ALBERT

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: Chronic red blood cell transfusion therapy is indicated for primary and secondary stroke prevention in children with sickle cell disease (SCD). The main transfusion goal is achievement of pre-transfusion sickle hemoglobin (HbS) levels of 30%. Unfortunately, there continues to be a population of patients with a history of stroke who have progressive vasculopathy and/or secondary stroke, despite chronic transfusion therapy. Predictive markers for vasculopathy and cerebral events are needed to identify patients at risk for disease progression. Increased reticulocytosis was previously associated with other sickle cell disease complications, and adherent reticulocytes may contribute to the vascular pathology. The objective of this study was to explore the hypothesis that pre-transfusion reticulocytosis may serve as a disease severity marker for cerebral vasculopathy among chronically transfused children with sickle cell disease. After obtaining consent and assent, reticulocytosis was studied in a cohort of pediatric sickle cell patients treated with chronic transfusions (n=33, ages 2-17 years). The group was stratified into three groups: group 1 with an abnormal transcranial doppler (TCD) study in the absence of magnetic resonance angiography (MRA) detected vasculopathy [TCD(+), MRA(-), n=14], group 2 with a history of a stroke in the absence of MRA-detected vasculopathy [Stroke(+), MRA(-), n=5], and group 3 with a history of abnormal TCD or stroke and more severe vasculopathy detected by magnetic resonance angiography [MRA(+), n=14]. Pre-transfusion blood samples were analyzed within 72 hours of collection. Steady-state blood samples were also examined from a control group of pediatric SCD patients (〉6 years of age) with normal TCD studies who were receiving supportive care in the absence of chronic transfusions or hydroxyurea (n=7). Hematologic data, including automated complete blood counts, absolute reticulocyte counts (ARC) with reticulocyte maturity were obtained. In addition, a flow cytometric approach was developed to further examine and quantitate reticulocyte subsets based upon staining with thiazole orange combined with CD36, CD45, CD49d, CD71, and CD235. The pre-transfusion HbS levels were not statistically different among the three transfused groups ([TCD(+), MRA(-)]: 30.2 ± 11.8%; [Stroke(+), MRA(-)]: 28.4 ± 3.3%; [MRA(+)]: 33.3 ± 9%, p〉0.3). The high levels of reticulocytosis in the pre-transfusion samples were similar to those measured in the control group (ARC: 451 ± 126 K/uL in the chronically transfused cohort; ARC: 369 ± 94 K/uL in the control group, p=0.11). Pre-transfusion reticulocytosis was detected in every chronically transfused subject (ARC range 151-701 K/ul). The mean ARC in the [TCD(+), MRA(-)] group was not significantly different from the [Stroke(+), MRA(-)] group (411 ± 135 K/uL and 396 ± 97 K/uL respectively, p=0.82). However, the mean ARC in the [MRA(+)] group (512 ± 107 K/uL) was significantly higher than the control group, the [TCD(+), MRA(-)] group and the [Stroke(+), MRA(-)] group (p

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: 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: Key Points The G9a methyltransferase inhibitor UNC0638 increased pancellular expression of HbF to levels greater than 30% in adult human erythroblasts. UNC0638 altered globin locus epigenetic status/protein occupancy favoring LCR interaction with fetal genes at the expense of adult genes.

Description: Globin gene expression undergoes developmental switching from embryonic (ε) through fetal (γ) to adult (δ and β) genes. Inherited mutations or deletions at the β-gene cause beta-thalassemia. One of the most propitious strategies of treatment for the disease is forced switching from mutated β-gene to unaffected fetal γ-gene expression in adult erythroid cells. Expression of globin genes is regulated by the upstream LCR enhancer. The LCR enhancer loops to globin gene promoters utilizing the LDB1/GATA-1/TAL1/LMO2 protein complex. Additionally histone-modifying enzymes play a significant role in regulation of globin gene expression. G9a methyltransferase, responsible for establishing H3K9me2 histone modification, is involved in repressing fetal and activating adult globin gene expression in mouse erythroid cells. Moreover, inhibition of G9a methyltransferase activity by the synthetic chemical compound UNC0638 activates γ- and represses β-gene expression in adult human hematopoietic precursor CD34(+) cells. Using ex vivo differentiation of primary CD34(+) adult human cells as a model system, we investigated the effect of UNC0638 on switching from β- to γ-globin gene expression, LDB1 complex occupancy and LCR/β-gene promoter looping patterns in adult erythroblast cells. Human peripheral blood CD34(+) progenitor cells from three healthy adult donors were differentiated for 21 days in a three phase serum-free media system. Based upon dose titration studies, 1µM UNC0638 was added to the medium during the most proliferative phase of culture (days 7-14) and compared to control cells grown without UNC0638. Under these conditions, a highly significant 5-fold increase in γ-globin gene expression was observed. UNC0638 treatment also caused a pronounced (3-fold) reduction in β-globin gene expression without substantial change in α-globin. At the end of the culture period, HPLC analyses also demonstrated that UNC0638 treatment resulted in a considerable increase in the cellular fetal hemoglobin (HbF / HbA + HbF: control: 2.9 +/- 1.2%; UNC0638: 30.9 +/- 2.5%, p=0.003). Chromatin immunoprecipitation and chromosome conformation capture assays were utilized to determine if the increase of fetal hemoglobin along with activation of γ-gene expression and concomitant reduction of β-gene expression were associated with epigenetic modification of the β-globin locus. UNC0638 erased H3K9me2 histone modification in the β-globin locus and caused changes in LCR looping from interaction with the β- to the γ-globin gene. Mirroring differences in looping pattern, LDB1 containing protein complex occupancy was significantly increased at the γ-globin gene and decreased at δ- and β-gene promoters. These results support a model whereby G9a establishes conditions preventing activation of γ-gene by interacting with the LCR and facilitating LCR looping with δ- and β-gene promoters and subsequent strong activation of adult globin genes expression during differentiation of adult erythroid progenitor cells. In this view, G9a inhibition represents a promising approach for treatment of β-hemoglobinopathies. 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.

Description: LIN28 proteins bind to RNA and regulate developmental timing events in multicellular organisms, in part, by reducing cellular levels of the let-7 family of microRNAs. High-level LIN28 expression in stem cells promotes their self-renewal. Over-expression of the LIN28 proteins causes suppression of let-7 in hematopoietic stem and early progenitor cell populations (CD34+) from adult donors and manifests a more fetal-like phenotype in the erythroid lineage. Here we explore LIN28expression that is restricted to erythroid cells, rather than stem or multi-potential progenitor cells. For this purpose, lentiviral transduction vectors were produced with LIN28A expression driven by erythroid-specific gene promoter regions of the human KLF1 or SPTA1 genes, as well as an internal ribosomal entry site for puromycin selection (vectors: KLF1-LIN28A-OE and SPTA1-LIN28A-OE). Viral supernatants from these constructs were compared with empty-vector controls in matched transductions of CD34+ cells from three adult human volunteers. The cells were transduced and cultured using a three-phase, serum-free model for ex vivo erythropoiesis. Erythroblast proliferation and differentiation were comparable between control and LIN28-transduced cells assessed by cell counting and flow cytometry with staining for CD71, glycophorin A and thiazole orange. To validate restricted expression of LIN28 in the erythroid lineage, colony formation assays were performed in semisolid methylcellulose containing 1.0 ug/ml puromycin. BFU-E, CFU-GM, CFU-G, CFU-M and GEMM colonies were enumerated 14 days after plating. Puromycin addition to KLF1-LIN28A-OE and SPTA1-LIN28A-OE transductions resulted in selection of the erythroid colonies (BFU-E as a percentage of total colonies: Control: 44.6 ± 6.1%; KLF1-LIN28A-OE: 98.4 ± 0.7%, p=0.003; SPTA1-LIN28A-OE: 95.2 ± 1.1%, p=0.005). LIN28A over-expression was confirmed by RT-QPCR (KLF1-LIN28A-OE: 2.1E+05 ± 7.0E+04 copies/ng; SPTA1-LIN28A-OE: 2.2E+05 ± 8.3E+04 copies/ng; Controls: below detection limits) and Western analyses after transduction. Suppression of all let-7 miRNA family members to less than 30% control levels were detected for both vectors resulting in a reduction in total let-7 miRNA (RT-QPCR: Control: 2.0E+07 ± 9.7E+05 copies/ng; KLF1-LIN28A-OE: 5.6E+06 ± 5.6E+05 copies/ng, p=0.003; SPTA1-LIN28A-OE: 4.6E+06 ± 6.2E+05 copies/ng, p=0.003). BCL11A expression levels were also measured by RT-QPCR and Western analyses. While BCL11A showed no significant change at the mRNA level (Control: 1.2E+03 ± 4.5E+02 copies/ng; KLF1-LIN28A-OE: 2.9E+02 ± 7.4E+01 copies/ng, p=0.07; SPTA1-LIN28A-OE: 4.2E+02 ± 3.3E+02 copies/ng, p=0.07), protein analyses of nuclear BCL11A showed moderately reduced levels after KLF1-LIN28A-OE and SPTA1-LIN28A-OE transductions. Globin mRNA and protein levels were investigated and compared with controls. Gamma-globin mRNA was significantly increased in LIN28A-OE samples (Control: 3.6E+06 ± 8.2E+05 copies/ng; KLF1-LIN28A-OE: 1.9E+07 ± 1.7E+06 copies/ng, p=0.007; SPTA1-LIN28A-OE: 1.7E+07 ± 8.9E+05 copies/ng, p=0.003). Fetal hemoglobin (HbF) production was measured at the end of the culture period using High Performance Liquid Chromatography, and was increased in the KLF1-LIN28A-OE and SPTA1-LIN28A-OE samples compared to the control (Control: 7.0 ± 1.4%; KLF1-LIN28A-OE: 31.9 ± 2.7%, p=0.004; SPTA1-LIN28A-OE: 43.0 ± 6.2%, p=0.004). Flow cytometry analyses demonstrated a pan-cellular HbF distribution. In contrast to promoting self-renewal in stem cells, these data suggest that adult erythroblast-restricted LIN28 functions to partially reverse the fetal-to-adult developmental transition in hemoglobin expression. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Hemophilia is a congenital bleeding disorder due to the deficiency of blood coagulation factor VIII or IX. The joint is the most common site of serious bleeding. Repeated episodes of hemarthrosis lead to hemophilic synovitis (HS) and destructive hemophilic arthropathy (HA). The molecular changes responsible for HS and HA are not known. Osteopontin (OPN) has been implicated in the process that leads to bone destruction in other forms of arthritis. We hypothesized that OPN plays a similar role in HA. Methods Hemophilic synovitis was induced in factor (F) VIII and IX deficient mice by causing traumatic (blunt trauma) and non-traumatic (needle puncture) hemarthrosis in the right knee joint of mice. The concentration of OPN was measured in plasma of mice five days after inducing hemorrhage. Expression of OPN in synovial tissue from affected knee joints was compared with control uninjured joints by DNA microarray and semi-quantitative RT-PCR analysis on different days following joint injury. Results OPN concentration in the plasma of both FVIII and FIX deficient mice sacrificed 5 days after a single capsular puncture was increased 92–98% compared to control mice. In other experiments, traumatic hemarthrosis was induced three times at weekly intervals in the right knee joints of FVIII deficient mice. On day 21, synovial tissue from the knee joints of 20 mice was isolated, pooled and RNA from affected and control joints probed using microarray. The expression of OPN in right knee tissue was 7.5-fold greater compared to the left. These data were confirmed by RT-PCR analysis which showed that OPN was highly expressed in synovial tissue from mice 14, 21 and 30 days after single puncture injury and 21 days following three traumatic injuries. Conclusion The concentration of OPN was increased in the plasma of mice following hemarthrosis and OPN expression was increased 7-fold in synovial tissue of mice with hemophilic arthropathy. Our data implicate OPN in the pathobiology of hemophilic arthropathy. Figure Figure Figure Figure