ALBERT

Description: Background: Hydroxyurea (HU) is highly clinically beneficial in patients with sickle cell disease (SCD). While HU reduces leukocytosis and inflammation, in general, patients with high levels of fetal hemoglobin (HbF) induction experience the greatest clinical benefit from HU. Despite over 25 years of use in patients with SCD, and intense study, the mechanism by which HU induces HbF has not been elucidated, nor the reasons for the considerable inter-individual variability in the amount of HU-mediated HbF induction. Proposed mechanisms of HbF induction by HU include erythropoietic stress response, production of nitric oxide, or reduction of BCL11A, KLF-1, and MYB, negative regulators of HbF. Various signaling pathways have been implicated in HU-mediated HbF induction, such as the activation of the ERK-1/ERK-2 by erythropoietin; Giα/JNK/Jun; p38/MAPK/CREB1; histone deacetylase (HDAC) and DNA methyl-transferase (DNMT) inhibitor-mediated epigenetic modification of γ-globin expression. To identify new genes and pathways involved in HU activity, we studied the in vivo effects of HU on the gene expression profile of paired samples obtained from the reticulocytes of children with SCD before and after HU treatment. Methods : 25 paired RNA samples were collected before HU treatment (pre-HU) and at the stable maximum tolerated dose (MTD) from peripheral blood CD71+ reticulocytes of pediatric patients from Texas Children's Hospital. Subjects were 1.2 to 19.1 years of age at the time of pre-HU sample collection (23 HbSS, 2 HbSβ0) and 48% were female. Hematologic indices were extracted from patient's medical record. RNA sequencing was performed through Illumina platform, paired-end 150 bps, with at least 20 million read in-depth. Quality control of raw sequence data was done with FastQC software. We used Kallisto to determine the compatibility of reads with targets and quantify abundances of transcripts. Then, we used Sleuth to implement statistical algorithms for differential analysis. After validation of results through relative quantity assessment of selected genes by RT-qPCR, the Enrichr web server was used for gene set enrichment (GSE) and pathway analysis. We chose two candidate genes, KLF4 and STAT5A, to analyze functionally based on differential expression, inclusion in enriched pathway analysis, and reported involvement in relevant biological processes, including erythroid maturation or HbF induction. siRNA knockdown of KLF4, and STAT5A in K562 cells was performed by chemical transfection (DharmaFECT) and was verified by RT-qPCR. K562 cells were treated with HU at 50 µM for 24 hours; RNA was extracted and analyzed for γ-globin expression levels by RT-qPCR. Results and conclusions: We compared our pre-HU and at MTD paired samples using a two-fold change expression cut-off and 0.1 FDR multiple comparison correction threshold. Using this threshold, 139 genes had statistically significant differential levels of expression.135 were upregulated and 4 downregulated in response to HU treatment. Pathway analysis primarily grouped the upregulated genes more into the EPO, IL-2, and IL-3 signaling pathways (adjusted p-value

Description: Sickle cell disease (SCD) affects 〉100,000 Americans and millions more worldwide. Symptoms and sequelae of SCD can be ameliorated by increasing fetal hemoglobin (HbF, α2γ2) levels. Unfortunately, up to 50% of adult SCD patients treated with hydroxyurea, the only FDA-approved and widely used HbF inducer, do not have a clinically meaningful response to the drug. Additional oral HbF inducing agents, especially those that require less intense laboratory monitoring, are urgently needed. Development of such drugs has been stymied by an incomplete understanding of γ-globin regulation. We hypothesized that natural human genetic variation can be used to identify genes that may be drug targets for HbF induction. To test this hypothesis, we performed whole exome sequencing on 171 pediatric SCD patients to identify variants associated with endogenous HbF levels. Gene-based analysis identified seven unique non-synonymous variations in a Forkhead box O transcription factor, FOXO3, as significantly associated with lower HbF (p=5.6x10-4, β-value of log transformed (ln) HbF= -0.66). Two variants in the α2 subunit of AMPK, a FOXO3 activator, were also associated with reduced HbF (p=1.56x10-4, β-value ln%HbF=-1.5). We then performed functional studies to verify the association between FOXO3 and endogenous HbF levels in an ex vivo model of erythroid differentiation from CD34+ cells isolated from peripheral blood of normal human blood donors. Lentiviral short hairpin RNA (shRNA) knockdown of FOXO3 reduced γ-globin expression from 1 to 0.4, p= 0.0005. While γ-globin expression and protein levels were reduced by FOXO3 knockdown, β-globin levels remained unchanged. These results suggest that FOXO3 is a positive regulator of γ-globin. Morphologic and flow cytometry analysis of primary erythroid culture with and without FOXO3 knockdown indicates that knockdown of FOXO3 delays erythroid maturation, while reducing γ-globin production. We therefore conclude that FOXO3 appears to regulate γ-globin through a specific mechanism rather than through alteration of erythroid maturation kinetics. FOXO3 is a viable therapeutic target for the treatment of individuals with SCD as well as those with quantitative hemoglobinopathies like β-thalassemia, who do not benefit from hydroxyurea due to its delay of erythropoiesis. FOXO3 expression is known to be increased by three drugs, metformin, phenformin, and resveratrol. We found that these drugs also cause FOXO3 to accumulate in the nucleus, where it is active, rather than in the cytoplasm, where FOXO3 is degraded. We have investigated the effects of these agents on FOXO3 and γ-globin expression in K562 cells. Metformin, phenformin and resveratrol increased FOXO3 and γ-globin transcription levels in a dose-dependent manner. We then treated primary erythroid culture cells with a range of metformin doses (20-200µM), with and without a stable dose of 30 µM hydroxyurea. Alone, metformin had a modest effect (1.5 fold) on γ-globin induction at all concentrations. In combination with hydroxyurea, 50 µM metformin increased γ-globin expression 3.7-fold compared to 2.5-fold with hydroxyurea alone when analyzed by RT-qPCR. β-globin levels were unchanged by hydroxyurea or metformin. γ-globin induction persisted through terminal maturation of the culture when measured serially every 5 days. Taken together, our results indicate that not only is FOXO3 is a positive regulator of γ-globin expression, but it is an excellent therapeutic target for HbF induction. Metformin, a well-studied, well tolerated oral agent, will be investigated in combination with hydroxyurea in a phase II trial as an adjunctive agent to increase HbF induction. Disclosures Off Label Use: This presentation will discuss off-label use of metformin as a possible HbF inducing agent..

Description: Abstract 241 Although they ostensibly have a monogenetic disease, individuals with sickle cell anemia exhibit wide variability in their laboratory and clinical phenotype, suggesting additional genetic modifiers exist beyond the sickle mutation. One of the most powerful and reproducible disease modifiers is fetal hemoglobin (HbF) level. The most widely used and safest method for increasing innate fetal hemoglobin levels in patients with sickle cell anemia is hydroxyurea. While hydroxyurea has disease modulating effects outside of HbF induction, the majority of its benefit is directly related to the %HbF produced in response to the drug. Unfortunately, the amount of the HbF produced in response to hydroxyurea is highly variable between individuals, with induced HbF levels ranging from 5 to 〉30% even for compliant patients on similar dosing regimens at the maximum tolerated dose (MTD). Hydroxyurea is an ideal target for pharmacogenomics investigation, since there is strong concordance of HbF response to hydroxyurea within sibling pairs, and amount of HbF produced at MTD is a quantifiable and objective phenotype. To address the hypothesis that genetic modifiers influence pharmacological induction of HbF, we investigated pediatric patients treated prospectively with hydroxyurea. We analyzed patients enrolled in the HUSTLE (NCT NCT00305175) and SWiTCH (NCT 00122980) studies (n=183); all patients received an identical dose escalation regimen to MTD and had the most reliable HbF phenotypes available. To best identify genetic modifiers of hydroxyurea induction of HbF, we categorized study subjects according to their HbF response. Of a total cohort of 183 treated subjects, we identified 55 pediatric patients who represented the extreme ends of HbF response to hydroxyurea: 30 high responders (final HbF 〉30% and 〉25% change from %HbF at baseline) and 25 low responders (final HbF

Description: Background: In sickle cell anemia (SCA), hemoglobin S (HbS) polymerizes upon deoxygenation, resulting in sickling of red blood cells (RBCs). These deoxygenated RBCs have strongly reduced deformability, which contributes to the etiology of vaso-occlusive crises and chronic hemolytic anemia. There are no widely available clinical laboratory tests to directly monitor effects of disease modifying therapies (i.e. hydroxyurea) on RBC deformability. RBC deformability can be measured using a Laser Optical Rotational Red Cell Analyzer (Lorrca) ektacytometer (RR Mechatronics, the Netherlands), which measures RBC deformability over a range of osmolalities. Recently, a new module was added which consists of a method to measure RBC deformability, expressed as Elongation Index (EI), during controlled deoxygenation. This test, termed oxygenscan, has 3 key read out parameters: 1) EImax, which represents RBC deformability at normoxia; 2) EImin represents deformability upon deoxygenation; and 3) the point of sickling (PoS), the point at which a 〉5% decrease in EI is observed during deoxygenation, reflecting the patient-specific pO2 at which sickling begins (Figure 1). In this study, we correlated laboratory parameters associated with SCA disease severity with oxygenscan parameters to establish the clinical utility of this test. Methods: The discovery cohort consisted of 15 SCA patients (median age 22.0 years, 33.3% on hydroxyurea (HU)) enrolled at University Medical Center Utrecht (UMCU). The validation cohort consisted of 21 patients with SCA (median age 12.5 years, 76.2% on HU) from Texas Children's Hematology Center (TCHC). Oxygenscans were carried out in duplicates at both sites. Percentage dense RBC (%DRBC) were measured using an ADVIA hematology analyzer (Siemens) at TCHC only. In this study, we used Pearson's correlation to test for linear correlations between oxygenscan parameters EImax, EImin and PoS and clinically relevant laboratory parameters: total hemoglobin (Hb), absolute reticulocyte count (ARC), %HbS and %HbF, and %DRBC. Results: In both cohorts PoS significantly positively correlated with ARC (Figure 2A-B). In the UMCU cohort, total Hb levels also significantly positively correlated with EImax (Figure 2C), which was validated in the TCHC cohort (Figure 2D). HbF positively correlated with the EImin in both cohorts (Figure 2E-F). EImin also significantly negatively correlated with HbS (r=-0.828 p=

Description: Background: Fetal hemoglobin (HbF, α2g2) induction has long been an area of investigation, as it is known to reduce the clinical complications of sickle cell disease (SCD) and beta thalassemia. Progress in identifying novel HbF inducing strategies has been stymied by an incomplete understanding of gamma-globin regulation. We used natural genetic variation to identify novel genes and pathways associated with HbF levels in patients with SCD. Our whole exome sequencing analysis of 1290 samples from patients with SCD identified the insulin signaling pathway to be related to HbF regulation. Functional studies performed in hematopoietic stem and progenitor cells (HSPCs) from patients with SCD established that FOXO3 is a positive regulator of HbF, and that metformin, a FOXO3 and AMPK activator, can induce HbF (Zhang et al, Blood 2018). We hypothesized that other proteins in the insulin signaling pathway, particularly AMPK, a direct activator of FOXO3, may contribute to HbF regulation and be a potential target for pharmacologic induction of HbF. Objectives: We now seek to determine the role of AMPK and AMPK activators such as piceatannol in HbF regulation through functional studies in HSPCs from patients with SCD. Methods: HSPCs from 3 unique patients with SCD were transduced with AMPK shRNA on day 5 of two phase primary erythroid culture. AMPK, FOXO3, gamma and beta globin gene expression were measured by RT-qPCR and HbF by HPLC respectively on day 14 of culture. HSPCs from 3 unique patients with SCD were treated with AICAR, piceatannol at 12.5µM and metformin at 100 µM on day 7 of erythroid culture. Cell lysate was collected on day 14, and AMPK, FOXO3, gamma and beta globin gene expression and protein levels measured by RT-qPCR and western blot respectively. Levels of pAMPK, at Thr172, were quantified by western blot. 1 µM Compound C was added with piceatannol and with metformin in separate erythroid cultures on day 7, and the effect on gamma globin and phosphorylation of AMPK at Thr172 was measured on day 14 by RT-qPCR and western blot respectively. Results: 70% knockdown of AMPK resulted in a 50% decrease in HbF (p

Description: Background: Fetal hemoglobin (HbF, α2g2) induction is known to reduce the clinical complications of sickle cell anemia (SCA). Progress in identifying novel HbF inducing strategies has been slowed by an incomplete understanding of gamma-globin regulation. We have used natural genetic variation to identify novel genes and pathways associated with HbF levels in patients with SCA, beginning with whole exome sequencing (WES). This approach identified FOXO3, a transcription factor important for insulin signaling and erythroid maturation, among other functions, as a positive regulator of HbF. We then confirmed the role of FOXO3 in HbF regulation with functional studies in erythroid culture (Zhang, Blood 2018). To overcome the limitations of WES, namely the absence of regulatory and promotor sequencing data, we performed whole genome sequencing (WGS) on 658 pediatric SCA patients, and analyzed the data for common variants predictive of HbF levels. Methods : WGS was performed on a cohort of 658 pediatric patients with HbSS and HbSβ0 with IRB approval from Texas Children's Hospital, and from St Jude Children's Research Hospital, as part of the Sickle Cell Clinical Research and Intervention Program (Hankins et al 2018). Baseline HbF levels, not on hydroxyurea therapy, was measured by HPLC. Subjects were between 6 months and 21 years of age from both institutions; 52% were male. We used mixed linear regression models to screen for variants associated with transformed HbF values, and performed ridge regression with 10-fold cross-validation to confirm associations after adjustment for HBG and BCL11A-associated variants, age, sex, and race (determined by principal components analysis). Hematopoietic stem and progenitor cells (HSPCs) from 3 patients (all HbSS) were treated with recombinant human IGFBP3 (1µg/ml) beginning on day 7 of two-phase culture. Effects of IGFBP3 on gamma-globin expression were evaluated by RT-qPCR, and on HbF levels by HPLC, on days 14 and 21 of culture. The effects of IGFBP3 on known modifiers of HbF and the FOXO3 pathway were assessed by RT-qPCR and western blot on day 21 of culture. Erythroid maturation was assessed by flow cytometry using anti-CD71, GPA, and Band3 on day 21 of culture. Results: Our whole genome sequencing data identified a strong association between all 11 variants 200 kb upstream of IGFBP3 and baseline HbF levels (p

Description: Elevated fetal hemoglobin (HbF, α2γ2) ameliorates the pathophysiology of sickle cell disease (SCD) by diluting HbS and inhibiting its polymerization in patient red blood cells. Hydroxyurea (HU) the only approved drug for SCD acts in part by raising HbF levels. However, up to 50% of adult SCD patients treated with hydroxyurea, do not have a clinically meaningful response, illustrating the need for new improved agents. We identified FOXO3, a potentially "druggable" transcription factor, as a candidate HbF inducer through analysis of rare variants from whole exome sequence data from SCD patients. Specifically, numerous rare variants that potentially impair FOXO3 activity and/or expression were associated with reduced HbF levels. FOXO3 was previously shown to facilitate erythroid precursor survival, maturation and resistance to oxidative stress. To investigate our findings from genomic studies further, we manipulated FOXO3 expression in normal human CD34+ hematopoietic stem and progenitor cells (HSPCs) induced to undergo erythroid differentiation and examined the effects on γ-globin RNA levels and HbF production. Transfection of CD34+ HSPCs with lentivirus expressing FOXO3 shRNA that reduced levels of the corresponding RNA by 80% reduced γ-globin (HBG1 and HBG2) mRNA by 60% compared to control cultures expressing scrambled shRNA (n= 3 separate experiments, p 〈 0.0005). A second non overlapping FOXO3 shRNA reduced the corresponding mRNA by 50% and reduced γ-globin by 31% (n=3 separate experiments, p 〈 0.0001). Reduction of FOXO3 mRNA was accompanied by delay in differentiation (11% GPA+ cells with 80%FOXO3 kd by day 14 compared to 72% GPA+ cells in the scramble kd). Metformin, an FDA-approved drug used for type 2 diabetes, is known to increase FOXO3 expression and therefore, we tested its effects on HbF expression in CD34+ HSPC-derived erythroblasts by treating with 30, 50 and 100 µM drug, beginning at day 5 of culture. At day 14 of culture, 59% late basophilic erythroblasts were present in control cultures, as evidenced by band3 and α4 integrin expression. In contrast, maturation was accelerated in metformin treated cultures, with 70% late basophilic erythroblasts, consistent with previous studies. Remarkably, metformin treatment also caused a dose dependent increase in HbF-immunostained cells ("F-cells", Figure 1) and in % HbF, as measured by HPLC (Figure 2). HPLC analysis at d14 showed that %HbF (HbF/(HbF + HbA) rose from 10.3% at baseline, to 19.8% and 30% at 50 and 100 µM metformin (n= 3, p=0.04 and 0.005). For dosage comparison patients on standard metformin therapy for diabetes typically exhibit plasma drug levels of 70 µM. When cultures were treated with metformin (100µM) and HU (30µM), %HbF was increased to 45% compared to 26% and 30% in cultures treated with HU or metformin respectively as single agents (n=3, p=

Description: Background: Sickle Cell Disease (SCD) is a devastating inherited disease, characterized by polymerization of sickle hemoglobin under deoxygenated conditions that can lead to acute pain crises, ischemia, and chronic organ damage. Pharmacologic anti-sickling agents that decrease polymerization are currently under investigation, however there is no consistent in vitro system to study these compounds; whole patient blood, subject to clinical variability and limited supply, is most often used. Human Umbilical Cord Derived Erythroid Progenitor 2 cells (HUDEP2) are an immortalized CD34+ hematopoietic stem cell (HSC) derived erythroid precursor cell line that can differentiate into red blood cells. We have engineered S-HUDEP2 cells to express sickle hemoglobin (HbS) via CRISPR/Cas9 gene editing. We hypothesized that this cell line will sickle under hypoxic conditions, produce dense red blood cells (DRBC, red cells with a density〉1.11 mg/mL that are dehydrated and prone to sickling. If these intrinsic, essential SCD RBC properties are found, we propose to use this novel cell line to screen drug compounds for anti-sickling capabilities. Methods: S-HUDEP cells were cultured as previously described (Kurita et al, 2013). %HbS and %HbA produced by parent HUDEP and S-HUDEP2 cells were measured by high performance liquid chromatography (HPLC). Hypoxia was induced by placing the cells at 2% O2 for four hours. Parent HUDEP and S-HUDEP2 cells were then fixed with glutaraldehyde and Giemsa stained. % sickling estimated at 40x magnification by a pathologist blinded to cell group counting sickle forms out of 1000 cells. The percentage of dense red blood cells (DRBCs) was quantified by an ADVIA hematology analyzer (Siemens). S-HUDEP2 cells were dosed with 0, 2.5 and 5 µM 5-hydroxymethylfurfural (5-HMF) and 75, 150, and 300 µM GBT440, two known anti-sickling agents, on day ten and day 14 of culture for one hour, subjected to hypoxic conditions and % sickling quantified as described above. Results: S-HUDEP2 cells express 98% HbS. Under hypoxia, 20% of S-HUDEPs sickle at day 10 of differentiation; 30% of S-HUDEP2 cells sickle at Day +14 of differentiation. Parent HUDEP-2 cells, which produce 98% HbA, did not sickle under hypoxic conditions at any stage of differentiation. 70% of S-HUDEP2 cells were determined to be DRBC under hypoxia at Day 10 and 14 time points; parent HUDEP-2 cells did not produce DRBC under hypoxia. Treatment of S-HUDEP2 cells with 5μM of 5-HMF and 150 μM of GBT440 reduced sickling by 40-50% under hypoxic conditions compared with untreated S-HUDEP2 cells (p

Description: Introduction: Over 300,000 infants are born with sickle cell disease (SCD) every year worldwide, including at least 1,000 in the US. Prenatal diagnosis by amniocentesis or chorionic villus sampling is available; but high cost, invasiveness, and risk of miscarriage limit their use. Recently, non-invasive prenatal testing (NIPT) has become commonplace for aneuploidies, including Trisomy 21. These non-invasive tests operate by genetic analysis of the cell-free fetal DNA (cffDNA) present in maternal blood. The safety and accuracy of NIPT have been key drivers for its rapid and widespread adoption. Yet, no NIPT for SCD or other hemoglobinopathies have been commercialized to date, despite the large numbers of patients affected in the US and worldwide. While de novo mutations can only be of fetal origin and can be identified by available next-generation sequencing (NGS) methods, NIPT for recessively inherited disorders is more challenging. This is because a mother who is a carrier for a recessive disorder contributes a high level of background pathogenic DNA molecules. Therefore, a key technical challenge of NIPT for recessive disorders is developing an assay sensitive enough to detect 1000 pre-clinical samples (mixtures of sheared SCT and SCD genomic DNA) to determine analytical sensitivity 〉98% and specificity 〉99%, even in the absence of paternal DNA. Conclusion: We have developed an assay for non-invasive prenatal testing of sickle cell disease. The results obtained to date indicate that the assay reliably detects fetal SCD when the fetal fraction is as low as 5%, the same limit as aneuploidy NIPT. A fetus with SCD has already been identified, and follow-up is ongoing with 〉20 pregnancies. Since the HBB NIPT is highly targeted, sequencing cost is

Description: Background: Many patients with sickle cell disease (SCD) require a surgical splenectomy for repeat splenic sequestration or hypersplenism, resulting in worsening anemia and/or thrombocytopenia, or abdominal discomfort. Higher rates of thrombosis, pain crises and acute chest syndrome (ACS) have been reported following surgical splenectomy, although the reasons for this are not known. We hypothesize that this clinical worsening post-splenectomy is due to hemorheological changes; studies of the effects of surgical splenectomy on hemorheology in non-SCD animal models found significant reductions in red cell deformability and increase in whole blood viscosity, or blood thickness, following splenectomy. Understanding the impact of surgical splenectomy on blood rheology is especially relevant for patients with SCD, who have many clinical complications as a result of their high whole blood viscosity for their given hemoglobin levels, and low hematocrit-to-viscosity ratio (HVR), a measure of oxygen carrying capacity. Another important measure of SCD rheology is percent dense red blood cells (%DRBC), red cells with a density〉1.11 mg/mL; they are typically the result of cellular dehydration, and are less deformable and more likely to sickle. We therefore sought to use our existing longitudinal rheology data, including measures of viscosity and %DRBC, to evaluate the impact of surgical splenectomy on our pediatric patients with SCD. Methods: We identified seven pediatric patients with multiple measurements of whole blood viscosity and %DRBC, collected before and after surgical splenectomy between November 2013 and April 2018 from SCD patients at Texas Children's Hospital on an IRB approved protocol. The cohort included 4 female and 3 male patients, ages 3-12 years old. Whole blood viscosity was measured using a cone and plate viscometer (DV3T Rheometer, AMETEK Brookfield, Middleboro, MA, USA) at 37 degrees Celsius within 4 hours of sample collection in an EDTA vacutainer tube. CBC data including %DRBC was measured on an ADVIA 120 Hematology System (Siemens Medical Solutions USA, Inc., Malvern, PA, USA). Samples collected 1 month before or after an emergency department visit or within 3 months of a packed red blood cell transfusion were omitted from analysis. Results: We found a significant rise in %DRBC following splenectomy (p=0.01). There was a significant increase in whole blood viscosity at 45 s-1 and 225 s-1 (p=0.006 and p=0.004, respectively) and a decline in hematocrit-to-viscosity ratio (HVR) at 45 s-1 and 225 s-1 (p=0.03 and p=0.03, respectively) (Table 1). Hemoglobin and hematocrit did not significantly change after splenectomy (p=0.6 and p=0.5, respectively), suggesting that the rise in viscosity was due to intrinsic changes in red cell rheology. Platelets increased markedly (p