Publications by authors named "Adam N Goldfarb"

24 Publications

  • Page 1 of 1

Iron control of erythroid microtubule cytoskeleton as a potential target in treatment of iron-restricted anemia.

Nat Commun 2021 03 12;12(1):1645. Epub 2021 Mar 12.

Department of Pathology, University of Virginia School of Medicine, Charlottesville, VA, USA.

Anemias of chronic disease and inflammation (ACDI) result from restricted iron delivery to erythroid progenitors. The current studies reveal an organellar response in erythroid iron restriction consisting of disassembly of the microtubule cytoskeleton and associated Golgi disruption. Isocitrate supplementation, known to abrogate the erythroid iron restriction response, induces reassembly of microtubules and Golgi in iron deprived progenitors. Ferritin, based on proteomic profiles, regulation by iron and isocitrate, and putative interaction with microtubules, is assessed as a candidate mediator. Knockdown of ferritin heavy chain (FTH1) in iron replete progenitors induces microtubule collapse and erythropoietic blockade; conversely, enforced ferritin expression rescues erythroid differentiation under conditions of iron restriction. Fumarate, a known ferritin inducer, synergizes with isocitrate in reversing molecular and cellular defects of iron restriction and in oral remediation of murine anemia. These findings identify a cytoskeletal component of erythroid iron restriction and demonstrate potential for its therapeutic targeting in ACDI.
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http://dx.doi.org/10.1038/s41467-021-21938-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7955080PMC
March 2021

levels in human hematopoietic progenitors are regulated by aging and dictate erythroid-myeloid balance.

Haematologica 2020 04 6;105(4):905-913. Epub 2019 Jun 6.

Department of Pathology, University of Virginia School of Medicine, Charlottesville, USA

Healthy bone marrow progenitors yield a co-ordinated balance of hematopoietic lineages. This balance shifts with aging toward enhanced granulopoiesis with diminished erythropoiesis and lymphopoiesis, changes which likely contribute to the development of bone marrow disorders in the elderly. In this study, RUNX3 was identified as a hematopoietic stem and progenitor cell factor whose levels decline with aging in humans and mice. This decline is exaggerated in hematopoietic stem and progenitor cells from subjects diagnosed with unexplained anemia of the elderly. Hematopoietic stem cells from elderly unexplained anemia patients had diminished erythroid but unaffected granulocytic colony forming potential. Knockdown studies revealed human hematopoietic stem and progenitor cells to be strongly influenced by RUNX3 levels, with modest deficiencies abrogating erythroid differentiation at multiple steps while retaining capacity for granulopoiesis. Transcriptome profiling indicated control by RUNX3 of key erythroid transcription factors, including and These findings thus implicate RUNX3 as a participant in hematopoietic stem and progenitor cell aging, and a key determinant of erythroid-myeloid lineage balance.
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http://dx.doi.org/10.3324/haematol.2018.208918DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7109730PMC
April 2020

Megakaryocyte ontogeny: Clinical and molecular significance.

Exp Hematol 2018 05 2;61:1-9. Epub 2018 Mar 2.

Department of Pathology, University of Virginia School of Medicine, Charlottesville, VA, USA. Electronic address:

Fetal megakaryocytes (Mks) differ from adult Mks in key parameters that affect their capacity for platelet production. However, despite being smaller, more proliferative, and less polyploid, fetal Mks generally mature in the same manner as adult Mks. The phenotypic features unique to fetal Mks predispose patients to several disease conditions, including infantile thrombocytopenia, infantile megakaryoblastic leukemias, and poor platelet recovery after umbilical cord blood stem cell transplantations. Ontogenic Mk differences also affect new strategies being developed to address global shortages of platelet transfusion units. These donor-independent, ex vivo production platforms are hampered by the limited proliferative capacity of adult-type Mks and the inferior platelet production by fetal-type Mks. Understanding the molecular programs that distinguish fetal versus adult megakaryopoiesis will help in improving approaches to these clinical problems. This review summarizes the phenotypic differences between fetal and adult Mks, the disease states associated with fetal megakaryopoiesis, and recent advances in the understanding of mechanisms that determine ontogenic Mk transitions.
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http://dx.doi.org/10.1016/j.exphem.2018.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5899671PMC
May 2018

Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition.

J Clin Invest 2017 Jun 8;127(6):2365-2377. Epub 2017 May 8.

Department of Pathology, University of Virginia School of Medicine, Charlottesville, Virginia, USA.

Hematopoietic transitions that accompany fetal development, such as erythroid globin chain switching, play important roles in normal physiology and disease development. In the megakaryocyte lineage, human fetal progenitors do not execute the adult morphogenesis program of enlargement, polyploidization, and proplatelet formation. Although these defects decline with gestational stage, they remain sufficiently severe at birth to predispose newborns to thrombocytopenia. These defects may also contribute to inferior platelet recovery after cord blood stem cell transplantation and may underlie inefficient platelet production by megakaryocytes derived from pluripotent stem cells. In this study, comparison of neonatal versus adult human progenitors has identified a blockade in the specialized positive transcription elongation factor b (P-TEFb) activation mechanism that is known to drive adult megakaryocyte morphogenesis. This blockade resulted from neonatal-specific expression of an oncofetal RNA-binding protein, IGF2BP3, which prevented the destabilization of the nuclear RNA 7SK, a process normally associated with adult megakaryocytic P-TEFb activation. Knockdown of IGF2BP3 sufficed to confer both phenotypic and molecular features of adult-type cells on neonatal megakaryocytes. Pharmacologic inhibition of IGF2BP3 expression via bromodomain and extraterminal domain (BET) inhibition also elicited adult features in neonatal megakaryocytes. These results identify IGF2BP3 as a human ontogenic master switch that restricts megakaryocyte development by modulating a lineage-specific P-TEFb activation mechanism, revealing potential strategies toward enhancing platelet production.
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http://dx.doi.org/10.1172/JCI88936DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5451240PMC
June 2017

Megakaryocytic irreversible P-TEFb activation.

Cell Cycle 2014 27;13(12):1827-8. Epub 2014 May 27.

Department of Pathology; University of Virginia School of Medicine; Charlottesville, VA USA.

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http://dx.doi.org/10.4161/cc.29324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4111741PMC
January 2015

Calpain 2 activation of P-TEFb drives megakaryocyte morphogenesis and is disrupted by leukemogenic GATA1 mutation.

Dev Cell 2013 Dec;27(6):607-20

Department of Pathology, University of Virginia School of Medicine, Charlottesville, VA 22908, USA. Electronic address:

Megakaryocyte morphogenesis employs a "hypertrophy-like" developmental program that is dependent on P-TEFb kinase activation and cytoskeletal remodeling. P-TEFb activation classically occurs by a feedback-regulated process of signal-induced, reversible release of active Cdk9-cyclin T modules from large, inactive 7SK small nuclear ribonucleoprotein particle (snRNP) complexes. Here, we have identified an alternative pathway of irreversible P-TEFb activation in megakaryopoiesis that is mediated by dissolution of the 7SK snRNP complex. In this pathway, calpain 2 cleavage of the core 7SK snRNP component MePCE promoted P-TEFb release and consequent upregulation of a cohort of cytoskeleton remodeling factors, including α-actinin-1. In a subset of human megakaryocytic leukemias, the transcription factor GATA1 undergoes truncating mutation (GATA1s). Here, we linked the GATA1s mutation to defects in megakaryocytic upregulation of calpain 2 and of P-TEFb-dependent cytoskeletal remodeling factors. Restoring calpain 2 expression in GATA1s mutant megakaryocytes rescued normal development, implicating this morphogenetic pathway as a target in human leukemogenesis.
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http://dx.doi.org/10.1016/j.devcel.2013.11.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3892434PMC
December 2013

Isocitrate ameliorates anemia by suppressing the erythroid iron restriction response.

J Clin Invest 2013 Aug 25;123(8):3614-23. Epub 2013 Jul 25.

Department of Pathology, University of Virginia, School of Medicine, Charlottesville, Virginia, USA.

The unique sensitivity of early red cell progenitors to iron deprivation, known as the erythroid iron restriction response, serves as a basis for human anemias globally. This response impairs erythropoietin-driven erythropoiesis and underlies erythropoietic repression in iron deficiency anemia. Mechanistically, the erythroid iron restriction response results from inactivation of aconitase enzymes and can be suppressed by providing the aconitase product isocitrate. Recent studies have implicated the erythroid iron restriction response in anemia of chronic disease and inflammation (ACDI), offering new therapeutic avenues for a major clinical problem; however, inflammatory signals may also directly repress erythropoiesis in ACDI. Here, we show that suppression of the erythroid iron restriction response by isocitrate administration corrected anemia and erythropoietic defects in rats with ACDI. In vitro studies demonstrated that erythroid repression by inflammatory signaling is potently modulated by the erythroid iron restriction response in a kinase-dependent pathway involving induction of the erythroid-inhibitory transcription factor PU.1. These results reveal the integration of iron and inflammatory inputs in a therapeutically tractable erythropoietic regulatory circuit.
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http://dx.doi.org/10.1172/JCI68487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3726169PMC
August 2013

Protein kinase D-HDAC5 signaling regulates erythropoiesis and contributes to erythropoietin cross-talk with GATA1.

Blood 2012 Nov 14;120(20):4219-28. Epub 2012 Sep 14.

Department of Pathology, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.

In red cell development, the differentiation program directed by the transcriptional regulator GATA1 requires signaling by the cytokine erythropoietin, but the mechanistic basis for this signaling requirement has remained unknown. Here we show that erythropoietin regulates GATA1 through protein kinase D activation, promoting histone deacetylase 5 (HDAC5) dissociation from GATA1, and subsequent GATA1 acetylation. Mice deficient for HDAC5 show resistance to anemic challenge and altered marrow responsiveness to erythropoietin injections. In ex vivo studies, HDAC5(-/-) progenitors display enhanced entry into and passage through the erythroid lineage, as well as evidence of erythropoietin-independent differentiation. These results reveal a molecular pathway that contributes to cytokine regulation of hematopoietic differentiation and offer a potential mechanism for fine tuning of lineage-restricted transcription factors by lineage-specific cytokines.
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http://dx.doi.org/10.1182/blood-2011-10-387050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3501719PMC
November 2012

Cyclic AMP signaling inhibits megakaryocytic differentiation by targeting transcription factor 3 (E2A) cyclin-dependent kinase inhibitor 1A (CDKN1A) transcriptional axis.

J Biol Chem 2012 Jun 17;287(23):19207-15. Epub 2012 Apr 17.

Department of Pathology, University of Virginia School of Medicine, Charlottesville, Virginia 22903, USA.

Signaling via the intracellular second messenger cyclic AMP (cAMP) has long been implicated in the repression of megakaryocytic differentiation. However, the mechanisms by which cAMP signaling impairs megakaryopoiesis have never been elucidated. In a human CD34(+) cell culture model, we show that the adenylyl cyclase agonist forskolin inhibits megakaryocytic differentiation in a protein kinase A-dependent manner. Using this system to screen for downstream effectors, we identified the transcription factor E2A as a key target in a novel repressive signaling pathway. Specifically, forskolin acting through protein kinase A-induced E2A down-regulation and enforced expression of E2A overrode the inhibitory effects of forskolin on megakaryopoiesis. The dependence of megakaryopoiesis on critical thresholds of E2A expression was confirmed in vivo in haploinsufficient mice and ex vivo using shRNA knockdown in human progenitors. Using a variety of approaches, we further identified p21 (encoded by CDKN1A) as a functionally important megakaryopoietic regulator residing downstream of E2A. These results thus implicate the E2A-CDKN1A transcriptional axis in the control of megakaryopoiesis and reveal the lineage-selective inhibition of this axis as a likely mechanistic basis for the inhibitory effects of cAMP signaling.
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http://dx.doi.org/10.1074/jbc.M112.366476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3365953PMC
June 2012

Aconitase regulation of erythropoiesis correlates with a novel licensing function in erythropoietin-induced ERK signaling.

PLoS One 2011 22;6(8):e23850. Epub 2011 Aug 22.

Department of Pathology, University of Virginia School of Medicine, Charlottesville, Virginia, United States of America.

Background: Erythroid development requires the action of erythropoietin (EPO) on committed progenitors to match red cell output to demand. In this process, iron acts as a critical cofactor, with iron deficiency blunting EPO-responsiveness of erythroid progenitors. Aconitase enzymes have recently been identified as possible signal integration elements that couple erythropoiesis with iron availability. In the current study, a regulatory role for aconitase during erythropoiesis was ascertained using a direct inhibitory strategy.

Methodology/principal Findings: In C57BL/6 mice, infusion of an aconitase active-site inhibitor caused a hypoplastic anemia and suppressed responsiveness to hemolytic challenge. In a murine model of polycythemia vera, aconitase inhibition rapidly normalized red cell counts, but did not perturb other lineages. In primary erythroid progenitor cultures, aconitase inhibition impaired proliferation and maturation but had no effect on viability or ATP levels. This inhibition correlated with a blockade in EPO signal transmission specifically via ERK, with preservation of JAK2-STAT5 and Akt activation. Correspondingly, a physical interaction between ERK and mitochondrial aconitase was identified and found to be sensitive to aconitase inhibition.

Conclusions/significance: Direct aconitase inhibition interferes with erythropoiesis in vivo and in vitro, confirming a lineage-selective regulatory role involving its enzymatic activity. This inhibition spares metabolic function but impedes EPO-induced ERK signaling and disturbs a newly identified ERK-aconitase physical interaction. We propose a model in which aconitase functions as a licensing factor in ERK-dependent proliferation and differentiation, thereby providing a regulatory input for iron in EPO-dependent erythropoiesis. Directly targeting aconitase may provide an alternative to phlebotomy in the treatment of polycythemia vera.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0023850PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3161794PMC
February 2012

Coordinating red cell differentiation with cell cycle arrest: GATA-1 activation of p21.

Authors:
Adam N Goldfarb

Cell Cycle 2010 Jun 1;9(11):2061. Epub 2010 Jun 1.

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June 2010

Iron control of erythroid development by a novel aconitase-associated regulatory pathway.

Blood 2010 Jul 20;116(1):97-108. Epub 2010 Apr 20.

Department of Pathology, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.

Human red cell differentiation requires the action of erythropoietin on committed progenitor cells. In iron deficiency, committed erythroid progenitors lose responsiveness to erythropoietin, resulting in hypoplastic anemia. To address the basis for iron regulation of erythropoiesis, we established primary hematopoietic cultures with transferrin saturation levels that restricted erythropoiesis but permitted granulopoiesis and megakaryopoiesis. Experiments in this system identified as a critical regulatory element the aconitases, multifunctional iron-sulfur cluster proteins that metabolize citrate to isocitrate. Iron restriction suppressed mitochondrial and cytosolic aconitase activity in erythroid but not granulocytic or megakaryocytic progenitors. An active site aconitase inhibitor, fluorocitrate, blocked erythroid differentiation in a manner similar to iron deprivation. Exogenous isocitrate abrogated the erythroid iron restriction response in vitro and reversed anemia progression in iron-deprived mice. The mechanism for aconitase regulation of erythropoiesis most probably involves both production of metabolic intermediates and modulation of erythropoietin signaling. One relevant signaling pathway appeared to involve protein kinase Calpha/beta, or possibly protein kinase Cdelta, whose activities were regulated by iron, isocitrate, and erythropoietin.
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http://dx.doi.org/10.1182/blood-2009-10-251496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2904585PMC
July 2010

Megakaryocytic programming by a transcriptional regulatory loop: A circle connecting RUNX1, GATA-1, and P-TEFb.

Authors:
Adam N Goldfarb

J Cell Biochem 2009 Jun;107(3):377-82

Department of Pathology, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.

Transcription factors originally identified as drivers of erythroid differentiation subsequently became linked to megakaryopoiesis, reflecting the shared parentage of red cells and platelets. The divergent development of megakaryocytic and erythroid progenitors relies on signaling pathways that impose lineage-specific transcriptional programs on non-lineage-restricted protein complexes. One such signaling pathway involves RUNX1, a transcription factor upregulated in megakaryocytes and downregulated in erythroid cells. In this pathway, RUNX1 engages the erythro-megakaryocytic master regulator GATA-1 in a megakaryocytic transcriptional complex whose activity is highly dependent on the P-TEFb kinase complex. The implications of this pathway for normal and pathological megakaryopoiesis are discussed.
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http://dx.doi.org/10.1002/jcb.22142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2775554PMC
June 2009

Discovering chemical modifiers of oncogene-regulated hematopoietic differentiation.

Nat Chem Biol 2009 Apr 26;5(4):236-43. Epub 2009 Jan 26.

Developmental Biology Laboratory, Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA.

It has been proposed that inhibitors of an oncogene's effects on multipotent hematopoietic progenitor cell differentiation may change the properties of the leukemic stem cells and complement the clinical use of cytotoxic drugs. Using zebrafish, we developed a robust in vivo hematopoietic differentiation assay that reflects the activity of the oncogene AML1-ETO. Screening for modifiers of AML1-ETO-mediated hematopoietic dysregulation uncovered unexpected roles of COX-2- and beta-catenin-dependent pathways in AML1-ETO function. This approach may open doors for developing therapeutics targeting oncogene function within leukemic stem cells.
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http://dx.doi.org/10.1038/nchembio.147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2658727PMC
April 2009

Cross-talk of GATA-1 and P-TEFb in megakaryocyte differentiation.

Blood 2008 Dec 9;112(13):4884-94. Epub 2008 Sep 9.

Department of Pathology, University of Virginia School of Medicine, Charlottesville, USA.

The transcription factor GATA-1 participates in programming the differentiation of multiple hematopoietic lineages. In megakaryopoiesis, loss of GATA-1 function produces complex developmental abnormalities and underlies the pathogenesis of megakaryocytic leukemia in Down syndrome. Its distinct functions in megakaryocyte and erythroid maturation remain incompletely understood. In this study, we identified functional and physical interaction of GATA-1 with components of the positive transcriptional elongation factor P-TEFb, a complex containing cyclin T1 and the cyclin-dependent kinase 9 (Cdk9). Megakaryocytic induction was associated with dynamic changes in endogenous P-TEFb composition, including recruitment of GATA-1 and dissociation of HEXIM1, a Cdk9 inhibitor. shRNA knockdowns and pharmacologic inhibition both confirmed contribution of Cdk9 activity to megakaryocytic differentiation. In mice with megakaryocytic GATA-1 deficiency, Cdk9 inhibition produced a fulminant but reversible megakaryoblastic disorder reminiscent of the transient myeloproliferative disorder of Down syndrome. P-TEFb has previously been implicated in promoting elongation of paused RNA polymerase II and in programming hypertrophic differentiation of cardiomyocytes. Our results offer evidence for P-TEFb cross-talk with GATA-1 in megakaryocytic differentiation, a program with parallels to cardiomyocyte hypertrophy.
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http://dx.doi.org/10.1182/blood-2008-03-145722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2597596PMC
December 2008

Regulation of RUNX1 transcriptional function by GATA-1.

Crit Rev Eukaryot Gene Expr 2007 ;17(4):271-80

Department of Pathology, University of Virginia School of Medicine, PO Box 800904, Charlottesville, VA 22908, USA.

Runt-related transcription factor 1 (RUNX1) and GATA-1 are both transcription factors known to play essential roles in hematopoiesis. Genetic alterations of each are associated with abnormal platelet development, as well as predisposition to leukemia. In addition, in vitro and animal studies indicate that both factors are involved in megakaryopoiesis. We and others have previously shown that RUNX1 and GATA-1 physically interact and cooperate in the activation of megakaryocytic promoters such as alpha IIb integrin and glycoprotein Ibalpha. Moreover, transcriptional cooperation of RUNX1 with GATA-1 is conserved back to Drosophila in which RUNX1 and GATA-1 homologs cooperate in crystal cell development. In this article, we will review the molecular and functional significance of the transcriptional cross talk between RUNX1 and GATA-1. In particular, we will elaborate on recent data which suggest that GATA-1 targets RUNX1 for modification, in particular phosphorylation by cyclin-dependent kinases. Furthermore, targeting of RUNX1 by GATA-1 for phosphorylation may convert RUNX1 from a repressor to an activator. This is a potential mechanism of transcriptional cooperation and may be an essential step in megakaryocytic differentiation.
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http://dx.doi.org/10.1615/critreveukargeneexpr.v17.i4.20DOI Listing
October 2007

Oncogenic pathways of AML1-ETO in acute myeloid leukemia: multifaceted manipulation of marrow maturation.

Cancer Lett 2007 Jun 27;251(2):179-86. Epub 2006 Nov 27.

Department of Pathology, University of Virginia School of Medicine, P.O. Box 800904, Charlottesville, VA 22908, USA.

The leukemic fusion protein AML1-ETO occurs frequently in human acute myeloid leukemia (AML) and has received much attention over the past decade. An initial model for its pathogenetic effects emphasized the conversion of a hematopoietic transcriptional activator, RUNX1 (or AML1), into a leukemogenic repressor which blocked myeloid differentiation at the level of target gene regulation. This view has been absorbed into a larger picture of AML1-ETO pathogenesis, encompassing dysregulation of hematopoietic stem cell homeostasis at several mechanistic levels. Recent reports have highlighted a multifaceted capacity of AML1-ETO directly to inhibit key hematopoietic transcription factors that function as tumor suppressors at several nodal points during hematopoietic differentiation. A new model is presented in which AML1-ETO coordinates expansion of the stem cell compartment with diminished lineage commitment and with genome instability.
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http://dx.doi.org/10.1016/j.canlet.2006.10.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1931834PMC
June 2007

Erythroid inhibition by the leukemic fusion AML1-ETO is associated with impaired acetylation of the major erythroid transcription factor GATA-1.

Cancer Res 2006 Mar;66(6):2990-6

Department of Pathology, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.

Human acute myeloid leukemias with the t(8;21) translocation express the AML1-ETO fusion protein in the hematopoietic stem cell compartment and show impairment in erythroid differentiation. This clinical finding is reproduced in multiple murine and cell culture model systems in which AML1-ETO specifically interferes with erythroid maturation. Using purified normal human early hematopoietic progenitor cells, we find that AML1-ETO impedes the earliest discernable steps of erythroid lineage commitment. Correspondingly, GATA-1, a central transcriptional regulator of erythroid differentiation, undergoes repression by AML1-ETO in a nonconventional histone deacetylase-independent manner. In particular, GATA-1 acetylation by its transcriptional coactivator, p300/CBP, a critical regulatory step in programming erythroid development, is efficiently blocked by AML1-ETO. Fusion of a heterologous E1A coactivator recruitment module to GATA-1 overrides the inhibitory effects of AML1-ETO on GATA-1 acetylation and transactivation. Furthermore, the E1A-GATA-1 fusion, but not wild-type GATA-1, rescues erythroid lineage commitment in primary human progenitors expressing AML1-ETO. These results ascribe a novel repressive mechanism to AML1-ETO, blockade of GATA-1 acetylation, which correlates with its inhibitory effects on primary erythroid lineage commitment.
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http://dx.doi.org/10.1158/0008-5472.CAN-05-2944DOI Listing
March 2006

AML-1-ETO-Mediated erythroid inhibition: new paradigms for differentiation blockade by a leukemic fusion protein.

Crit Rev Eukaryot Gene Expr 2005 ;15(3):207-16

Department of Pathology, University of Virginia School of Medicine, Charlottesville, 22908, USA.

The chromosomal translocation t(8;21), generating the AML1-ETO fusion protein, is frequently associated with French-American-British (FAB) type M2 acute myeloid leukemia (AML). t(8;21) fuses the runt domain from the hematopoietic transcription factor RUNX1 with almost the entire transcriptional repressor ETO. AML1-ETO inhibits normal definitive hematopoiesis and blocks erythroid differentiation. Several mechanistic models for the role of AML1-ETO in leukemia development have emerged over the last decade. Most of these models have emphasized the capacity of the fusion protein to redirect repressive cofactors, such as histone deacetylases (HDACs) and DNA methyltransferases (DNMTs), to RUNX target genes, thereby reversing the hematopoietic transcriptional program activated by wild-type RUNX1a phenomenon referred to collectively in this review as the "classical" corepressor model. Because erythropoiesis occurs in a RUNX-independent manner, this dominant-negative "classical" model cannot explain the prominent repression of red-cell development by AML1-ETO. This review will consider the clinical and mechanistic significance of erythroid inhibition by AML1-ETO. Additional models to account for this mysterious oncogenic function are proposed.
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http://dx.doi.org/10.1615/critreveukargeneexpr.v15.i3.30DOI Listing
March 2006

ERK expands its empire.

Authors:
Adam N Goldfarb

Leuk Res 2005 Nov;29(11):1235-6

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http://dx.doi.org/10.1016/j.leukres.2005.05.021DOI Listing
November 2005

Jun blockade of erythropoiesis: role for repression of GATA-1 by HERP2.

Mol Cell Biol 2004 Sep;24(17):7779-94

University of Virginia School of Medicine, P.O. Box 800904, Charlottesville, VA 22908, USA.

Although Jun upregulation and activation have been established as critical to oncogenesis, the relevant downstream pathways remain incompletely characterized. In this study, we found that c-Jun blocks erythroid differentiation in primary human hematopoietic progenitors and, correspondingly, that Jun factors block transcriptional activation by GATA-1, the central regulator of erythroid differentiation. Mutagenesis of c-Jun suggested that its repression of GATA-1 occurs through a transcriptional mechanism involving activation of downstream genes. We identified the hairy-enhancer-of-split-related factor HERP2 as a novel gene upregulated by c-Jun. HERP2 showed physical interaction with GATA-1 and repressed GATA-1 transcriptional activation. Furthermore, transduction of HERP2 into primary human hematopoietic progenitors inhibited erythroid differentiation. These results thus define a novel regulatory pathway linking the transcription factors c-Jun, HERP2, and GATA-1. Furthermore, these results establish a connection between the Notch signaling pathway, of which the HERP factors are a critical component, and the GATA family, which participates in programming of cellular differentiation.
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http://dx.doi.org/10.1128/MCB.24.17.7779-7794.2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC506977PMC
September 2004

RUNX1 and GATA-1 coexpression and cooperation in megakaryocytic differentiation.

Blood 2003 Jun 6;101(11):4333-41. Epub 2003 Feb 6.

Department of Pathology, University of Virginia, Charlottesville, VA 22908-0904, USA.

Megakaryocytic and erythroid lineages derive from a common bipotential progenitor and share many transcription factors, most prominently factors of the GATA zinc-finger family. Little is known about transcription factors unique to the megakaryocytic lineage that might program divergence from the erythroid pathway. To identify such factors, we used the K562 system in which megakaryocyte lineage commitment is dependent on sustained extracellular regulatory kinase (ERK) activation and is inhibited by stromal cell contact. During megakaryocytic induction in this system, the myeloid transcription factor RUNX1 underwent up-regulation, dependent on ERK signaling and inhibitable by stromal cell contact. Immunostaining of healthy human bone marrow confirmed a strong expression of RUNX1 and its cofactor, core-binding factor beta (CBFbeta), in megakaryocytes and a minimal expression in erythroblasts. In primary human hematopoietic progenitor cultures, RUNX1 and CBFbeta up-regulation preceded megakaryocytic differentiation, and down-regulation of these factors preceded erythroid differentiation. Functional studies showed cooperation among RUNX1, CBFbeta, and GATA-1 in the activation of a megakaryocytic promoter. By contrast, the RUNX1-ETO leukemic fusion protein potently repressed GATA-1-mediated transactivation. These functional interactions correlated with physical interactions observed between GATA-1 and RUNX1 factors. Enforced RUNX1 expression in K562 cells enhanced the induction of the megakaryocytic integrin proteins alphaIIb and alpha2. These results suggest that RUNX1 may participate in the programming of megakaryocytic lineage commitment through functional and physical interactions with GATA transcription factors. By contrast, RUNX1-ETO inhibition of GATA function may constitute a potential mechanism for the blockade of erythroid and megakaryocytic differentiation seen in leukemias with t(8;21).
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http://dx.doi.org/10.1182/blood-2002-09-2708DOI Listing
June 2003

Stromal inhibition of megakaryocytic differentiation is associated with blockade of sustained Rap1 activation.

Blood 2003 Mar 5;101(5):1744-51. Epub 2002 Sep 5.

Department of Pathology, University of Virginia Health Sciences Center, Charlottesville, MD 22908, USA.

Coculture with stromal cells tends to maintain normal hematopoietic progenitors and their leukemic counterparts in an undifferentiated, proliferative state. An example of this effect is seen with megakaryocytic differentiation, wherein stromal contact renders many cell types refractory to potent induction stimuli. This inhibitory effect of stroma on megakaryocytic differentiation correlates with a blockade within hematopoietic cells of protein kinase C-epsilon (PKC-epsilon) up-regulation and of extracellular signal-regulated kinase/mitogen-activated protein (ERK/MAP) kinase activation, both of which have been implicated in promoting megakaryocytic differentiation. In this study K562DeltaRafER.5 cells, expressing an estradiol-responsive mutant of the protein kinase Raf-1, were used to determine the relevance and stage of ERK/MAPK pathway blockade by stromal contact. Activation of DeltaRafER by estradiol overrode stromal blockade of megakaryocytic differentiation, implicating the proximal stage of the ERK/MAPK pathway as a relevant control point. Because stromal contact blocked delayed but not early ERK activation, the small guanosine triphosphatase (GTPase) Rap1 was considered as a candidate inhibitory target. Activation assays confirmed that Rap1 underwent sustained activation as a result of megakaryocytic induction, as previously described. As with ERK activation, stromal contact selectively blocked delayed but not early Rap1 activation, having no effect on Ras activation. Enforced expression of either wild-type Rap1 or the GTPase (GAP) resistant mutant Rap1 V12 failed to override stromal inhibition, suggesting that the inhibitory mechanism does not involve GAP up-regulation but rather may target upstream guanine nucleotide exchange factor (GEF) complexes. Accordingly, coimmunoprecipitation demonstrated stromally induced alterations in a protein complex associated with c-Cbl, a scaffolding factor for Rap1-GEF complexes.
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http://dx.doi.org/10.1182/blood-2002-04-1278DOI Listing
March 2003