Glucose Phosphate Isomerase Deficiency In 2 Patients With Novel Mutations Presenting As Severe Neurologic Abnormalities and Transfusion Dependent Hemolytic Anemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 947-947 ◽  
Author(s):  
Mammen Puliyel ◽  
Patrick G. Gallagher ◽  
Vasilios Berdoukas ◽  
Bertil Glader ◽  
Thomas Coates
Keyword(s):  
Developmental Delay ◽  
Hemolytic Anemia ◽  
Conflicts Of Interest ◽  
Cerebral Atrophy ◽  
Global Developmental Delay ◽  
Glucose Phosphate Isomerase ◽  
Hematopoietic Stem ◽  
Neurologic Symptoms ◽  
Glucose Phosphate

Abstract Introduction Glucose phosphate isomerase (GPI) deficiency is the third most common red cell enzymopathy. GPI is an enzyme that reversibly catalyzes the conversion of glucose-6-phosphate into fructose 6-phosphate in the second step of glycolysis. Patients afflicted by GPI deficiency have chronic hemolysis and may also suffer from acute hemolytic crises. There are 184 known mutations of the GPI gene and to date, a neurological deficit is found in only five patients and only two of these have been characterized at a molecular level. We report 2 patients with previously unknown mutations of the GPI gene associated with, severe neurologic abnormalities and hemolytic anemia. Case 1 He was born at 38 weeks gestation; marked pallor and hepatosplenomegaly were noted at birth. The bilirubin was elevated at birth (indirect 7.5 mg/dl and direct 2.2mg/dl) requiring phototherapy. He has transfusion dependent anemia since birth. Enzymes studied were performed which showed GPI levels of 2.02 EU/gm hb ( normal range 16.3-24.7 ) and elevated glucose 6 phosphate dehydrogenase , pyruvate kinase and hexokinase. The pyrimidine 5'-nucleotidase screen was normal. In his subsequent course, he started to have seizures at 6 months of age, refractory to anticonvulsant therapy. He has severe hypotonia and global developmental delay. Magnetic resonance imaging of the brain showed generalized cerebral atrophy with no evidence of kernicterus. Case 2 He was noted to have anemia and marked hepatosplenomegaly at birth. He required exchange transfusions and phototherapy in the neonatal period. He has subsequently suffered from lifelong transfusion-dependent hemolytic anemia. He also suffers from developmental delay, ataxia, spasticity, and seizures. Of note, MRI did not exhibit evidence of kernicterus. The mutations were predicted to be pathogenic (probably damaging) by PolyPhen. Both these mutations were in a highly conserved residue. Genetic probe for preimplantation diagnosis is being used for selection of an embryo which is does not have GPI deficiency and is also a potential HLA match with the hope of undergoing hematopoietic stem cell transplantation to avoid the complications of chronic transfusions and iron overload. Discussion GPI has many functions. In dimeric form, it exhibits its catalytic function. In monomeric form, it acts as a neurotrophic growth factor, neuroleukin, which in vitro promotes survival of neurons. Abnormalities in neuroleukin have been found in motor neuron disease and in patients with central nervous system abnormalities in patients with acquired immunodeficiency disease. These effects of GPI/neuroleukin and relative deficiency in brain and neurons of this protein may explain the neurologic presentation. Decreased phosphatide phosphatase 1 activity, a lipogeneic enzyme due to mTOR activation by accumulated glucose-6-phosphate has been suggested to contribute to the neurologic symptoms. Why some of these mutation are associated with neurologic deficits while most others are not is not known. It has been speculated that the mutations which affect the folding may cause altered structure and function causing neurologic symptoms as well as hemolytic anemia while mutations affecting the catalytic site presents only as hemolytic anemia without neurologic symptoms. Disclosures: No relevant conflicts of interest to declare.

Investigation of the determinative state of the mouse inner cell mass

Development ◽  
1975 ◽  
Vol 33 (4) ◽  
pp. 979-990
Author(s):  
J. Rossant
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
In Utero ◽  
Glucose Phosphate Isomerase ◽  
Trophoblast Cells ◽  
Electrophoretic Variants ◽  
Glucose Phosphate

Inner cell masses (ICMs) were dissected from 3½- and 4½-day blastocysts and cultured in contact with 2½-day morulae. Blastocysts and morulae were homozygous for different electrophoretic variants of the enzyme glucose phosphate isomerase (GPI). Aggregation of ICMs and morulae was observed, and such aggregates were able to form blastocysts in vitro and morphologically normal foetuses in utero. GPI analysis of these conceptuses revealed that most were chimaeric. However, donor ICM-type isozyme was only detected in the embryonic and extra-embryonic fractions of the chimaeras and never in the trophoblastic fraction. Thus, ICM cells appear unable to form trophoblast derivatives even when exposed to ‘outside’ conditions as experienced by developing trophoblast cells. This is evidence that ICM cells, although not overtly differentiated, are determined by 3½ days.

Red cell glucose phosphate isomerase (GPI): a molecular study of three novel mutations associated with hereditary nonspherocytic hemolytic anemia

2006 ◽  
Vol 27 (11) ◽  
pp. 1159-1159 ◽  
Author(s):  
Ada Repiso ◽  
Baldomero Oliva ◽  
Joan-Lluis Vives-Corrons ◽  
Ernest Beutler ◽  
José Carreras ◽  
...  
Keyword(s):  
Hemolytic Anemia ◽  
Molecular Study ◽  
Red Cell ◽  
Glucose Phosphate Isomerase ◽  
Novel Mutations ◽  
Glucose Phosphate ◽  
Cell Glucose

The bHLH Transcription Factors SCL and LYL1 Awaken Hematopoietic Stem Cells by Repression of p21

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 214-214
Author(s):  
David J. Curtis ◽  
Nhu-Y Nguyen ◽  
Jessica Salmon
Keyword(s):  
Cell Cycle ◽  
Transcription Factors ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Short Term ◽  
Hematopoietic Stem ◽  
Bhlh Factors ◽  
Mild Defect ◽  
Bhlh Transcription Factors

Abstract Abstract 214 The basic helix-loop-helix (bHLH) transcription factors SCL (TAL1) and LYL1 are regulators of adult hematopoietic stem cell (HSC) activity with significant functional redundancy: HSCs lacking SCL (SCLδ/δ) have a mild defect in short-term repopulating activity whilst HSCs lacking LYL1 (LYL1−/−) have normal repopulating activity. In contrast, we have shown previously that HSCs lacking both SCL and LYL1 (DKO) are unable to grow in vitro and have no in vivo repopulating activity. Phenotypic and expression analyses of SCLδ/δ, LYL1−/− and DKO mice were performed to determine how bHLH factors regulate HSC activity. Consistent with the short-term repopulating defects of SCLδ/δ HSC, Lineage negative Sca-1+ c-Kit+ (LSK) bone marrow cells from SCLδ/δ mice had reduced in vitro replating activity associated with increased quiescence – 90% in G0 compared with 70% in normal LSK. Increased quiescence was associated with delayed hematopoietic recovery following treatment of mice with 5-Fluorouracil. Consistent with the increased quiescence, expression of the cell cycle inhibitor, Cdkn1a (p21) was increased three-fold in SCLδ/δ and LYL1−/− LSK. Moreover, p21 levels in LSK isolated from DKO mice were increased 50-fold. To determine the functional relevance of the elevated levels of p21 in DKO HSCs, we generated DKO mice on a p21-deficient (p21−/−) background. Remarkably, loss of p21 rescued in vitro cell growth of DKO progenitors. More importantly, primary and secondary competitive repopulation assays demonstrated multi-lineage repopulating activity of p21−/− DKO HSCs. These results suggest the bHLH factors SCL and LYL1 function as repressors of p21, allowing HSCs to enter cell cycle during stress hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Innate Lymphoid Cell-Derived IL-22 Mediates Endogenous Thymic Repair Under the Control of IL-23

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 143-143
Author(s):  
Jarrod A Dudakov ◽  
Alan M Hanash ◽  
Lauren F. Young ◽  
Natalie V Singer ◽  
Mallory L West ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Critical Role ◽  
Lymphoid Cells ◽  
Thymic Epithelial Cells ◽  
Immune Competence ◽  
Strong Negative Correlation ◽  
Hematopoietic Stem ◽  
Stimulation Of

Abstract Abstract 143 Despite being exquisitely sensitive to insult, the thymus is remarkably resilient in young healthy animals. Endogenous regeneration of the thymus is a crucial function that allows for renewal of immune competence following infection or immunodepletion caused by cytoreductive chemotherapy or radiation. However, the mechanisms governing this regeneration remain poorly understood. Thymopoiesis is a highly complex process involving cross-talk between developing thymocytes and their supporting non-hematopoietic stromal microenvironment, which includes highly specialized thymic epithelial cells (TECs) that are crucial for T cell development. IL-22 is a recently identified cytokine predominantly associated with maintenance of barrier function at mucosal surfaces. Here we demonstrate for the first time a critical role for IL-22 in endogenous thymic repair. Comparing IL-22 KO and WT mice we observed that while IL-22 deficiency was redundant for steady-state thymopoiesis, it led to a pronounced and prolonged loss of thymus cellularity following sublethal total body irradiation (SL-TBI), which included depletion of both thymocytes (p=0.0001) and TECs (p=0.003). Strikingly, absolute levels of IL-22 were markedly increased following thymic insult (p<0.0001) despite the significant depletion of thymus cellularity. This resulted in a profound increase in the production of IL-22 on a per cell basis (p<0.0001). These enhanced levels of IL-22 peaked at days 5 to 7 after SL-TBI, immediately following the nadir of thymic cellularity. This was demonstrated by a strong negative correlation between thymic cellularity and absolute levels of IL-22 (Fig 1a). In mucosal tissues the regulation of IL-22 production has been closely associated with IL-23 produced by dendritic cells (DCs) and ex vivo incubation of cells with IL-23 stimulates the production of IL-22. Following thymic insult there was a significant increase in the amount of IL-23 produced by DCs (Fig 1b) resulting in similar kinetics of intrathymic levels of IL-22 and IL-23. We identified a population of radio-resistant CD3−CD4+IL7Ra+RORg(t)+ thymic innate lymphoid cells (tILCs) that upregulate both their production of IL-22 (Fig 1c) and expression of the IL-23R (p=0.0006) upon exposure to TBI. This suggests that they are responsive to IL-23 produced by DCs in vivo following TBI and, in fact, in vitro stimulation of tILCs by IL-23 led to upregulation of Il-22 production by these cells (Fig 1d). We found expression of the IL-22Ra on cortical and medullary TECs (cTECs and mTECs, respectively), and uniform expression across both mature MHCIIhi mTEC (mTEChi) and immature MHCIIlo mTECs (mTEClo). However, in vitro stimulation of TECs with recombinant IL-22 led to enhanced TEC proliferation primarily in cTEC and mTEClo subsets (p=0.002 and 0.004 respectively). It is currently unclear if IL-22 acts as a maturation signal for mTECs, however, the uniform expression of IL-22Ra between immature mTEClo and mature Aire-expressing mTEChi, together with the preferential promotion of proliferation amongst mTEClo and cTEC seem to argue against IL-22 as a maturational signal but rather as promoter of proliferation, which ultimately leads to terminal differentiation of TECs. Of major clinical importance, administration of exogenous IL-22 led to enhanced thymic recovery (Fig. 1e) following TBI, primarily by promoting the proliferation of TECs. Consistent with this, the administration of IL-22 also led to significantly enhanced thymopoiesis following syngeneic BMT. Taken together these findings suggest that following thymic insult, and specifically the depletion of developing thymocytes, upregulation of IL-23 by DCs induces the production of IL-22 by tILCs and regeneration of the supporting microenvironment. This cascade of events ultimately leads to rejuvenation of the thymocyte pool (Fig. 1f). These studies not only reveal a novel pathway underlying endogenous thymic regeneration, but also identify a novel regenerative strategy for improving immune competence in patients whose thymus has been damaged from infection, age or cytoreductive conditioning required for successful hematopoietic stem cell transplantation. Finally, these findings may also provide an avenue of study to further understand the repair and regeneration of other epithelial tissues such as skin, lung and breast. Disclosures: No relevant conflicts of interest to declare.

Pak A Proteins Are Essential for Hematopoietic Stem/Progenitor Cell (HSC/P) Engraftment Through Regulation of Signaling Pathways Controlling HSC/P Proliferation, Survival, Migration, and the Actin Cytoskeleton

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 917-917
Author(s):  
Adrienne M. Dorrance ◽  
Rachelle Kosoff ◽  
Meaghan McGuinness ◽  
Chad Harris ◽  
Serena De Vita ◽  
...  
Keyword(s):  
Cell Migration ◽  
Conflicts Of Interest ◽  
Effector Proteins ◽  
Proliferating Cells ◽  
Hematopoietic Stem ◽  
Time Points ◽  
Empty Vector ◽  
Group A

Abstract Abstract 917 Rho GTPases, including Rac, integrate multiple extracellular signals and play important regulatory roles in HSC/P functions such as engraftment, retention, migration, adhesion, proliferation, and survival (Gu et al. Science, 2003). Our studies now focus on identifying potential Rac downstream effector proteins important for normal HSC/P function(s). The p21-activated kinases (Pak) are serine/threonine kinases that interact with and are major downstream targets of Rac and Cdc42. There are six human Paks (Pak1–6), which are grouped based on homology into Group A (Pak 1–3) and Group B (4–6) Paks. Paks regulate cytoskeletal organization including stress fiber dissolution, lamellipodia formation and focal adhesion disassembly and mediate activation of MAPK pathways. To identify the possible role(s) of Pak proteins in engraftment, freshly isolated LSK cells from WT (CD45.1+/CD45.2+) BM were transduced with retrovirus containing the Pak Inhibitory Domain (PID), which inhibits Group A Pak protein function or empty vector control (Mieg3); both constructs co-express GFP. 1.0×105 GFP+ LSK+ cells were then isolated and co-transplanted with 5.0×105 BoyJ (CD45.1+) whole bone marrow (WBM) into lethally irradiated C57Bl/6J (CD45.2+) recipients. Percent chimerism was measured at 3- to 24- weeks post BMT. PID transduced LSK+ cells were incapable of contributing to recipient hematopoietic reconstitution (Table 1). To explore the underlying mechanism of this engraftment failure we performed in vivo homing assays and found a 4- and 16- fold decrease, respectively, in BM homing of PID transduced LSK+ vs controls at 12 and 48 hours (p<0.05, for both time points). Altered cell migration of LSK+ cells was confirmed by live imaging microscopy which showed a 4-fold decrease in overall cell displacement in SDF-1-stimulated directed migration in the PID-expressing LSK+ compared to controls and was associated with a two-fold increase in random cell migration of PID-transduced LSK+ cells in transwell migration assays. PID-expressing LSK+ cells also demonstrated abnormal lamellipodia associated with significant increases in both cell surface area and cell perimeter. Because cytoskeletal changes may be linked to alterations in cell growth, we next examined the effect of Pak inhibition on cell survival and proliferation. PID-expressing LSK+ cells had decreased proliferation (17.7% vs 36.8% of cells in S-phase, p<0.05) and increased apoptosis (48.1% vs 16.7% AnnexinV+ cells, p<0.05) when compared to controls, respectively. These phenotypic changes were associated with decreased pERK and pAKT in PID-expressing LSK+. To confirm the importance of Pak activation of these proteins in HSC/P, we performed experiments to rescue the observed engraftment defect by co-transducing PID or Mieg3 with a constitutively active-ERK (ca-MEK1) or ca-AKT. We found ca-MEK1, but not ca-AKT, was able to increase proliferation in vitro (% proliferating cells for PID + empty vector = 6.1% and PID+ ca-MEK1 = 9.5%; p<0.05) and partially but only transiently rescue Pak-deficient HSC/P engraftment (% donor cells for LSK+ transduced with: PID + empty vector =1.5%, PID + ca-MEK1 =15.8%, and PID + ca-AKT =0.5% at 3-weeks post-BMT; p<0.05 for empty vector vs ca-MEK1). Finally, to determine which PakA pathway is critical in HSC engraftment we studied Pak genetic knock-out cells. We found that Pak2Δ/Δ -but not Pak1−/− -cells resulted in a profound HSC/P engraftment defect (% Pak2Δ/Δ vs Pak2flox/flox and Pak1−/− vs Pak1wt/wt: 1.0% vs 26.5% and 35.8% vs 37.4%; p<0.05 and p=ns, respectively at 3-weeks). Taken together, these data suggest that Pak A proteins regulate multiple HSC/P functions and link Rac GTPases to actin cytoskeletal rearrangements, directed cell migration, and proliferation/survival of HSC/P during engraftment.TABLE 1:Percentage of GFP+ cells in peripheral blood of recipient mice at indicated time points post BMT3-weeks6-weeks10-weeks14-weeks24-weeksWT-Mieg331.6% (±6.69)27.1% (±8.6)34.6% (±15.0)43.2% (±16.3)22.2% (±10.7)WT-PID0.22 (±0.19)0.07% (0.06)0%0%0%**Data represent mean ± s.d., n=10 recipients per group, p<0.05 for all time points, two independent experiments. Disclosures: No relevant conflicts of interest to declare.

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.

Treatment of Acute Graft-Versus-Host Disease by Means of Multipotent Mesenchymal Stromal Cells: Role of Immunomodulating Soluble Factors Expressed by These Cells in Vitro

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4554-4554
Author(s):  
Natalia A. Petinaty ◽  
Larisa A. Kuzmina ◽  
Irina N. Shipounova ◽  
Oxana A Zhironkina ◽  
Alexey Bigildeev ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Acute Gvhd ◽  
Conflicts Of Interest ◽  
Multipotent Mesenchymal Stromal Cells ◽  
Expression Level ◽  
Relative Level ◽  
Hematopoietic Stem ◽  
Steroid Resistant ◽  
Graft Versus Host

Abstract Abstract 4554 Background Severe graft-versus-host disease (GvHD) is a life-threatening complication after allogeneic hematopoietic-stem cell transplantation (allo-HSCT). Steroids are the first-line treatment for established GvHD with a response rate of 30–50%. The efficacy of multipotent mesenchymal stromal cells (MMSCs) in the treatment of steroid-resistant GvHD was reported to be 71–94%. Although it has been shown that the immunomodulatory effect of MMSCs mainly occurs through the secretion of soluble mediators, the exact mechanism of their action remains controversial. Aim The aim of the study was to investigate the influence of immunomodulating factors expressed by donor's MMSCs in vitro on the efficacy of MMSCs in the treatment of steroid-resistant GvHD. IL-6, IL-10, colony stimulating factor 1 (CSF1), indoleamine 2, 3 dioxygenase (IDO1), prostaglandin E synthase (PTEGS) and complement factor H (CFH) were measured as factors playing substantial role in GvHD development. Methods Five patients with steroid resistant acute GvHD were treated with bone marrow derived MMSCs from hematopoietic stem cells donors. MMSCs were infused intravenously at the dose 1×106 per kg of body weight. The efficiency of GvHD therapy by means of MMSCs was scored as: complete response – 3, partial response - 2, clinical improvement – 1, no response – 0. MMSCs were cultured in aMEM with 4% human platelet lysate for 2–5 passages. Total RNA was extracted from MMSCs at 2–3 passages by standard protocol. Relative level of gene expression was estimated by the real-time PCR with previous reverse transcription in MMSCs from 31 donors and determined by normalizing the expression of each target gene to b-actin and GAPDH, calculated using ΔΔCt method for each MMSCs sample. Results Characteristics of patients, donors and graft MMSCs are shown in the table 1. Correlation between relative level of gene expression and efficacy of MMSCs infusion was investigated. The existence of inverse negative relationship between IL-6, CSF1 expression level and treatment score was revealed. The increased level of IL-6 in donor's MMSCs in patients with low and no response (treatment score 1 – 0) could be related to main functions of this factor in inflammation. Increased level of CSF1 in donors' MMSCs could further enhance macrophage activation resulting in GvHD progression instead of inhibition. The augmentation of PTGES expression could be associated with improvement of response to MMSCs therapy, but the correlation was not found. There were no relationship between the expression levels of IL-10, IDO1 and CFH and efficiency of MMSCs infusion. In a patient with no response the relative expression level of all studied factors was altered in comparison with average level of their expression in MMSCs from studied donors: IL-6 and CSF1 increased about 2 fold, while the expression level of IL-10, CHF and PTGES decreased 1.7, 12 and 11 fold correspondingly. So not all MMSCs fit to GvHD therapy that could be associated with their characteristics. Conclusions The data demonstrate the influence of IL-6, CSF1 and PTGES expression on the efficacy of MMSCs in the treatment of steroid-resistant GvHD. As most clinical trials involve non-relative allogeneic MMSCs, analysis of expression level of IL6, CSF1 and PTGES in available MMSCs before infusion could predict the efficiency of acute GvHD therapy and permit the choice of the most proper samples. For successful clinical use of MMSCs further investigation of their properties on cellular and molecular levels are needed. Disclosures: No relevant conflicts of interest to declare. Disclosures: No relevant conflicts of interest to declare.

A Critical Role of Mir-9 in Myelopoesis and EVI1-Induced Leukemogenesis.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2332-2332
Author(s):  
Vitalyi Senyuk ◽  
Yunyuan Zhang ◽  
Yang Liu ◽  
Ming Ming ◽  
Jianjun Chen ◽  
...  
Keyword(s):  
Cell Transformation ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Ectopic Expression ◽  
Myeloid Differentiation ◽  
Dna Hypermethylation ◽  
Hematopoietic Stem

Abstract Abstract 2332 MicroRNA-9 (miR-9) is required for normal neurogenesis and organ development. The expression of miR-9 is altered in several types of solid tumors suggesting that it may have a function in cell transformation. However the role of this miR in normal hematopoiesis and leukemogenesis is unknown. Here we show that miR-9 is expressed at low levels in hematopoietic stem/progenitor cells (HSCs/HPCs), and that it is upregulated during hematopoietic differentiation. Ectopic expression of miR-9 strongly accelerates terminal myelopoiesis, while promoting apoptosis in vitro and in vivo. In addition, the inhibition of miR-9 in HPC with a miRNA sponge blocks myelopoiesis. EVI1, required for normal embryogenesis, and is considered an oncogene because inappropriate upregulation induces malignant transformation in solid and hematopoietic cancers. In vitro, EVI1 severely affects myeloid differentiation. Here we show that EVI1 binds to the promoter of miR-9–3 leading to DNA hypermethylation of the promoter as well as repression of miR-9. We also show that ectopic miR-9 reverses the myeloid differentiation block that is induced by EVI1. Our findings suggest that inappropriately expressed EVI1 delays or blocks myeloid differentiation, at least in part by DNA hypermethylation and downregulation of miR-9. It was previously reported that FoxOs genes inhibit myeloid differentiation and prevent differentiation of leukemia initiating cells. Here we identify FoxO3 and FoxO1 as new direct targets of miR-9 in hematopoietic cells, and we find that upregulation of FoxO3 in miR-9-positive cells reduces the acceleration of myelopoiesis. These results reveal a novel role of miR-9 in myelopoiesis and in the pathogenesis of EVI1-induced myeloid neoplasms. They also provide new insights on the potential chromatin-modifying role of oncogenes in epigenetic changes in cancer cells. Disclosures: No relevant conflicts of interest to declare.

DNMT3A Mutation Leads to Hematopoietic Dysregulation.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2382-2382
Author(s):  
Jie Xu ◽  
Wei-na Zhang ◽  
Tao Zhen ◽  
Yang Li ◽  
Jing-yi Shi ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Epigenetic Modification ◽  
Hematopoietic Cells ◽  
In Vitro Assays ◽  
Clinical Samples ◽  
Hematopoietic Stem

Abstract Abstract 2382 Epigenetic modification process is required for the development of hematopoietic cells. DNA methyltransferase DNMT3A, responsible for de novo DNA methylation, was newly reported to have a high frequency of mutations in hematopoietic malignancies. Conditional knock-out of DNMT3A promoted self-renewal activity of murine hematopoietic stem cells (HSCs). However, the role of mutated DNMT3A in hematopoiesis and its regulative mechanism of epigenetic network mostly remain unknown. Here we showed that the Arg882His (R882H) hotspot locus on DNMT3A impaired the normal function of this enzyme and resulted in an abnormal increase of primitive hematopoietic cells. In both controlled in vivo and in vitro assays, we found that the cells transfected by R882H mutant promoted cell proliferation, while decreased the differentiation of myeloid lineage compared to those with wild type. Analysis of bone marrow (BM) cells from mice transduced by R882H reveals an expansion of Lin−Sca-1+C-kit+ populations and a reduction of mature myeloid cells. Meanwhile, a cluster of upregulated genes and downregulated lineage-specific differentiation genes associated with hematopoiesis were discovered in mice BM cells with R882H mutation. We further evaluated the association of mutated DNMT3A and HOXB4 which was previously detected to be highly expressed in clinical samples carrying R882 mutation. Compared with wildtype DNMT3A, R882H mutation disrupted the repression of HOXB4 by largely recruiting tri-methylated histone 3 lysine 4 (H3K4). Taken together, our results showed that R882H mutation disturbed HSC activity through H3K4 tri-methylation, and transcriptional activation of HSC-related genes. Disclosures: No relevant conflicts of interest to declare.

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