ALBERT

Description: Key Points Conditional Gata2-deficient mice have profoundly reduced DC populations. Gata2 deficiency in DC progenitors reduced the expression of myeloid-related genes and increased that of T-lymphocyte–related genes.

Description: (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.

Description: Abstract 3443 Background: Developmental control mechanisms often utilize multimeric complexes containing transcription factors, coregulators, and additional non-DNA binding components. It is challenging to ascertain how such components contribute to complex function at endogenous loci. LMO2 (LIM-only protein 2) is a non-DNA binding transcriptional coregulator, and is an important regulator of hematopoietic stem cell development and erythropoiesis, as mice lacking this gene show defects in blood formation as well as fetal erythropoiesis (Warren et al. Cell. 1994). In the context of erythropoiesis, LMO2 has been demonstrated to be a part of multimetric complex, including master regulators of hematopoiesis (GATA-1 and SCL/TAL1), chromatin looping factor LDB1 and hematopoietic corepressor ETO2 (referred as GATA-SCL/TAL1 complex). As LMO2 controls hematopoiesis, its dysregulation is leukemogenic, and its influence on GATA factor function is still not evident, we investigated here the transcriptional regulatory mechanism via LMO2 in erythroid cells. Methods: For LMO2 knockdown, anti-LMO2 siRNA (Thermo Scientific Dharmacon) and pGIPZ lentiviral shRNAmir system (Open Biosystems) were used. Western blotting and Quantitative ChIP analysis were performed using antibodies for GATA-1, LMO2 (abcam), GATA-2, TAL1 and LDB1 (Santa Cruz). To obtain human primary erythroblasts, CD34-positive cells isolated from cord blood were induced in liquid suspension culture. For transcription profiling, human whole expression array was used (Agilent), and the data was analyzed with GeneSpring GX software. To induce erythroid differentiation of K562 cells, hemin was treated at a concentration of 30 uM for 24h. Results: siRNA-mediated LMO2 knockdown in hemin-treated K562 cells results in significantly decreased ratio of benzidine-staining positive cells, suggesting that LMO2 has an important role in the erythroid differentiation of K562 cells. Next, we conducted microarray analysis to characterize LMO2 target gene ensemble in K562 cells. In contrast to the predominantly repressive role of LMO2 in murine G1E-ER-GATA-1 cells (Fujiwara et al. PNAS. 2010), the analyses (n = 2) demonstrated that 177 and 78 genes were upregulated and downregulated (〉1.5-fold), respectively, in the LMO2-knockdowned K562 cells. Downregulated gene ensemble contained prototypical erythroid genes such as HBB and SLC4A1 (encodes erythrocyte membrane protein band 3). To test what percentages of LMO2-regulated genes could be direct target genes of GATA-1 in K562 cells, we merged the microarray results with ChIP-seq profile (n= 5,749, Fujiwara et al. Mol Cell. 2009), and demonstrated that 26.4% and 23.1% of upregulated and downregulated genes, respectively, contained significant GATA-1 peaks in their loci. Furthermore, whereas LMO2 knockdown in K562 cells did not affect the expression of GATA-1, GATA-2 and SCL/TAL1 based on quantitative RT-PCR as well as Western blotting, the knockdown resulted in the significantly decreased chromatin occupancy of GATA-1, GATA-2, SCL/TAL1 and LDB1 at beta-globin locus control region and SLC4A1 locus. We subsequently analyzed the consequences of LMO2 knockdown in primary erythroblasts. Endogeneous LMO2 expression was upregulated along with the differentiation of cord blood cell-derived primary erythroblasts. shRNA-mediated knockdown of LMO2 in primary erythroblasts resulted in significant downregulation of HBB, HBA and SLC4A1. Conclusion: Our results suggest that LMO2 contributes to the expression of GATA-1 target genes in a context-dependent manner, through modulating the assembly of the components of GATA-SCL/TAL1 complex at endogeneous loci. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction. Monocytes/macrophages play an essential role in systemic iron homeostasis by their recycling and storage capacity of iron. Under chronic inflammatory condition, pro-inflammatory cytokines such as TNF-α and IL-1 stimulate production of hepcidin by liver cells, which in turn down-regulates iron exporter ferroportin expression on monocytes/macrophages. Thus, hepcidin/ferroportin axis contributes to the pathogenesis of anemia in chronic inflammation by restricting cellular iron export into plasma. Pro-inflammatory cytokine IL-1β is mainly produced by monocytes/macrophages througha molecular platform, called inflammasome. One of the best characterized members is NLRP3 inflammasome, which plays a central role in sterile inflammation in response to wide range of danger stimuli, including uric crystal, cholesterol crystal, silica and asbestos. Given that excess cellular iron increases the level of redox-active labile iron pool which is responsible for iron-mediated cytotoxicity, cellular labile iron might be sensed as a danger signal in monocytes/macrophages. In this study we addressed whether cellular labile iron activates NLRP3 inflammasome. Methods. Peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation from healthy donors. After 4 h priming with 10 ng/ml LPS, PBMCs were stimulated with various dose of ferric ammonium citrate (FAC) for 4 h. IL-1β production in culture supernatants was determined by ELISA. Cleaved caspase-1 and mature IL-1β was analyzed by western blot. Calcein-AM assay was used to determine cellular labile iron pool.To investigate the mechanism of NLRP3 inflammasome activation, total reactive oxygen species (ROS) detection assay and JC-10 mitochondrial membrane potential assay were performed. To evaluate lysosomal membrane permeabilization, PMA-differentiated THP-1 cells stained with Lyso Tracker were analyzed by fluorescence microscope. Results. FAC induced the concentration-dependent increase of the labile iron pool and secretion of IL-1β in LPS-primed human monocytes. Ferrous iron chelator, bipyridine significantly inhibited the IL-1β production, highlighting importance of cellular chelatable iron pool for IL-1β production. IL-1β production induced by FAC was abrogated by pan-caspase inhibitor as well as by caspase-1 specific inhibitor, suggesting that inflammasome-mediated caspase-1 activation is required for this process. Consistently, cleaved caspase-1 and mature IL-1β was confirmed by western blot analysis. We next addressed whether NLRP3 inflammasome is activated in response to cellular labile iron. NLRP3 inhibitor, glyburide significantly inhibited IL-1β production. Furthermore, FAC treatment induced IL-1β production in THP-1 cells, but not in NLRP3-deficient THP-1 cells, indicating that cellular iron activates NLRP3 inflammasome. Next, we addressed how cellular iron activates NLRP3 inflammasome. Potassium efflux is thought to be a common trigger of NLRP3 inflammasome activation. Consistently, IL-1β production was completely abrogated in high-potassium media, indicating that potassium efflux is required for iron-mediated NLRP3 inflammasome activation. Since cellular labile iron is involved in ROS generation through Fenton reactions, we next investigated the role of ROS in iron-mediated NLRP3 inflammasome activation. FAC treatment increased ROS levels of monocytes in concentration-dependent manner. Accordingly, the percentage of monocytes with decreased mitochondorial membrane potential was increased. N-acetyl cysteine inhibited FAC-induced IL-1β production, suggesting that ROS-mediated mitochondorial damage is involved in iron-mediated NLRP3 inflammasome activation. In addition, ROS-induced lysosomal membrane permeabilization was observed in FAC-treated THP-1 cells. A cathepsin B inhibitor, CA-074 methyl ester, inhibited IL-1β production, suggesting that ROS-mediated lysosomal damage is also involved in iron-mediated NLRP3 inflammasome activation. Conclusion. In this study, we found that cellular labile iron activates NLRP3 inflammasome through its redox activity. Because IL-1β has been reported to induce hepcidin synthesis, excess cellular iron in monocytes/macrophages might be implicated in a positive feedback loop for inflammation through NLRP3 inflammasome and hepcidin/ferroportin axis. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1360 Introduction: The tyrosine kinase inhibitor (TKI) imatinib is used as the first-line therapy for newly diagnosed chronic myeloid leukemia (CML). However, some patients fail to respond or become intolerant to imatinib. Nilotinib is a second-generation TKI with higher selectivity and more potent inhibitory effects on BCR-ABL than imatinib. Several studies have shown hematologic and cytogenetic responses to nilotinib in patients with imatinib-resistant or intolerant CML. Purpose: To investigate the safety and efficacy of nilotinib for patients with imatinib-resistant or intolerant, chronic (CP)- or accelerated (AP)-phase CML from the East Japan CML Study Group (EJCML) trial by evaluating molecular responses in terms of the BCR-ABL1 mutational status and plasma trough concentration of nilotinib. Methods: In this multicenter phase II clinical trial, nilotinib (400 mg bid) was administered orally for one year and the molecular responses were monitored by means of the international scale of quantitative PCR (IS-PCR). BCR-ABL1 mutations were analyzed by direct sequencing at the baseline and 12 months or at the time of the event for discontinuation of the treatment (i.e., progressive disease, insufficient effects, or severe adverse events). The plasma trough concentration of nilotinib was measured by high-performance liquid chromatography 3 months after nilotinib administration. Results: From March 2009 through February 2011, 51 patients were registered in this study, and data of 49 patients whose molecular responses were evaluated by the IS-PCR were analyzed (imatinib-resistant CML = 33, imatinib-intolerant CML = 16; CP CML = 46, AP CML = 3). The median follow-up period was 12.0 months (range = 0.1–13.3 months). At 6 and 12 months, the major molecular response (MMR; ≤0.1% IS) rates were 52.5% and 67.6%, respectively, and the complete cytogenetic response (CCyR)-equivalent (≤1.0% IS) rates were 75.0% and 85.3%, respectively. Five types of BCR-ABL1 mutations (M244V, F317L, N358D, F359V, and E459K) were detected in 6 patients (12.2%) at the baseline, but the M244V, N358D, and E459K mutations disappeared after the nilotinib treatment. Acquired BCR-ABL1 mutations (Y253H, I418V, and exon 8/9 35bp insertion) were detected in 3 patients (8.6%) at 12 months or at the time of the event; these patients did not achieve a CCyR or an MMR. No patients showed an acquired mutation of T315I. Most patients except 11 subjects (22.4%) still received the treatment. The reasons for discontinuation were progressive disease in one patient with an F317L mutation, insufficient effects in one patient without any mutation, and adverse events in 9 patients (thrombocytopenia in 5 patients, hyperbilirubinemia in 2 patients, headache in one patient, and heart disease in one patient). Among 30 patients without BCR-ABL1 mutations, the plasma trough concentration of nilotinib was significantly higher in 21 patients with an MMR than in those without an MMR by 12 months (median = 1255.1 ng/mL vs. 372.8 ng/mL, P = 0.0012 by Mann–Whitney U-test; see the figure). The concentration of 761 ng/mL was significantly associated with an MMR by 12 months in a receiver-operating characteristic (ROC) curve analysis of the best sensitivity (76.2%) and specificity (77.8%). Conclusion: The patients with imatinib-resistant or intolerant, CP or AP CML, even those having BCR-ABL1 mutations M244V, N358D, and E459K, achieved an MMR by 12 months of nilotinib treatment. The plasma trough concentration of the drug was related to the MMR by 12 months, and the plasma threshold of nilotinib should be set above 761 ng/mL. These findings suggest that nilotinib shows good efficacy and tolerability in Japanese patients with imatinib-resistant or intolerant, CP or AP CML. (ClicalTrials.gov, UMIN ID 000002201) Disclosures: No relevant conflicts of interest to declare.

Description: Stromal cells in the bone marrow provide an optimal microenvironment for hematopoiesis through cell-to-cell interaction as well as cytokine production. We have previously established 33 bone marrow stromal cell lines from SV40 T-antigen transgenic mice, and demonstrated the lineage-specific activity of supporting hematopoiesis (J Cell Physiol1995: 164; 55). Of the 33 cell lines, 27 clones showed stimulatory abilities to support large erythroid colony formation in the presence of erythropoietin, while 6 cell lines did not. Since these activities were not dependent on the amount of produced cytokines, the existence of unknown molecules responsible for the selective activity of erythropoiesis has been implicated. To identify molecules expressed on bone marrow stromal cells which are responsible for determining the stromal cell dependent erythropoiesis, we compared the gene expression profiling by cDNA microarray between cell lines which support erythropoiesis (E+; TBR 9, 184, 31-2) and cell lines which do not (E-; TBR 17, 33, 511). Out of 7226 genes examined, 138 genes were up-regulated 3-fold or more, and 282 genes were down-regulated 3-fold or more, in E+ cell lines. Among these genes, we have selected one of the up-regulated genes, tenascin-C, as a candidate for erythroid progenitor supporting genes, and examined its expression and function in detail. First, we confirmed the expression of tenascin-C by real time quantitative RT-PCR. The expression of tenascin-C adjusted by the expression of GAPDH in the three E+ cell lines were all higher than the three E- cell lines with a mean of 3.6 times. Next, we examined the effect of suppressing tenascin-C expression by small interfering RNA (siRNA) on the erythroid colony formation. siRNAs were synthesized by in vitro transcription method, and were transfected into TBR cell lines by lipofection. At 24 hours after the transfection of tenascin-C siRNA, the expression of tenascin-C was reduced to 10 to 40% of the control siRNA as determined by real time quantitative RT-PCR. The number of erythroid colony dependent on TBR 9 and TBR 184 in the presence of tenascin-C siRNA was significantly lower than that in the presence of control siRNA (13.0±6.5 versus 0.75±0.95 colonies/104 fetal liver cells; p

Description: (Introduction) During erythroid differentiation, the level of erythroid-specific genes increases synchronizing with the intracellular heme content. In addition, heme has been shown to play a role in transcription and protein synthesis. Based on these evidences, it is possible that heme widely regulates the expression of erythroid specific genes. With this hypothesis, we compared the gene expression profile between wild-type and heme-deficient erythroblasts generated from wild-type and ALAS2 (−) ES cells in in vitro, and identified 4 heme-regulated erythroid-specific genes (UCP2, CNBP, NuSAP and unknown EST1), (BBRC2006;340:105–110). Among them, unknown EST1 is consisted of 110 a.a. with a conserved acetyl-CoA binding domain, which was characteristic of GNAT (GCN5-related N-acetyltransferase) superfamily. Thus, it is likely that EST1 is a novel acetyltransferase. In the present study, we focused two genes, EST1 and NuSAP, and investigated their function during erythoid differentiation. (Methods) First, the expression and regulation of NuSAP gene during erythroid differentiation was examined. For the expression analysis, in vivo erythroblasts were fractionated according to the surface expression of TER119/CD71, and the level of expression of NuSAP mRNA was examined by quantitative RT-PCR. For the promoter analysis, the promoter region of mouse NuSAP gene was cloned, and the regulatory cis-element was determined by luciferase assay and EMSA. Next, for defining the properties of EST1, EST1 was constitutively expressed using Flag/HA tagged retroviral vector into mouse erythroleukemia (MEL) cell line, and nuclear extract of EST1-expessed MEL cells was purified by affinity chromatography, which was loaded on an SDS/PAGE gel and subjected to electrophoresis. In addition, for in vitro histone acetyltransferase (HAT) assay, free histone and purified EST1 protein were incubated with [3H]acetyl-CoA, and acetyltransferase activity was measured by scintigraphy. (Results) (1) NuSAP mRNA was more significantly abundant in the subset corresponding to immature erythroblasts (TER119+CD71high) than mature erythroblasts (TER119+CD71low), and it was significantly increased in TER119+ cells from in vivo phlebotomized mice compared with control mice. Furthermore, during erythroid maturation of MEL cells by dimethylsulfoxide, NuSAP mRNA was increased at 24–72 hrs. Promoter analyses of NuSAP gene demonstrated that duplicated CCAAT boxes located at −81/−85 and −30/−34 were essential for promoter activity, which was trans-activated by NF-YA. These results suggested that NuSAP might contribute to the expansion of immature erythroblast pool under the control of NF-Y. (2) By the gel electrophoresis, it was revealed that EST1 protein forms a multimeric protein complex. Furthermore, whereas recombinant EST1 protein did not show HAT activity, EST1 complex could acetylate free histones in vitro, suggesting that EST1 might be a component of HAT complex. (Conclusion) The novel functions of EST1 and NuSAP suggest that heme regulates erythroid differentiation by controlling the expression of variety of genes.

Description: Aplastic anemia (AA) is characterized by reduced hematopoiesis resulting in pancytopenia. It is suggested that a certain immunological attack to hematopoietic stem cells play an important role in developing AA. However, limited information is available for the intrinsic characteristics of stem cells in AA. Previous work in our laboratory showed decreased expression of GATA-2 gene in CD34 positive cells in AA, suggesting that there is an aberrant expression of stem cell-specific genes in stem cells in AA. Recently it is emerged that some genes such as HOXB4 and BMI-1, function for the proliferation and maintenance of stem cells. In this study, we examined expression levels of HOXB4 and BMI-1 in CD34 positive cells by quantitative PCR in 10 patients with AA and 13 with idiopathic thrombocytopenic purpura (ITP). Between these two factors, the expression level of HOXB4 was markedly decreased in AA compared with in ITP, whereas that of BMI-1 was not. Moreover, the expression level of GATA-2 in these populations was significantly correlated to HOXB4 gene expression (Spearman’s rank correlation, r=0.6573 p0.05). As they functions as a transcription factor, these results raise the possibility that GATA-2 and HOXB4 regulate each other. To explore this possibility, first, we cloned HOXB4 5′flanking region by PCR and performed promoter analysis. Since the previous report showed that the region from −164 to −116 was important for promoter activity of HOXB4 gene, we focused on a GATA element located at −160. When this element was deleted, the reporter activity was decreased to 60% of wild-type in K562 cells. Furthermore, co-transfection of GATA-2 expression vector significantly activates the reporter gene in a dose dependent manner. EMSA revealed that GATA-2 binds specifically to this element. On the other hand, the active region both of exon 1S and 1G promoter of GATA-2 gene, which was identified by the promoter analysis, did not contain the consensus sequence recognized by HOXB4. These findings suggest that in stem cells in AA, the decreased expression of GATA-2 gene lead to the reduced HOXB4 gene expression, which may responsible for the development of the disease.