ALBERT

Description: AML1-ETO (A/E) is the fusion product of a chromosomal translocation, t(8;21) frequently associated with FAB M2 acute myeloid leukemia (AML). The fusion combines the runt domain of the hematopoietic transcription factor RUNX1 with almost the entire transcriptional repressor ETO. Clinical cases of AML with t(8;21) are distinguished by blockade in erythroid differentiation. In addition, enforced expression of A/E in primary human erythroid progenitors impairs differentiation. Existing paradigms postulate that A/E exerts its leukemogenic effects through recruitment to RUNX binding sites of cofactors such as corepressors, histone deacetylases (HDACs), and DNA methyltransferases (DNMTs), causing repression of RUNX target genes. However, this paradigm fails to explain effects of A/E on erythropoiesis as erythroid genes generally lack functional RUNX sites. We have published a physical and functional interplay between RUNX1 and the erythroid master regulator GATA-1 (Blood 101:4333). Furthermore, A/E physically interacted and functionally interfered with GATA-1. In the current studies we have examined domain and cofactor requirements for A/E inhibition both of GATA-1 function and of erythroid differentiation. Deletional mutagenesis of A/E demonstrated that the zinc finger (NH4) and runt domains were absolutely required for GATA-1 inhibition. Treatment with HDAC and DNMT inhibitors failed to affect A/E repression of GATA-1. RNAi knockdown of all known NH4 interactors, HDACs 1-3, N-CoR, SMRT-A, and SMRT-B also failed to affect A/E inhibition of GATA-1. Inducible expression of A/E in MEL cells caused downregulation of endogenous GATA-1 protein and mRNA, an effect dependent on induction of erythroid differentiation. A coexpressed GATA-1-GFP fusion showed downregulation with identical kinetics to endogenous GATA-1. Interestingly, proteasome-specific inhibitors effectively prevented the downregulation of endogenous GATA-1 and GATA-1-GFP caused by induction of A/E coupled with erythroid differentiation. Fluorescence microscopy showed a striking relocation of GATA-1-GFP from the nucleus to discrete, paranuclear bodies upon joint induction of A/E expression and erythroid differentiation. Our findings indicate that A/E inhibition of GATA-1 occurs through a previously undescribed mechanism that involves GATA-1 redistribution to novel cellular structures followed by proteasome-mediated degradation. These findings expand the paradigm of A/E leukemogenicity to include a non-transcriptional mechanism in which a growth inhibitor/tumor suppressor, GATA-1, is targeted by A/E for proteolytic degradation in a manner reminiscent of human papilloma virus E6 targeting of p53 for degradation in cervical carcinogenesis.

Description: Programming of megakaryocytic differentiation requires precise coordination of multiple signal transduction and transcription pathways. Previous in vivo and in vitro studies have implicated RUNX1 and GATA-1 as transcription factors that collaborate in the execution of this program. Analysis of the mechanism for the synergy of these two factors revealed induction of RUNX1 hyperphosphorylation by GATA-1 coexpression. A pharmacologic screen identified roscovitine as an inhibitor of the transcriptional cooperation, implicating a cyclin-dependent kinase (Cdk). A screen employing a panel of dominant-negative Cdk mutants identified Cdk9 as a critical component of the GATA-1-RUNX1 cooperation. In addition, HEXIM1, an endogenous Cdk9 inhibitor, similarly blocked transcriptional synergy. Furthermore, two kinase inhibitory compounds, DRB and flavopiridol, also blocked GATA-1-RUNX1 cooperation at concentrations specific for Cdk9 inhibition. Regarding the mechanism for GATA-1 induction of RUNX1 phosphorylation, coimmunoprecipitation experiments showed GATA-1 binding to both Cdk9 and cyclinT1. To examine the role of P-TEFb in primary megakaryocytic differentiation, human CD34+ cells in megakaryocytic cultures underwent treatment with 50 nM flavopiridol, a dose selective for Cdk9 inhibition. This treatment blocked megakaryocytic polyploidization while having no effect on the cell cycle properties of the non-megakaryocytic cells in the cultures. The treatment also impaired upregulation of CD41. Extending these findings to an in vivo model system, mice underwent treatment with daily low dose flavopiridol (5–7 mg/kg/day), a regimen previously shown to have no toxicity. Wild type C57BL/6 (wt BL/6) mice were compared with the ΔneoΔHS strain (GATA-1Lo) which has diminished GATA-1 expression in megakaryocytes. After only 1 week of treatment, the GATA-1Lo mice developed worsening thrombocytopenia associated with new-onset anemia, with several dying after 2 weeks of treatment. Flow cytometry on marrow from the treated GATA-1Lo mice revealed a marked expansion of abnormal megakaryocytes showing coexpression of CD71 plus CD41 and loss of polyploidization. Marrow and spleen histology showed extensive replacement by immature-appearing megakaryocytes with hypolobulated nuclei, as well as frequent pyknotic megakaryocytes. The control mice, flavopiridol treated wt BL/6 and saline treated GATA-1Lo, displayed none of these abnormalities. Additional experiments determined the flavopiridol effect on the GATA-1Lo mice to be completely reversible, with normalization of all parameters 2 weeks after ending treatment. In aggregate, these data implicate P-TEFb recruitment by GATA-1 in mediating cooperative activation of megakaryocytic promoters with RUNX1. This pathway may depend in part on the direct phosphorylation of RUNX1 by Cdk9. In mice, a synthetic lethal relationship between megakaryocytic GATA-1 deficiency and Cdk9 inhibition exists, manifesting as a fulminant but reversible megakaryocytic proliferative disorder reminiscent of the Down syndrome-associated megakaryocyte proliferations. A model is proposed in which P-TEFb, as a component of GATA-1-RUNX1 transcriptional complexes, plays an integral role in the specific programming of megakaryocytic differentiation, with particular importance in the unique cell cycle changes associated with this lineage.

Description: 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 β (CBFβ), in megakaryocytes and a minimal expression in erythroblasts. In primary human hematopoietic progenitor cultures, RUNX1 and CBFβ up-regulation preceded megakaryocytic differentiation, and down-regulation of these factors preceded erythroid differentiation. Functional studies showed cooperation among RUNX1, CBFβ, 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 αIIb and α2. 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).

Description: Abstract 289 The regulation of megakaryopoiesis by cyclic AMP (cAMP) signaling forms the basis for treatment approaches to thrombocytosis in patients with myeloproliferative neoplasms. Anagrelide, the longstanding treatment of choice, is an inhibitor of the cAMP catabolizing enzyme phosphodiesterase III, and acts to lower platelet counts by inhibiting early stages of megakaryocytic differentiation. Essential for programming megakaryocytic differentiation is the transcriptionally activating pentameric complex consisting of GATA-1, SCL/TAL-1, an E-protein (either E2A or the similar HEB), and the bridging factors Lmo2 and Ldb1. This complex becomes repressive upon the recruitment of the co-repressor ETO-2 (also called MTG16 and CBFA2T3) by either SCL or E-protein components. ETO-2 has also been described as an A-kinase floating protein (AKAP). AKAPs bind the regulatory subunits of Protein kinase A (PKA) and tether the kinase within a specific compartment of the cell, ensuring proximity to appropriate substrates following kinase activation. The current study addresses the hypothesis that elevations in cAMP, such as those induced by anagrelide, inhibit megakaryocytic differentiation through the activation of a novel PKA signaling pathway involving ETO-2 and other components of the pentameric complex. Using peripheral blood mobilized human CD34+ progenitors cultured under conditions promoting megakaryopoiesis, we have confirmed that 500 nM anagrelide or 10 μ M forskolin, a direct adenylyl cyclase agonist, suppress upregulation of CD41 by two to three-fold as well as polyploidization, as determined by FACS on day 6 cultures. Pre-treatment of cultures with 10 μ M H89, a specific inhibitor of PKA, prevented the inhibitory effect of forskolin on megakaryopoiesis, as reflected by CD41 upregulation at day 6. Analysis of the kinetics of PKA activation, by immunoblotting for phospho-CREB and for phospho-PKA substrates (Cell Signaling), surprisingly showed only transient PKA induction by forskolin, with signals returning to baseline level after 4 hours. Additionally, washout experiments in which cells were treated with forskolin for only the first 4 hours of megakaryocytic culture showed severely blunted CD41 upregulation on day 6. These findings argue that elevations in cAMP inhibit megakaryocytic differentiation via activation of PKA and that a brief window of PKA activation during early megakaryopoiesis suffices for a stable blockade of differentiation. To determine a potential role for ETO-2, expression levels were analyzed at various times during megakaryocytic culture. In contrast to the rapid downregulation described in fetal murine megakaryocyte culture (Hamlett I. et al. Blood 112:2738-2749, 2008), human CD34+ progenitors showed no downregulation of ETO-2 mRNA or protein during megakaryocytic differentiation, nor were levels significantly affected by forskolin treatment. To study the functional role of ETO-2, we utilized shRNA knockdown in CD34+ hematopoietic progenitor cells undergoing megakaryocytic culture. In contrast to cells transduced with control vector, cells expressing shRNA targeting ETO-2 showed resistance to the inhibitory effects of elevated cAMP (n = 3). In examining potential targets within the pentameric complex, we found that elevations in cAMP reproducibly lowered the protein and mRNA levels of E2A by 50% while not affecting mRNA levels of another E protein, HEB. Altogether, these data suggest that cAMP mediates inhibition of megakaryopoiesis via activation of PKA, which utilizes ETO-2, possibly as an AKAP, to modulate the composition of an essential activating complex by downregulating E2A. Interestingly, E2A+/− mice are impaired in their ability to generate megakaryocytic progenitors from bipotent megakaryocyte-erythroid precursors (Semerad CL et al. PNAS 106:1930-1935, 2009). This novel signaling pathway contributes to an understanding of the mechanism of action of anagrelide and also further elucidates the basic regulatory circuitry governing determination of the megakaryocytic lineage. Disclosures: No relevant conflicts of interest to declare.

Description: 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 Cα/β, or possibly protein kinase Cδ, whose activities were regulated by iron, isocitrate, and erythropoietin.

Description: 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.

Description: Abstract 166 The erythroid iron deprivation response results from lineage-selective inactivation of aconitase enzymes, causing diminished erythropoietin (Epo) responsiveness in erythroid progenitors. Provision of exogenous isocitrate in either cell culture or murine models of iron deficiency restores Epo responsiveness and abrogates the erythropoietic block characteristic of the iron deprivation response. Although isocitrate administration can restore erythropoiesis in iron deficient mice, the response is transient, and iron administration is required for sustained correction. In anemias other than those due to iron deficiency, inappropriate activation of this iron deprivation pathway might also contribute to suppression of erythropoiesis. Such anemias are predicted to respond to isocitrate administration. A major area of clinical controversy is the degree to which iron restriction plays a role anemia of chronic inflammation (ACI). In support of such a role, inflammatory stimuli promote increased hepcidin production by the liver, and diminished serum iron represents a consistent finding in patients with ACI. Challenging such a role, a recently published series of elderly patients with ACI showed no evidence of increased hepcidin production (Ferrucci et al., Blood 115:3810, 2010). Furthermore, ACI usually manifests as a normochromic, normocytic anemia in contrast to the microcytic, hypochromic anemia of iron deficiency. Finally, administration of anti-TNF antibody to patients with rheumatoid arthritis corrected their associated anemia, suggesting a role for direct cytokine repression of erythropoiesis (Papadaki et al., Blood 100:474, 2002). To experimentally examine the role of the erythroid iron deprivation response in ACI, we determined the effects of isocitrate administration in a classic rat arthritis model of ACI. 6 week-old female Lewis rats received a single IP injection (15 μ g rhamnose/g) of Streptococcal cell wall peptidoglycan-polysaccharide (PG-PS). Normochromic, normocytic anemia developed at 2 weeks post injection. Specifically, the mean red cell count (RBC) in rats receiving PG-PS was 7.18 ± 0.22 × 106 cells/μ l vs 8.65 ± 0.39 × 106 cells/μ l in rats not receiving PG-PS (P = 0.014). At this time, the anemic rats underwent daily IP injections with either trisodium isocitrate (200 mg/kg/day) or with equivalent volumes of saline. Rats receiving isocitrate showed correction of anemia after 3 days of treatment, with a mean RBC of 8.14 ± 0.06 × 106 cells/μ l as opposed to a mean RBC in the control group of 6.42 ± 0.45 × 106 cells/μ l (P = 0.018). This correction was maintained after 5 additional days of treatment: RBC in isocitrate-treated group of 8.36 ± 0.24 × 106 cells/μ l versus RBC in saline-treated group of 7.01 ± 0.19 × 106 cells/μ l (P = 0.004). No differences in neutrophil or platelet counts were observed at any point in rats receiving isocitrate vs saline. These results support a role for the erythroid iron deprivation response in the impaired erythropoiesis associated with ACI. These results also support our previous in vitro findings that iron deprivation sensitizes erythroid progenitors to inhibition by inflammatory cytokines (i.e. IFNγ or TNFα) (Richardson et al., ASH 2009 #159). More recent in vitro studies on the mechanisms underlying this sensitization have addressed whether iron restriction synergizes with inflammatory cytokines in promoting aconitase inactivation. Using a gel-based enzymatic assay, we confirmed the inhibitory effect of iron deprivation on both cytosolic and mitochondrial aconitase isoforms, but could find no inhibitory effects associated with either IFNγ or TNFα treatment. In subsequent experiments, a functional screen for elements integrating the erythroid iron deprivation response with inflammatory signaling implicated a calmodulin-regulated factor. Specifically, the calmodulin inhibitor, KN62 reversed the cell death observed with the combination of iron deprivation plus inflammatory cytokines but had minimal effects on viability of cells subjected to either iron deprivation or inflammatory cytokines separately (n = 4). These data thus delineate an iron-regulated signaling element downstream of aconitase which employs calmodulin to modulate erythroid responsiveness to inflammatory cytokines. Pharmacologic targeting of this element, as with isocitrate administration, may provide a new avenue for clinical management of ACI. Disclosures: No relevant conflicts of interest to declare.

Description: Hematopoietic transitions that accompany fetal development, for example erythroid globin chain switching, play important roles in normal physiology and disease development. In the megakaryocyte lineage, human fetal progenitors show impaired execution of the 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 also contribute to inferior platelet recovery after cord blood stem cell transplantation and to inefficient platelet production by megakaryocytes (Mk) derived from pluripotent stem cells. In this study, comparison of neonatal versus adult human progenitors identified a blockade in the specialized positive transcription elongation factor b (P-TEFb) activation mechanism known to drive adult Mk morphogenesis. A central feature of this pathway is known to involve sustained high amplitude activation of the P-TEFb kinase (Cdk9/cyclin T1). This cascade is initiated by downregulation of core components of the repressive complex of P-TEFb: the 7SK snRNP consisting of the 7SK small nuclear RNA (7SK) and its stabilizing factors MePCE and LARP7. The resulting high amplitude activation of P-TEFb drives multiple features of Mk differentiation: induction of cytoskeletal morphogenetic factors (ACTN1, FLNA, MKL1, HIC5), silencing of erythroid genes, promotion of histone H2B K120 monoubiquitiniation (H2BUb1), and phosphorylation of Spt5 at T806 (pSpt5 T806). Critical, rate-limiting steps triggering this pathway comprise MePCE proteolysis by calpain 2 and downregulation of LARP7, both resulting in destabilization of 7SK. In comparison to Mk derived from adult peripheral blood stem cells (adult Mk), Mk derived from umbilical cord blood stem cells (neonatal Mk) showed evidence of decreased P-TEFb activation with decreases in: 1) the expression of the cytoskeletal morphogenetic factors, 2) global H2BUb1, and 3) pSpt5 T806. In addition, neonatal Mk retained expression of the erythroid marker glycophorin A (GPA). Surprisingly, neonatal Mk failed to downregulate 7SK despite the downregulation of its stabilizers MePCE and LARP7, suggesting the existence of alternative 7SK stabilizing factor/s unique to neonatal Mk. Our screening identified the oncofetal RNA-binding protein IGF2BP3 to be expressed in neonatal but not adult Mk, and ectopic expression of IGF2BP3 in adult Mk conferred neonatal phenotypic features including reduction in size, increased proliferation and leaky erythroid antigen expression. Immunoprecipitation and glycerol gradient studies indicated the participation of IGF2BP3 in the 7SK snRNP complex. Mining of endogenous IGF2BP3 iCLIP data indicated that 7SK is one of the top direct targets of IGF2BP3 and further mapped binding to the 7SK fourth hairpin, a critical stability determinant. In loss of function studies, the knockdown of IGF2BP3 in neonatal Mk resulted in destabilization of 7SK and upregulation of the Mk morphogenetic cytoskeletal factors, as well as increased levels of H2BUb1, pSpt5 T806, and hyperphosphorylated RNA Pol II. Phenotypically, the knockdown of IGF2BP3 in neonatal Mk elicited adult features including increased size, enhanced polyploidization, reduced proliferation and silencing of erythroid antigen expression. Collectively, these findings suggest that the block in P-TEFb activation in neonatal Mk results from ontogentic stage-specific expression of IGF2BP3 which prevents the 7SK destabilization normally associated with adult megakaryocytic P-TEFb activation. We also identified a pharmacologic approach to inhibit IGF2BP3 expression, through inhibition of bromodomain and extra- terminal (BET) proteins, which reproducibly promoted adult features in neonatal Mk including enlargement, inhibition of erythroid antigen expression, upregulation of morphogenetic cytoskeletal factors, and increased platelet formation in vitro. Enforced expression of IGF2BP3 in neonatal Mk significantly blunted the effects of BET inhibitors indicating the specificity of their action in downregulating IGF2BP3. These results identify IGF2BP3 as a human ontogenic masterswitch that restricts megakaryocyte development through modulating a lineage-specific P-TEFb activation mechanism, revealing new strategies toward enhancing platelet production. Disclosures No relevant conflicts of interest to declare.

Description: RUNX1 and GATA-1 both play essential roles in the transcriptional programming of normal mammalian megakaryocytic development, deficiencies of either factor having similar phenotypic consequences. We have previously characterized physical and functional interactions between these two factors, and others have confirmed analogous cooperations in Drosophila and Danio homologs. We now present data on molecular mechanisms for the cooperation of these two factors in the transcriptional activation of the megakaryocytic aIIb integrin promoter. In these studies, GATA-2 also physically interacted with RUNX1 but failed to cooperate in transcriptional activation. In fact, increasing amounts of GATA-2 repressed the functional interplay between GATA-1 and RUNX1. Through generation of GATA-2/GATA-1 chimeras, we identified a conserved subdomain within the GATA-1 amino terminus that was both necessary and sufficient for transcriptional cooperation with RUNX1. Coexpression of wild type GATA-1 or of cooperating GATA-2/GATA-1 chimeras, but not of GATA-2 or of non-cooperating chimeras, induced a mobility shift in wild type RUNX1. Using immunoprecipitation followed by immunoblot with a panel of phosphospecific antibodies, we found GATA-1 to induce RUNX1 phosphorylation at recognition sites for cyclin-dependent kinases (cdks). Treatment of cells with roscovitine, a specific cdk inhibitor, blocked the transcriptional cooperation of GATA-1 with RUNX1 and eliminated the RUNX1 mobility shift caused by GATA-1 coexpression. Mutagenesis of RUNX1 identified a cluster of serine/threonine-proline (S/TP) sites collectively required for the transcriptional augmentation and mobility shift induced by GATA-1. In addition, intact DNA binding by RUNX1 was required for cooperation with GATA-1. These results provide a new paradigm for cooperation of interacting transcription factors, in which one partner recruits a kinase leading to phosphorylation and activation of the other partner. Furthermore, these results provide a biochemical basis for the previously inexplicable functional differences between GATA-1, which promotes megakaryocytic maturation, and GATA-2 which promotes proliferation without maturation.

Description: Erythroid iron deficiency, whether due to dimished body stores or histiocytic retention, diminishes marrow responsiveness to erythropoietin (Epo). Conversely, intravenous iron infusion augments marrow responsiveness to Epo, even in anemia patients with adequate pre-existing iron stores. Iron regulation of Epo-driven erythropoiesis affects proliferation and differentiation of early progenitors, prior to the commitment to heme production. Thus, while iron is essential for all cells, erythroid precursors manifest an exquisite sensitivity to iron deficiency, most likely as a rationing mechanism to protect other, more vital iron-dependent functions. Using primary human hematopoietic cultures with defined levels of transferrin saturation, we have confirmed the existence of a critical threshold of iron deprivation, at which erythroid progenitors display proliferative and maturation blockade while granulopoiesis and megakaryopoiesis remain unaffected. Extensive pharmacologic and genetic screening for components of this erythroid iron response pathway have identified the iron-sulfur cluster containing aconitase enzymes as a critical signaling node. Mitochondrial and cytosolic aconitase (mAcon & cAcon) interconvert citrate and isocitrate as a key step in the Krebs cycle. Firstly, treatment of iron deprived erythroid cultures with exogenous isocitrate, but not citrate, completely restored differentiation, as judged by glycophorin A (GPA) expression and hemoglobinization. By contrast, both citrate and isocitrate enhanced erythroid differentiation under iron replete conditions. Secondly, treatment of iron replete erythroid cultures with a specific aconitase inhibitor, fluorocitrate, induced a lineage-selective maturation blockade identical to that seen with iron deprivation. Thirdly, enzymography showed erythroid-lineage specific inactivation of both cAcon and mAcon in response to iron deprivation; immunoblotting showed no change mAcon protein levels as a function of either lineage or iron status. Fourthly, a retroviral genetic screen identified HBLD2, an iron-sulfur cluster assembly factor, as a protein whose overexpression reversed the erythroid maturation blockade associated with iron deprivation. Enzymography confirmed that overexpression of HBLD2 enhanced both cAcon and mAcon activities. Fifthly, administration to wild type, iron replete C57BL/6 mice of isocitrate at 200 mg/kg/day for 5 days caused a significant increase in peripheral red cell hemoglobinization, reflected by MCHC and Hb levels. Taken together, these results identify isocitrate as a positive regulator of Epo-mediated erythroid differentiation. In Epo-independent CD34+ cell culture systems, isocitrate did not enhance erythropoiesis. Therefore aconitase enzymes serve as a critical nexus in iron and Epo regulation of erythropoiesis, integrating cellular metabolism with developmental programming. The unique sensitivity of the erythroid lineage to iron deprivation appears to derive from a cellular milieu effect on the aconitase enzymes, promoting their inactivation under iron deprivation. Notably, IRP repression of mAcon translation does not contribute to the erythroid iron deprivation response pathway. This pathway may have relevance for future clinical approaches to Epo-refractory chronic anemias and polycythemia vera.