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Background Sickle cell disease (SCD) is one of the most common and devastating inherited blood disorders characterized by a single nucleotide mutation in the beta-globin chain leading to the production of mutant hemoglobin S (HbS). HbS polymerizes upon deoxygenation causing red blood cells (RBC) to sickle which results in extremely painful episodes of vaso-occlusive crisis (VOC), severe hemolytic anemia, chronic multiorgan failure and a reduced life span. An important metabolic feature directly associated with RBC sickling is increased intracellular levels of the glycolytic intermediate 2,3-diphosphyglycerate (2,3-DPG) which promotes deoxygenation by lowering the oxygen affinity of hemoglobin (Hb). Pyruvate kinase R (PKR) is a key enzyme in RBC metabolism, generating adenosine triphosphate (ATP) to maintain energy homeostasis, membrane integrity and deformability, and modulates 2,3-DPG levels. Mitapivat (AG-348) is an oral, small molecule allosteric activator of PKR and shows promise as an anti-sickling agent in addition to its effect in PK deficiency and thalassemia. The safety and efficacy of mitapivat in subjects with SCD was evaluated in the dose finding period of this ongoing phase 2 study. Methods The ESTIMATE study is a phase 2, open-label study in which subjects ≥16 years with SCD (HbSS, HbS/β0, HbS/β+) with a baseline hemoglobin >6.1 g/dL and ≤11.1 g/dL, no chronic transfusion and adequate organ function were eligible. In the 8-week Dose Finding Period, initial dosing of mitapivat was 20 mg twice daily (BID). Subjects received a maximum of two sequential dose escalations of mitapivat (i.e. from 20 mg BID to 50 mg BID and 100 mg BID) depending on safety. The primary endpoints were safety, evaluated by frequency and severity of adverse events (AEs), and efficacy of mitapivat on RBC sickling. RBC sickling was evaluated by change in Point of Sickling (PoS), the pO2 at which sickling occurs as measured by oxygen gradient ektacytrometry on the Lorrca (RR Mechatronics). Secondary endpoints included changes in hematological parameters, levels of 2,3-DPG and ATP, Hb-oxygen affinity (p50) and surrogate markers of organ damage and mortality. Subjects who safely tolerated mitapivat and showed evidence of clinical improvement, were eligible to continue a 52-week follow-up period (Fixed Dose Extension Period). Results Six subjects have been enrolled as of September 2020 and completed the Dose Finding Period. All had homozygous HbSS except one patient who had HbS/β0-thalassemia. Baseline characteristics were: median age of 36 years (range 20-59 years), 4 (66.7%) were female and 5 (83.3%) were on stable-dose hydroxyurea. All subjects received dose escalation to a maximum dose of 100 mg BID. No serious adverse events (SAEs) occurred. Adverse events (AEs) were mild and often transient, with the most common treatment emergent AEs: transaminase increase (n=3 [50.0%], Grade 1), gastrointestinal disorders including dyspepsia, diarrhea and abdominal discomfort (n=3 [50.0%], Grade 1) and headache (n=2 [33.3%], Grade 1). One VOC occurred without hospital admission and did not require dose reduction or discontinuation. Table 1 summarizes the anti-sickling effect as well as the hematological and biochemical response to mitapivat treatment. Sickling occurred at lower pO2 levels in all 6 patients during the Dose Finding Period reflected by a significant decrease in treatment week 8 mean PoS compared to baseline. 5/6 subjects (83.3%) achieved a Hb increase of ≥1 g/dL during this period, which was accompanied by a decrease in hemolytic markers. Consistent with activation of PKR, 2,3-DPG levels decreased and ATP levels increased. Additional results including biomarker data will be presented. Conclusion Mitapivat demonstrated an adequate safety profile during the 8-week Dose Finding Period in patients with SCD. The data show promising efficacy in terms of a decrease in the pO2 at which RBCs start to sickle, as well as increase in Hb from baseline and a concomitant decrease in markers reflecting hemolysis. The observed changes in 2,3-DPG and ATP levels are consistent with the proposed mechanism of the drug. The study is ongoing and further data including follow-up data, patient-reported outcomes, PKR activity and thermostability will be reported at a later stage. Figure 1 Figure 1. Disclosures van Dijk: Agios Pharmaceuticals: Research Funding; Axcella Health: Research Funding. Rab: Axcella Health: Research Funding; Agios Pharmaceuticals: Research Funding. Rijneveld: Servier: Research Funding; Amgen: Research Funding. Nur: Celgene: Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Roche: Speakers Bureau. Schutgens: CSL Behring: Research Funding; Novo Nordisk: Research Funding; OctaPharma: Research Funding; Pfizer: Research Funding; Shire/Takeda: Research Funding; Bayer: Research Funding. Wijk: Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Axcella health: Research Funding; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Van Beers: Pfizer: Research Funding; Novartis: Research Funding; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; RR Mechatronics: Research Funding.

Background-Aim: Infection from SARS-CoV-2 has emerged as new pathological entity within the global medical community. One of the earliest questions was in relation to the ability of the immunocompromised patients to clear the infection. In COST EuNet-INNOCHRON we were interested in the impact of SARS-CoV-2 infection in patients with different types of chronic neutropenia (CNP). The aim of the current study is to understand the impact of SARS-CoV-2 infection and to identify any possible characteristic patterns of the clinical course in patients with CNP. Patients and Methods: The COST EuNet-INNOCHRON Action in collaboration with the European Haematology Association - Scientific Working Group (EHA-SWG) on Granulocytes and Constitutional Marrow Failure Syndromes has conducted an online survey on SARS-CoV-2 infection in patients with CNP. The EuNet-INNOCHRON participants from different countries got access to an on-line platform fulfilling the General Data Protection Regulation (GDPR) and could register adult and paediatric CNP patients who had been infected by SARS-CoV-2 from March 2020 to June 2021. Data on demographic characteristics, type of CNP, patients' background and SARS-CoV-2 infection history (symptoms, laboratory features, radiological appearance, therapeutic approach and outcome) were collected. Results: Twenty-six patients with diagnosis of CNP, 7 males and 19 females were registered. Patient age distribution as follows: 16 patients >18 years old (y.o.)5 patients 5-18 y.o, 4 patients < 5 y.o whereas age was not available for one of the patients. Nine of the patients were diagnosed with idiopathic CNP, 7 patients with congenital neutropenia (6 of them with severe congenital neutropenia), 3 with secondary CNP, 2 with suspected autoimmune neutropenia of infancy (although antineutrophil Ab were negative), one with autoimmune neutropenia, one with drug induced neutropenia and 3 with other types of CNP. Twelve patients were on treatment with G-CSF and 6 patients had a history of previous viral or bacterial infections. Clonal Cytopenia(s) of Undetermined Significance (CCUS) was excluded in the eight patients who were investigated. Twenty-four out of 26 patients had positive PCR and one was found incidentally with positive antibodies for SARS-CoV-2. One more patient was symptomatic with history of close contact with SARS-CoV-2 infected family members. The commonest observed symptoms were fever >38 oC (19 patients), cough (10 patients), rhinorrhoea (10 patients), sore throat (6 patients), musculoskeletal pains (7 patients), taste/smell loss (5 patients), headache (5 patients), dyspnoea (4 patients), chest pain (one patient) and none of them had gastrointestinal symptoms. No other associated respiratory viral or bacterial infections were reported. Four patients who had one or more underlying conditions (immune deficiency, heart/respiratory/kidney disease) were admitted in hospital and needed anti SARS-CoV-2 treatment. Two of them had non-invasive ventilation and one of them needed admission in intensive care unit (ICU); both recovered. Another patient with Fallot's tetralogy needed mechanical ventilation in ICU and sadly passed away. No other deaths were observed. Deterioration of the pre-existing neutropenia was seen in two patients, two patients developed thrombocytopenia, one patient developed worsening lymphopenia and one anaemia. Twelve patients had chest X-ray and consolidation was found in two of them. All three patients who had chest CT scans were found with ground-glass changes. During the observation period (up to two months), no re-infection from SARS-CoV-2 was found. The Stockholm, Sweden experience is similar to the above data. One hundred fifty-four patients with CNP were followed up, for 10 months (March 1 to December 31, 2020) for SARS-CoV-2. Seventeen of these (i.e. 11 %) were infected. None needed hospitalization and there were no fatalities. Conclusion: Although the relative susceptibility of neutropenic patients to contract SARS-CoV-2 needs to be assessed with further studies, the clinical course and severity of SARS-CoV-2 infection doesn't seem to be worse in CNP patients (regardless the type of neutropenia and the need for GCSF treatment) compared to the general population. Also, like what has been observed in non-neutropenic patients, underlying comorbidities is a significant risk factor for severe disease and adverse outcome. Disclosures Dale: X4 Pharmaceuticals: Consultancy, Honoraria, Research Funding. Palmblad: Chiesi Ltd Sweden: Honoraria; Roche Sweden: Speakers Bureau; Chiesi Ltd Candada,: Honoraria.

Introduction: Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome (IBMFS) characterized by hypoplastic anemia, congenital malformations and an increased risk to develop malignancies.Until now, treatment of DBA consists of red blood cell (RBC) transfusions, glucocorticoids (GC) and allogeneic hematopoietic stem cell transplantation in a selection of patients. Whereas RBC transfusions are the main cause of IO, elevated iron parameters have also been reported in non-transfusion-dependent DBA patients. Here we investigated the incidence and severity of IO in a well-described cohort of transfusion-dependent and non-transfusion-dependent DBA patients in order to gain more insight in the regulation of iron metabolism in DBA, and to provide clinical guiding to improve the diagnosis and management of IO in DBA. Methods: In this retrospective, observational study we have included twenty-nine pediatric and adult DBA patients for whom at least one serum ferritin level and/or MRI result was available. Ten patients (34%) were classified as transfusion-dependent (TD) (ten or more transfusions during the twelve months prior to evaluation). Non-transfusion-dependent (NTD) patients (66%) were treated with either GC, incidental transfusions or received no treatment. Transfusion burden (transfusion history) was assessed via medical records. Serum ferritin levels ≥250 ng/mL in males and ≥150 ng/mL in females were considered to be elevated. Results of MRI were expressed as liver iron content (LIC) and as cardiac T2* in milliseconds (ms). LIC ≥3 mg/g indicates significant hepatic IO, and LIC ≥7 mg/g is associated with clinical morbidity. Cardiac T2* ≤20 ms indicates significant cardiac IO. Results: In 15/29 (52%) MRI analysis of IO was performed. Hepatic IO (LIC >3 mg/g) was present in 9/29 (31%) of DBA patients, of which 8/9 (89%) had moderate to severe IO (LIC>7mg/g), despite the fact that all but one were treated with chelation therapy. Overall serum ferritin levels and LIC correlated significantly (r=0.7907, p1000 ng/mL (Figure 1A). Interestingly, in the NTD group, hepatic IO was present in 2/7 patients (29%), who both only had mildly elevated serum ferritin levels (263 ng/mL and 277 ng/mL) and were not treated with iron chelation therapy. Based on total transfusion burden since birth, patients were classified in distinct groups: nine patients who received ³10 transfusions during life (9/10) were diagnosed with hepatic IO, whilst none of the patients who received Discussion: We demonstrate that IO is common in DBA yet can be easily overlooked in NTD patients that were treated with transfusions in the past. While serum ferritin levels significantly correlated with LIC values, this parameter cannot be used exclusively to screen for IO or titrate iron chelation therapy. We conclude that in clinical practice, biochemical parameters in combination with transfusion history justify a low-threshold to perform an MRI-based evaluation of IO, and to start adequate chelation therapy. Figure 1 Figure 1. Disclosures Van Beers: Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Novartis: Research Funding; RR Mechatronics: Research Funding. Wijk: Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Axcella health: Research Funding; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Background: In sickle cell disease (SCD), hemoglobin S (HbS) polymerizes upon deoxygenation, reducing red blood cell (RBC) deformability. RBC deformability can be measured over a gradient of oxygen tensions (pO2) with the Laser Optical Rotational Red Cell Analyzer (Lorrca) ektacytometer (RR Mechatronics). Oxygen gradient ektacytometry generates 3 key parameters: 1) EImax, RBC deformability at normoxia; 2) EImin, minimum RBC deformability upon deoxygenation; and 3) the point of sickling (PoS): the oxygen tension at which a 5% decrease in deformability is observed during deoxygenation, reflecting the patient-specific pO2 at which sickling begins (Figure 1A). Previously we showed that oxygen gradient ektacytometry-derived biomarkers correlate with measures of SCD disease severity and hemolytic rate (Rab et al. Blood 2018), and is associated with vaso-occlusive crisis (VOC) frequency (Rab et al, Blood 2019). In this study, we confirm these observations in 2 independent cohorts and extend it to occurrence of acute chest syndrome (ACS), stroke including silent cerebral infarction (SCI), and transcranial Doppler (TCD) outcome. Methods: We analyzed 2 cohorts of SCD patients; an adult patient cohort of 53 SCD patients, enrolled at either University Medical Center Utrecht, The Netherlands (UMCU, n=25) or Hospital Lyon France (LIBM, n=28), and a pediatric patient cohort of 190 SCD patients enrolled at Texas Children's Hospital, USA (TCH). Subjects were HbSS or HbS/β-thalassemia, with a substantial number of subjects on hydroxyurea (HU) therapy (adult cohort 66% and pediatric cohort 86%), and not on chronic transfusion therapy. Correlations between oxygen gradient ektacytometry-derived biomarkers and the clinical complications of stroke or silent infarcts (SCI), ACS, VOC were assessed in both pediatric and adult patients. Patient groups generally did not significantly differ significantly by age, gender or HU treatment in the adult cohort except for age, which was lower in the ACS+ group (25.3years (y) compared to 32.0y) and also lower in the VOC+ group (27.1y compared to 35.8y). In the pediatric cohort, patient groups differed significantly in the ACS+ group compared to the ACS- group by age (ACS- group 8.37, ACS+ group 10.9y) and HU treatment (ACS- group 76%, ACS+ group 93%). Similarly, age was significantly higher in the Stroke+ group compared to the Stroke- group (14.0y compared to 9.3y), which was also found when studying VOC (VOC+ group 11.6y, VOC- group 8.2y). Results: In the pediatric cohort, PoS was significantly higher in patients with ACS (mean 40.3 compared to 34.9 mmHg, p=0.0001, Figure 1B). In the adult cohort, PoS was also higher in those with ACS although this did not reach significance (p=0.053, Figure 1C). In the pediatric cohort, PoS was higher in patients with stroke or SI (mean 43.0 mmHg compared to mean 37.3 mmHg, p In the adult and pediatric patient cohort, PoS was higher in patients with recent VOC (both p Differences in mean PoS between pediatric and adult patient cohorts could be due to differences in treatment, age, genetic background or technical differences between Lorrca devices. Lower significance levels found in the adult patient cohort could be due to smaller sample size. Conclusion: We show that oxygen gradient ektacytometry provides functional, clinically relevant next generation biomarkers that are associated with ACS, stroke and VOC. This study therefore further validates the clinical usefulness of these biomarkers, in particular in relation to cerebral vasculopathy. Since our results merely describe an association and not causality further validation is warranted to establish how well oxygen gradient ektacytometry can assess disease severity. However, its parameters could already provide the clinician with information about patient RBC characteristics and sickling propensity that could aid in clinical decision making. Our results provide a rationale for further development of these biomarkers in the evaluation of novel therapies as part of clinical care, or clinical trial endpoints. Disclosures Rab: RR Mechatronics: Research Funding. Bos:RR Mechatronics: Research Funding. Cnossen:Takeda: Research Funding; Shire: Research Funding; Baxter: Research Funding; Bayer: Research Funding; Sobi: Research Funding; CSL behring: Research Funding; Nordic Pharma: Research Funding; Novo Nordisk: Research Funding; Pfizer: Research Funding. Wijk:Agios Pharmaceuticals Inc.: Research Funding; RR mechatronics: Research Funding. Sheehan:Emmaus: Research Funding; Global Blood Therapeutics: Research Funding; Novartis: Research Funding. van Beers:Pfizer: Research Funding; RR mechatronics: Research Funding; Agios: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding.

Background: Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome (IBMFS) marked by erythroid hypoplasia, reticulocytopenia and macrocytosis, and associated with congenital malformations and an increased risk of developing malignancies. From a clinical and molecular perspective this disease is highly heterogeneous, and no clear genotype-phenotype correlations can be found. Since the majority of molecular defects have been found in ribosomal protein (RP) genes, DBA is regarded a "ribosomopathy". While the molecular basis has been studied intensively, the pathophysiology of DBA is still not fully understood. One of the major unresolved issues is how RP gene mutations result in the specific erythroid defect seen in DBA, with macrocytic erythrocytes and increased adenosine deaminase activity. In addition, the highly heterogeneous and variable clinical presentation and disease course, even in patients with similar molecular defects, remains enigmatic. Hence, in order to investigate the cellular defect, and increase our understanding of disease pathophysiology and clinical heterogeneity, novel tools are needed. In this study, we explore the potential of untargeted metabolomics on dried blood spots and report for the first time a metabolic fingerprint for DBA. Aims: Defining a metabolic signature for DBA in order to: 1. Increase our understanding of cellular determinants of impaired ribosome biogenesis, and 2. Extend the toolbox for diagnostic evaluation. Methods: Untargeted metabolic profiling was performed on DBS samples obtained from 18 DBA patients and 45 healthy controls using direct infusion high resolution mass spectrometry following a previously established approach1. Statistical analysis was performed in MetaboAnalyst and predictive modeling was executed within R-software. Results: In total, 1917 unique metabolite features were identified in DBS samples from patients and controls. Multivariate analysis yielded distinct metabolic profiles, reflected by natural separation detected by principal component analysis (Figure 1A), and emphasized by clear distinction with partial least square discriminant analysis (Figure 1B). This 'metabolic fingerprint' was incorporated into a machine learning algorithm, and subsequently a binary classification (or prediction) model was constructed by randomly dividing patient and controls into 'training' (32 HC, 13 DBA) or 'test' set (13 HC, 5 DBA). Accurate class assignment was achieved for all patients and controls in the training set (Figure 1C). Prominent metabolites in the fingerprint and classification algorithm were a.o. menadione (a vitamin K precursor), 4-hydroxyproline (collagen component) and methylmalonylcarnitine (an acylcarnitine). In addition a large number of interesting metabolites were identified that could provide novel starting points for studying/understanding downstream effects of RP defects in DBA and therapeutic mechanisms (Figure 1D). Conclusion: In this study we performed untargeted metabolomics on dried blood spots from a substantial cohort of DBA patients and report for the first time a metabolic fingerprint of this disease. By incorporating this fingerprint in a machine learning algorithm we underlined the diagnostic potential of this approach. Moreover, the metabolites identified in this fingerprint, provide promising starting points for further studies to increase our insights in disease pathophysiology, including the mechanism involved in elevated eADA activity, as well as the development of new therapeutic strategies. References: 1. de Sain-van der Velden MGM, van der Ham M, Gerrits J, et al. Quantification of metabolites in dried blood spots by direct infusion high resolution mass spectrometry. Anal Chim Acta. 2017;979:45-50. Disclosures Wijk: Agios Pharmaceuticals Inc.: Research Funding; RR mechatronics: Research Funding.

C/EBPε, a member of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors, is exclusively expressed in myeloid cells and regulates transition from the promyelocytic stage to the myelocytic stage of neutrophil development, being indispensable for secondary and tertiary granule formation. Knowledge concerning the functional role of C/EBPε posttranslational modifications is limited to studies concerning phosphorylation and sumoylation. In the current study, using ectopic expression and ex vivo differentiation of CD34(+) hematopoietic progenitor cells, we demonstrate that C/EBPε is acetylated, which was confirmed by mass spectrometry analysis, identifying 4 acetylated lysines in 3 distinct functional domains. Regulation of C/EBPε acetylation levels by the p300 acetyltransferase and the sirtuin 1 deacetylase controls transcriptional activity, which can at least in part be explained by modulation of DNA binding. During neutrophil development, acetylation of lysines 121 and 198 were found to be crucial for terminal neutrophil differentiation and the expression of neutrophil-specific granule proteins, including lactoferrin and collagenase. Taken together, our data illustrate a critical role for acetylation in the functional regulation of C/EBPε activity during terminal neutrophil development.

Background: The group of rare hereditary anemias includes a large variety of intrinsic defects of the red blood cell, as well as erythropoiesis. They include hemolytic anemias (e.g. enzyme deficiencies), hemoglobinopathies, hypoplastic anemias (e.g. Diamond-Blackfan Anemia, DBA), and dyserythropoietic anemias. As a result of the rapid developments in genetic testing and the subsequent increased knowledge of molecular defects underlying hereditary anemias, our understanding of the pathophysiology of rare anemias has increased during the last decade. However, in a substantial number of patients, the clinical phenotype does not fit classical criteria of a disease, response to therapy is less than expected, or a molecular defect cannot be found. In addition, in patients with well-described molecular defects, there is often no clear genotype-phenotype correlation. In order to better understand the underlying pathophysiological mechanisms driving ineffective erythropoiesis in patients and to improve their classification and clinical evaluation, novel functional tests are needed. Metabolomics is the large-scale, unbiased study of metabolites and their interactions within a biological system, directly reflecting the underlying biochemical activity and state of cells. Metabolomics can be used to identify novel disease biomarkers, study deregulated cellular pathways, and to determine the cellular responses to therapeutic interventions. In this study we demonstrate that dried blood spots (DBS) can be used as a minimal invasive and validated technical approach to perform large scale metabolomics in a variety of rare hereditary anemias. Methods: DBS samples from >100 patients suffering from a variety of rare anemiaswere collected during regular hospital visits. Quantification of metabolites was performed by direct infusion high resolution mass spectrometry (DI-HRMS) followed by an untargetedmetabolomics pipeline. For annotation, the Human Metabolome Database (HMDB) was used. Results were compared with DBS samples of 70 healthy adult controls and 35 pediatric patients negatively screened for metabolic diseases Results: For each patient sample, Z-scores were calculated for all mass peaks annotated with metabolites (HMDB, 3930). Mass peak, intensity and corresponding Z-scores were compared with two distinct groups of controls (∆Z-scores): pediatric patients who were screened for metabolic diseases but were found negative, and healthy adult controls. For data interpretation, two strategies were used. First, by untargeted statistical analysis in Metabo-analyst, we identified metabolites (and/or isomers) that showed either increased or decreased intensity. For the second strategy we specifically focused on red blood cell metabolic pathways, including glycolysis, the pentose phosphate pathway, ascorbate and glutathione metabolism, arginine and polyamine metabolism, and erythrocyte membrane turnover and transport. We corrected for a potential hematocrit effect and performed subgroup analyses correcting for reticulocyte counts. Our preliminary data indicate potential biomarkers for distinct disease entities, including altered polyamine metabolism (DBA, SCD), glycolysis (DBA, HS), and aberrant arginine metabolism (SCD) (Figure 1). Further in-depth pathway analyses, and targeted validation of biomarker profiles are currently being performed. Conclusion: Untargeted metabolomics using dried blood spots provides a novel functional tool to identify disease biomarkers and common and distinct deregulated cellular pathways. This will improve diagnostic evaluation and clinical management of patients with rare hereditary anemias, contribute to a better understanding of disease pathophysiology, and aid in the development of therapeutic strategies. Disclosures van Beers: Agios Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Research Funding; RR Mechatronics: Research Funding. van Wijk:RR Mechatronics: Research Funding; Agios Pharmaceuticals: Consultancy, Research Funding.

Background: In sickle cell disease (SCD), hemoglobin S (HbS) polymerizes upon deoxygenation, reducing red blood cell (RBC) deformability. RBC deformability can be measured over a range of oxygen concentrations using a Laser Optical Rotational Red Cell Analyzer (Lorrca) ektacytometer (RR Mechatronics, Zwaag, the Netherlands) with Oxygenscan module. The Oxygenscan measures 3 key parameters: 1) EImax, RBC deformability at normoxia; 2) EImin, RBC deformability upon deoxygenation; and 3) the point of sickling (PoS): the oxygen tension at which a 5% decrease in normoxic EI is observed during deoxygenation, reflecting the patient-specific pO2 at which sickling begins (Figure 1A). Previously we showed that Oxygenscan parameters correlate with measures of SCD disease severity and hemolysis (absolute reticulocyte count, %fetal hemoglobin, %HbS, total Hb, %dense RBC, (Rab et al. Blood 2018). In this study, we investigated the relationship between oxygenscan parameters and incidence of vaso-occlusive crisis (VOC), and we tested the ability of the Oxygenscan parameters to assess response to hydroxyurea (HU) and chronic transfusion (CTF) in patients with SCD. Methods: We analyzed 2 cohorts: a European cohort (EUC) of 62 SCD patients (all HbSS or HbS/β-thalassemia), enrolled at either University Medical Center Utrecht (UMCU, n= 42) or Hospital Lyon (LIBM, n=20), and a US cohort (USC) of 97 SCD patients enrolled at Texas Children's Hospital (TCH). Differences in Oxygenscan parameters in SCD patients without/with VOC (requiring a doctor's evaluation) in the past 2 years were measured in 46 adult EUC SCD patients and 80 pediatric USC SCD patients. EUC patients without VOC (n=18, median age 40.6 years (y); 11 female (F), 44% on HU, 6% on CTF, 28% on both treatments), were compared to patients with a positive history of VOC (n=28, median age 23.9y, 13F, 39% on HU, 11% on CTF and 11% on both). Patients without VOC in the USC (n= 34, median age 8.0y, 15F, 53% on HU, 15% on CTF and 21% on both treatments) were compared to patients with VOC (n=46, median age 11.8y, 20F, 74% on HU, 13% on CTF and 11% on both treatments). To establish treatment related Oxygenscan parameter changes, we analyzed RBCs from 9 SCD patients from UMCU (median age 19y, 5F), before and during HU treatment (measurements performed at baseline, and 1, 3 and 6 months after starting HU), 7 SCD patients from UMCU (median age 26.7y; 6F) before and after transfusion and 17 SCD patients from TCH (median age 10.8y; 6F) on HU before and after transfusion. Results: In the EUC, PoS differed significantly between patients without VOC in the last 2 years (median 41.6mmHg) and patients with VOC in the last 2 years (median 53.7 mmHg, p Transfusion improved EImax, EImin, and PoS (USC: p Conclusion: Our results show that RBC from SCD patients without VOC in the last two years were able to tolerate lower oxygen concentrations before sickling (PoS). RBCs from patients without VOC were also more deformable when deoxygenated (EImin) compared to patients who had experienced one or more VOCs in the last two years. In contrast, RBC deformability, when oxygenated (EImax) was not different in patients with or without VOC in the last two years. All 3 Oxygenscan parameters significantly improved with standard of care SCD treatments, namely CTF and/or HU. We therefore conclude that the PoS, EImax and EImin are useful biomarkers of clinical severity and treatment response, and may be essential in monitoring novel SCD treatments as part of a clinical trial as a surrogate endpoint. Disclosures Rab: RR Mechatronics: Research Funding. van Wijk:Agios Pharmaceuticals: Consultancy, Research Funding; RR Mechatronics: Research Funding. Bos:RR Mechatronics: Research Funding. Nur:Novartis Pharmaceuticals: Consultancy. Cnossen:NWO: Other: Governmental grants , ZonMW, Innovation fund and Nationale Wetenschapsagenda 2018; Roche: Other: Travel Grants; Takeda: Other: Travel Grants, Research Funding; Shire: Other: Travel Grants, Research Funding; Baxter: Other: Travel Grants, Research Funding; Sobi: Research Funding; CSL Behring: Other: Travel Grants, Research Funding; Nordic Pharma: Research Funding; Novo Nordisk: Research Funding; Bayer: Other: Travel Grants, Research Funding; Pfizer: Other: Travel Grants, Research Funding. van Beers:Agios Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Research Funding; RR Mechatronics: Research Funding.

Background/Objective: Paroxysmal nocturnal hemoglobinuria (PNH) is an extremely rare disease and is even more rare in children; pediatric cases are estimated to account for Methods: The analysis included eculizumab-treated patients enrolled in the Registry as of May 2017. Patients were stratified by age at baseline (defined as the date of eculizumab initiation; age categories, Results: Of 4903 patients enrolled in the Registry as of May 1, 2017, 1725 had been treated with eculizumab and had data available on demographics and enrollment date (5 times the upper limit of normal at baseline to normal or near normal range at last follow-up in both age cohorts. There were no TEs reported in the pediatric cohort after baseline, but 2/43 patients (4.7%) with no history of MAVEs at baseline experienced non-TE MAVEs after baseline. Among adult patients, 19/1497 (1.3%) with no history of TEs and 7/1497 (0.5%) with history of TEs experienced TEs after baseline. Non-TE MAVEs after baseline in patients with no history of MAVEs were experienced by 17/1492 patients (1.1%) and non-TE MAVEs after baseline in patients with history of MAVEs were experienced by 15/1492 patients (1.0%). The proportion of patients requiring transfusion was lower at last follow-up in both cohorts compared with baseline (23.0% at last follow-up vs 49.3% at baseline for adults and 32.3% at last follow-up vs 48.4% at baseline for pediatric patients). A similar proportion of patients became transfusion-independent after treatment with eculizumab in both age cohorts (32.3% [10/31] in the pediatric cohort compared with 33.8% [370/1095] in the adult cohort). The proportion of patients with PNH-related symptoms decreased after baseline in both cohorts for most of the symptoms assessed, although fewer than 10 pediatric patients had symptom data available at both baseline and last follow-up. Conclusions: The effectiveness of eculizumab for reducing intravascular hemolysis and transfusion requirements, and preventing MAVEs (including TEs) was similar in these large cohorts of pediatric patients with PNH and adults with PNH. The higher proportion of patients transfusion-dependent among the pediatric cohort may be associated with the higher likelihood of underlying aplastic anemia in pediatric patients with PNH. Table. Table. Disclosures Urbano-Ispizua: Alexion Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees. Kulasekararaj:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Other: Travel Support . Bartels:Alexion Pharmaceuticals, Inc.: Honoraria, Other: Travel Support and is site investigator for clinical trials with the company, Research Funding. Patriquin:Ra Pharmaceuticals: Consultancy, Research Funding; Octapharma: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Other: Travel Support and is site investigator for clinical trials with the company. Hoechsmann:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding. Wilson:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership. Gustovic:Alexion Pharma GmbH: Employment, Equity Ownership. Schrezenmeier:Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding.

Abstract 215 Dysregulation of myeloid differentiation can result in the development of a variety of pathological conditions ranging from bone marrow failure to myelodysplastic syndromes and leukemia. Neutrophil development is tightly regulated by key transcription factors including CCAAT enhancer-binding protein-alpha (C/EBPα) and CCAAT enhancer-binding protein-epsilon (C/EBPε). In recent years it has become clear that the expression and function of such proteins is regulated by post-translational modifications. Here, we have investigated the regulation and functional role of C/EBPε acetylation. Our results demonstrate that C/EBPε is indeed acetylated and that this can be increased by the lysine acetyltransferases (KAT) TIP60 and p300, as well as the sirtuin 1(SIRT1) inhibitor nicotinamide (NAM). In agreement with this, acetylation was decreased upon co-transfection of SIRT1. Despite normal expression levels and the capacity to form homo- and heterodimers, the C/EBPε-lysine dead mutant (C/EBPε K15xR) was transcriptionally inactive in luciferase reporter assays, suggesting that acetylation of C/EBPε is functionally important. Moreover, co-transfection of SIRT1 inhibited C/EBPε transcriptional activation. In order to investigate the functional relevance of acetylaton of C/EBPε, we retrovirally transduced CD34+ hematopoietic progenitors with C/EBPε or C/EBPε K15xR and differentiated sorted progenitors towards mature neutrophils. We observed a significant decrease in the percentage of mature neutrophils after transduction with C/EBPε K15xR compared to C/EBPε or control cells. Cytospin analysis demonstrated an immature phenotype of the C/EBPε K15xR-transduced cells, suggesting a differentiation block at the promyelocytic stage. In order to determine the functionally important acetylated lysine residues, we performed mass spectometry and identified four C/EBPε acetylation sites including two sites in the repression domain and one site in the basic region, which includes the DNA-binding domain. To investigate the functional role of acetylation of these lysine residues, we designed specific C/EBPε lysine mutants. Utilizing the previously described ectopic expression system, we observed reduced levels of total C/EBPε acetylation upon transfection of two specific lysine mutants (K121R and K198R). Acetylation of these specific residues was confirmed by performing add-back experiments in the C/EBPε K15xR background. Furthermore, upon transfection of C/EBPε K121R and C/EBPε K198R in a luciferase reporter assay, we observed reduced transcriptional activation by C/EBPε, suggesting that acetylation of lysine 121 and lysine 198 is important for C/EBPε function. Currently we are investigating the functional role of acetylation of these specific lysine residues during neutrophil differentiation. There is increasing knowledge concerning the role of epigenetic modifications in the development of myeloid malignancies. However, the specific role of acetylation of non-histone proteins, including key-transcription factors involved in myelopoiesis, remains largely unknown. We demonstrate, for the first time, the functional importance of C/EBPε acetylation during neutrophil differentiation. Together our data provide new insights in the regulation of both normal and aberrant myeloid differentiation. Disclosures: No relevant conflicts of interest to declare.

Epigenetic modifications play an important role in the pathogenesis of myeloid malignancies of which alterations in cellular acetylation profiles have proven to contribute substantially. The clinical use of chromatin modulating drugs, such as histone deacetylase (HDAC) inhibitors, has more recently been evaluated in several clinical trials. Nonetheless, little is known concerning the effects of HDAC inhibitors on myelopoiesis or their specific targets in human hematopoietic stem cells. In order to investigate this, we utilized a differentiation system in which umbilical cord blood derived CD34+ cells were differentiated ex-vivo in liquid and semi-solid cultures. We investigated the effect on lineage development, colony-forming potential, proliferation and differentiation using trichostatin A (TSA; group I/II/IV, 5–25nM), sodium butyrate (SB; group I/IIa, 100–500μM) and valproic acid (VPA; group I/IIa, 100–500μM). In our liquid culture system we observed that TSA increased the numbers of CD34+ hematopoietic progenitors resulting in a dose dependent delay in granulocyte/macrophage progenitor (GMP) differentiation. Furthermore, TSA promoted differentiation towards megakaryocyte/erythroid progenitors (MEP) in the absence of thrombopoietin (TPO) and erythropoietin (EPO). SB promoted a shift towards the granulocyte/macrophage lineage, and inhibited proliferation and differentiation of CD34+ progenitors. VPA increased the number of CD34+ hematopoietic progenitors in a dose dependent manner, with increased numbers of MEP and a delay in GMP differentiation. To investigate the colony-forming potential of hematopoietic progenitors and further define the effect on lineage choice decisions in the presence of HDAC inhibitors, we also used a semi-solid culture system. In agreement with the results of the progenitor analysis, 100–200mM VPA stimulated granulocyte/macrophage colony formation which was also observed after 100mM SB treatment, although colonies were smaller and less differentiated. All three HDAC inhibitors showed a dose dependent inhibition of erythroid colony-forming potential and differentiation. We also used the liquid culture system to examine proliferation during neutrophil development. TSA moderately increased CD34+ progenitor expansion in a dose dependent manner, while 100–500mM SB and 500mM VPA abrogated expansion, eventually leading to decreased cell survival. In contrast, 100–200mM VPA increased cell expansion without affecting differentiation potential. Neutrophil differentiation was improved, although not significantly, by TSA, while SB inhibited neutrophil differentiation showing dysplastic features and signs of early apoptosis. Treatment with 500μM VPA also inhibited neutrophil differentiation reflected by an increased amount of common myeloid progenitors (CMP), a differentiation block in immature neutrophils and decreased cell survival. In order to determine the effects of HDAC inhibition on histone acetylation we performed Western Blot analysis on protein lysates from hematopoietic progenitors. SB and VPA strongly increased acetylation of total H4 with hyperacetylation of H4K16 and this effect was observed to a lesser extent with TSA. During myeloid differentiation, all three HDAC inhibitors clearly increased total H3 acetylation as represented by H3K9 hyperacetylation. Taken together, we observed comparable effects on histone acetylation profiles, but distinct effects on myeloid progenitor function and neutrophil development. These data suggest that the effects observed in hematopoietic progenitor function are likely to include effects of HDAC inhibitors on non-histone targets. Furthermore, the differences between the effects of TSA, SB and VPA suggest inhibitor specific effects (HDAC preference) or cell specific responses. We are currently investigating the molecular mechanisms underlying these observations.