Different effects of an N-phenylimide herbicide on heme biosynthesis between human and rat erythroid cells

Heme is a prosthetic group comprising ferrous iron (Fe2+) and protoporphyrin IX and is an essential cofactor in various biological processes such as oxygen transport (hemoglobin) and storage (myoglobin) and electron transfer (respiratory cytochromes) in addition to its role as a structural component of hemoproteins. Heme biosynthesis is induced during erythroid differentiation and is coordinated with the expression of genes involved in globin formation and iron acquisition/transport. However, erythroid and nonerythroid cells exhibit distinct differences in the heme biosynthetic pathway regulation. Defects of heme biosynthesis in developing erythroblasts can have profound medical implications, as represented by sideroblastic anemia. This review will focus on the biology of heme in mammalian erythroid cells, including the heme biosynthetic pathway as well as the regulatory role of heme and human disorders that arise from defective heme synthesis.

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Heme Oxygenase 1 Plays An Unexpected Role During Erythroid Differentiation

Blood ◽

10.1182/blood.v118.21.344.344 ◽

2011 ◽

Vol 118 (21) ◽

pp. 344-344

Author(s):

Daniel Garcia Santos ◽

Matthias Schranzhofer ◽

José Artur Bogo Chies ◽

Prem Ponka

Keyword(s):

Red Blood Cells ◽

Heme Oxygenase ◽

Blood Cells ◽

Erythroid Differentiation ◽

Heme Biosynthesis ◽

Erythroid Cell ◽

Heme Oxygenase 1 ◽

Erythroid Cells ◽

Wild Type ◽

Protein Levels

Abstract Abstract 344 Red blood cells (RBC) are produced at a rate of 2.3 × 106 cells per second by a dynamic and exquisitely regulated process known as erythropoiesis. During this development, RBC precursors synthesize the highest amounts of total organismal heme (75–80%), which is a complex of iron with protoporphyrin IX. Heme is essential for the function of all aerobic cells, but if left unbound to protein, it can promote free radical formation and peroxidation reactions leading to cell damage and tissue injury. Therefore, in order to prevent the accumulation of ‘free' heme, it is imperative that cells maintain a balance of heme biosynthesis and catabolism. Physiologically, the only enzyme capable of degrading heme are heme oxyganase 1 & 2 (HO). Red blood cells contain the majority of heme destined for catabolism; this process takes place in splenic and hepatic macrophages following erythrophagocytosis of senescent RBC. Heme oxygenase, in particular its heme-inducible isoform HO1, has been extensively studied in hepatocytes and many other non-erythroid cells. In contrast, virtually nothing is known about the expression of HO1 in developing RBC. Likewise, it is unknown whether HO1 plays any role in erythroid cell development under physiological or pathophysiological conditions. Using primary erythroid cells isolated from mouse fetal livers (FL), we have shown that HO1 mRNA and protein are expressed in undifferenetiated FL cells and that its levels, somewhat surprisingly, increase during erythropoietin-induced erythroid differentiation. This increase in HO1 can be prevented by succinylacetone (SA), an inhibitor of heme synthesis that blocks 5-aminolevulinic acid dehydratase, the second enzyme in the heme biosynthesis pathway. Moreover, we have found that down-regulation of HO1 via siRNA increases globin protein levels in DMSO-induced murine erythroleukemic (MEL) cells. Similarly, compared to wild type mice, FL cells isolated from HO1 knockout mice (FL/HO1−/−) exhibited increased globin and transferrin receptor levels and a decrease in ferritin levels when induced for differentiation with erythropoietin. Following induction, compared to wild type cells, FL/HO1−/− cells showed increased iron uptake and its incorporation into heme. We therefore conclude that the normal hemoglobinization rate appears to require HO1. On the other hand, MEL cells engineered to overexpress HO1 displayed reduced globin mRNA and protein levels when induced to differentiate. This finding suggests that HO1 could play a role in some pathophysiological conditions such as unbalanced globin synthesis in thalassemias. Disclosures: No relevant conflicts of interest to declare.

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Endothelial Cell-Selective Adhesion Molecule (ESAM) Is Required for the Ontogeny of Definitive Hematopoietic System in Mice

Blood ◽

10.1182/blood.v128.22.3874.3874 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3874-3874

Author(s):

Tomoaki Ueda ◽

Takafumi Yokota ◽

Yasuhiro Shingai ◽

Yukiko Doi ◽

Tomohiko Ishibashi ◽

...

Keyword(s):

Endothelial Cell ◽

Adhesion Molecule ◽

Research Funding ◽

Heme Biosynthesis ◽

Mrna Levels ◽

Severe Anemia ◽

Erythroid Cells ◽

Differentiation Potential ◽

Life Threatening ◽

Definitive Erythropoiesis

Abstract We previously reported that endothelial cell-selective adhesion molecule (ESAM), which was initially identified as an endothelial cell-specific antigen, is an effective lifelong hematopoietic stem cell (HSC) marker in mice and humans (Yokota Blood 2009; Ishibashi Exp Hematol 2016). Prior to the advent of the first definitive HSC, ESAM was already expressed on hemogenic endotherium in the developing aorta of murine embryos. We also reported that ESAM expression on HSCs is functionally important for adult hematopoiesis because ESAM deficiency causes life-threatening myelo-suppression, especially severe anemia, after administration of 5-fluorouracil (5-FU) (Sudo J Immunol 2012, PLoS One 2016). Collective data obtained from the genotyping of newborn ESAM knockout (KO) mice suggested that the number of homozygous (homo) ESAM KO mice was about half of that expected as per the Mendelian ratio. The functional significance of ESAM in the development of hematopoiesis, however, has yet to be determined. Thus, in the present study we have analyzed how ESAM deletion affects hematopoietic development in fetuses of ESAM KO mice. Unexpectedly, the frequency and the size of ESAM homo KO fetuses were comparable to those of wildtype (WT) or heterozyqous KO littermates at embryonic day (E) 14.5. However, we found that the liver of ESAM homo KO fetuses contained significantly fewer mononuclear cells. FACS analyses revealed that all the tested hematopoietic cell populations, including lineage- Sca1+cKitHigh (LSK) and LSK CD150+ CD48- HSCs, B220+ B cells, Gr1+ myeloid cells, and Ter119+ erythroid cells, were significantly decreased in the ESAM homo KO fetal liver. Erythroid differentiation was thought to be delayed in ESAM homo KO fetuses because Ter119+ mature erythroid cells significantly decreased whereas CD71+ Ter119- immature cells significantly increased. HSC-enriched LSK cells from E14.5 ESAM homo KO mice produced fewer numbers of blood cells in MS5 co-culture than those from the others, particularly B-lineage cells, suggesting that the growth and differentiation potential of HSCs is impaired in the absence of ESAM. Although ESAM-deficient fetuses grew without an apparent malfunction in developing organs until E14.5, we found that life-threatening events occurred in 3 days following E14.5. Approximately half of homo KO fetuses exhibited severe anemia at E15.5 and died before E17.5. Quantitative real-time PCR analyses from E16.5 ESAM KO homo fetal livers revealed a significant reduction in messenger RNA (mRNA) levels for adult globins (α and β major). In addition, the mRNA level for an erythroid-specific isoenzyme of 5-aminolevulinic acid synthase 2 (ALAS2), the first and rate-limiting enzyme in the heme biosynthesis pathway, was also found to be reduced in the liver of E16.5 ESAM KO homo fetuses. To learn more about molecular mechanisms involved in the developmental failure of hematopoiesis in ESAM KO fetuses, we performed RNA sequencing (RNA-seq) of LSK cells sorted from E14.5 WT and ESAM KO homo mice. We found that, while transcripts for embryonic globins (ζ and Ey) remained substantially, those for adult globins (α, β major, and β minor) were markedly down-regulated in ESAM-KO HSCs. The results suggested that ESAM deficiency disturbs the globin switch from embryonic to adult type. ALAS2 was insufficiently induced in LSK cells of ESAM KO fetal livers, which presumably results in defects in heme biosynthesis. During the embryonic development, rapid and explosive production of erythroid cells is imperative to support the growth and survival of fetuses. To meet the physiological requirement, definitive erythropoiesis occurs in the developing liver and replaces primitive erythropoiesis. Our data suggest that ESAM expression is indispensable for the development of definitive erythropoiesis. In conclusion, we have revealed that ESAM plays a critical role in the development of definitive hematopoiesis. Approximately half of ESAM KO homo fetuses died between E15.5 and E17.5, at least partly due to the delay of adult hemoglobin synthesis in the absence of ESAM. Disclosures Yokota: SHIONOGI & CO., LTD.: Research Funding. Doi:Yakult Honsha Co.,Ltd.: Speakers Bureau. Shibayama:Novartis Pharma: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding, Speakers Bureau; Takeda: Speakers Bureau; Chugai Pharmaceutical: Speakers Bureau; Ono Pharmaceutical: Speakers Bureau. Kanakura:Chugai Pharmaceutical: Research Funding; Pfizer: Research Funding; Shionogi: Research Funding; Kyowa Hakko Kirin: Research Funding; Fujimotoseiyaku: Research Funding; Toyama Chemical: Research Funding; Bristol Myers: Research Funding; Alexionpharma: Research Funding; Nippon Shinyaku: Research Funding; Astellas: Research Funding; Eisai: Research Funding.

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Regulation of heme biosynthesis: Distinct regulatory features in erythroid cells

Stem Cells ◽

10.1002/stem.5530110607 ◽

1993 ◽

Vol 11 (S1) ◽

pp. 24-35 ◽

Cited By ~ 22

Author(s):

Prem Ponka ◽

Herbert M. Schulman

Keyword(s):

Heme Biosynthesis ◽

Erythroid Cells ◽

In Erythroid Cells

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Transcriptome Analysis of Erythroid Cells Cultured from Diamond Blackfan Anemia Patients with Ribosomal and GATA1 Mutations Reveals Dysregulation of Inflammatory Response Genes

Blood ◽

10.1182/blood.v126.23.3605.3605 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3605-3605

Author(s):

Kelly O'Brien ◽

Adrianna Vlachos ◽

Stacie M. Anderson ◽

Crystiana Tsujiura ◽

Lionel Blanc ◽

...

Keyword(s):

Inflammatory Response ◽

Ribosomal Protein ◽

Blot Analysis ◽

Full Length ◽

Heme Biosynthesis ◽

Biosynthesis Pathway ◽

Erythroid Cells ◽

Diamond Blackfan Anemia ◽

Cd34 Cells ◽

Gata1 Mutation

Abstract Diamond Blackfan anemia (DBA) is a rare, congenital bone marrow failure syndrome characterized by red cell aplasia, usually without perturbation of other hematopoietic lineages. DBA patients are generally diagnosed during infancy or early childhood, have a high frequency of congenital anomalies, and a predisposition to cancer. Approximately 65% of DBA patients have identifiable heterozygous gene mutations or deletions in ribosomal protein genes. Additionally, mutations in GATA1, a key transcription factor in erythropoiesis, have been demonstrated in DBA patients (Sankaran VG et al. JCI. 122: 2439-43, 2012; Ludwig LS et al. Nat Med 20(7):748-53, 2014; Klar J et al. Br J Haem 166(6): 949-51, 2014). Despite our knowledge of the molecular pathology, the mechanism underlying the erythroid failure in DBA is not well understood, largely due to the inherent difficulties in studying primary erythroid cells from DBA patients. Furthermore, DBA patients with GATA1 mutations have not been well characterized and it remains unclear whether the pathogenic mechanisms of GATA1 DBA are similar to that of ribosomal DBA. We designed a two-step, 14 day in vitro culture system to generate erythroid cells from CD34+ stem/progenitor cells isolated from <10ml of peripheral blood (PB) collected from patients enrolled in the DBA Registry of North America. Patients with RPL5, RPS17(n=2), RPS24 and RPL35a, GATA1 mutations (n=2), and patients without identifiable mutations (n=3) were studied and compared to healthy control PB CD34+ cells. At day 14, we routinely obtain at least 10x fewer CD235+ erythroid cells (proerythroblasts and basophilic erythroblasts) in DBA cultures vs. controls (1x106 vs. 1x107 from 1x104 CD34+ cells). Further, a 2-7 day delay in the acquisition of CD235 was observed. Gene expression analyses was performed by Affymetrix GeneChip Human Gene ST Arrays and RNASeq to analyze protein coding and long non-coding RNA transcripts in cells from day 14 of erythroid cultures. Ingenuity pathway analysis (IPA) of the dysregulated genes between patients with ribosomal mutations, GATA1 mutations and controls revealed that many of the genes dysregulated in DBA were involved in multiple leukocyte migration and inflammatory signaling pathways, including the IL8, IL1R1, CXCR4, ICAM3, MPO, TNFSF10, and TLR4 genes with IL6, TNF, and lipopolysaccharide as top upstream regulators. Notably, the dysregulated genes in GATA1 patient cells largely overlapped that of the DBA patients with ribosomal mutations, including disruption of the leukocyte migration and inflammatory response genes. Patients with GATA1 and ribosomal protein mutations shared a number of dysregulated erythroid genes including AHSP, FAM132B, HEMGN, and TRIM10, however GATA1 patient cells showed GATA1 as the top upstream regulator and additionally showed dysregulation of heme biosynthesis pathway genes, including the ALAS2, FECH, CPOX, PPOX, and UROS. Few studies have looked at the pathogenesis of DBA in patients with GATA1 mutations. The DBA-associated splice donor mutation in GATA1 results in the exclusive expression of the short form of the GATA1 protein (GATA1s), which lacks the transactivation domain. Western blot analysis of control erythroid cells showed expression of both full length GATA1 and GATA1s, with the full length protein predominating. In cells from the patients with the GATA1 mutation, only GATA1s protein was expressed and the level of GATA1s exceeded the combined level of both GATA1 isoforms in control cells. Erythroid cells from a patient with an RPL5 mutation showed GATA1 protein levels comparable to controls, indicating the DBA phenotype of the RPL5 patient is not due to altered translation of full length GATA1. Northern blot analysis demonstrated that the GATA1 mutation did not affect ribosomal RNA processing. In summary, our transcriptome analyses have revealed novel insights into the molecular pathogenesis of ribosomal and GATA1-mutated DBA. Significant dysregulation of inflammatory gene pathways in DBA patients with both ribosomal protein and GATA1 mutations were observed, suggesting a shared pathological mechanism. Furthermore, the heme biosynthesis pathway was uniquely disrupted in patients with GATA1 mutations. Further investigating the inflammatory pathways in DBA may reveal novel targets for therapeutic development. Disclosures No relevant conflicts of interest to declare.

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Erythropoietin Signaling Regulates Heme Biosynthesis

Blood ◽

10.1182/blood.v128.22.543.543 ◽

2016 ◽

Vol 128 (22) ◽

pp. 543-543

Author(s):

Jacky Chung ◽

Johannes G. Wittig ◽

Alireza Ghamari ◽

Manami Maeda ◽

Harvey Lodish ◽

...

Keyword(s):

Conflicts Of Interest ◽

Protoporphyrin Ix ◽

Janus Kinase ◽

Heme Biosynthesis ◽

Erythroid Cells ◽

Downstream Target ◽

Hematologic Disorder ◽

Hemoglobin Production ◽

Dependent Mechanism

Abstract Heme plays a fundamental role in a diverse array of cellular processes and is required for the survival of all cells. During erythropoiesis, heme production is drastically upregulated to support the production of oxygen-carrying hemoglobin. This increase in heme production is mediated by transcriptional induction of heme metabolism genes including ferrochelatase (FECH), which is the enzyme that catalyzes the rate-limiting insertion of ferrous iron into protoporphyrin IX in the mitochondria of erythroid cells. However, how heme production is coordinately regulated by extracellular cues is currently unknown. Here, using complementary biochemical and genetics approaches, we show that erythropoietin (EPO) signaling regulates heme biosynthesis via a protein kinase A (PKA)-dependent mechanism. In its inactive state, PKA is a tetrameric complex consisting of two catalytic subunits (C) that are bound to and inhibited by two regulatory subunits (R). The C subunits become activated to phosphorylate downstream target proteins when they dissociate from the R subunits. We demonstrate that EPO-induced JAK2 (janus kinase 2) activity leads to release of the C subunits from the R subunits. We also find that phosphorylated STAT5 (signal transducer and activator of transcription 5) forms a molecular complex with PKA-C. This suggests that phospho-STAT5 can outcompete PKA-R to release PKA-C to directly phosphorylate FECH at a highly conserved threonine residue located in the catalytic site. We examined the importance of FECH phosphorylation in vivo by taking advantage of CRISPR/Cas9-mediated genome editing to knock-in the analogous Thr115Ala substitution into the endogenous Fech gene in murine RBCs. Erythroid cells harboring the homozygous Thr115Ala Fech mutation exhibited a block in hemoglobin production and concomitant intracellular accumulation of upstream protoporphyrin intermediates. Strikingly, this phenotype bears resemblance to erythropoietic protoporphyria (EPP), a human hematologic disorder typically associated with FECHmutations. Together, our results support a model where EPO signaling during erythroid maturation activates PKA by a previously unrecognized JAK2/STAT5-dependent mechanism. Phosphorylation of FECH is required for full activity to support elevated heme biosynthesis and hemoglobin production. Furthermore, our data implicates aberrant EPO/JAK2/PKA signaling in the pathogenesis of human EPP. Figure Figure. Disclosures No relevant conflicts of interest to declare.

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KLF1 Modulates Delta-Aminolevulinic Acid Dehydratase (ALAD) Gene Transcription in a Context-Specific Manner

Blood ◽

10.1182/blood.v118.21.2094.2094 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2094-2094

Author(s):

Aurelie Desgardin ◽

Tatiana Abramova ◽

Jenny Lin ◽

Eun-Hee Shim ◽

John M Cunningham

Keyword(s):

Cell Line ◽

Gene Transcription ◽

Heme Biosynthesis ◽

Erythroid Cell ◽

Specific Gene ◽

Regulatory Sequences ◽

Mrna Levels ◽

Erythroid Cells ◽

Molecular Events ◽

In Erythroid Cells

Abstract Abstract 2094 Krüppel-like factor 1 (KLF1) is a zinc finger-encoding transcription factor that recognizes CACC elements, and is essential for maximal erythroid-specific gene transcription. Several critical mechanisms dependent on KLF1 and required for gene activation have been elucidated, predominantly using the beta-globin locus. KLF1 has been associated with the ordered recruitment of SWI/SNF and RNA polymerase-II complexes, necessary for chromatin remodeling and gene transcription respectively. KLF1 has also been reported to influence erythroid-specific heme biosynthesis. Studies in KLF1-null fetal erythroblasts and a KLF-1 deficient cell line have demonstrated that mRNA levels of the first three enzymes of the biosynthetic pathway are underrepresented. However, although in vitro studies of the rate-limiting enzymes ALAS2 and PBGD suggested a potential regulatory role for KLF1, in vivo studies failed to validate these findings. ALAD is the second enzyme of the pathway. Complete loss of ALAD expression in erythroid cells results in catastrophic events during zebrafish ontogeny. Interestingly, no human erythropoietic defect has been reported as a consequence of aberrant ALAD expression. To extend the analysis of KLF1's regulation of heme biosynthesis, we evaluated KLF1 binding of enzyme regulatory sequences by EMSA and ChIP studies, identifying a KLF1 binding CACC element in the erythroid-specific ALAD promoter. This regulatory element was transactivated specifically by a KLF1 transgene in KLF1-deficient cells. Using a unique 4-OH-Tamoxifen (4-OHT) mediated KLF1-inducible erythroid cell line (K1-ERp), we identified KLF1 as an essential, and early (within 2 hours of induction) activator of transcription of the endogenous ALAD, but not ALAS2 or PBGD genes. Further studies in K1-ERp cells, including DNAseI hypersensitivity and ChIP assays revealed that KLF1 occupancy at the erythroid-specific ALAD promoter triggers a series of molecular events including histone modifications, and enhanced recruitment of the sequence-specific transcription factors, GATA-1, NF-E2 and the TAL-1/SCL multiprotein complex. Importantly, we identified differences in the kinetics of recruitment of the closely related histone acetyltransferases proteins CBP and p300 and the SWI/SNF ATPase Brg1. The latter complex was recruited subsequent to KLF1 binding, although the ALAD promoter was already DNAseI hypersensitive. These results suggest strongly that KLF1 plays a major role in the regulation of heme biosynthesis in erythroid cells. Furthermore, our data challenges a model in which an identical temporal cascade of molecular events are required for transcription at KLF1-dependent promoters. Disclosures: No relevant conflicts of interest to declare.

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Heme-Related Blood Disorders

Blood ◽

10.1182/blood.v122.21.sci-18.sci-18 ◽

2013 ◽

Vol 122 (21) ◽

pp. SCI-18-SCI-18

Author(s):

Hervé Puy ◽

Karim Zoubida ◽

Lyoumi Said ◽

Lydie M. Da Costa ◽

Gouya Laurent

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Modifier Gene ◽

Heme Biosynthesis ◽

Type I ◽

Erythroid Cells ◽

Inherited Disorders ◽

Sideroblastic Anemia ◽

Hematopoietic Stem ◽

In Erythroid Cells

Abstract Heme biosynthesis in erythroid cells is intended primarily for the formation of hemoglobin. As in every cell, this synthesis requires a multi-step pathway that involves eight enzymes including the erythroid-specific δ-aminolevulinate synthase (ALAS2, the first regulated enzyme that converts glycine and succinyl CoA into ALA) and the ubiquitous ferrochelatase (FECH, the final enzyme). Heme biosynthesis also requires membrane transporters that are necessary to translocate glycine, precursors of heme, and heme itself between the mitochondria and the cytosol. Defects in normal porphyrin and/or heme synthesis and transport cause four major erythroid inherited disorders, which may or may not be associated with dyserythropoiesis (e.g., sideroblastic, microcytic anemia and/or hemolytic anemia): "X-linked" sideroblastic anemia (XLSA) and X-linked dominant protoporphyria (XLDPP) are two different and opposing disorders but related to altered gene encoding ALAS2 only. Defective activity of this enzyme due to mutations in the ALAS2 gene causes the XLSA phenotype, including microcytic, hypochromic anemia with abundant ringed sideroblasts in the bone marrow. Vice versa, gain-of-function mutations of ALAS2 are responsible of the XLDPP characterized by predominant accumulation of the hydrophobic protoporphyrin (PPIX, the last heme precursor) in the erythrocytes without anemia or sideroblasts. Furthermore, the glycine transporter (SLC25A38) and Glutaredoxin 5 genes are reported to be involved in human non-syndromic sideroblastic anemia. Congenital erythropoietic porphyria (CEP) is the rarest autosomal recessive disorder due to a deficiency in uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme biosynthetic pathway. CEP leads to excessive synthesis and accumulation of type I isomers of porphyrins in the reticulocytes, followed by intravascular hemolysis and severe anemia. The ALAS2 gene may act as a modifier gene in CEP patients (Figueras J et al, Blood. 2011;118(6):1443-51). Erythropoietic protoporphyria (EPP) results from a partial deficiency of FECH and leads similarly to XLDPP, to deleterious accumulation of PPIX in erythroid cells. Most EPP patients present intrans to a FECH gene mutation an IVS3-48C hypomorphic allele due to a splice mutation. Abnormal spliced mRNA is degraded which contributes to the lowest FECH enzyme activity and allowed EPP phenotype expression. We have identified an antisense oligonucleotide (ASO) to redirect splicing from cryptic to physiological site and showed that the ASO-based therapy mediates normal splice rescue of FECH mRNA and reduction by 60 percent of PPIX overproduction in primary cultures of EPP erythroid progenitors. Therapeutic approaches to target both ALAS2 inhibition and heme-level reduction may be useful in other erythroid disorders such as thalassemia (where reduced heme biosynthesis was shown to improve the clinical phenotype) or the Diamond-Blackfan anemia (DBA). Indeed, in some DBA patients, an unusual mRNA splicing of heme exporter FLVCR has been found, reminiscent of Flvcr1-deficient mice that develop a DBA-like phenotype with erythroid heme accumulation. Thus, FLVCR may act as a modifier gene for DBA phenotypic variability. Recent advances in understanding the pathogenesis and molecular genetic heterogeneity of heme-related disorders have led to improved diagnosis and treatment. These advances include DNA-based diagnoses for all the porphyrias and some porphyrins and heme transporters, new understanding of the pathogenesis of the erythropoietic disorders, and new and experimental treatments such as chronic erythrocyte transfusions, bone marrow or hematopoietic stem cell transplants, and experimental pharmacologic chaperone and stem cell gene therapies for erythropoietic porphyrias. Disclosures: No relevant conflicts of interest to declare.

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IDH1 mutation contributes to myeloid dysplasia in mice by disturbing heme biosynthesis and erythropoiesis

Blood ◽

10.1182/blood.2020007075 ◽

2020 ◽

Author(s):

Yu Gu ◽

Risheng Yang ◽

Ying Yang ◽

Yuanlin Zhao ◽

Andrew Wakeham ◽

...

Keyword(s):

Erythroid Differentiation ◽

Genetic Alterations ◽

Idh1 Mutation ◽

Heme Biosynthesis ◽

Heme Oxygenase 1 ◽

Erythroid Cells ◽

Hematopoietic Stem ◽

Idh Mutations ◽

Mutant Idh1 ◽

New Treatments

Isocitrate dehydrogenase (IDH) mutations are common genetic alterations in myeloid disorders, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Epigenetic changes, including abnormal histone and DNA methylation, have been implicated in the pathogenic build-up of hematopoietic progenitors, but it is still unclear whether and how IDH mutations themselves affect hematopoiesis. Here, we show that IDH1-mutant mice develop myeloid dysplasia in that these animals exhibit anemia, ineffective erythropoiesis, increased immature progenitor and erythroblast. In erythroid cells of these mice, D-2-hydroxyglutarate (D-2HG), an aberrant metabolite produced by the mutant IDH1 enzyme, inhibits oxoglutarate dehydrogenase (OGDH) activity and diminishes succinyl-CoA production. This succinyl-CoA deficiency attenuates heme biosynthesis in IDH1-mutant hematopoietic cells, thus blocking erythroid differentiation at the late erythroblast stage and the erythroid commitment of hematopoietic stem cells (HSC), while the exogenous succinyl-CoA or 5-ALA rescues erythropoiesis in IDH1-mutant erythroid cells. Heme deficiency also impairs heme oxygenase-1 (HO-1) expression, which reduces levels of important heme catabolites such as biliverdin and bilirubin. These deficits result in accumulation of excessive reactive oxygen species (ROS) that induce the cell death of IDH1-mutant erythroid cells. Our results clearly demonstrate the essential role of IDH1 in normal erythropoiesis and show how its mutation leads to myeloid disorders. Our data thus have important implications for the devising of new treatments for IDH-mutant tumors.

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Stimulation of Transferrin Receptor Expression by Enhanced Heme Biosynthesis in Murine Erythroleukemia Cells.

Blood ◽

10.1182/blood.v104.11.3200.3200 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3200-3200

Author(s):

Chun-Nam Lok ◽

Prem Ponka

Keyword(s):

Aminolevulinic Acid ◽

Regulatory Protein ◽

Binding Activity ◽

Receptor Expression ◽

Heme Biosynthesis ◽

Mrna Levels ◽

Erythroid Cells ◽

Iron Chelate ◽

Heme Synthesis ◽

Murine Erythroleukemia

Abstract In erythroid cells iron uptake from transferrin (Tf) is utilized largely for heme synthesis. Here we provide evidence that Tf receptor (TfR) expression and cellular uptake of iron from Tf is stimulated by enhanced heme synthesis. Incubation of murine erythroleukemia (MEL) cells with 5-aminolevulinic acid (ALA) resulted in an increase in TfR expression accompanied by enhanced uptake of iron from Tf and incorporation of iron into heme. ALA-mediated enhancement of TfR mRNA expression was completely prevented by succinylacetone, an inhibitor of ALA dehydratase, and N-methylprotoporphyrin, an inhibitor of ferrochelatase, indicating that the effect of ALA required its metabolism to heme. Treatment of cells with ALA was associated with enhanced iron regulatory protein-2 (IRP-2) binding activity, which could be blocked by inhibitors of heme synthesis and supplementation of the culture medium with a permeable iron chelate or Tf. In all cases, IRP-2 activities were correlated exactly with TfR mRNA levels. Thus, in addition to the previously characterized transcriptional up-regulation of TfR expression in differentiating erythroid cells, increased TfR expression mediated by enhanced heme biosynthesis may ensure sufficient iron availability for optimal heme synthesis and prevent possible protoporphyrin accumulation under conditions of inadequate iron supply.

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