Coordinate Loss of a MicroRNA Mir 145 and a Protein-Coding Gene RPS14 Cooperate in the Pathogenesis of 5q- Syndrome.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 947-947 ◽  
Author(s):  
Madhu Kumar ◽  
Anupama Narla ◽  
Atsushi Nonami ◽  
Brian Ball ◽  
Christine Chin ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Macrocytic Anemia ◽  
Erythroid Differentiation ◽  
Deletion Syndrome ◽  
Disease Genes ◽  
Reciprocal Effect ◽  
Protein Coding ◽  
Molecular Abnormalities ◽  
Human Malignancy

Abstract Abstract 947 Large hemizygous chromosomal deletions are among the most common molecular abnormalities in cancer, but the identification of critical haploinsufficiency disease genes within the deleted regions has been difficult. The 5q- syndrome, a subtype of myelodysplastic syndrome (MDS), is a well-studied chromosomal deletion syndrome characterized by a consistent clinical phenotype with macrocytic anemia and thrombocytosis. We have previously shown that while hemizygous loss of RPS14 recapitulates the failed erythroid differentiation seen in 5q- syndrome, it does not account for the thrombocytosis. Evaluation of the effects of all protein coding genes in the CDR on hematopoietic differentiation showed no genes other than RPS14 altered the ratio of megakaryocytic to erythroid cells, either alone or in combination with RPS14. We therefore examined the 5q- syndrome CDR for non-coding RNAs and identified a microRNA, miR-145, which targets Fli-1, a transcriptional factor that regulates megakaryocyte development. Patients with del(5q) MDS have decreased expression of miR-145 and increased expression of Fli-1. Overexpression of miR-145 or inhibition of Fli-1 in CD34+ cells decreases megakaryocyte production, while inhibition of miR-145 or overexpression of Fli-1 has the reciprocal effect. These findings have been validated in vivo using transgenic mice. Moreover, the combined loss of miR-145 and RPS14 cooperate to alter erythroid-megakaryocytic differentiation in a manner similar to the 5q- syndrome. Taken together, these findings demonstrate for the first time that coordinate deletion of a microRNA and a protein-coding gene contributes to the phenotype of a human malignancy, the 5q- syndrome. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Coordinate loss of a microRNA and protein-coding gene cooperate in the pathogenesis of 5q− syndrome

Blood ◽  
2011 ◽  
Vol 118 (17) ◽  
pp. 4666-4673 ◽  
Author(s):  
Madhu S. Kumar ◽  
Anupama Narla ◽  
Atsushi Nonami ◽  
Ann Mullally ◽  
Nadya Dimitrova ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Erythroid Differentiation ◽  
Deletion Syndrome ◽  
Chromosomal Deletion ◽  
Reciprocal Effect ◽  
Protein Coding ◽  
Megakaryocytic Differentiation ◽  
Molecular Abnormalities ◽  
Human Malignancy ◽  
The Common

Abstract Large chromosomal deletions are among the most common molecular abnormalities in cancer, yet the identification of relevant genes has proven difficult. The 5q− syndrome, a subtype of myelodysplastic syndrome (MDS), is a chromosomal deletion syndrome characterized by anemia and thrombocytosis. Although we have previously shown that hemizygous loss of RPS14 recapitulates the failed erythroid differentiation seen in 5q− syndrome, it does not affect thrombocytosis. Here we show that a microRNA located in the common deletion region of 5q− syndrome, miR-145, affects megakaryocyte and erythroid differentiation. We find that miR-145 functions through repression of Fli-1, a megakaryocyte and erythroid regulatory transcription factor. Patients with del(5q) MDS have decreased expression of miR-145 and increased expression of Fli-1. Overexpression of miR-145 or inhibition of Fli-1 decreases the production of megakaryocytic cells relative to erythroid cells, whereas inhibition of miR-145 or overexpression of Fli-1 has a reciprocal effect. Moreover, combined loss of miR-145 and RPS14 cooperates to alter erythroid-megakaryocytic differentiation in a manner similar to the 5q− syndrome. Taken together, these findings demonstrate that coordinate deletion of a miRNA and a protein-coding gene contributes to the phenotype of a human malignancy, the 5q− syndrome.

TIF1γ Knockdown Enhances Hematopoietic Stem Cell Self Renewal with Preferential Myeloid Differentiation and Delayed Erythropoiesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4829-4829
Author(s):  
David C Dorn ◽  
Wei He ◽  
Joan Massague ◽  
Malcolm A.S. Moore
Keyword(s):  
Transforming Growth Factor ◽  
Conflicts Of Interest ◽  
Erythroid Differentiation ◽  
Transforming Growth Factor Β ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Abstract 4829 The role of TIF1γ in hematopoiesis is still incompletely understood. We previously identified TIF1γ as a novel binding factor for Smad2/3 in the Transforming Growth Factor-β (TFGβ)-inducible signaling pathway implicated in the enhancement of erythropoiesis. To investigate the function of TIF1γ in regulation of hematopoietic stem cells we abrogated TIF1γ signaling by shRNA gamma-retroviral gene transfer in human umbilical cord blood-derived CD34+ hematopoietic stem/ progenitor cells (HCS/ HPCs). Upon blocking TIF1γ the self-renewal capacity of HSCs was enhanced two-fold in vitro as measured by week 5 CAFC assay and three-fold in vivo as measured by competitive engraftment in NOD/ SCID mice over controls. This was associated with a delay in erythroid differentiation and enhanced myelopoiesis. These changes were predominantly observed after TIF1γ knockdown and only mildly after Smad2 depletion but not after Smad3 or 4 reduction. Our data reveal a role for TIF1γ-mediated signaling in the regulation of HSC self-renewal and differentiation. Disclosures: No relevant conflicts of interest to declare.

DMT1-Mutant Erythrocytes Have Shortened Life Span, Accelerated Glycolysis and Increased Oxidative Stress

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 957-957
Author(s):  
Zuzana Zidova ◽  
Pavla Pospisilova ◽  
Renata Mojzikova ◽  
Katarina Kapralova ◽  
Dalibor Dolezal ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Life Span ◽  
Conflicts Of Interest ◽  
Transmembrane Protein ◽  
Erythroid Differentiation ◽  
Compound Heterozygous ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter

Abstract Divalent metal transporter 1 (DMT1, also known as NRAMP2 and SLC11A2) is a transmembrane protein important for intestinal iron (Fe2+) absorption and erythroid iron utilization. Homozygous or compound heterozygous mutations in DMT1 are associated with moderate to severe hypochromic microcytic anemia in human patients and a mouse model - mk/mk mice. We have previously reported that DMT1 deficiency leads to an impaired erythroid differentiation hallmarked by accumulation of immature forms of erythroblast which also showed increased rate of apoptosis. For human samples we observed suppression of colony-forming capacity of erythroid progenitors that can be corrected by the addition of iron saturated chelate Fe-SIH. Later we proved this result also for mk/mk progenitors and showed reduced number of mk/mk CFU-E (164±25 vs. 283±50) and BFU-E (9±4 vs. 22±5) colonies in comparison to the colonies of wild-type (wt) mice and improvement of the colony growth with Fe-SIH. In our following studies we focused on mature erythrocytes, the last stage of erythroid differentiation that has not been analyzed yet. We first determined the in vivo half-life of red blood cells (RBC). Isolated RBCs from mk/mk mice and wt controls were in vitro labeled with CFSE fluorescent dye and injected into the wt mice. The intensity of RBCs fluorescence was measured on the 1st, 7th, 10th, 14th, 19th, 26th and 30th day following the injection. We observed an accelerated clearance of CFSE-labeled mk/mk RBCs from circulating blood when compared to wt RBCs, which indicates increased destruction of DMT1-mutant erythrocytes in vivo. It is known, that mature RBCs retain the ability to undergo stress-induced death (eryptosis), characterized by their shrinkage, membrane blebbing and phosphatidylserine surface exposure. This process may be triggered by iron deficiency. To determine the involvement of eryptosis in mk/mk RBCs clearance, RBCs were exposed to different stress conditions in vitro. A significantly increased number of Annexin V-positive RBCs was detected for mk/mk RBCs when compared to wt RBCs after 5 and 7 hour exposure to hyperosmotic shock (400mM sucrose) and glucose depletion, respectively. These results indicate shortened life span of DMT1-mutant erythrocytes and their reduced ability to cope with stress. To unravel the possible underlying mechanisms we focus on two processes important for RBC survival; anti-oxidative defense and anaerobic glycolysis. We observed 1.5 to 2-fold higher activity of glutathione peroxidase, catalase and methemoglobin reductase and elevated levels of methemoglobin in mk/mk RBCs in comparison to wt RBCs, indicating increased oxidative stress in mk/mk RBCs. Increased activity of hexokinase (2.5 times) and pyruvatkinase (2.4 times) together with reduced ratio of ATP/ADP in mk/mk mice compared with wt mice (from 2.89±0.56 μmol/L to 1.71±0.49 μmol/L) shows enhanced demand for glycolytically derived ATP to maintain the stability of RBC membrane in mk/mk mice. Our analyzes suggest that DMT1 deficiency negatively affects metabolism and life span of mature erythrocytes; two other aspects of defective erythropoiesis contributing to the pathophysiology of the disease. Grant support Czech Grant Agency, grant No. P305/11/1745; Ministry of Health Czech Republic Grant No. NT11208 and Internal Grant of Palacky University Olomouc (LF_2013_010). Disclosures: No relevant conflicts of interest to declare.

A Screen In Primary Erythroblasts Reveals a MicroRNA-Centered Homeostatic Mechanism for Regulating Cyclin E Activity.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1543-1543
Author(s):  
Yanfei Xu ◽  
Tanushri Sengupta ◽  
Alexander C. Minella
Keyword(s):  
Cell Cycle ◽  
Ubiquitin Ligase ◽  
Conflicts Of Interest ◽  
Cyclin E ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Mrna Levels ◽  
Homeostatic Mechanism ◽  
Regulated Expression

Abstract Abstract 1543 A growing body of evidence highlights the importance of microRNAs in regulating the expression of mediators of cell cycle progression. A theme emerging from these studies is that microRNAs participate in feedback or feed-forward circuits to provide bistability for key transition points in the cell cycle. We previously have shown that proper regulation of cyclin E activity is required for normal erythroid cell maturation in vivo, using cyclin ET74AüT393A knock-in mice, which have markedly dysregulated cyclin E due to its failure to interact with the Fbw7 ubiquitin ligase complex. We hypothesized that we could identify novel, microRNA-based molecular circuitry for maintaining appropriate levels of cyclin E activity by screening cyclin E knock-in erythroblasts for alterations in microRNA expression. We analyzed data we obtained from multiplex real-time PCR arrays comparing the expression of over 500 microRNAs in cyclin ET74A T393A knock-in versus wild-type erythroblasts (Ter119+/CD71+) and found down-regulated expression of a number of microRNAs targeting CDK inhibitors. We also identified down-regulated expression of potential microRNA regulators of Fbw7 expression. We found that overexpression of miR-223, in particular, significantly reduces Fbw7 mRNA levels, increases endogenous cyclin E protein and activity levels, and increases genomic instability. We next confirmed that miR-223 targets the Fbw7 3’ untranslated region. We then found that reduced miR-223 expression leads to increased Fbw7 expression and decreased cyclin E activity. Finally, we found that miR-223 expression in K562 cells is responsive to acute alterations in cyclin E regulation by the Fbw7 pathway and that dysregulated Fbw7 expression alters the erythroid differentiation capacity of these cells. Mir-223 plays an important role in myeloid and erythroid differentiation by regulating multiple substrates involved in these maturation programs. Here, we identify Fbw7 as a novel target of miR-223. Our data also indicate that miR-223 modulates Fbw7 expression as part of a homeostatic mechanism to regulate cyclin E activity and provide the first evidence that activity of the SCFFbw7 ubiquitin ligase can be controlled by the microRNA pathway. Disclosures: No relevant conflicts of interest to declare.

Expression Of Mutant Spliceosomal Protein SF3B1 Results In Dysregulated Hematopoietic Maturation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2773-2773
Author(s):  
Alexander C. Minella ◽  
Oscar Ramirez ◽  
Yanfei Xu ◽  
Tushar Murthy ◽  
Xiaodong Yang ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Erythroid Differentiation ◽  
Retroviral Vectors ◽  
Peptide Sequence ◽  
Causative Role ◽  
Wild Type ◽  
Erythroid Maturation

Abstract Whole genome sequencing has recently revealed the prevalence of mutations in proteins directing splicing of RNA in up to half of the patients with Myelodysplastic Syndrome (MDS). Mutations in the protein SF3B1 are particularly common in MDS patients with the phenotypic abnormality termed ring sideroblasts (dysplastic erythroid precursors with perinculear rings formed by iron-laden mitochondria). The most common SF3B1 mutation in MDS patients results in a change from lysine to glutamic acid at amino acid position 700 (K700E). Given that splicing of RNA is a ubiquitous phenomenon, it is unclear how these mutations result in clonal proliferation and dysplastic hematopoiesis; two hallmark features of MDS. Furthermore, direct experimental evidence demonstrating a causative role for SF3B1 mutations in MDS-related phenotypes is lacking. To better understand how mutations of spliceosomal proteins contribute to MDS pathogenesis, we sought to define how expression of mutant SF3B1 changes erythroid maturation in vitro and in vivo. Native SF3B1 cDNA constructs are not amenable to bacterial propagation due to toxicity of its HEAT-domain repeats. We overcame this problem by codon optimization (changing the DNA sequence while preserving the native peptide sequence). Human cord blood derived CD34+ cells were transduced with retroviral vectors to express either the wild-type or K700E mutant of SF3B1. After a week of expansion in cytokines (IL-3, SCF and IL6), cells were induced to erythroid differentiation by addition of erythropoietin (EPO) and analyzed for surface markers of erythroid differentiation (CD 71, CD117, CD105, CD45 and CD235A) at regular intervals. K700E mutant expressing cells were found to have significantly reduced expression of CD105 when compared to wild-type SF3B1-expressing cells (average 50% recuction, n =8). CD105 or endoglin is a TGF-beta receptor accessory receptor expressed at high levels during intermediate stages of erythroid maturation. A more modest reduction of CD71 expression was also noted in K700E-SF3B1 cells. MDS bone marrow is known to express low levels of both CD105 and CD71 making our results clinically relevant. To further characterize how mutant SF3B1 may cause dysplastic hematopoiesis, we studied transduced and transplanted murine progenitor cells in vivo and in colony forming assays. Murine data demonstrate significantly reduced K700E-transduced hematopoietic progenitors (as defined by flow-cytometry) in vivo and impaired erythroid colony formation in vitro. Together, our results suggest that enforced expression of K700E-SF3B1 induces aberrant erythroid maturation and impairs homeostasis of hematopoietic precursor cells. Thus, we provide direct evidence that MDS-associated SF3B1 mutations perturb normal hematopoiesis and offer rationale for using our complementary experimental approach as a platform for elucidating the molecular mechanisms through which mutations in RNA splicing factors promote hematologic disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Dysregulation of RUNX3 in Aged Human HSPCs May Contribute to Perturbations in Erythropoiesis and Balanced Lineage Output

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2553-2553
Author(s):  
Peter Balogh ◽  
Brian Capaldo ◽  
Sandeep Singh ◽  
Kamaleldin Elagib ◽  
Hui Li ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Colony Formation ◽  
Conflicts Of Interest ◽  
Erythroid Differentiation ◽  
Driver Mutations ◽  
Globin Genes ◽  
Rna Seq ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem

Abstract Major progress in understanding the pathobiology of human bone marrow disorders associated with aging has come from identifying recurrent, acquired mutations in the hematopoietic stem and progenitor cell (HSPC) compartment. However, causal roles for some mutations, and mechanistic pathways in cases lacking mutations, remain unclear. Complex changes in the transcriptional repertoire and the epigenome may contribute independently of driver mutations. A key HSPC alteration observed in aging, and exaggerated in marrow disorders, consists of lineage skewing toward myeloid output, usually at the expense of erythropoiesis - the basis of which remains unknown. From mining of validated RNA-seq datasets, we discovered RUNX3 as a factor down-regulated with aging in human and murine HSPCs, correlated with diminished expression of key erythroid genes Gata1, Klf1, Gypa, and Epor. While widely characterized in solid malignancies as a tumor suppressor, RUNX3 in hematopoiesis has been minimally examined. However, overlapping function with Runx1 in hematopoiesis has been described in zebrafish and murine models. Runx3 deficiency in zebrafish blocked transition to definitive hematopoiesis during development, recapitulating Runx1 findings. Murine HSC knockout studies exhibited an age-dependent granulocytic hyperplasia with a myeloproliferative phenotype, and when combined with Runx1 knockout, rapid-onset marrow failure involving Mac1+ granulocyte progenitor expansion, and severely blunted erythropoiesis. To explore the role of RUNX3 in human hematopoiesis, CD34+ HSPC underwent expression analysis and lentiviral shRNA knockdown (kd). Notably, in unmanipulated progenitors, immunoblot showed RUNX3 to be expressed in undifferentiated CD34+ cells as well as in CD235a+ erythroid cells. Immunofluorescence revealed an initial cytoplasmic predominance followed a nuclear shift upon erythroid induction. In vivo expression in erythroid progenitors was confirmed by immunostaining of human marrow samples. In uni-lineage cultures monitored by flow cytometry, and in colony formation assays, RUNX3 kd of ~60% blocked erythroid output, while sparing granulopoiesis. When cells were maintained in HSPC expansion medium, RUNX3 kd had no effect on growth or viability but suppressed both features on transfer of cells to erythroid medium. To stage the defect in RUNX3-deficient HSPC, multi-parametric flow cytometry and mass cytometry (CyTOF) interrogated progenitor composition. In these studies, RUNX3 kd blocked entry into the erythroid lineage and retained cells in a GMP-like state, based on diminished CD36 and CD71 expression, and increased CD45RA and CD123 expression, respectively. RNA sequencing of control and RUNX3-deficient progenitors briefly cultured in expansion or erythroid media revealed diminished expression of erythroid master regulators such as GATA1, KLF1, and several globin genes, as well as an increase in the myeloid master regulator GFI1. These findings recapitulate RNA-seq data from aged murine HSPCs. Because of its persistent expression during erythroid differentiation, RUNX3 also underwent functional analysis in committed progenitors including the pro-erythroblastic HUDEP-2 line and primary sorted human CD36+ cells. RUNX3-deficient HUDEP-2 cells lost their capacity for inducible hemoglobinization, and RUNX3-deficient CD36+ progenitors displayed a similar inability to execute erythroid maturation, based on a failure to upregulate CD235a. These data suggest an additional later role in erythroid differentiation. As evidence of its human clinical relevance, RUNX3 expression was found to be diminished in HSPCs purified from elderly individuals with Unexplained Anemia (UA), as compared with age-matched non-anemic control subjects. UA HSCs showed significant impairment in erythroid colony formation, with no changes in granulopoieis. The frequency of MEPs was found to be increased in UA marrow, and UA MEPs subjected to colony formation showed blunted CFU-E outgrowth in response to TGFb, signaling of which is known to be dependent on RUNX3 in other cell types. Our findings thus highlight RUNX3 as a human hematopoietic transcription factor downregulated in aging, and critical in the maintenance of balanced lineage output. We further suggest that its deficiency may contribute to aging-associated HSPC perturbations. Disclosures No relevant conflicts of interest to declare.

Defining the Pathophysiology of Pure Red Cell Aplasia Due to Feline Leukemic Virus Subgroup C Receptor (FLVCR) Dysfunction: Does Heme Toxicity Account for the Erythroid Marrow Failure?

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 175-175
Author(s):  
Sioban B Keel ◽  
Marilyn Sanchez-Bonilla ◽  
Li Liu ◽  
Yan Wang ◽  
Janis L. Abkowitz
Keyword(s):  
Preliminary Data ◽  
Conflicts Of Interest ◽  
Globin Gene ◽  
Macrocytic Anemia ◽  
Erythroid Differentiation ◽  
Luciferase Assay ◽  
Heme Synthesis ◽  
Free Heme ◽  
Terminal Erythroid Differentiation ◽  
In Cells

Abstract Abstract 175 Heme is critical to all aerobic cells both as the prosthetic group for a diverse number of hemoproteins and as a regulatory molecule in cellular processes. In addition, in erythroid cells, heme acts not only as structural component of hemoglobin but also initiates globin transcription and translation. However, because heme is highly reactive and directly toxic to cells, its intracellular level must be tightly controlled, particularly in cells with high heme requirements, like erythroid precursors. We previously determined that Feline Leukemia Virus, subgroup C, Receptor (FLVCR), is a heme export protein required for erythroid differentiation (Cell 885,757–66;2004 & Science 1206,825–8;2008). Conditional deletion of Flvcr in mice results in a severe hypoproliferative, macrocytic anemia with a block in proerythroblast maturation. These findings suggest that erythroid failure in mice lacking FLVCR is mediated by a transient excess of free heme at or around the proerythroblast stage, when heme synthesis exceeds its utilization and/or catabolism. However, we lacked proof to establish this cause and effect relationship. To test our hypothesis, we first measured intracellular free heme as sensed by the globin LCR. We adapted a luciferase assay system based on the transcriptional control of ß-globin expression by heme (J Biol Chem 279,5480–5487;2004). Specifically, we generated a lentiviral vector that contains a 838-bp fragment of the human ß-globin gene promoter and a 3-kb fragment of the LCR (which includes a heme-responsive MARE site) upstream of the firefly luciferase gene. We validated the assay's heme-specificity in a murine fetal liver erythroid culture and then transduced human CD34+ stem/progenitor cells and followed the expression of luciferase when erythroid differentiation was induced. Free heme was detected prior to Glycophorin A expression. We also determined (by q RT-PCR) that heme oygenase-1 (HMOX1), an inducible enzyme which catabolizes heme, is expressed in primary murine marrow cells and its expression decreases during terminal erythroid differentiation, potentially when ß-globin is available to bind heme. As Hmox1 expression is transcriptionally-upregulated by heme, these data suggest that heme excess occurs early in erythroid differentiation. To definitively determine whether excess heme causes the erythroid marrow failure in mice lacking FLVCR, we used a genetic approach to reduce heme synthesis. We bred Flvcrfloxflox;Mx-cre mice to mice with a recessive, severe loss of function mutation in the ferrochelatase gene, the final enzyme in the heme synthetic pathway. Fechm1Pas/m1Pas mice have a mild microcytic anemia compared to wild-type controls (Blood 109,811–818;2007, preliminary data: hemoglobin 13.4±0.6 vs. 16.2±0.8 g/dL, MCV 41.1± 0.9 vs. 46.5±1.5 fL, n=2 in each group, mean±SEM). Notably, in our first cohort of mice, the adult Flvcr-deleted mouse expressing mutant ferrochelatase had a near normal hemoglobin. This contrasted the severe anemia of the control Flvcr-deleted mice. Specifically, seven weeks after poly(I)poly(C) deletion of the floxed Flvcr allele, the hemoglobin in the Fechm1Pas/m1Pas;Flvcrfloxflox;Mx-cre mouse was 10.1 g/dL, while the hemoglobins in the Fechwildtype/wildtype;Flvcrfloxflox;Mx-cre and Fechwildtype/ m1Pas;Flvcrfloxflox;Mx-cre mice were 4.4 and 4.5 g/dL, respectively. Terminal erythroid differentiation was also corrected as assessed by flow cytometry (immunostained for CD44, Ter119, and CD71). Additional cohort studies are underway. As a second approach to reduce intracellular free heme in cells lacking FLVCR, we transduced Flvcrflox/flox;Mxcre bone marrow with a retroviral vector expressing Hmox-1, and transplanted the cells into irradiated recipients. After hematopoietic recovery, the mice were treated with poly(I)poly(C) to delete Flvcr specifically in engrafted cells. These studies are ongoing. Together, our preliminary data show that FLVCR protects proerythroblasts from excess intracellular heme and heme toxicity. This pathophysiology may be relevant to other models of ineffective or failed erythropoiesis where heme synthesis and globin expression are discordant. Disclosures: No relevant conflicts of interest to declare.

FLVCR1a But Not FLVCR1b Is Required For Effective Erythropoiesis In Adult Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 308-308
Author(s):  
Raymond T Doty ◽  
Marilyn Sanchez-Bonilla ◽  
Siobán B. Keel ◽  
Janis L. Abkowitz
Keyword(s):  
Conflicts Of Interest ◽  
Macrocytic Anemia ◽  
Microcytic Anemia ◽  
Model Systems ◽  
Over Expression ◽  
Post Transplant ◽  
Hypochromic Microcytic Anemia ◽  
Adult Mice ◽  
And Control

Abstract There are two characterized isoforms of FLVCR. The long form, FLVCR1a, is a broadly expressed cell surface heme exporter (Cell 118: 757, 2004) which exports heme from the cytoplasm to an extracellular carrier protein (JBC 285: 28874, 2010). The short isoform, FLVCR1b, is transcribed off an internal promoter and localizes to the mitochondria where it exports heme out of mitochondria into the cytoplasm (JCI 112: 4569, 2012). Adult mice with an induced deletion of both isoforms of FLVCR develop severe macrocytic anemia (Science 319: 825, 2008), indicating that FLVCR is essential for erythropoiesis. Mice constitutively lacking both isoforms of FLVCR die midgestation with craniofacial and digit abnormalities and lack definitive erythropoiesis while mice lacking only FLVCR1a die midgestation with craniofacial and digit abnormalities yet appear to have intact erythropoiesis (Science 319: 825, 2008, JCI 112: 4569, 2012). These results suggest that FLVCR1b, and not FLVCR1a, is required for erythropoiesis. To definitively test whether FLVCR1a or FLVCR1b is sufficient for red cell development, we transplanted mice with marrow lacking both isoforms of FLVCR that had been transduced with either FLVCR1a or FLVCR1b along with a GFP marker. Both cohorts of mice had comparable marking frequency in granulocytes (31.1±16.4%, N=15, FLVCR1a vs 32.7±16.9%, N=8, FLVCR1b) at 4 weeks post-transplant. By 7 wks post-transplant, 71.3±19.7% of the RBC in mice that received FLVCR1a were derived from transduced cells, while only 1.0±0.8% of the RBC in mice that received FLVCR1b were derived from transduced cells. Mice that received FLVCR1a are healthy and have normal CBC parameters (WBC 8.34±4.7 k/ml, RBC 7.95±1.2 M/ml, HGB 12.5±1.7 g/dl, MCV 47.9±2.0 fl , PLT 879±387 k/ml) which persist 9 months later, while mice that received FLVCR1b are severely anemic (WBC 1.9±1.3 k/ml, RBC 1.5±0.6 M/ml, HGB 2.0±0.8 g/dl, MCV 36.5±0.8 fl , PLT 1442±1232 k/ml) and die by 8 weeks post-transplant. This demonstrates that only the FLVCR1a isoform is capable of reconstituting erythropoiesis in adult mice lacking both isoforms in hematopoietic cells. One possible way to reconcile these data with the reported role of FLVCR1b, would be if FLVCR1b were needed during fetal, but not adult, erythropoiesis. As mentioned above, adult mice with an induced deletion of FLVCR develop severe macrocytic anemia (HBG 6.5±2.2 g/dl, MCV 67.9±7.3 fL; vs controls 14.6±0.7, 44.7±3.6), in contrast, over expression of FLVCR1a results in mild hypochromic microcytic anemia (HBG 13.2±1.4 g/dl, MCV 41±5.2 fL; vs controls 15.4±0.5, 49.3±1.3). Because hypochromasia and microcytosis only result from heme or hemoglobin deficiency, FLVCR1a must export heme from differentiating erythroblasts in vivo. To confirm this, we sorted developing erythroblasts from FLVCR-deleted and control mice and measured heme content at each stage. Terminally differentiating erythroid precursors (populations I-IV, PNAS 106: 17413, 2009) from FLVCR-deficient mice have significantly more heme than those from control mice (I&II 359.6±99.9 pg/cell, III 702.2±302.2 pg/cell, IV 657.8±292.9 pg/cell, versus controls 131.4±65.2, 153.9±27.1, and 269.1±102.7 respectively, all p<0.05) and have significantly more apoptosis. To definitively demonstrate that heme toxicity causes proerythroblast apoptosis and macrocytic anemia, we are using existing mouse model systems to modulate impact heme synthesis or degradation and test whether they alter the effect of FLVCR deletion on erythropoiesis. The presence of the ferrochelatase mutation, Fechm1Pas/m1Pas, does not rescue FLVCR deficiency, most likely because the accumulation of toxic precursor products which are also substrates of FLVCR (JBC 285: 28874, 2010). We are currently evaluating whether over-expression of HO-1 or restricted expression of the transferrin receptor can mitigate the effect of FLVCR deletion. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Regulatory Reprogramming of Erythropoiesis By DNMT3A Mutation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4343-4343
Author(s):  
Janghee Woo ◽  
Sandra Stehling-Sun ◽  
H. Joachim Deeg ◽  
Thalia Papayannopoulou ◽  
Fyodor D Urnov ◽  
...  
Keyword(s):  
Dna Methyltransferase ◽  
Catalytic Domain ◽  
Conflicts Of Interest ◽  
Erythroid Differentiation ◽  
Regulatory Elements ◽  
Rna Seq ◽  
Hematopoietic Stem ◽  
Regulatory Landscapes

Abstract DNA methyltransferase 3A (DNMT3A) regulates diverse epigenetic processes, and DNMT3A mutations occur frequently in myelodysplastic syndromes (MDS), including in founding clones of MDS samples. Most DNMT3A mutations affect Arg882 (R882) in the catalytic domain of DNMT3A, and are found almost exclusively in a heterozygous state. To resolve the relationship between the genetic and epigenetic architectures of R882H+ MDS, we engineered primary human CD34+ hematopoietic stem and progenitor cells (HSPCs) to carry heterozygous DNMT3A R882H and performed temporally resolved, genome-wide regulatory mapping via DNase-seq combined with RNA-seq during erythroid differentiation in vitro, and in an in vivo transplantation model. Compared with isogenic controls, heterozygous R882H HSPCs cells exhibited markedly impaired erythroid differentiation, accumulation of early myeloid progenitors, and diverse maturation defects. Transplantation of R882H HSPCs into W41 NSG mice revealed both impaired erythroid differentiation and preferential survival of mutant alleles in multiple hematopoietic lineages compatible with an early progenitor defect. Regulatory profiling of DNMT3A R882H heterozygous cells during differentiation via combined DNase- and RNA-seq revealed global and sequential alterations in the regulatory landscapes in mutant cells, most prominently decommissioning of thousands of regulatory regions normally found in primitive cells that mark gene loci destined for expression during later differentiation stages. Decommissioned regulatory elements in R882H heterozygotes were concentrated around genes involved in both regulation of erythropoiesis and cell-cycle control, biasing HSPC differentiation away from erythropoiesis. Similar findings were observed in CD34+-selected bone marrows from 33 patients with MDS, comparing heterozygous DNMT3A R882H and wild type. Collectively, our results indicate that DNMT3A R882H mutation reprograms early myeloid regulatory landscapes by preferentially targeting elements that control genes destined to be expressed at later stages of differentiation, resulting in a combined phenotype of impaired myeloid differentiation, impaired erythroid maturation, and preferential survival of R882H+ cells. The results provide novel mechanistic insights into the chromatin programming of erythroid differentiation and its connection with MDS. Disclosures No relevant conflicts of interest to declare.

scholarly journals CBFβ-SMMHC Impairs Erythroid Differentiation and Induces Expansion of Aberrant Megakaryocytic/Erythroid Progenitors Capable of Leukemia Initiation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2149-2149
Author(s):  
Qi Cai ◽  
Robin Jeannet ◽  
Hongjun Liu ◽  
ya-Huei Kuo
Keyword(s):  
Mouse Model ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Erythroid Differentiation ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Differentiation Potential ◽  
Altered Expression

Abstract Approximately 12% of human acute myeloid leukemia (AMLs) harbor a recurrent chromosomal rearrangement inv(16)(p13q22). Inv(16) creates a fusion gene Cbfb-MYH11, encoding the fusion protein CBFß-SMMHC. Expressing CBFß-SMMHC in hematopoietic cells using a constitutive knock-in mouse model (Cbfb+/Cbfb-MYH11) or a conditional knock-in mouse model (Cbfb56M/+/Mx1-Cre; 129SvEv strain) causes defects in lymphoid and myeloid differentiation, and predisposes mice to AML. Previous studies with the constitutive knock-in mouse model showed impaired primitive erythropoiesis, however, Cbfb-MYH11 knocked-in cells were able to contribute to erythropoiesis in chimeric mice. To further delineate the effect of CBFß-SMMHC in adult erythropoiesis in the conditional knock-in mouse, we backcrossed Cbfb56M/+/Mx1-Cre into C57BL/6 and a Rosa26mT/mG Cre reporter strain. Induced expression of CBFß-SMMHC in adult mice leads to cell number dependent development of AML, consistent with previous studies in 129SvEv strain. Analysis of pre-leukemic bone marrow 2 weeks after induction revealed a 5.7-fold expansion of immunophenotypic pre-megakaryocyte/erythrocyte (Pre-Meg/E; Lin-cKit+Sca1-CD16-/loCD150+CD105-), and a 4.7 fold decrease of the erythroid progenitor (EP; Lin-cKit+Sca1-CD16-/loCD105hi) subset compared to similarly treated control mice. Both methylcellulose-based erythroid colony forming assay and in vitro erythroid differentiation culture showed that pre-leukemic Pre-Meg/Es expressing CBFß-SMMHC had an impaired differentiation potential for erythroid lineage. Using the Rosa26mT/mG Cre reporter allele, we tracked the proportions of CBFß-SMMHC- expressing cells (GFP+) in the Pre-Meg/E and EP subsets. We observed that the contribution of GFP+ cells sharply decreased in EPs but not in Pre-Meg/Es from primary pre-leukemic mice. Similar results were seen in transplant recipients engrafted with sorted GFP+ pre-leukemic Lin-cKit+Sca1+ cells. These results further confirmed that CBFß-SMMHC impairs cell-autonomous erythroid differentiation in vivo. Consistent with the impaired differentiation of Pre-Meg/Es, we observed altered expression pattern of erythroid regulatory genes, including Fog1, Gata2, and Gfi1b. The pre-leukemic Pre-Meg/Es exhibited increased colony forming and replating capacity in vitro and enhanced proliferation and survival in vivo. To determine whether these phenotypic Pre-Meg/E cells could be the cellular origin for leukemic transformation, we expressed a known cooperative onco-protein Mpl by retroviral transduction followed by transplantation. The majority of mice (83%) receiving 100,000 Pre-Meg/E cells developed leukemia with a medium onset of 92 days, suggesting that Pre-Meg/Es indeed are capable of leukemia initiation. In conclusion, the expression of CBFß-SMMHC impairs adult erythropoiesis at the transition of Pre-Meg/E to EPs, causing an expansion of Pre-Meg/E cells. These pre-leukemic Pre-Meg/Es could be the target cell of additional mutations contributing to leukemia transformation. Disclosures No relevant conflicts of interest to declare.

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