ALBERT

Description: Key Points NRF2 knockout inhibits fetal hemoglobin expression during gestational erythropoiesis in SCD mice. Loss of the cellular antioxidant response mediated by NRF2 exacerbates spleen damage, inflammation, and oxidative stress in SCD mice.

Description: Background: Sickle cell anemia (SCA) is a monogenic disease resulting in polymerization of hemoglobin in hypoxic conditions. This leads to red blood cell (RBC) membrane damage and sickling, causing vaso-occlusion and hemolysis. Continual, excessive release of lysed RBC contents within the vascular space can activate the inflammasome, a multiprotein oligomer that promotes maturation and secretion of pro-inflammatory cytokines, including interleukin 1-beta (IL-1β). The intravascular inflammation associated with SCA, e.g., increased serum c-reactive protein (CRP) and absolute counts of neutrophils and monocytes, is predictive of long-term morbidity and mortality. Inflammation is a major component of many of the clinical complications of SCA, including vaso-occlusive pain episodes, acute chest syndrome, vascular-endothelial dysfunction, renal disease and other forms of end organ damage. Standard of care for SCA is hydroxyurea, which augments fetal hemoglobin levels and may have some anti-inflammatory effects by reducing neutrophil and monocyte counts. Canakinumab is a fully human monoclonal antibody targeting IL-1ß and blocking its downstream pro-inflammatory activities with potential to ameliorate the inflammatory complications of SCA. Objective:To clinically validate in pediatric and young adult SCA patients the hypothesis that IL-1ß blockade by canakinumab is safe and provides clinical benefits. Methods: A multi-center, randomized, parallel group, double-blind, placebo-controlled trial recruited SCA patients (HbSS or HbS/ß0thalassemia) with history of ≥2 major pain episodes/year, screening baseline detectable pain (using pain e-diaries) and serum high sensitivity CRP level ≥1.0 mg/L. Patients were randomized with 1:1 ratio to receive six monthly s.c. injections of either canakinumab 300 mg (4 mg/kg for patients ≤40 kg) or placebo. The concurrent use of hydroxyurea was a stratification factor at randomization. Outcomes were measured at baseline and at weeks 4, 8, 12, 16, 20, 24, after which all patients moved to open label canakinumab treatment for additional 6 months. Electronic patient reported outcomes included daily pain intensity with a 0-10 cm visual analog scale, school/work absences secondary to SCA, fatigue and analgesic use. The primary outcome was change from baseline in the 4-week average daily pain intensity at week 12. Other secondary and exploratory outcomes included daily activity measured by wrist actigraphy, rate of hospitalization and adverse events, serious adverse events, transcranial Doppler velocities, percent oxygen saturation and laboratory markers of inflammation and hemolysis. Results: A planned interim analysis for futility and safety was performed on the first 30 enrolled patients (canakinumab, n=16; placebo, n=14), of whom 26 patients completed the Week 12 assessments (canakinumab, n=14; placebo, n=12), and 13 patients completed the Week 24 assessments. Enrolled patients (median age 17 years, range 12-20; 19 male, 11 female) were evenly distributed between treatment arms. All except one patient were maintained on a stable hydroxyurea regimen. Baseline overall disease activity levels (median, [range]) included average daily pain 3.93 [0.29, 6.57]; high sensitivity CRP 3.93 mg/L [1, 64.7]; transcranial Doppler velocities 85.0 m/s [23, 267]; hemoglobin 94.8 g/L [73.5, 121]. Futility criteria were not met and no canakinumab-associated safety issues were identified in this first interim analysis. Conclusions: Canakinumab was well tolerated and not associated with any major side effects in SCA. Results from a second interim analysis of study outcomes for all currently enrolled patients (n=49) completing the blinded, 24-week treatment period will be available in November 2019. Disclosures Rees: Agios: Other: Grants; TauRx (methylene blue): Other: Data monitoring committees; Astra Zeneca (ticagrelor): Other: Data monitoring committees; Novartis: Other: Strategic advisory role,Principal investigator,sickle cell disease2.6 Investigator; Emmaus: Other: Strategic advisory role; Celgene: Other: Strategic advisory role; Global Blood Therapeutics: Other: Strategic advisory role; Alnylam: Other: Principal investigator. Dampier:Micelle Biopharma: Consultancy, Research Funding; Merck: Research Funding; Hudson Publishing Company: Consultancy; Global Blood Therapeutics: Consultancy; Ironwood: Consultancy; Epizyme: Consultancy; Modus Therapeutics: Consultancy; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Mahlangu:Sanofi Genzyme: Research Funding, Speakers Bureau; Pfizer: Consultancy, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau; Novartis: Research Funding; Baxalta: Consultancy, Research Funding, Speakers Bureau; LFB: Consultancy; Biomarin: Research Funding; uniQure: Research Funding; Spark: Consultancy, Speakers Bureau; Chugai: Consultancy; CSL Behring: Consultancy, Research Funding, Speakers Bureau; Freeline Therapeutics: Research Funding; NovoNordisk: Consultancy, Research Funding, Speakers Bureau; Roche: Consultancy, Research Funding, Speakers Bureau; Shire: Consultancy, Research Funding, Speakers Bureau; Sobi: Research Funding, Speakers Bureau; World Federation of Haemophilia: Speakers Bureau. Mortier:Novartis Pharma AG: Employment. McNamara:Novartis Pharma AG: Employment. Li:Novartis Pharma AG: Employment. Oliver:Novartis Pharma AG: Employment, Equity Ownership. OffLabel Disclosure: canakinumab use in the treatment of sickle cell anemia.

Description: We previously demonstrated that signal transducer and activator of transcription 3β (Stat3β) acts as a negative regulator of γ-globin gene expression. We speculate that Stat3β participates in the γ to β-globin switch recapitulated during erythroid maturation. Regulatory elements located between nucleotide −52 and +36 relative to the γ-globin cap site may define a silencing domain where Stat3β exerts its negative effect. We analyzed DNA-protein interactions in a putative Stat3 binding site at +9γ and an overlapping Stat3/GATA-1 consensus sequence at +26γ. The +9γ motif was required for Stat3β mediated γ-gene silencing in K562 cells and normal erythroid progenitors. Mutating the +26γ site disrupted both Stat3 and GATA-1 binding which was confirmed by electrophoretic mobility shift assay. We demonstrated for the first time direct interaction between Stat3 and GATA-1 using immunoprecipitation techniques and protein extracts isolated from K562 and mouse erythroleukemia cells. We speculate that developmental stage specific DNA-protein interactions in the −52 to +36 region are required for γ-gene silencing. To investigate this hypothesis, we tested the ability of Stat3β to bind in vivo using chromatin immunoprecipitation (ChIP) assay. K562 cells were treated with Interleukin-6 (100ng/ml) to activate Stat3β and then used for ChIP analysis with phosphorylated-Stat3β antibody. Specific PCR primers were designed to amplify the region between nucleotide −52 and +36 relative to the γ-globin cap site. We observed increased Stat3β binding to the γ-globin silencing domain in vivo after Interleukin-6 treatment. To compliment this approach, we modified our ChIP assay as follows. A trypsin digest step was added after cross-linking proteins to the γ-globin silencing domain in vitro. The samples will be analyzed by nanospray LC/MS/MS mass spectrometry to characterize the transcription factors that bind the silencing domain and interact with Stat3β in this region. Understanding the DNA-protein interactions required for γ-globin regulation will provide insights into mechanisms for globin gene silencing.

Description: Hydroxyurea (HU) has been shown to induce fetal hemoglobin (HbF) synthesis through activation of the soluble guanosine cyclase/cyclic guanosine monophosphate signaling pathway. The release of NO from HU by heme iron is thought to be involved in this mechanism of HbF induction. Studies completed in sickle cell patients confirmed increased serum NO levels after oral HU therapy but NO generation in red blood cells and the effect on γ-gene transcription have not been extensively investigated. Therefore, we performed studies to quantify NO generated by HU in K562 cells and normal erythroid progenitors as a mechanism for γ-globin activation. NO levels were measured after drug treatments using the Nitric Oxide Assay Kit (Calbiochem) and γ-globin mRNA was measured using quantitative PCR. HU (100μM) increased NO 1.4 to 1.8-fold at 24–72 hrs in K562 cells compared to a 2.0 to 2.5-fold increase in NO produced by the known NO donor, deta-nonoate (DE; 400μM). NO levels were also measured in erythroid progenitors grown in liquid cultures; a 1.6-fold increase in NO was produced by 30μM HU after 48 hrs with comparable increases produced by 200μM DE. To understand the effects of HU on normal NO synthesis from L-arginine through the action of NO synthase (NOS), we performed studies with the NOS inhibitor, NG-Monomethyl-L-arginine (L-NMMA). Interestingly, HU increased NO levels 2.5-fold at 24 hrs when combined L-NMMA compared a 1.4-fold increase produced by HU alone; this pattern persisted up to 72 hrs. Parallel with these findings γ-globin activation by HU was augmented approximately 25% by L-NMMA; DE combined with L-NMMA did not produce the same effect. These data suggest a novel mechanism for NOS regulation by HU compared to DE. Subsequent studies were completed to determine if HbF synthesis could be augmented by combining NO donors since they have different mechanisms of action. HbF levels in K562 cells were measured by ELISA (Bethyl Laboratories) and normalized by total hemoglobin and protein. Treatment with HU or DE increased HbF 3.6-fold and 4.6-fold respectively; when HU was combined with DE an additive 7.6-fold increase in HbF was produced. These data confirm that HU treatment lead to NO generation in K562 cells and normal erythroid progenitors which plays a role in its mechanism of γ-globin activation. HU combined with DE had an additive effect on HbF synthesis. These findings are relevant to current research efforts to develop novel HbF inducers for therapy in sickle cell patients.

Description: High-level expression of fetal (Υ) globin reduces clinical complications in sickle cell disease and this is achieved with hydroxyurea (HU) in young children. However, non-cytotoxic high-potency therapeutics, particularly which can be utilized in combination with HU, are needed for most adolescent and adult patients who have continued serious clinical events. We have identified additional pharmaceutical candidates which induce HbF without cytotoxicity, using a Υ-globin gene promoter linked to GFP for robotic high-throughput screening, and screening five diverse chemical libraries. From a library of US and EU drugs which are approved for treatment of other medical conditions, a small panel of approved therapeutics were found to induce Υ globin expression, and have benign safety profiles, are orally active, and are suitable for long-term use. Three orally active candidates were evaluated in anemic baboons, and two, DLT and PB-04, induced Υ-globin mRNA by 15- to 33-fold over baseline levels. In 3/3 beta-globin locus YAC transgenic mice, one candidate (PB-04; 20 mg/kg) given by intra-peritoneal (IP) injections (for experimental feasibility) 3 times/ week for 5 wks significantly increased F-cells from 0.1 to 9%, 0.4 to 18%, and 0.13 to 12% respectively; and mean fluorescence intensity (MFI) increased by 10- to 33-fold. Responses were observed within one week. In hydroxyurea treated mice (100 mg/kg; IP, 5 days/ wk) F-cells increased from 0.3 to 2.3% on average (p

Description: Abstract 1000 Objective: The use of small molecules to reactive fetal hemoglobin (HbF) expression in patients with sickle cell disease (SCD) is a promising clinical strategy. Erythroid Kruppel-like factor (KLF1) and B cell lymphoma 11A (BCL11A) are key regulatory components of the developmental switch from fetal to adult hemoglobin. Current models suggest that KLF1 positively regulates the expression of the γ-globin gene repressor BCL11A by interacting with the BCL11A promoter. Knockdown studies of KLF1 in adult erythroid progenitors are consistent with this model and result in low BCL11A levels and derepression of γ-globin gene expression. Based on the critical role of KLF1 in regulating HbF production, we performed a limited screen of small molecules for their ability to suppress KLF1 expression. We identified resveratrol, a small polyphenolic compound, as the most potent suppressor of KLF1. The following studies were performed to confirm RSV's mechanism of action. Methods: Cell Culture. KU812 leukemia cells express both fetal and adult hemglobin and erythroid markers similar to days 7–14 primary erythroid cells, which makes them a useful in vitro model for testing the activity of HbF-inducing compounds. KU812 cells (ATTC) were cultured at a density of 5×105 cells/mL for 72h in the presence of vehicle or RSV (50 μM). Flow cytometry. Fixed KU812 cells were labeled with monoclonal anti-human γ-globin (Invitrogen) and β-globin (Santa Cruz) antibodies to determine the γ- to β-globin mean fluorescent ratio. Western Blot. Whole cell lysates were prepared in RIPA buffer containing protease inhibitors and quantified by BCA protein assay. Protein samples (40 μg per lane) were resolved on a 4–15% SDS-PAGE gradient gel and transferred onto a PVDF membrane. Blots were incubated on at 4°C in primary antibody (KLF1, 1:200, Santa Cruz; BCL11A, 1:1000, Novus Biologicals) followed by 1h incubation with an HRP-conjugated secondary antibody at RT. Protein bands were visualized by chemiluminescence and analyzed by densitometry using Image J software. Transient transfections. KU812 cells were transfected with a constitutively active KLF1 expression construct (pCMVSPORT6-KLF1) or an empty vector control (pCMV-EV) using the Amaxa 4D Nucleofector kit (Lonza). 12 h post transfection, KU812 cells (5×105 cells/mL) were incubated with vehicle or RSV (50 μM) for 72 h. Statistical analysis. One-Way ANOVA followed by Student-Newman-Kuels (Sigma Stat). Data are reported as the mean ± SEM. A p-value of

Description: Elevated fetal hemoglobin (HbF) expression ameliorates the clinical severity of sickle cell disease (SCD) by inhibiting hemoglobin S polymerization. Differences in HbF levels are attributed to inherited DNA genetic variations that regulate γ-globin transcription; however the role of microRNA (miRNA) genes in HbF regulation has not been investigated using clinical samples. miRNAs are small non-protein-coding RNA molecules that negatively regulate gene expression through inhibition of mRNA translation. Our goal is to identify miRNA genes with altered expression in sickle cell patients with elevated HbF levels, to elucidate mechanisms of γ-globin gene regulation. After IRB approval, peripheral blood was collected from SCD patients (not on hydroxyurea therapy), followed in the pediatric and adult Sickle Cell Clinics at Georgia Regents University. HbF levels measured by high performance liquid chromatography and complete blood and reticulocyte counts were obtained. Twelve blood samples, six each from SCD subjects with high HbF (19.9±2.1%) or low HbF (4.4±0.9%) levels were analyzed. We observed more severe anemia and higher reticulocyte counts in the low HbF group. After Histopaque separation, red blood cells were processed on a MACS column with anti-CD71 antibody to isolate reticulocytes, followed by total RNA extraction using Trizol. RNA (750ng) was hybridized to a genome-wide miRCURY LNA microRNA Array (Exiqon) containing 1,921 human probes. The microarray raw data were collected on an Agilent G2565BA Microarray Scanner System and normalized by Model-Based Background Correction and Principle Component Analysis. Characterization of miRNA profiles for low HbF compared to high HbF groups identified 327 differentially expressed genes including multiple miR-144 isoforms. We subsequently explored the function of miR-144 because it targets Nrf2 which mediates drug-induced HbF expression and Nrf2 has an antioxidant protective effect in SCD. Therefore, we conducted supervised learning of the normalized microarray data based on miR-144 expression. Interestingly, in the low HbF group we observed two subphenotypes: 1) associated with 8-fold increased miR-144 expression (3 subjects) and 2) associated with no change in miR-144 level (3 subjects) when compared to the high HbF group suggesting a role of miR-144 in HbF regulation. In the supervised learning analysis, there were 62 up-regulated and 33 down-regulated miRNAs (〉2-fold; p

Description: The histone deacetylase inhibitors (HDA-CIs) butyrate and trichostatin A activate γ-globin expression via a p38 mitogen-activating protein kinase (MAPK)-dependent mechanism. We hypothesized that down-stream effectors of p38 MAPK, namely activating transcription factor-2 (ATF-2) and cyclic AMP response element (CRE) binding protein (CREB), are intimately involved in fetal hemoglobin induction by these agents. In this study, we observed increased ATF-2 and CREB1 phosphorylation mediated by the HDACIs in K562 cells, in conjunction with histone H4 hyperacetylation. Moreover, enhanced DNA-protein interactions occurred in the CRE in the Gγ-globin promoter (G-CRE) in vitro after drug treatments; subsequent chromatin immunoprecipitation assay confirmed ATF-2 and CREB1 binding to the G-CRE in vivo. Enforced expression of ATF-2 and CREB produced Gγ-promoter trans-activation which was abolished by a 2-base pair mutation in the putative G-CRE. The data presented herein demonstrate that γ-gene induction by butyrate and trichostatin A involves ATF-2 and CREB1 activation via p38 MAPK signaling.

Description: Orally bioactive compounds that induce γ globin gene expression at tolerable doses are needed for optimal treatment of the β-hemoglobinopathies. Short-chain fatty acids (SCFAs) of 2 to 6 carbons in length induce γ globin expression in animal models, and butyrate, phenylbutyrate, and valproate induce γ globin in human patients. The usefulness of these compounds, however, is limited by requirements for large doses because of their rapid metabolism and their tendency to inhibit cell proliferation, which limits the pool of erythroid progenitors in which γ globin can be induced. Selected short-chain fatty acid derivatives (SCFADs) were recently found to induce γ globin and to stimulate the proliferation of hematopoietic cells in vitro. These SCFADs are now evaluated in vivo in nonanemic transgenic mice containing the human β globin gene locus and in anemic phlebotomized baboons. In mice treated with a SCFAD once daily for 5 days, γ globin mRNA increased 2-fold, reticulocytes increased 3- to 7-fold, and hematocrit levels increased by 27%. Administration of 3 SCFADs in anemic baboons increased F-reticulocytes 2- to 15-fold over baseline and increased total hemoglobin levels by 1 to 2 g/dL per week despite ongoing significant daily phlebotomy. Pharmacokinetic studies demonstrated 90% oral bioavailability of 2 SCFADs, and targeted plasma levels were maintained for several hours after single oral doses equivalent to 10% to 20% of doses required for butyrate. These findings identify SCFADs that stimulate γ globin gene expression and erythropoiesis in vivo, activities that are synergistically beneficial for treatment of the β hemoglobinopathies and useful for the oral treatment of other anemias.

Description: Abstract 3444 CD34+ fetal stem cells (FSC) are primitive cells which can be isolated from umbilical cord blood. They have hematopoietic potential and can reconstitute the different cell lineages of the bone marrow similar to adult CD34+ stem cells. There are gaps in knowledge related to mechanisms of FSC differentiation which can be used to develop therapy for genetic diseases such as the hemoglobinopathies. Insights into fetal erythropoiesis can be gained by understanding mechanisms of globin gene regulation in the human β-globin locus where five functional genes (ε, Aγ, Gγ, δ, β-globin) are expressed in a developmentally regulated fashion. Globin gene expression during development is controlled in part by the locus control region however genomic controls during fetal erythropoiesis have not been clearly elucidated. Therefore, we utilized FSC isolated from umbilical cord blood induced to undergo differentiation as a model to characterize the transcriptome associated with the γ/β globin switch during fetal erythropoiesis. FSC (0.5 million) were grown in the one-phase liquid culture system containing stem cell factor (50ng/ml), interleukin-3 (10ng/ml) and erythropoietin (4U/ml). We observed a 30-fold higher proliferative capacity of FSC compared to adult progenitors reaching 10 billion cells by day-56 in culture. Erythroid differentiation was confirmed by Giemsa staining and increased expression of the differentiation biomarkers CD71 and CD235a and decline of CD34 was observed. Moreover, the γ/β globin switch occurred around day 45 in FSC compared to day 21 for adult stem cells (Li et al. BMC Genomics13:153, 2012). These data suggest that the γ-globin gene is activated for a longer period in the FSC environment in contrast to adult progenitors supporting different mechanisms of gene regulation in the two systems. To gain further insights, microarray analysis was performed on the HumanHT-12 v4 Expression BeadChip containing 47,000 probes, using triplicate RNA samples harvested on day 21, 42, 49 and 56. After data normalization by model-based background correction method, profile-1 genes with decreased expression from day-21 to day-56 (similar to γ-globin silencing) or profile-2 similar to β-globin activation from day-21 to day-56 were defined using Principal Component Analysis. Sixty-five profile-1 genes were subsequently identified by Gene Set Enrichment Analysis (GSEA) for erythroid-specific gene subsets. DAVID (Database for Annotation Visualization and Integrated Discovery) Ontology analysis confirmed 23 out of 65 erythroid genes including known regulators of γ-globin such as KLF4, KLF11, BCL11A and SIRT3. A similar analysis of profile-2 genes demonstrated 155 erythroid genes by GSEA and 28 factors were confirmed by DAVID platform related with β-globin regulation such as KLF1, GATA1, MXl1 and SOX6. To define transcription factor networks related to globin gene expression in FSC, the confirmed gene subsets were analyzed using the Michigan Molecular Interaction plugin for Cytoscape. A transcription factor network centered on KLF4 and GATA2 was defined; KLF4 positively regulates CREB, KLF6, GATA2, POU1F1, JUN and STAT3 while repressing Sp1. We previously demonstrated the ability of KLF4 to trans-activate γ-globin in adult stem cells. How these factors regulate γ-globin expression in FSC will be explored further. Similar studies for profile-2 genes revealed KLF1 and GATA1 as a major network hub. The special characteristics of fetal stem cell including high proliferative capacity and prolonged growth and γ-globin expression combined with a novel transcription factor network will be used to define genomic mechanisms of fetal erythropoiesis. Disclosures: No relevant conflicts of interest to declare.