed and correlated with gene promoter methylation for each lineage at different time points. DNMT expression (all isoforms) was increased during thrombopoiesis whereas elevated DNMT1 level were seen during erythropoiesis. Associations between aberrant promoter methylation and DNMT expression were found in high-risk MDS for all lineages and during erythropoiesis. Hypermethylation of p15, p16, p73, survivin, CHK2, RARb and DAPK were associated with elevated DNMT isoform expression. No general overexpression of DNMT subtype was detected during MDS hematopoiesis. However a negative association of DNMT3a and 3b expression with MDS disease risk (IPSS) could be observed. Our data indicate that all mammalian DNMT isoforms may be involved in the aberrantly methylated phenotype in MDS but seem also to be essential for the differentiation of normal hematopoietic stem cells. In particular elevated DNMT1 expression may in particular contribute to ineffective erythropoiesis in MDS.

28 Increased apoptosis as a mechanism of ineffective erythropoiesis in myelodysplastic syndromes

28 Increased apoptosis as a mechanism of ineffective erythropoiesis in myelodysplastic syndromes

The soluble transferrin receptor in dysplastic erythropoiesis in myelodysplastic syndrome

In individuals without iron deficiency, the soluble transferrin receptor (sTfR) directly reflects the erythropoietic activity. This study investigated sTfR concentrations in ineffective, dysplastic erythropoiesis in myelodysplastic syndrome (MDS). To exclude influences of other myeloid cells on sTfR, only patients with refractory anemia (RA), refractory anemia with ringed sideroblasts (RARS) and 5q(-) syndrome were included. sTfR was measured nephelometrically (normal range 0.81-1.75 mg/L). Thirty-four untreated MDS patients (RA = 14, RARS = 10, 5q(-) syndrome = 10) were enrolled and analysed. The mean sTfR value of all MDS patients (1.30 +/- 0.8 mg/L, range 0.2-3.8) did not differ from our control group. In 5q(-) syndrome, the mean sTfR concentration (0.80 +/- 0.5 mg/L) was significantly lower than in RA (1.32 +/- 0.4 mg/L, P = 0.02) and RARS (1.75 +/- 1.1 mg/L, P = 0.03). Subdividing MDS according to their amount of erythroid mass in bone marrow a significant difference of sTfR between patients with decreased (0.70 +/- 0.4 mg/L), normal (1.32 +/- 0.4 mg/L) and increased (2.06 +/- 0.9 mg/L) erythropoiesis was observed. MDS patients with sTfR values below the reference range of 0.81 mg/L required transfusions in 90% of cases and showed higher erythropoietin levels compared to MDS patients with sTfR levels > or =0.81 mg/L (P = 0.01). There was a good agreement between sTfR and the amount of polychromatic erythroblasts observed (r = 0.68, P < 0.001). In conclusion, the serum concentration of sTfR reflects erythropoietic activity in MDS, but it is in particular determined by the degree of erythroid maturation and the severity of ineffective erythropoiesis. Low sTfR values in MDS are associated with a reduced, poorly differentiated erythropoiesis and requirement of blood transfusions.

Lenalidomide: Targeted Anemia Therapy for Myelodysplastic Syndromes

Lenalidomide, an IMiD drug (a novel type of immunomodulating drug) was recently approved by the US Food and Drug Administration for the treatment of transfusion-dependent anemia in patients with myelodysplastic syndromes (MDS) and interstitial deletions of chromosome 5q [del(5q)]. This review examines the clinical experience from the MDS-001 and MDS-003 clinical trials that led to this approval, the results of biological correlates supporting the targets of drug action, and the results from a non-del(5q) multicenter study (MDS-002). Lenalidomide treatment resulted in both erythroid and cytogenetic responses in the majority of patients with del(5q), accompanied by reductions in inflammatory cytokine generation and marrow microvessel density and improvement in primitive hematopoietic progenitor recovery. Central pathology review showed that resolution of cytologic dysplasia was common in patients with del(5q) but was infrequent in erythroid-responding patients without the chromosome 5 deletion. These findings indicate that lenalidomide promotes erythropoiesis in lower-risk MDS, with two apparently distinct mechanisms of action: suppression of the ineffective del(5q) clone and promotion of effective erythropoiesis in non-del(5q) MDS progenitors. These studies identified lenalidomide as a highly active erythropoietic- and cytogenetic-remitting agent in lower-risk MDS patients who otherwise would not be expected to benefit from recombinant erythropoietin therapy. The most common adverse reactions include dose-dependent neutropenia and thrombocytopenia that are more pronounced in patients with del(5q) in whom early suppression of the clone is expected.