ALBERT

Description: Here we investigated in primary human erythroid tissues a downstream element of the heterochroniclet-7miRNA pathway, the insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), for its potential to affect the hemoglobin profiles in human erythroblasts. Comparison of adult bone marrow to fetal liver lysates demonstrated developmental silencing in IGF2BP1. Erythroid-specific overexpression of IGF2BP1 caused a nearly complete and pancellular reversal of the adult pattern of hemoglobin expression toward a more fetal-like phenotype. The reprogramming of hemoglobin expression was achieved at the transcriptional level by increasedgamma-globincombined with decreasedbeta-globintranscripts resulting ingamma-globinrising to 90% of total beta-like mRNA.Delta-globinmRNA was reduced to barely detectable levels.Alpha-globinlevels were not significantly changed. Fetal hemoglobin achieved levels of 68.6 ± 3.9% in the IGF2BP1 overexpression samples compared with 5.0 ± 1.8% in donor matched transduction controls. In part, these changes were mediated by reduced protein expression of the transcription factor BCL11A. mRNA stability and polysome studies suggest IGF2BP1 mediates posttranscriptional loss of BCL11A. These results suggest a mechanism for chronoregulation of fetal and adult hemoglobin expression in humans.

Description: Highly-conserved LIN28 proteins regulate certain developmentally-timed events in multicellular organisms by decreasing the levels of let-7 miRNAs. It was recently reported that increased expression of LIN28 proteins or decreased expression of the target let-7 miRNAs in human erythroblasts cultured from healthy adult humans causes increased fetal hemoglobin expression. Here LIN28A expression in sickle cell donors’ cells was explored for its potential to regulate fetal and sickle hemoglobin, and to affect the morphological sickling of the mature erythrocytes. After obtaining consent and assent, CD34(+) cells from five pediatric research subjects with HbSS genotype (ages 9-16 years old) were harvested from discarded whole blood following partial manual exchange transfusions. Transgenic expression of LIN28A was accomplished using lentiviral transduction of human CD34(+) sickle cells cultivated ex vivo in serum-free medium for a total of 21 days. On culture day 14, LIN28A over-expression (LIN28A-OE) was confirmed by Q-RT-PCR (control: 8.6E+00 ± 8.1E+00 copies/ng, LIN28A-OE: 2.3E+05 ± 2.1E+05 copies/ng) and Western blot analyses. Erythroblast differentiation and terminal maturation were not affected by LIN28A-OE. Enucleation, as assessed by thiazole orange (TO) staining, was equivalent between the LIN28A-OE cells and control transductions (LIN28A-OE enucleation 40.8 ± 17.0% compared to control 49.9 ± 23.4%, p=0.19). LIN28A-OE strongly suppressed all members of the let-7 family of miRNAs, with average reductions from 66% to 96% for let-7a, let-7b, let-7c, let-7d, let-7e, let-7f-2, let-7g and let-7i. LIN28A-OE caused reduced expression of BCL11A, a known repressor of gamma-globin gene expression. Gamma-, beta (sickle)- and alpha-globin mRNA levels were also investigated by Q-RT-PCR. Gamma-globin mRNA expression levels were significantly increased in LIN28A-OE samples (control: 2.0E+06 ± 7.0E+05 copies/ng, LIN28A-OE: 2.0E+07 ± 6.0E+06 copies/ng, p=0.006), and beta (sickle)-globin mRNA significantly decreased in LIN28A-OE samples (control: 2.0E+07 ± 5.2E+06 copies/ng, LIN28A-OE: 1.6E+07 ± 6.3E+06 copies/ng, p=0.024). Differences in alpha-globin mRNA expression were not statistically significant. Hemoglobin chromatography (HPLC) demonstrated that LIN28A-OE significantly increased the proportion of fetal hemoglobin (HbF control: 10.8 ± 7.1%, LIN28A-OE: 40.1 ± 14.0%; p=0.003) that was balanced by a significant decrease in the proportion of sickle hemoglobin. HbA was not detected. For investigation of the sickling phenotype, enucleated [TO(-)] sickle erythrocytes from LIN28A-OE and control transductions of two subjects’ cells were sorted at the end of the culture period into duplicate tissue culture wells. The sorted erythrocytes were incubated in hypoxia (2% oxygen) for 16 hours, and imaged using inverted microscopy within three minutes after removal from the hypoxia incubator. Four random microscopic field images from each well were acquired. Blinded observers then scored the images from the control and LIN28A-OE transductions according to non-sickled versus sickled morphologies. Cultured erythrocytes from the control transductions demonstrated 86.3 ± 9.5% with sickled morphologies. By comparison, a significant reduction in sickling morphology was observed in the LIN28A-OE cells (56.2 ± 23.1% sickled morphologies; p=0.000009). These results demonstrate that transgenic expression of LIN28A during ex vivo erythropoiesis causes increased gamma-globin gene and protein expression balanced with decreased beta (sickle)-globin at levels that are sufficient to ameliorate hypoxia-related sickling of mature erythrocytes. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 683 Mitochondrial specialization in erythroblasts is important for efficient heme synthesis, with defects or reduced expression of several mitochondrial proteins causing anemia. Trafficking kinesin binding 2 (TRAK2) is known to participate in mitochondrial movement along microtubule by interacting with kinesin motor protein and making a complex with Miro that is localized on the mitochondrial outer membrane. Transcriptome data suggest that TRAK2 is highly and specifically expressed in early erythroid cells. Here the role of TRAK2 was studied among human CD34+ cells that were grown in ex vivo serum-free cultures supplemented with erythropoietin (EPO, total culture period 21 days). Quantitative PCR studies indicated that TRAK2 expression is highly regulated during erythropoiesis. Its expression pattern was nearly identical to aminolevulinate synthase 2, the erythroid specific enzyme for the committed step of the heme biosynthetic pathway, and mitoferrin 1, the erythroid specific mitochondrial iron transporter. Western analyses revealed that TRAK2 protein is detected as a doublet band with molecular weights of 130kD and 105kD. Mitochondrial co-localization of TRAK2 was verified by confocal microscopy in TRAK2-overexpressing K562 cells. To study a potential role of TRAK2 in erythropoiesis, TRAK2 expression was reduced in cultured human erythroid cells using lentiviral shRNA transduction. TRAK2 knockdown (TRAK2-KD) was confirmed by Western analysis in K562 cells. In primary erythroblasts, TRAK2-KD caused slight reduction of CD36+ immature erythroblasts at culture day 7 prior to the addition of EPO (CD36+ population 58% in control vs 40% in TRAK2-KD). After the addition of erythropoietin to the culture medium, TRAK2-KD severely restricted erythroblast proliferation (5.0 million cells/ml in control vs 0.25 million cells/ml in TRAK2-KD on culture day 18). Flow cytometric analyses showed that 90% in control cultures. Annexin-V staining indicated that more than 90% of cells had undergone apoptosis by day 14. These data suggest that TRAK2 expression is required for erythroid differentiation. As such, defects in TRAK2 expression should be considered in cases of unexplained anemia. The data also support the notion that mitochondrial location or mobility within erythroblasts may be important for iron trafficking or heme synthesis. Disclosures: No relevant conflicts of interest to declare.

Description: HMGA2 is a member of the high-mobility group A family and plays a role in the regulation of gene transcription and chromatin structure. HMGA2 is a validated target of the let-7 family of miRNAs. Let-7 miRNAs are highly regulated in erythroid cells during the fetal-to-adult developmental transition (1). Recent studies demonstrated that the LIN28 -let-7 axis mediated up-regulation of fetal hemoglobin (HbF) expression to 〉30% of the total globin levels in cultured erythroblasts from adult humans (2) and the amelioration of hypoxia-related sickling of cultured mature erythrocytes from pediatric patients with sickle cell disease (3). Interestingly, increased expression of endogenous HbF in a patient receiving gene therapy was also associated with truncated HMGA2 protein expression after lentiviral integration and disruption of let-7 targeting at the HMGA2 gene locus (4). Therefore, we hypothesized that HMGA2 may be involved in fetal hemoglobin regulation as a downstream target of the let-7 miRNAs. To study the effects of HMGA2 upon erythropoiesis and globin expression, lentiviral constructs were designed for let-7 resistant expression of HMGA2 driven by the erythroid-specific gene promoter region of the human SPTA1 gene (HMGA2 -SPTA1-OE), with a matched empty vector control. Transductions were performed in CD34+ cells from four adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Overexpression of HMGA2 was confirmedby Q-RT-PCR (control: below detection limits; HMGA2 -SPTA1-OE: 2.51E+04 ± 3.44E+04 copies/ng) and Western blot analyses at culture day 14. Cell counting revealed no significant changes between HMGA2 -SPTA1-OE and control (empty vector) transductions at culture day 14. Terminal maturation with loss of CD71 from the erythroblast cell surface and enucleation assessed by thiazole orange staining were analyzed in the control and HMGA2 -SPTA1 -OE samples at the end of the culture period. Globin genes expression levels were evaluated for HMGA2 -SPTA1-OE by Q-RT-PCR. HMGA2 -SPTA1-OE caused a significant increase in gamma-globin mRNA expression levels compared to controls (control: 5.02E+05 ± 8.62E+04 copies/ng; HMGA2 -SPTA1-OE: 1.45E+06 ± 7.31E+05 copies/ng; p=0.037). Consistent with the increase in gamma-globin mRNA levels, HPLC analyses at culture day 21 demonstrated modest but significant increases in HbF levels in HMGA2 -SPTA1-OE compared to controls (HbF control: 5.41 ± 2.15%; HMGA2 -SPTA1-OE: 16.53 ± 4.43%; p=0.006). Possible effect(s) and downstream mechanism(s) triggered by HMGA2 -SPTA1-OE were investigated. Q-RT-PCR analyses demonstrated no significant changes in the let-7 family of miRNAs in HMGA2 -SPTA1-OE compared to controls. Expression patterns of several transcription factors such as BCL11A, KLF1, SOX6 and GATA1 were investigated by Q-RT-PCR and no significant changes were detected in HMGA2 -SPTA1-OE compared to controls. While BCL11A mRNA levels were decreased by HMGA2 -SPTA1 -OE, the differences did not reach statistical significance (control: 4.26E+02 ± 8.18E+01 copies/ng; HMGA2 -SPTA1 -OE: 2.84E+02 ± 1.48E+02 copies/ng; p=0.104). However, nuclear BCL11A protein levels assessed by Western analysis were suppressed in HMGA2 -SPTA1 -OE. In summary, these results demonstrate that HMGA2, a validated target of let-7 miRNAs, causes moderately increased gamma-globin gene and protein expression in human erythroblasts, and reduces levels of BCL11A protein. These data thus support the notion that suppression of let-7 miRNAs increases fetal hemoglobin, in part, by the targeting of erythroblast HMGA2 mRNA. (1) Noh SJ et al. J Transl Med. 7:98 (2009). (2) Lee YT et al. Blood. 122:1034-41 (2013). (3) Vasconcellos JF et al. PLoS One. 9:e106924 (2014). (4) Cavazzana-Calvo M et al. Nature. 467:318-22 (2010). Disclosures No relevant conflicts of interest to declare.

Description: MicroRNAs (miRNAs) are a class of small, noncoding RNAs that bind and regulate target messenger RNAs (mRNAs). The let-7 family consists of twelve genes encoding nine highly conserved miRNAs that are involved in developmental timing events in multicellular organisms. Previous studies showed regulation during the fetal-to-adult transition in the erythroid lineage with significant increases in let-7 miRNAs from adult compared to umbilical cord blood reticulocytes (1). Further studies indicated that reduced expression of let-7 in adult CD34+ cells by “sponge” targeting the miRNA family seed region caused increased fetal hemoglobin (HbF), but the mean level of HbF remained less than 20% of the total hemoglobin (2). Increased expression of LIN28A (a major regulator of all let-7 miRNAs) caused greater increases in HbF (greater than 30% of the total) in cultured erythrocytes from pediatric patients with HbSS genotype (3). However, these studies did not address the potential for targeting an individual let-7 miRNA family member to regulate HbF expression. For this purpose, we initially determined the expression levels of mature let-7 family members in purified cell populations sorted from peripheral blood. The total levels of let-7 miRNAs in peripheral blood cells were as follows: reticulocytes: 1.7E+08 ± 1.0E+08 copies/ng; neutrophils: 2.0E+07 ± 1.1E+07 copies/ng; lymphocytes: 1.1E+07 ± 6.2E+06 copies/ng and monocytes: 3.5E+06 ± 2.7E+06 copies/ng. Among the individual species, let-7a was identified as a predominantly expressed let-7 family member in reticulocytes. As such, we hypothesized that specifically targeting let-7a may be sufficient to regulate HbF levels. To study the effects of let-7a miRNAs upon erythropoiesis and globin expression, a lentiviral construct that incorporated the tough decoy (TuD) design to target let-7a was compared with empty vector controls. Transductions were performed in CD34+ cells from five adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Down-regulation of let-7a was confirmed by Q-RT-PCR at day 14 (control: 1.4E+07 ± 2.4E+06 copies/ng; let-7a-TuD: 1.6E+06 ± 4.6E+05 copies/ng; p=0.0003). Cell proliferation and differentiation were comparable in let-7a-TuD versus control transductions. Expression levels of globin genes were evaluated upon let-7a-TuD by Q-RT-PCR. Let-7a-TuD transductions caused significantly increased gamma-globin mRNA expression levels compared to control transductions (control: 1.2E+06 ± 6.8E+05 copies/ng; let-7a-TuD: 1.1E+07 ± 4.5E+06 copies/ng; p=0.004). HPLC analyses at the end of the culture period demonstrated robust increases in HbF levels after let-7a-TuD transduction (HbF control: 4.7 ± 0.6%; let-7a-TuD: 38.2 ± 3.8%; p=0.00003). In addition, the expression patterns of the erythroid transcription factors BCL11A, KLF1 and SOX6 were investigated. Let-7a-TuD decreased BCL11A mRNA expression levels (control: 1.7E+03 ± 4.5E+02 copies/ng; let-7a-TuD: 4.3E+02 ± 1.8E+02 copies/ng; p=0.003), but major changes in KLF1 or SOX6 were not detected. In summary, we report here that the let-7 miRNA family is differentially expressed in purified cell populations from adult human blood, and that let-7a is a predominantly expressed species in reticulocytes. Further, targeted reduction of let-7a in erythroblasts is sufficient to cause robust increases in gamma-globin mRNA expression and HbF to mean levels around 35-40% of the total hemoglobin produced. Targeting of individual let-7 genes or RNA transcripts may be useful for therapeutic induction of HbF expression in patients with sickle cell disease or other beta-hemoglobinopathies. 1) Noh SJ et al. J Transl Med. 7:98 (2009). 2) Lee YT et al. Blood. 122:1034-41 (2013). 3) Vasconcellos JF et al. Blood. 122: Abstract 313 (2013). Disclosures No relevant conflicts of interest to declare.

Description: Iron deficiency affects billions worldwide, and anemia develops when iron stores become insufficient to maintain normal erythropoiesis. However, iron depleted erythropoiesis is incompletely understood. In this study, an ex vivo model of iron depleted erythropoiesis was developed using dosed titrations of the iron chelator deferoxamine (DFO) in CD34+ cell cultures. All experiments were performed in triplicate with cells from three separate donors. Hemoglobinization and expression patterns of erythroid markers (GPA/CD71) demonstrated minimal changes in DFO supplemented medium. DFO caused dose-related suppression of cell counts after 14 days (20uM DFO: 42% reduction, 30uM DFO: 86% reduction, 40uM DFO: 96% reduction of cell counts compared to 0uM DFO controls). Cultures supplemented with 30uM DFO were used for subsequent studies, and the DFO-mediated effects were completely reversed by addition of 30uM ferric chloride. To investigate whether apoptosis caused the reduction in cell counts, surface Annexin V was measured by flow cytometry. No significant increases in levels of apoptosis among the proliferating erythroblasts were detected (Annexin V positive cells; 0uM DFO 6.2 ± 0.95%, 30uM DFO 9.0 ± 1.55%, p = 0.058). Analyses of p53 protein quantitation and p53 DNA-binding activity further suggested that the suppression of cell counts by iron chelation was not due to p53 related apoptosis. In the absence of apoptosis or maturation arrest, we hypothesized that the growth suppressing effects of DFO were not due to ineffective erythropoiesis. To test this hypothesis, a recently-discovered marker of ineffective erythropoiesis named GDF15 (Tanno et al. Nat. Med. 2007) was measured in the culture supernatants, and no increase was detected (GDF15 concentration per 1×104 cells: 0uM DFO; 79.7 ± 4.4 pg, 30uM; 43.9 ± 4.3 pg). In the clinic, serum GDF15 levels from 17 blood donors with significantly reduced ferritin and iron saturation (279 ± 98 pg/ml) were not increased when compared to serum GDF15 levels in 17 healthy volunteers with normal iron parameters (410 ± 119 pg/ml). To determine if cell cycling, instead of apoptosis, caused the reduction in cell counts, carboxyfluorescein diacetate succinimidyl diester (CFSE) staining studies were performed during the first and second weeks of culture. CFSE is an intracellular fluorescent stain, the intensity of which halves with each cell division. Three or four fewer erythroblast cell divisions were detected in 30uM DFO during the 14-day culture period when compared to controls. Based upon the reduction in cell divisions, it was estimated that the average cell cycle time was prolonged by 40–70% in DFO. Propidium iodide analyses additionally demonstrated changes in the cell cycle kinetics including a significant reduction in the percentage of S-phase erythroblasts at the peak phase of proliferation (30uM DFO: 37.0 ± 1.8% vs. control: 48.7 ± 3.4%, p = 0.0063). These results suggest that iron depletion inhibits the overall growth of erythroblasts by prolonging the cell cycle rather than causing apoptosis or maturation arrest. Due to the large amount of iron required for hemoglobin production, slower cell cycles may facilitate effective erythropoiesis by allowing extra time for uptake when extracellular iron is scarce.

Description: Key Points LIN28B regulates HbF expression in erythroblasts that are cultured from umbilical cord and adult human blood. LIN28B expression manifested a more fetal-like phenotype among adult human erythroblasts.

Description: Abstract 827 The highly-conserved Lin28 genes regulate cellular metabolism as well as the timing of developmental events and cell fates in multicellular organisms. Lin28 protein acts primarily by negatively regulating biogenesis of let-7 RNA, a microRNA family whose targets include growth-related signaling and transcription factor proteins. Published studies showed significantly increased expression of let-7 in purified adult blood reticulocytes compared to umbilical cord blood reticulocytes (1). This pattern correlates inversely with Lin28B expression. While present in the fetal liver and umbilical cord blood, Lin28B decreased to undetectable levels in adult bone marrow (2). Based upon the association of human ontogeny with hemoglobin switching, Lin28 was explored to identify novel mechanisms for hemoglobin regulation that may be useful for therapeutic application among patients with thalassemia or other hemoglobinopathies. To study the effects of Lin28B upon erythropoiesis and hemoglobin, ectopic expression of Lin28B was accomplished using retroviral transduction of human CD34+ cells cultivated ex vivo in erythropoietin-supplemented, serum-free cultures for 21 days. All experiments were performed in triplicate using cells from three separate adult volunteers. Lin28B over-expression (Lin28B-OE) was confirmed by Q-RT-PCR (control: 0.14 ± 0.37 copies/ng, Lin28B-OE: 1.8E+04 ± 353.8 copies/ng, p=0.01). Western analyses confirmed protein expression, and confocal microscopy revealed Lin28B predominantly in the cytoplasm of the transduced cells. Proliferation, maturation and morphology assays revealed that Lin28B-OE did not inhibit erythropoiesis when compared to control (empty vector) transductions. Terminal maturation with loss of CD71 from the erythroblast surface and enucleation by culture day 21 was detected in the control and Lin28B-OE samples. Expression levels of globin genes were evaluated upon Lin28B-OE by Q-RT-PCR. Lin28B-OE enhances gamma-globin mRNA expression (control: 5.14E+06 ± 2.6E+06 copies/ng, Lin28B-OE: 1.81E+07 ± 5.82E+06 copies/ng, p=0.038). Protein analysis confirmed the increased expression of gamma-globin. Fetal hemoglobin (HbF) levels were also increased in the Lin28B-OE cultures (control: 5.82 ± 4.54%, Lin28B-OE: 33.63 ± 9.38%; p=0.011). The increased HbF expression was maintained throughout differentiation including enucleated populations of culture-generated erythrocytes. Possible mechanism(s) for the increased expression of HbF caused by Lin28B-OE were investigated. Q-RT-PCR analyses demonstrated suppression of the let-7 microRNA family with greater-than 70% reductions of let-7a, let-7b, let-7c, let-7d, let-7e, let-7f-2, let-7g and let-7i. Expression patterns of several transcription factors including BCL11A, KLF1, SOX6 and GATA1 were explored. No major changes were detected with the exception of BCL11A. Lin28B-OE caused a 65% reduction in BCL11A expression (control: 3.07E+03 ± 1.5E+02 copies/ng, Lin28B-OE: 1.07E+03 ± 18 copies/ng; p=0.02). Western blot analyses of Lin28B-OE showed a consistent reduction of BCL11A protein. By comparison with Lin28B-OE, separately performed studies of BCL11A knockdown in adult CD34+ cells produced comparable increases in gamma-globin expression, but Lin28B expression in those cells was not affected. In addition to a more general role in development and metabolism, these experimental results suggest that Lin28B increases fetal hemoglobin and regulates BCL11A in human erythroblasts. Lin28B is thus identified as the first defined link between the regulation of a developmental clock and hemoglobin switching in humans. Disclosures: No relevant conflicts of interest to declare.

Description: Recent studies demonstrated that IGF2BP1 over-expression (IGF2BP1-OE) in adult erythroblasts has robust effects on fetal hemoglobin (HbF; 〉65% of the total globin levels), accompanied by reversal of the beta-like globin expression patterns to a fetal-like phenotype. Here we investigated if another member of the insulin-like growth factor 2 mRNA-binding protein family, IGF2BP3, also has potential for HbF regulation that may be useful for therapeutic application among patients with beta-hemoglobin disorders. The developmental pattern and expression levels for IGF2BP3 were initially determined in cord blood versus adult blood CD34(+) samples cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. RNA samples were collected at culture day 14 and expression levels were measured by qRT-PCR. IGF2BP3 showed a developmentally regulated expression pattern similar to IGF2BP1 (IGF2BP1: cord blood: 1.3.E+03 ± 4.3.E+02 and adult blood: below detection limits; IGF2BP3: cord blood: 5.8.E+02 ± 2.4.E+02 and adult blood: below detection limits). These results were confirmed in vivo by comparing human fetal liver to adult bone marrow samples (IGF2BP1: fetal liver: 3.5.E+02 ± 5.7.E+01, adult bone marrow: below detection limits and IGF2BP3: fetal liver: 2.0.E+01 ± 2.7.E+00, adult bone marrow: below detection limits). To investigate the effects of IGF2BP3 upon erythropoiesis and globin expression, a lentiviral construct was designed for expression of IGF2BP3 driven by the erythroid-specific gene promoter region of the human SPTA1 gene (IGF2BP3-OE), with a matched empty vector control. Transductions were performed in CD34(+) cells from four adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Over-expression of IGF2BP3 was confirmedby qRT-PCR and Western blot analyses at culture day 14. IGF2BP3-OE cells maintained their ability to differentiate and enucleate ex vivo compared to donor-matched controls. The expression levels of globin genes were evaluated at culture day 14 by qRT-PCR and showed that IGF2BP3-OE caused significantly increased gamma-globin expression levels compared to control transductions (control: 7.7.E+05 ± 1.7.E+05; IGF2BP3-OE: 8.4.E+06 ± 3.2.E+06; p=0.018). Consistent with increased gamma-globin, HbF rose to moderately high levels upon IGF2BP3-OE (control: 4.0 ± 2.1%; IGF2BP3-OE: 18.6 ± 1.0%; p=0.0021). In addition, the expression pattern of the erythroid transcription factor BCL11A was investigated by qRT-PCR at culture day 14 and no significant changes were observed (control: 5.6.E+02 ± 2.7.E+02; IGF2BP3-OE: 6.7.E+02 ± 3.5.E+02; p=0.694). However, minor decreases in BCL11A protein levels were detected by Western analysis. These results demonstrate that IGF2BP3 is developmentally regulated in human erythroid tissues with silencing during the fetal-to-adult transition. However, the effects of IGF2BP3-OE on HbF levels were less robust when compared to IGF2BP1-OE in cultured adult erythroblasts. Disclosures No relevant conflicts of interest to declare.

Description: Epigenetic modification of chromatin in erythroid cells represents an active field of study aimed, in part, toward increased expression of fetal hemoglobin in patients with beta-thalassemia. The homologous methyltransferases G9a and GLP regulate globin gene transcription by catalyzing mono- and dimethylation at Lys 9 and dimethylation at Lys 27 of histone H3. Inhibition of these methyltransferases by the small molecule named UNC0638 was recently shown to increase gamma-globin gene expression in adult human hematopoietic precursor and stem cells. Here UNC0638 was explored further to include fetal hemoglobin expression among more mature erythroid cells cultured from CD34(+) cells of three healthy adult human donors in a serum-free culture medium. According to this culture model, the main erythroblast population on culture days 0-7 consists of CD36(+), CD45(+), CD71(moderate), CD235a(-) erythroid progenitor cell. On culture days 7-14, the progenitor cells differentiate in the presence of erythropoietin to become CD36(+), CD45(-), CD71(high), CD235a(+) precursor cells. During the final week in culture, the erythroblasts undergo nuclear condensation, enucleation, and loss of RNA combined with the loss of CD36 and CD71 on the plasma membrane to become mature erythrocytes. To investigate different stages of erythroblast maturation, the cells were cultured in medium containing 1µM UNC0638 for periods of seven days (culture days 0-7, 7-14, or 14-21) and compared to control cultures without UNC0638. The effects of UNC0638 were determined by flow cytometry, Q-RT-PCR and hemoglobin chromatography (HPLC). Unexpectedly, fetal hemoglobin expression was highly-dependent upon the differentiation stage of the cells in the presence of UNC0638. When cultured in UNC0638 supplemented medium on culture days 0-7 or 14-21, the cells underwent terminal maturation, but there was no significant increase in the fetal hemoglobin content of the mature cells (see abstract figure). In contrast, UNC0638 added on culture days 7-14, caused a significant increase in fetal hemoglobin (HbF; control: 3.9 ± 3.5% vs. day 7-14 UNC0638: 32.6 ± 0.95%, p=0.007). The increase in HbF was associated with a similar increase in gamma-globin mRNA (control: 1.5E+06 ± 1.7E+05 copies/ng vs. day 7-14 UNC0638: 7.5E+06 ± 1.4E+06 copies/ng, p=0.021). Additionally, terminal maturation and enucleation were partially inhibited when compared to the other conditions or controls. These data suggest that UNC0638 causes a robust increase in fetal hemoglobin as the cells undergo maturation. Fetal hemoglobin increases were more pronounced after exposure to UNC0638 during the erythropoietin-dependent transition from CD235a(-) to CD235a(+) erythroblasts. The results suggest that fetal hemoglobin regulation by G9a and GLP may be differentiation stage dependent. Disclosures: No relevant conflicts of interest to declare.