Characterization of Congenital Sideroblastic Anemia Model Due to ABCB7 Defects: How Do Defects in Iron-Sulfur Cluster Metabolism Lead to Ring Sideroblast Formation?

Backgroun d: The sideroblastic anemias (SAs) are a group of congenital and acquired bone marrow disorderscharacterized by bone marrow ring sideroblasts (RSs). The disease commonly presents as myelodysplastic syndrome with RS (MDS-RS), known as an acquired clonal SA that is strongly correlated with a specific somatic mutation inSF3B1 (splicing factor 3b subunit 1), which is involved in RNA splicing machinery. Thus far, several studies have consistently revealed compromised splicing and/or expression of ABCB7 (ATP-binding cassette subfamily B member 7) in MDS-RS harboring the SF3B1 mutation. ABCB7 encodes an ATP-binding cassette family transporter localizing to the inner mitochondrial membrane, and its loss-of-function mutation causes a syndromic form of congenital SA, which is associated with cerebellar ataxia. The substrates transported by ABCB7 are predicted to be iron-sulfur clusters (ISCs), which are essential for the function of multiple mitochondrial and extramitochondrial proteins, such as ferrochelatase and aconitase (its apo-form without ISC is called IRP1; iron regulatory protein 1). However, the detailed molecular mechanisms by which defects in ISC metabolism resulting from ABCB7 defects contribute to RS formation remains to be fully elucidated. Methods: Endogenous ABCB7 was depleted based on pGIPZ lentiviral shRNAmir (Dharmacon) in human umbilical cord blood-derived erythroid progenitor (HUDEP)-2 cells (Kurita et al., PLoS ONE, 2013). Puromycin (Sigma) was used for the selection of transduced cells. To induce terminal erythroid differentiation, HUDEP-2 cells were co-cultured with OP9 stromal cells (ATCC) in Iscove's modified Dulbecco's medium supplemented with fetal bovine serum, erythropoietin, dexamethasone, monothioglycerol, insulin-transferrin-selenium, ascorbic acid, and sodium ferrous citrate (Saito and Fujiwara et al., MCB, 2019). For transcription profiling, Human Oligo Chip 25K (Toray) was used. Results: We first conducted ABCB7 knockdown in HUDEP-2 cells based on two independent shRNA plasmids. When the knockdown cells were induced to undergo erythroid differentiation,the majority of the erythroblasts exhibited aberrant mitochondrial iron deposition. Thus, we sought to clarify the potential causative link between ABCB7 defects and RS formation. Expression profiling revealed >1.5-fold up- and down-regulation of 33 and 44 genes, respectively, caused by the ABCB7 knockdown. Intriguingly, 43% of the downregulated gene ensemble (19/44 genes) included multiple ribosomal genes, such as RPS2, RPL11,and RPS12. The downregulated genes also included HMOX1 (heme oxygenase 1), implying that heme biosynthesis would be compromised by the knockdown. Gene ontology (GO) analysis revealed significant (p< 0.01) enrichment of genes associated with nuclear-transcribed mRNA catalytic process, cytoplasmic translation, and cellular iron ion homeostasis. Whereas the mRNA expression for ALAS2 (erythroid-specific 5-aminolevulinate synthase), encoding a rate-limiting enzyme of heme biosynthesis and one of the responsible genes for congenital SA, was not affected, its protein expression was noticeably decreased by ABCB7 knockdown, indicating that compromised transport of ISC from mitochondria to the cytosol may result in decreased ALAS2 translation by the binding of IRP1 to the iron-responsive element located in the 5'-UTR of ALAS2 mRNA.We are currently conducting detailed biological analyses to elucidate the causative link between defects in ISC metabolism due to ABCB7 defects and RS formation. Conclusion: We have first demonstrated the emergence of RS by ABCB7 depletion in human erythroblasts. Further characterization of the established SA model would aid in the clarification of its molecular etiology and the establishment of novel therapeutic strategies. Furthermore, our results may lead to a better understanding of the role of ISC in affecting cerebellar symptoms. Disclosures Fukuhara: Gilead: Research Funding; Nippon Shinkyaku: Honoraria; Zenyaku: Honoraria; AbbVie: Research Funding; Takeda Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Mundi: Honoraria; Ono Pharmaceutical Co., Ltd.: Honoraria; Bayer: Research Funding; Celgene Corporation: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria; Eisai: Honoraria, Research Funding; Janssen Pharma: Honoraria; Kyowa-Hakko Kirin: Honoraria; Mochida: Honoraria; Solasia Pharma: Research Funding. Onishi:Novartis Pharma: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Astellas Pharma Inc.: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Janssen Pharmaceutical K.K.: Honoraria; MSD: Honoraria, Research Funding; Sumitomo Dainippon Pharma: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Takeda Pharmaceutical Co., Ltd.: Research Funding; Nippon Shinyaku: Honoraria; Pfizer Japan Inc.: Honoraria; Kyowa-Hakko Kirin: Honoraria; Celgene: Honoraria. Yokoyama:Astellas: Other: Travel expenses.

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Generation and Molecular Characterization of Human Ring Sideroblasts

Blood ◽

10.1182/blood-2018-99-111066 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3613-3613

Author(s):

Kei Saito ◽

Tohru Fujiwara ◽

Shunsuke Hatta ◽

Chie Suzuki ◽

Noriko Fukuhara ◽

...

Keyword(s):

Bone Marrow ◽

Molecular Characterization ◽

Ferrous Iron ◽

Research Funding ◽

Erythroid Differentiation ◽

Wild Type ◽

Sideroblastic Anemia ◽

Iron Transporter ◽

Ring Sideroblasts

Abstract (Background) Sideroblastic anemias are heterogeneous congenital and acquired refractory anemias characterized by bone marrow ring sideroblasts, reflecting excess mitochondrial iron deposition. While the disease is commonly associated with myelodysplastic syndrome, the congenital forms of sideroblastic anemias comprise a diverse class of syndromic and non-syndromic disorders, which are caused by the germline mutation of genes involved in iron-heme metabolism in erythroid cells. Although the only consistent feature of sideroblastic anemia is the bone marrow ring sideroblasts, evidence on the detailed molecular characteristics of ring sideroblasts is scarce owing to a lack of the biological models. We have recently established ring sideroblasts by inducing ALAS2 gene mutation based on human-induced pluripotent stem cell-derived erythroid progenitor (HiDEP) cells (ASH 2017) and have further extended the molecular characterization of human ring sideroblasts to gain new biological insights. (Method) We targeted the GATA-1-binding region of intron 1 of the human ALAS2 gene in HiDEP cells and established two independent clones [X-linked sideroblastic anemia (XLSA) clones]. A co-culture with OP9 stromal cells (ATCC) was conducted with IMDM medium supplemented with FBS, erythropoietin, dexamethasone, MTG, insulin-transferrin-selenium, and ascorbic acid. To obtain human primary erythroblasts, CD34-positive cells isolated from cord blood were induced in a liquid suspension culture (Fujiwara et al. JBC 2014). Bone marrow glycophorin A (GPA)-positive erythroblasts of patients with XLSA and normal individuals were separated using the MACS system (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) after obtaining written informed consent. For transcription profiling, Human Oligo chip 25K (Toray) was used. (Results) We previously demonstrated that co-culture with OP9 cells in the medium supplemented with 100 uM sodium ferrous citrate (SFC) promoted erythroid differentiation of XLSA clones, which enabled the establishment of ring sideroblasts (ASH 2017). To confirm the importance of SFC in terminal erythroid differentiation, we further demonstrated that the addition of SFC, and not transferrin-loaded iron, induced the frequency of GPA+ cells and TfR1-GPA+ mature erythroid population, based on primary erythroblasts derived from human CD34-positive cells. Subsequently, to reveal the molecular mechanism by which abnormal iron mitochondrial iron accumulation occurs by co-culture with SFC, we evaluated the expressions of various metal transporters, demonstrating that the addition of SFC significantly increased the expressions of mitoferrin 1 (MFRN1; a ferrous iron transporter in mitochondria), divalent metal transporter 1 (DMT1), and Zrt- and Irt-like protein 8 (ZIP8; a transmembrane zinc transporter, recently known as a ferrous iron transporter) in the XLSA clone than the wild-type cells, which would have contributed to the formation of ring sideroblasts. Moreover, we performed expression analyses to elucidate the biochemical characteristics of ring sideroblasts. After co-culture with OP9 in the presence of SFC, ring sideroblasts exhibited more than two-fold upregulation and downregulation of 287 and 143 genes, respectively, than the wild-type cells. Interestingly, when compared with the expression profiling results before co-culture (ASH 2017), we noticed prominent upregulation of gene involved in anti-apoptotic process (p = 0.000772), including HSPA1A, superoxide dismutase (SOD) 1, and SOD2. In addition, we conducted a microarray analysis based on GPA-positive erythroblasts from an XLSA patient and a normal individual. The analysis revealed significant upregulation of genes involved in the apoptosis process, as represented by apoptosis enhancing nuclease, DEAD-box helicase 47, and growth arrest and DNA-damage-inducible 45 alpha, and anti-apoptotic genes, such as HSPA1A and SOD2. Concomitantly, when the XLSA clone was co-cultured with OP9 in the presence of SFC, the apoptotic cell frequency as well as DNA fragmentation were significantly reduced compared with the XLSA clone co-cultured without SFC, indicating that ring sideroblasts avoid cell death by inducing anti-apoptotic properties. (Conclusion) Further characterization of the XLSA model would help clarify its molecular etiology as well as establish novel therapeutic strategies. Disclosures Fukuhara: Celgene: Research Funding; Chugai: Research Funding; Daiichi-Sankyo: Research Funding; Boehringer Ingelheim: Research Funding; Eisai: Honoraria, Research Funding; GlaxoSmithKline: Research Funding; Janssen: Honoraria, Research Funding; Japan Blood Products Organization: Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding; Mitsubishi Tanabe: Research Funding; Mundipharma: Honoraria, Research Funding; MSD: Research Funding; Nippon-shinyaku: Research Funding; Novartis pharma: Research Funding; Ono: Honoraria, Research Funding; Otsuka Pharmaceutical: Research Funding; Pfizer: Research Funding; Sanofi: Research Funding; Symbio: Research Funding; Solasia: Research Funding; Sumitomo Dainippon: Research Funding; Taiho: Research Funding; Teijin Pharma: Research Funding; Zenyaku Kogyo: Honoraria, Research Funding; Takeda: Honoraria; Baxalta: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Bayer Yakuhin: Research Funding; Alexionpharma: Research Funding; AbbVie: Research Funding; Astellas: Research Funding; Nihon Ultmarc: Research Funding.

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Expression and Characterization of the Peptidase Domain ofStreptococcus pneumoniaeComA, a Bifunctional ATP-binding Cassette Transporter Involved in Quorum Sensing Pathway

Journal of Biological Chemistry ◽

10.1074/jbc.m512516200 ◽

2005 ◽

Vol 281 (8) ◽

pp. 4726-4731 ◽

Cited By ~ 26

Author(s):

Seiji Ishii ◽

Takato Yano ◽

Hideyuki Hayashi

Keyword(s):

Quorum Sensing ◽

Atp Binding ◽

Atp Binding Cassette Transporter ◽

Atp Binding Cassette

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Expression of some ATP-binding cassette transporters in acute myeloid leukemia

Hematology Reports ◽

10.4081/hr.2017.7406 ◽

2017 ◽

Vol 9 (4) ◽

Cited By ~ 5

Author(s):

Antonella Maria Salvia ◽

Flavia Cuviello ◽

Sabrina Coluzzi ◽

Roberta Nuccorini ◽

Immacolata Attolico ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Abc Transporters ◽

Healthy Subjects ◽

Myeloid Leukemia ◽

Resistance Mechanisms ◽

Atp Binding ◽

Atp Binding Cassette Transporters ◽

Acute Myeloid ◽

Atp Binding Cassette

Hematopoietic cells express ATP binding cassette (ABC) transporters in relation to different degrees of differentiation. One of the known multidrug resistance mechanisms in acute myeloid leukemia (AML) is the overexpression of efflux pumps belonging to the superfamily of ABC transporters such as ABCB1, ABCG2 and ABCC1. Although several studies were carried out to correlate ABC transporters expression with drug resistance, little is known about their role as markers of diagnosis and progression of the disease. For this purpose we investigated the expression, by real-time PCR, of some ABC genes in bone marrow samples of AML patients at diagnosis and after induction therapy. At diagnosis, ABCG2 was always down-regulated, while an up regulated trend for ABCC1 was observed. After therapy the examined genes showed a different expression trend and approached the values of healthy subjects suggesting that this event could be considered as a marker of AML regression. The expression levels of some ABC transporters such as ABCC6, seems to be related to gender, age and to the presence of FLT3/ITD gene mutation.

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