Erythroid Phenotype Research Articles

Identification of Biologically Relevant Enhancers in Human Erythroid Cells

Identification of cell type-specific enhancers is important for understanding the regulation of programs controlling cellular development and differentiation. Enhancers are typically marked by the co-transcriptional activator protein p300 or by groups of cell-expressed transcription factors. We hypothesized that a unique set of enhancers regulates gene expression in human erythroid cells, a highly specialized cell type evolved to provide adequate amounts of oxygen throughout the body. Using chromatin immunoprecipitation followed by massively parallel sequencing, genome-wide maps of candidate enhancers were constructed for p300 and four transcription factors, GATA1, NF-E2, KLF1, and SCL, using primary human erythroid cells. These data were combined with gene expression analyses, and candidate enhancers were identified. Consistent with their predicted function as candidate enhancers, there was statistically significant enrichment of p300 and combinations of co-localizing erythroid transcription factors within 1-50 kb of the transcriptional start site (TSS) of genes highly expressed in erythroid cells. Candidate enhancers were also enriched near genes with known erythroid cell function or phenotype. Candidate enhancers exhibited moderate conservation with mouse and minimal conservation with nonplacental vertebrates. Candidate enhancers were mapped to a set of erythroid-associated, biologically relevant, SNPs from the genome-wide association studies (GWAS) catalogue of NHGRI, National Institutes of Health. Fourteen candidate enhancers, representing 10 genetic loci, mapped to sites associated with biologically relevant erythroid traits. Fragments from these loci directed statistically significant expression in reporter gene assays. Identification of enhancers in human erythroid cells will allow a better understanding of erythroid cell development, differentiation, structure, and function and provide insights into inherited and acquired hematologic disease.

L-Leucine Improves the Anemia of DBA and the 5q- Syndrome Via Activation of the mTOR Pathway in a p53-Independent Manner

Abstract 1257Haploinsufficiency of ribosomal proteins (RP) has been shown to be the common basis for the anemia observed in ribosomopathies such as Diamond-Blackfan anemia (DBA) and myelodysplastic syndrome with loss of chromosome 5q (del(5q) MDS). DBA is a congenital bone marrow failure syndrome characterized by a profound macrocytic anemia. More than half the patients with DBA have been shown to have a heterozygous loss of an RP gene, with RPS19 being the most frequently mutated. The “5q- syndrome” is a subtype of myelodysplastic syndrome (MDS) also characterized by severe anemia that is caused by heterozygous loss of the RPS14 gene on chromosome 5q. The p53 pathway is known to play a critical role in the pathophysiology of the ribosomopathies. The leading hypothesis is that ribosomal haploinsufficiency leads to disrupted ribosome biogenesis with an accumulation of free ribosomal proteins that bind MDM2. MDM2 is an E3 Ubiquitin ligase that normally binds to and targets p53 for proteosomal degradation. The consequent accumulation of p53 leads to cell cycle arrest and apoptosis, which ultimately results in anemia. Several animal models have shown that the anemia associated with RP haploinsufficiency is almost completely alleviated in a p53 null background. However, we and others have shown that p53-independent pathway(s) also contribute to the anemia associated with RP haploinsufficiency.We have previously modeled DBA and del (5q) MDS in zebrafish using antisense morpholinos to rps19 and rps14 respectively, and have demonstrated that, as in humans, haploinsufficient levels of these proteins lead to a profound anemia. We have further demonstrated that treatment of Rps19 and Rps14 deficient embryos with the amino acid L-Leucine, a known activator of mRNA translation, results in a marked improvement in anemia. This observation was confirmed in primary human CD34+ cells, following shRNA knockdown of RPS19 and RPS14. Furthermore, we showed that L-leucine treatment activates the mTOR pathway in zebrafish embryos deficient in Rps19 or Rps14. In order to determine if the effect of L-Leucine on RP deficient erythroid cells is p53 dependent, we injected rps19 and rps14 morpholinos into zebrafish embryos and treated them with L-Leucine. Total RNA was collected 48hpf and evaluated for expression of p53 by qPCR analysis. As expected, the expression of p53 and its downstream targets (p21 and PUMA) were upregulated in Rps14 and Rps19 deficient embryos. P53 expression levels remained elevated even after L-Leucine treatment. Levels of p21, a direct transcriptional target of p53, remained unchanged in L-Leucine treated RP deficient zebrafish embryos; however, expression of PUMA increased following L-Leucine treatment. The expression of the PUMA gene has previously been shown to have a p53-independent regulatory component. Preliminary studies in the A549 cell line, which harbors wild type p53, also showed increased levels of p53 expression upon shRNA mediated downregulation of both RPS19 and RPS14, which remained unaltered following L-Leucine treatment. These observations are currently being confirmed in primary human CD34+ cells, following shRNA knockdown of RPS19 and RPS14.Our preliminary studies show that the effect of L-Leucine in improving the anemia in models of DBA and del(5q) MDS occurs independently of p53. This supports our hypothesis that the erythroid phenotype in these disorders has a p53-independent component. Our finding that L-Leucine treated RP deficient cells are likely to express elevated rather than diminished levels of p53 in spite of improved anemia also has important implications for the clinical management of patients, since p53 inactivation is associated with tumor growth. A trial using L-Leucine for patients with DBA will be opening soon in the United States. Disclosures:Ebert:Celgene: Consultancy; Genoptix: Consultancy.

Cyclin D3 coordinates the cell cycle during differentiation to regulate erythrocyte size and number

Genome-wide association studies (GWASs) have identified a genetic variant of moderate effect size at 6p21.1 associated with erythrocyte traits in humans. We show that this variant affects an erythroid-specific enhancer of CCND3. A Ccnd3 knockout mouse phenocopies these erythroid phenotypes, with a dramatic increase in erythrocyte size and a concomitant decrease in erythrocyte number. By examining human and mouse primary erythroid cells, we demonstrate that the CCND3 gene product cyclin D3 regulates the number of cell divisions that erythroid precursors undergo during terminal differentiation, thereby controlling erythrocyte size and number. We illustrate how cell type-specific specialization can occur for general cell cycle components-a finding resulting from the biological follow-up of unbiased human genetic studies.

Primary hematopoietic cells from DBA patients with mutations in RPL11 and RPS19 genes exhibit distinct erythroid phenotype in vitro

Diamond-Blackfan anemia (DBA) is caused by aberrant ribosomal biogenesis due to ribosomal protein (RP) gene mutations. To develop mechanistic understanding of DBA pathogenesis, we studied CD34+ cells from peripheral blood of DBA patients carrying RPL11 and RPS19 ribosomal gene mutations and determined their ability to undergo erythroid differentiation in vitro. RPS19 mutations induced a decrease in proliferation of progenitor cells, but the terminal erythroid differentiation was normal with little or no apoptosis. This phenotype was related to a G0/G1 cell cycle arrest associated with activation of the p53 pathway. In marked contrast, RPL11 mutations led to a dramatic decrease in progenitor cell proliferation and a delayed erythroid differentiation with a marked increase in apoptosis and G0/G1 cell cycle arrest with activation of p53. Infection of cord blood CD34+ cells with specific short hairpin (sh) RNAs against RPS19 or RPL11 recapitulated the two distinct phenotypes in concordance with findings from primary cells. In both cases, the phenotype has been reverted by shRNA p53 knockdown. These results show that p53 pathway activation has an important role in pathogenesis of DBA and can be independent of the RPL11 pathway. These findings shed new insights into the pathogenesis of DBA.

Involvement of miRNA in Erythroid Differentiation

miRNAs are a family of small ncRNAs that regulate gene expression by targeting mRNAs in a sequence-specific manner, inducing translational repression or mRNA degradation. In this review, we present and discuss the available literature on the expression of miRNAs in erythroid cells. There are several experimental systems that can be employed for studies focusing on the relationship between miRNAs and erythroid differentiation, including human embryonic stem cells forced to erythroid differentiation, K562 and UT-7 cells induced to hemoglobin production by chemical compounds, erythropoietin-treated erythroid precursor cells from normal subjects or patients affected by hematological disease and in vivo systems, such as zebrafish embryos. Several miRNAs were identified as deeply involved in the erythroid phenotype, including miR-15a, miR-16-1, miR-126, miR-144, miR-451 and miR-210. Several functions related with erythroid cells were demonstrated to be regulated by these miRNAs, including maturation and proliferation of early erythroid cells, expression of fetal γ-globin genes and enucleation. These identified erythroid specific miRNAs represent the starting point to develop new protocols for miRNA therapeutics, based on both anti-miR molecules or miRNA replacement.

Ribosomal deficiencies in Diamond-Blackfan anemia impair translation of transcripts essential for differentiation of murine and human erythroblasts

AbstractDiamond-Blackfan anemia (DBA) is associated with developmental defects and profound anemia. Mutations in genes encoding a ribosomal protein of the small (eg, RPS19) or large (eg, RPL11) ribosomal subunit are found in more than half of these patients. The mutations cause ribosomal haploinsufficiency, which reduces overall translation efficiency of cellular mRNAs. We reduced the expression of Rps19 or Rpl11 in mouse erythroblasts and investigated mRNA polyribosome association, which revealed deregulated translation initiation of specific transcripts. Among these were Bag1, encoding a Hsp70 cochaperone, and Csde1, encoding an RNA-binding protein, and both were expressed at increased levels in erythroblasts. Their translation initiation is cap independent and starts from an internal ribosomal entry site, which appeared sensitive to knockdown of Rps19 or Rpl11. Mouse embryos lacking Bag1 die at embryonic day 13.5, with reduced erythroid colony forming cells in the fetal liver, and low Bag1 expression impairs erythroid differentiation in vitro. Reduced expression of Csde1 impairs the proliferation and differentiation of erythroid blasts. Protein but not mRNA expression of BAG1 and CSDE1 was reduced in erythroblasts cultured from DBA patients. Our data suggest that impaired internal ribosomal entry site–mediated translation of mRNAs expressed at increased levels in erythroblasts contributes to the erythroid phenotype of DBA.

Establishing Rps6 hemizygous mice as a model for studying how ribosomal protein haploinsufficiency impairs erythropoiesis

Diamond-Blackfan anemia is a congenital hypoproliferative macrocytic anemia and 5q- syndrome myelodysplastic syndrome is an acquired hypoproliferative macrocytic anemia. Their common erythroid phenotype reflects a shared pathophysiology-haploinsufficiency of one of many ribosomal proteins and somatic deletion of one allele of the ribosomal protein S14 gene, respectively. Although these abnormalities lead to defective ribosome biogenesis, why ribosomal protein hemizygosity results in anemia is not certain. Here, we characterize the hematopoietic phenotype of mice lacking one allele of the ribosomal protein S6 gene. The mice have an erythroid phenotype similar to both Diamond-Blackfan anemia and the 5q- syndrome and lenalidomide therapy improves their anemia.

Regulation of Erythroid Cell Maturation Is Mediated by a Foxo3-mTOR Cross Talk: Outcome for Beta-Thalassemic Erythropoiesis

Abstract 176Differentiation of erythroid progenitors to mature red blood cells requires erythropoietin receptor (EpoR) signaling. Stimulation of EpoR results in Jak2-mediated activation of mainly AKT, ERK/MAPK and STAT5 signaling pathways. Although alteration of these pathways is involved with the pathophysiology of major erythroid disorders such as beta-thalassemia mechanisms by which these signals impact transcriptional programs of erythroid cell maturation are largely unknown. We have shown previously that AKT signaling is required for Epo-mediated erythroid cell maturation and identified Foxo3 transcription factor, that is negatively regulated by AKT, as a critical regulator of erythroid cell cycle, maturation and lifespan mostly through the control of oxidative stress (Marinkovic et al., JCI, 2007). In addition to Foxo3, AKT regulates several proteins including the mammalian target of rapamycin (mTOR). Here we asked how Foxo3 regulation of oxidative stress impacts erythroid cell maturation. We found that AKT/mTOR signaling pathway is constitutively activated, possibly as part of a feedback loop, in primary Foxo3−/− erythroid precursors. In addition, Epo stimulation of primary Foxo3−/− erythroid precursors led to hyperphosphorylation of Jak2, AKT, mTOR and its target p70S6 Kinase (S6K) as compared to control cells. Since Foxo3 controls levels of reactive oxygen species (ROS) in erythroid cells, and ROS are known to modify signaling proteins, we asked whether ROS are involved in the hyperactivation of AKT/mTOR signaling pathway in Foxo3−/− erythroid precursors. Combined in vivo and in vitro treatment of Foxo3−/− erythroid precursors with ROS scavenger N-Acetyl-Cysteine (NAC) reduced significantly the hyper-phosphorylation of AKT, mTOR and S6K in response to Epo. These results strongly suggest that ROS mediate the hyperactivation of AKT/mTOR signaling pathway in Foxo3−/− erythroid precursors. Next we addressed whether the imbalanced production versus maturation of Foxo3−/− erythroid precursors (Marinkovic et al., JCI, 2007) is due to the constitutive activation of AKT/mTOR signaling. This was indeed the case since in vivo treatment of Foxo3−/− mice for three weeks with the mTOR inhibitor rapamycin shifted the balance from immature towards mature erythroid cells. Interestingly while rapamycin treatment decreased cycling of Foxo3−/− erythroid progenitors as anticipated, it resulted in highly increased proliferation of Foxo3−/− mature erythroblasts as analyzed by in vivo BrdU assay. Importantly, the described Foxo3−/− erythroid phenotype was maintained on two distinct genetic backgrounds (C57BL/6 and BALB/c) in mice. These results strongly suggest that the oxidative stress-induced activation of mTOR signaling pathway mediates the imbalanced production of mature erythroid cells in Foxo3−/− mice. Given that both oxidative stress and delayed erythroid cell differentiation as seen in Foxo3−/−erythroid precursors, contribute significantly to beta-thalassemia, we asked whether the mTOR signaling is involved in the pathogenesis of this disease. Rapamycin treatment improved erythroid cell maturation in the bone marrow as analyzed by cell size, CD44, TER 119 and CD71 surface markers, and resulted in significant increase in total peripheral blood red cells and hemoglobin (1 to 1.5 g/dl increase), significant reduction in reticulocyte production as well as decrease in the spleen size of beta-thalassemic intermedia (th3/+) mice similar to what was seen in Foxo3−/− mice. Collectively these results indicate an important function for the Foxo3-mTOR cross talk in the regulation of erythroid cell maturation and suggest that rapamycin may be considered for treatment of beta-thalassemia. Disclosures:No relevant conflicts of interest to declare.

Ineffective Erythropoiesis and Production of Normoblasts with a Beta Thalassemia Major Phenotype Using CD34+ Cells From Healthy Donors

Abstract 1085Reversal of anemia is the major target of thalassemia research, but studies of the molecular and cellular basis of the ineffective erythropoiesis of thalassemia are limited by access to donor progenitor cells. Here we demonstrate that thalassemic erythropoiesis may be recapitulated ex vivo by reducing the expression of hemoglobin in cultured CD34+ cells. Using lentiviral transduction of progenitor cells obtained from three healthy adult human donors, shRNA molecules were screened for their ability to reduce beta-globin gene and protein expression over 21 days in culture. Cells transduced with a scrambled vector served as donor-matched controls. Among the screened shRNA, one named HBB caused a consistent and significant reduction in beta-globin mRNA and protein. Beta-globin mRNA was reduced to levels <10% (p<0.001) compared to that of the controls (day14/21), while maintaining expression of gamma- and alpha-globin mRNA. HPLC was performed on an equivalent number of cells sampled on culture day 21 for hemoglobin type (HbA vs. HbF) and quantitation (area under each HPLC peak). The HbA peak was reduced by 96%, and there was a minor increase in the HbF peak (1.6 fold) after HBB transduction. Based upon these quantitative changes in hemoglobin, HbF represented 49.3±9.3% in the HBB transduced population compared with 2.9±0.7% (p<0.01) in controls. On culture day 14, there was no significant difference in glycophorin A (CD235), transferrin receptor (CD71), or cellular morphology despite the reduction in beta-globin mRNA. However, impaired terminal differentiation was detected by retainment of surface CD71 and a lack of enucleation during the third week of culture. Cell counts were lower in HBB transduced cells during the final stages of erythroid differentiation with a 61% (p=0.03) reduction in total cell counts by day 21 when compared to controls. Annexin V assay on day 21 also demonstrated increased phosphatidylserine expression in the HBB transduced cells [HBB=55.7±14.4% vs. Control=25.0±3.0%] in association with the decreased terminal differentiation. GDF15 quantitation demonstrated a significant (p=0.006) increase in the culture supernatants of HBB transduced cells. Sorted cytospin preparations revealed a distinct population of mature normoblasts containing a highly condensed nucleus surrounded by a thin ring of hypochromic cytoplasm. Reduction of erythroblast beta-globin gene and protein expression to levels associated with beta thalassemia major in humans causes ineffective erythropoiesis ex vivo by reducing cell production, increasing surface expression of phosphatidylserine, and impairing enucleation during terminal maturation. Efforts are now underway to use the culture system to explore mechanisms whereby reduced hemoglobin synthesis causes normoblast defects, and for screening of chemical and genetic rescue therapies for the thalassemic erythroid phenotype. Disclosures:No relevant conflicts of interest to declare.

Delayed fetal hemoglobin switching in subjects with KLF1 gene mutation

Variations at the KLF1 gene have been associated with a series of human erythroid phenotypes including the In-(Lu) phenotype, hereditary persistence of fetal hemoglobin, congenital dyserythropoietic anemia, borderline HbA 2 and increased red blood cell protoporphyrin. Natural mutations have shown that KLF1 regulates gamma globin gene expression and its role in the switching from fetal to adult globin expression has been suggested by experimental studies. In this paper we report that subjects with S270X KLF1 mutations show a decrease of HbF levels with increasing age, supporting in vivo the role of KLF1 in hemoglobin switching in humans.

Erythroid phenotypes associated with KLF1 mutations

Erythroid Kruppel-Like Factor (KLF1; previously known as EKLF) is an essential erythroid-specific transcription factor that was first identified by Miller and Bieker in 1993.[1][1] It binds the CACCC motif, an important DNA binding site in the regulatory elements of many erythroid genes including

Interactions of mouse glycophorin A with the dRTA-related mutant G719D of the mouse Cl–/HCO3–exchanger Ae1This paper is one of a selection of papers published in a Special Issue entitled CSBMCB 53rd Annual Meeting — Membrane Proteins in Health and Disease, and has undergone the Journal’s usual peer review process.

The AE1 mutation G701D, associated with recessive distal renal tubular acidosis (dRTA), produces only minimal erythroid phenotype, reflecting erythroid-specific expression of stimulatory AE1 subunit glycophorin A (GPA). GPA transgene expression could theoretically treat recessive dRTA in patients and in mice expressing cognate Ae1 mutation G719D. However, human (h) GPA and mouse (m) Gpa amino acid sequences are widely divergent, and mGpa function in vitro has not been investigated. We therefore studied in Xenopus oocytes the effects of coexpressed mGpa and hGPA on anion transport by erythroid (e) and kidney (k) isoforms of wild-type mAe1 (meAe1, mkAe1) and of mAe1 mutant G719D. Coexpression of hGPA or mGpa enhanced the function of meAe1 and mkAe1 and rescued the nonfunctional meAe1 and mkAe1 G719D mutants through increased surface expression. Progressive N-terminal truncation studies revealed a role for meAe1 amino acids 22-28 in GPA-responsiveness of meAe1 G719D. MouseN-cyto/humanTMD and humanN-cyto/mouseTMD kAE1 chimeras were active and GPA-responsive. In contrast, whereas chimera mkAe1N-cyto/hkAE1 G701DTMD was GPA-responsive, chimera hkAE1N-cyto/mkAe1 G719DTMD was GPA-insensitive. Moreover, whereas the isolated transmembrane domain (TMD) of hAE1 G701D was GPA-responsive, that of mAe1 G719D was GPA-insensitive. Thus, mGpa increases surface expression and activity of meAe1 and mkAe1. However, the G719D mutation renders certain mAe1 mutant constructs GPA-unresponsive and highlights a role for erythroid-specific meAe1 amino acids 22-28 in GPA-responsiveness.

New Mutation in Erythroid-Specific Delta-Aminolevulinate Synthase as the Cause of X-Linked Sideroblastic Anemia Responsive to Pyridoxine

Background/Aims: Congenital sideroblastic anemias (SA) are characterized by the presence of ringed sideroblasts in the bone marrow. The most common form is X-linked SA, which results from mutations in erythroid-specific δ-aminolevulinate synthase (ALAS2), the first enzyme in heme biosynthesis. In addition, autosomal recessive mutations in the erythroid-specific mitochondrial transporter SLC25A38 and glutaredoxin 5 (GLRX5) have recently been identified in SA patients with isolated erythroid phenotype. Materials and Methods: We studied 5 young males with congenital SA from the Czech Republic. Mutation analysis was performed on the complete coding regions of 3 candidate genes (ALAS2, SLC25A38 and GLRX5), and the enzyme activity of ALAS2 was measured by a continuous spectrophotometric assay. Results: We found the previously published R452H and R452C ALAS2 mutations in 3 patients. A novel K156E substitution in ALAS2 was discovered in 1 pyridoxine-responsive patient. The functional study showed that this substitution severely decreases ALAS2 enzyme activity. In 1 pyridoxine-refractory patient, no mutations were detected in ALAS2, SLC25A38 or GLRX5. Conclusion: Our report extends the list of known ALAS2 mutations, with the addition of a novel K156E substitution that is responsive to pyridoxine treatment and contributes to the general knowledge of congenital SA cases characterized worldwide.

Characterization of the Hematologic Phenotype of rpS6 Heterozygous Null Mice as a Model of the Erythroid Failure In Diamond-Blackfan Anemia

AbstractAbstract 2033Diamond-Blackfan Anemia (DBA) is a congenital form of pure red cell aplasia characterized by a hypoproliferative, macrocytic anemia, congenital anomalies, and a predisposition to cancer. DBA, along with a growing number of human diseases, is linked to defects in ribosome biogenesis. Mutations in at least 10 ribosomal protein genes of both the 40S and 60S ribosomal subunits have now been identified in over 50% of patients with DBA (Narla A, et al. Blood 2010; 115) resulting in ribosomal protein haploinsufficency and in turn a defect in ribosome biogenesis. It remains, however, unknown how these events culminate in erythroid marrow failure. The study of this pathophysiology has been hindered by a lack of animal models. We became aware of the Rps6-deleted mouse as a potential murine model of DBA (Volarevic S, et al. Science 2000; 288). RPS6 is another 40S ribosomal subunit protein required for ribosomal subunit assembly. Haploinsufficiency of RPS6 causes a phenotype reminiscent of DBA during embryogenesis (Panic L, et al. Mol Cell Biol 2006; 26), however, the erythropoietic phenotype of the conditionally-deleted Rps6 heterozygous mouse was unknown. The purpose of these studies is to fully characterize the erythroid phenotype of this mouse as a model of DBA. We demonstrate that deletion of one Rps6 allele in mice results in a macrocytic anemia and leukopenia (an absolute neutropenia and lymphocytopenia, Table 1). Though this finding is not typical, neutropenia has been described in DBA. Like DBA, the anemia is hypoproliferative (corrected reticulocyte counts were equivalent in rpS6 heterozygous and control mice: 3.3% ± 0.21, n= 3 vs. 3.6 ± 0.33, n=3; two-tailed Student's t-test, p= 0.08, which is an inappropriately low value given the deleted animals' anemia).Table 1Hematologic parameters of Rps6 heterozygous mice. Control mice n= 24, Rps6 heterozygous deleted mice n= 18; mean ± SEM, two-tailed Student's t-test, 5–6 weeks old, 6–7 weeks post deletion. Mice were a gift from George Thomas, University of Cincinnati.rpS6hetscontrolspWBC, k/ul3.7 ± 0.39.0 ± 0.4<4E-14ANC, k/ul0.9 ± 0.12.1 ± 0.1<4E-10ALC, k/ul2.4 ± 0.26.2 ± 0.3<1E-12RBC, m/ul5.6 ± 0.39.3 ± 0.1<3E-12HGB, g/dl9.2 ± 0.413.9 ± 0.2<2E-9HCT, %32.4 ± 1.445.2 ± 0.4<3E-8MCV, fL58.4 ± 0.848.7 ± 0.4<2E-11MCH, pg16.6 ± 0.315.0 ± 0.2<5E-5MCHC, %28.4 ± 0.330.4 ± 0.50.005RDW, %27.3 ± 1.119.1 ± 0.5<9E-7Platelets, k/ul1679.5 ± 81.9900.5 ± 24.8<2E-8Flow cytometric analyses of bone marrow and spleen double-stained for Ter119 and transferrin receptor (CD71) demonstrate impaired early erythroid differentiation, evidenced by a relative expansion in the proerythroblast and basophilic erythroblast populations. Hematopoietic colony assays confirm this early defect. These data suggest that haploinsufficiency of rpS6 impacts both erythropoiesis and granulopoiesis, and since the mice are not thrombocytopenic, the effect appears lineage specific, rather than occurring in a common progenitor cell. Polysome profiles to confirm a defect in ribosome biogenesis are pending. Since heterozygous mice recapitulate the erythroid phenotype of DBA, we treated the mice with standard and potential DBA therapies. Specifically, mice received 2 mg/kg/day of prednisone for 12 weeks. There was no improvement in the hemoglobin or MCV in treated animals. As DBA and 5q- syndrome myelodysplastic syndrome (MDS) share an erythroid phenotype and both result from a haploinsufficiency of a ribosomal protein, we also tested whether the macrocytic anemia in rpS6 heterozygous mice responds to lenalidomide (Revlimid®, gift from Celgene Corporation, San Diego, CA). Mice received 3 mg/kg/day of lenalidomide by oral gavage for 12 weeks. The hemoglobin increased in control mice and markedly increased in rpS6 heterozygous mice after 12 weeks of therapy (13.5 ± 0.4 to 14.9 ± 0.2, p= 0.0 and 7.9 g/dL ± 0.9 to 10.3 ± 0.8, p= 0.01, respectively; mean ± SEM, Student's t-test, paired). Additionally, the MCV decreased with therapy in both groups (49.1 fL ± 1.4 to 41.1 ± 0.2, p=0.005 and 57.4 ± 1.1 to 53.77 ± 1.4, p=0.08). With the caveat that we did not monitor drug levels achieved in vivo, these data suggest that lenalidomide improves hemoglobinization and deserves further study in DBA. Disclosures:No relevant conflicts of interest to declare.

Codanin-1 Mutations In Congenital Dyserythropoietic Anemia Type 1 Affect HP1α Localization In Erythroblasts

AbstractAbstract 1003 Introduction:The Congenital dyserythropoietic anemias (CDAs) are a heterogeneous group of rare inborn disorders mainly affecting erythropoiesis. Distinct from other inherited bone marrow failure syndromes, they are marked by morphological abnormalities of the erythroblasts leading to ineffective erythropoiesis/dyserythropoiesis, while the other hematopoietic lineages appear to be unaffected. Based largely on the dysplastic changes observed in erythroblasts by light and electron microscopy, the CDAs have been divided into 3 major types (CDA-1, 2 and 3). Causative genes have been identified for CDA-1 (CDAN1/15q15) and CDA-2 (CDAN2/20p11), while the chromosomal locus is known for CDA-3 (15q22). The majority of CDA-1 cases are associated with CDAN1 missense mutations, leading to single amino-acid substitutions in the 134 kDa Codanin-1 protein. Electron microscopy of CDA-1 erythroblasts reveals a characteristic pattern of spongy (“Swiss cheese”) heterochromatin. We investigated the chromatin in CDA-1 patients, the localization, distribution and interactions of Codanin-1 and generated a murine knock-out model for CDAN1. Results:Given the grossly abnormal chromatin ultrastructure in CDA-1 erythroblasts, we examined its overall content. No gross differences between normal and patient samples were seen, both in the amounts of histone proteins or various epigenetic marks of histone tails, suggesting that histone signatures, involved in maintenance of chromatin structure and epigenetic regulation, are globally maintained CDA-1. We confirmed the latter by ChIP-on-chip analysis where immunoprecipitated H4 acetylated, H3K4 dimethylated, H3K9 trimethylated and H3K27 trimethylated chromatin from CDA-1 erythroblasts and normal controls was hybridized to ENCODE genome-wide microrrays. Using a hybridoma technique, we produced 3 novel monoclonal antibodies to Codanin-1. These consistently demonstrate its distribution in both nucleus and cytoplasm of cells by immunofluorescence and Western Blotting. While Codanin-1 is found in the nucleus, it is more abundant in the cytoplasm in primary human erythroblasts. This localization pattern was unchanged in CDA-1 erythroblasts. Within the nucleus, Codanin-1 is localized in foci, while in the cytoplasm it shows an aggregated distribution, with the endoplasmic reticulum and Golgi apparatus being the most likely locations. No differences were found in the distribution patterns of RNA-polymerase-II, PML, nucleophosmin, HP1β and HP1γ between patients' and control erythroblasts. However, the localization of HP1α, a key component of heterochromatin, was found to be markedly perturbed. HP1α accumulates in the Golgi apparatus of CDA-1 but not normal erythroblasts. This was not seen in lymphoblasts derived from CDA-1 patients, nor in erythroblasts from a patient with CDA-2. We confirmed that the abnormal localization of HP1α in CDA-1 patients is confined to the intermediate erythroblast maturation stage, where the characteristic ultrastructural chromatin pattern of CDA-1 is observed. Immunoprecipitation of erythroblast protein extracts with anti- HP1α antibodies co-precipitated Codanin-1, suggesting that an abnormality in Codanin-1 could be responsible for the aberrant localization of HP1α. By confocal immunofluorescence, we also found Codanin-1 to co-localize partially with SEC23B, the protein mutated in CDA-2, suggesting a molecular link between the two major types of CDA. We generated the first murine Cdan1 gene-trap model demonstrating its widespread expression during embryonic development. Cdan1gt/gt homozygotes die in-utero before the onset of primitive erythropoiesis, suggesting that Cdan1 has other critical roles during embryogenesis. Adult heterozygous Cdan1wt/gt mice had normal hematopoietic function and morphology, normal life expectancy and fertility. No extra-hematopoietic macroscopic phenotype could be identified. Conclusions:Our results show that the absence of the highly conserved and ubiquitously expressed Codanin-1 is embryonic lethal, and suggest that missense CDAN1 mutations are responsible for the erythroid specific phenotype of CDA-1 via the abnormal cellular trafficking of the heterochromatin protein HP1α. Moreover, our findings suggest that common molecular pathways underlie the CDAs. Disclosures:No relevant conflicts of interest to declare.

Mouse Models of Myeloproliferative Neoplasms and Their Use In Preclinical Drug Testing

AbstractAbstract SCI-35The myeloproliferative neoplasms (MPNs) and related conditions, many of which are characterized by dysregulated tyrosine kinase (TK) signalling, can be modelled in mice by expressing the relevant mutant signalling molecules (for example, BCR-ABL1 or mutant JAK2) in mouse hematopoietic cells. There are two strategies to accomplish this: retroviral- or lentiviral gene transfer into hematopoietic cells followed by transplantation, and expression via a chromosomal transgene. Each method has advantages and disadvantages for modeling MPNs and for pre-clinical evaluation of molecularly targeted therapies. For BCR-ABL1, such preclinical studies have proven useful in predicting clinical responses to TK inhibitors in patients (Hu et al., Nat. Genet. 2004;36:453).The retroviral strategy has been used to model MPNs induced by JAK2V617F, JAK2 exon 12 mutants, and MPL W515L/K mutants (Lacout et al., Blood 2006;108:1652; Zaleskas et al., PLoS ONE 2006;1:e18; Pikman et al., PLoS Med. 2006;3:e270). For JAK2V617F, the models recapitulate predominantly the erythroid phenotype of PV, with polycythemia, splenomegaly, endogenous erythroid colonies (EEC), and progression to myelofibrosis (MF). Treatment with small molecule JAK2 inhibitors reverses polycythemia and splenomegaly, but the effects on EEC frequency, JAK2V617F allele burden, and MF are in general less profound, possibly reflecting a lack of discrimination between endogenous and mutant JAK2 by these drugs in vivo. MPL W515L induces more fulminant MPN and MF in mice, and while early therapy with a JAK2 inhibitor can prolong survival and decrease MF (Koppikar et al., Blood 2010;115:2919), it is less clear whether established MF responds to treatment.With the transgenic approach, a TK can be expressed at more physiologic levels. As JAK2V617F must associate with EpoR or MPL for signaling activity, competition between endogenous and mutant JAK2 might influence disease phenotype. Several transgenic models of JAK2V617F MPN have been published recently (Tiedt et al., Blood 2008;111:3931; Shide et al., Leukemia 2008;22:87), including “knock-in” models where JAK2V617F is conditionally expressed from the endogenous promoter (Akada et al., Blood 2010; 15:3589; Marty et al., Blood 2010;Epub May 14; Li et al., Blood 2010;Epub May 20). These models lend some experimental support for the concept that expression of JAK2V617F at levels similar to or higher than endogenous JAK2 causes erythrocytosis whereas lower expression favors thrombocytosis, but several variables, including origin of JAK2 and mouse strain, may confound the picture. The response of these transgenic mice to treatment with JAK2 inhibitors may differ from MPN patients in that they lack a normal population of HSC in their marrow and spleen, and are wholly dependent on JAK2V617F–associated hematopoiesis for blood cell production. Overall, these models should prove useful for guiding clinical trials of targeted therapies in the Ph− MPNs. Disclosures:Van Etten:AstraZeneca Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cephalon Oncology: Membership on an entity's Board of Directors or advisory committees. Off Label Use: INCB18424 (JAK2 inhibitor) TG101348 (JAK2 inhibitor) AZD1480 (JAK2 inhibitor).

Treatment of Zebrafish Models of Ribosomopathies (Diamond Blackfan Anemia (DBA) and 5q- Syndrome) with L-Leucine Results In An Improvement of Anemia and Developmental Defects: Evidence for a Common Pathway?

AbstractAbstract 195A growing number of disorders associated with impaired ribosome biogenesis and function have been recently recognized and termed ribosomopathies. The first of these to be identified was Diamond Blackfan Anemia (DBA), a congenital autosomal dominant bone marrow failure syndrome of childhood manifested as profound macrocytic anemia. DBA is characterized by enhanced sensitivity of hematopoietic progenitors to apoptosis with evidence of stress erythropoiesis. In addition to bone marrow defects, DBA patients often have craniofacial, genitourinary, cardiac and limb abnormalities and are at an increased risk of developing hematopoietic malignancies and osteosarcoma. Twenty-five percent of patients with DBA have heterozygous mutations in the ribosomal protein S19 (RPS19) gene, which encodes a component of the 40S ribosomal subunit. Additionally, a growing percentage of DBA patients lacking a mutation in the RPS19 gene have been shown to have mutations in other ribosomal protein genes. A second ribosomopathy, 5q- syndrome is a subtype of myelodysplasia, with an erythroid phenotype strikingly similar to DBA. The erythroid phenotype of 5q- syndrome has been recently demonstrated to be attributable to acquired haploinsufficiency of the ribosomal protein RPS14 gene associated with clonal loss of the long arm of chromosome 5. In both DBA and 5q- syndrome, it is unclear why defects in ribosomal proteins, which would be expected to have widespread consequences, have such a specific effect on erythroid maturation. It has been demonstrated that the efficiency of mRNA translation is significantly depressed in cells derived from DBA patients, consistent with a pathogenic ribosomal defect. L-leucine is an essential branched chain amino acid that is known to modulate protein synthesis by enhancing translation. L-leucine has been used to treat DBA patients, and has been shown to lead to improvement in hemoglobin levels and to transfusion independence in some patients. Based on this observation we hypothesized that a common pathway associated with defective mRNA translation underlies both DBA and 5q- MDS, and that activation of translation by L-Leucine would alleviate the anemia associated with both ribosomopathies. In order to address this hypothesis, we have modeled DBA and 5q- MDS in zebrafish embryos using an antisense morpholino appraoach and have demonstrated that similar to the human diseases, Rps19 and Rps14 deficiency lead to defective erythropoiesis and to developmental abnormalities (specific to DBA). Treatment of Rps19 and Rps14 deficient embryos with L-Leucine resulted in partial reversal of both the anemia and the developmental defects. L-Leucine is thought to regulate protein synthesis by acting as a nutrient signal involving the mTOR (mammalian target of rapamycin) signaling pathway. It has been suggested that L-Leucine enhances translation by activating translation initiation factors and by specific up-regulation of the ribosomal protein S6 kinase. Activation of the mTOR pathway is currently being analyzed in the L-Leucine treated Rps19 and Rps14 deficient zebrafish embryos. Additionally, our observations are also being validated in in vitro human models for DBA and 5q-MDS using human hematopoietic progenitor CD34+ cells and shRNAs specific for Rps19 and Rps14 genes followed by L-Leucine treatment. Our studies should provide evidence for activation of pathways involved in mRNA translation and validate the use of the orally administered amino acid L-Leucine as a therapeutic agent in the treatment of DBA as well as 5q- syndrome without the side-effects associated with current therapies for both diseases. Disclosures:No relevant conflicts of interest to declare.

Activation of p53 Pathway In Erythroid Precursor Cells of Diamond-Blackfan Anemia Leads to Two Distinct Phenotypes

AbstractAbstract 2225Identification of mutations in a number of ribosomal genes has established DBA as a disease of aberrant biogenesis of ribosomes. We have previously documented that knockdown of RPS19, RPL5 and RPL11 mRNA by shRNAs in CD34+ cells from cord blood leads to two distinct invitro erythroid phenotypes. The haploinsufficient RPS19 precursors exhibited decreased erythroid proliferation, normal erythroid differentiation, activation of p53 pathway with cell cycle arrest in G0/G1 and no apoptosis. In contrast, RPL5 or RPL11 haploinsufficient precursors showed a larger decrease in erythroid proliferation, a delay in erythroid differentiation, activation of p53 pathway with cell cycle arrest in G0/G1 in association with increased apoptosis. To validate these findings, we studied in vitro erythroid differentiation of primary CD34+ cells isolated from peripheral blood of DBA patients. CD34+ cells from 12 DBA individuals carrying mutations in RPS19 gene, 7 DBA individuals with no mutations in RPS19 and 19 normal individuals. CD34+ cells isolated from peripheral blood were cultured in two-phases, first in methyl-cellulose for 7 days and then in liquid culture for additional 5 days. Cells were harvested at Day 7 (D7), D10 and D12 and analyzed for cell clonogenicity, erythroid cell differentiation (May Grünwald Giemsa or flow cytometry), apoptosis (Tunel assay or flow cytometry), and mRNA and protein expression levels. From D0 (CD34+ cells) to D7 (CFU-e/proerythroblasts), to D10 (basophilic erythroblasts) and D12 (orthophilic erythroblasts), we observed a decrease in the extent of cell amplification of CD34+ cells from all DBA individuals. Interestingly, this decrease was more pronounced for CD34+ cells not carrying a mutation of RPS19 gene than for cells harbouring a mutation of RPS19. While a 9.5 fold amplification was noted between D0 to D7, for normal CD34+ cells, it was reduced to 6.1 fold for DBA cells with mutant RPS19 and further reduced to 3.8 fold for DBA cells not carrying RPS19 mutations. While we did not find a difference in cell amplification between D10 and D12 for DBA cells with RPS19 mutations compared to normal cells, we noted a large decrease in cell amplification for DBA cells without RPS19 mutations. We also studied 6 additional DBA patients (one with a mutant RPS19 gene, 2 with mutations in RPL11 gene and 3 in whom no RP gene mutations could be identified to date) using the two phase liquid culture system. Once again we noted that CD34+ cells with RPL11 mutations or with yet to be defined mutations exhibited a larger decrease in cell amplification from D0 to D7 and from D10 to D12 compared to CD34+ cells harbouring the RPS19 mutation. While erythroid differentiation was normal on both MGG and by flow cytometry in RPS19 mutant erythroid precursors, we observed a delay in erythroid differentiation in non RPS19 mutated precursors, in particular at D7 or D10 with increased number of CD34+ cells. Importantly, we noted increased apoptosis of cells not carrying RPS19 mutations compared to cells with RPS19 mutations. Strikingly, in all 6 DBA patients, p53 pathway (p53, p21 at the mRNA level and bax) was activated in the erythroid precursor cells. We confirmed an increase in apoptosis in the DBA patient erythroid precursors with non-mutant RPS19 as evidenced by decreases in Hsp70 and procaspase 3, and an increase in cleaved caspase 3. We confirmed cell cycle arrest in G0/G1 in cells from both DBA individuals studied. Reinforcing these findings, phosphoRb was found to be decreased in cells from all 6 DBA patients studied. In summary, using primary CD34+ cells from DBA individuals, we confirmed our earlier finding of the two distinct DBA phenotypes with cell arrest in conjunction with either presence or absence of apoptosis depending on whether the cells carry mutations in RPS19 or not. Strikingly, p53 pathway was activated in primary cells of all DBA patients studied. Our results suggest that erythroblastopenia, the main characteristics of DBA is likely the result of p53 activation leading to cell cycle arrest and apoptosis. Disclosures:No relevant conflicts of interest to declare.

Exportin 7 (RanBP16) Plays An Essential Role In Terminal Erythroid Differentiation

AbstractAbstract 3877Members of the nuclear transport receptor family of importins and exportins regulate the passage of proteins between the nucleus and cytoplasm. Although evolutionarily conserved across several species, Exportin 7 (Xpo7 or RanBP16) and its cargo are not well understood. In our study, we find that Xpo7 is highly erythroid-specific, as all other exportins are downregulated during terminal erythroid differentiation, a process including the induction of a highly specialized erythroid expression program, a set number of 3–5 terminal cell divisions, and chromatin condensation and eventual enucleation. Xpo7, in contrast, is highly induced during terminal erythropoiesis. Using retroviral shRNA knockdown of Xpo7 in in vitro fetal liver erythroid cell cultures, we demonstrate that exportin 7 is necessary for normal cellular proliferation and terminal erythroid differentiation, specifically for normal enucleation. Through microarray and biocomputational analysis of mRNA isolated from the knockdown cultures, we have found that the promoters of genes that are dysregulated after Xpo7 knockdown are enriched for binding sites for the activating transcription factor 4 (ATF4). Given that the erythroid phenotype of the ATF4 knockout mouse is very similar to the specific erythroid defects we observe in our in vitro knockdown analysis, our data suggests that either ATF4 or its binding protein may be Xpo7's cargo during terminal erythroid differentiation. Ongoing studies aimed at confirming this mechanism, the interaction between ATF4 and Xpo7, and the role and cargo of Xpo7 in terminal erythroid differentiation, are underway. Disclosures:No relevant conflicts of interest to declare.

EPO Receptor Gain-of-Function Causes Hereditary Polycythemia, Alters CD34+ Cell Differentiation and Increases Circulating Endothelial Precursors

BackgroundGain-of-function of erythropoietin receptor (EPOR) mutations represent the major cause of primary hereditary polycythemia. EPOR is also found in non-erythroid tissues, although its physiological role is still undefined.Methodology/Principal FindingsWe describe a family with polycythemia due to a heterozygous mutation of the EPOR gene that causes a G→T change at nucleotide 1251 of exon 8. The novel EPOR G1251T mutation results in the replacement of a glutamate residue by a stop codon at amino acid 393. Differently from polycythemia vera, EPOR G1251T CD34+ cells proliferate and differentiate towards the erythroid phenotype in the presence of minimal amounts of EPO. Moreover, the affected individuals show a 20-fold increase of circulating endothelial precursors. The analysis of erythroid precursor membranes demonstrates a heretofore undescribed accumulation of the truncated EPOR, probably due to the absence of residues involved in the EPO-dependent receptor internalization and degradation. Mutated receptor expression in EPOR-negative cells results in EPOR and Stat5 phosphorylation. Moreover, patient erythroid precursors present an increased activation of EPOR and its effectors, including Stat5 and Erk1/2 pathway.Conclusions/SignificanceOur data provide an unanticipated mechanism for autosomal dominant inherited polycythemia due to a heterozygous EPOR mutation and suggest a regulatory role of EPO/EPOR pathway in human circulating endothelial precursors homeostasis.