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Papers by Betty Pace

Blood, Apr 28, 2022

Excessive intravascular release of lysed cellular contents from damaged red blood cells (RBCs) in... more Excessive intravascular release of lysed cellular contents from damaged red blood cells (RBCs) in patients with sickle cell anemia (SCA) can activate the inflammasome, a multiprotein oligomer promoting maturation and secretion of proinflammatory cytokines, including interleukin-1β (IL-1β). We hypothesized that IL-1β blockade by canakinumab in patients with SCA would reduce markers of inflammation and clinical disease activity. In this randomized, double-blind, multicenter phase 2a study, patients aged 8 to 20 years with SCA (HbSS or HbSβ0-thalassemia), history of acute pain episodes, and elevated high-sensitivity C-reactive protein >1.0 mg/L at screening were randomized 1:1 to received 6 monthly treatments with 300 mg subcutaneous canakinumab or placebo. Measured outcomes at baseline and weeks 4, 8, 12, 16, 20, and 24 included electronic patient-reported outcomes, hospitalization rate, and adverse events (AEs) and serious AEs (SAEs). All but 1 of the 49 enrolled patients were receiving stable background hydroxyurea therapy. Although the primary objective (prespecified reduction of pain) was not met, compared with patients in the placebo arm, patients treated with canakinumab had reductions in markers of inflammation, occurrence of SCA-related AEs and SAEs, and number and duration of hospitalizations as well as trends for improvement in pain intensity, fatigue, and absences from school or work. Post hoc analysis revealed treatment effects on weight, restricted to pediatric patients. Canakinumab was well tolerated with no treatment-related SAEs and no new safety signal. These findings demonstrate that the inflammation associated with SCA can be reduced by selective IL-1β blockade by canakinumab with potential for therapeutic benefits. This trial was registered at www.clinicaltrials.gov as #NCT02961218.

Cellular & Molecular Biology Letters, 1998

Journal of clinical and translational science, 2023

Although mentoring is critical for career advancement, underrepresented minority (URM) faculty of... more Although mentoring is critical for career advancement, underrepresented minority (URM) faculty often lack access to mentoring opportunities. We sought to evaluate the impact of peer mentoring on career development success of URM early career faculty in the National Heart Lung and Blood Institute-sponsored, Programs to Increase Diversity Among Individuals Engaged in Health-Related Research-Functional and Translational Genomics of Blood Disorders (PRIDE-FTG). The outcome of peer mentoring was evaluated using the Mentoring Competency Assessment (MCA), a brief open-ended qualitative survey, and a semi-structured exit interview. Surveys were completed at baseline (Time 1), 6 months, and at the end of PRIDE-FTG participation (Time 2). The following results were obtained. Between Time 1 and Time 2, mentees' self-assessment scores increased for the MCA (p < 0.01) with significant increases in effective communication (p < 0.001), aligning expectations (p < 0.05), assessing understanding (p < 0.01), and addressing diversity (p < 0.002). Mentees rated their peer mentors higher in the MCA with significant differences noted for promoting development (p < 0.027). These data suggest that PRIDE-FTG peer mentoring approaches successfully improved MCA competencies among URM junior faculty participants with faculty ranking peer mentors higher than themselves. Among URM faculty, peer mentoring initiatives should be investigated as a key strategy to support early career scholar development.

Blood Cells Molecules and Diseases, Mar 1, 2007

PUBLISHED BY IMPERIAL COLLEGE PRESS AND DISTRIBUTED BY WORLD SCIENTIFIC PUBLISHING CO. eBooks, 2007

Blood, Dec 8, 2017

Abstract The hemoglobinopathies including sickle cell disease (SCD) and β-thalassemia are the mos... more Abstract The hemoglobinopathies including sickle cell disease (SCD) and β-thalassemia are the most common genetic disorders in the world producing significant morbidity and mortality. Drug-mediated fetal hemoglobin (HbF) induction ameliorates the clinical severity of SCD and improves long-term survival. To define mechanisms of γ-globin regulation, previously we demonstrated the role of the Gγ-globin cAMP response element, in drug-mediated HbF induction (Sangerman et al., Blood 2006). Subsequently, data generated by the ENCODE project led us to investigate a region 4 kb upstream of Gγ-globin, which harbors a DNase hypersensitive site and enrichment of the histone 3 lysine 4, mono-methylated (H3K4Me1) enhancer mark, that we named the Fetal Chromatin Domain (FCD). Recently, a synthetic zinc finger DNA-binding domain targeting this region was shown to reduce γ-globin gene expression in K562 cells (Shen et al., Blood 128:320, 2016). Initially, we performed multispecies sequence alignments which revealed the FCD is located in a genomic region shared by Old World monkeys (humans and Baboons) with a delayed fetal to adult globin switch that occurs after birth. By contrast, the FCD region is absent in New World monkeys where the switch occurs during the fetal developmental stage. These findings support involvement of the FCD region in γ-globin regulation during hemoglobin switching. Subsequent studies were performed to define the functional role of the FCD during erythropoiesis. ENCODE long RNA-sequencing assay detected PolyA RNA species throughout the FCD region in K562 cells. Based on these findings, we designed two small-interference RNA molecules targeting the core FCD (siFCD). Both siFCD molecules were transfected into KU812 cells and individual γ-globin gene expression measured using RT-qPCR with gene-specific primers. The level of Gγ-globin mRNA increased by 64%, while Aγ-globin transcription was reduced 38%. These results suggest the FCD facilitates differential regulation of the γ-globin genes. To further characterize FCD function, a series of γ-globin promoter luciferase reporters were constructed with the sense or antisense 500-bp core FCD region, cloned downstream of the luciferase gene. In KU812 stable cell lines, the sense FCD construct enhanced Aγ-globin promoter activity 10-fold, whereas the antisense construct repressed promoter activity. By contrast, the FCD in both orientations silenced Gγ-globin promoter activity. These results support unidirectional gene-specific enhancer properties of the FCD. Subsequent pulldown assay with a 34-bp biotinylated FCD probe confirmed NFE2, BHLHE40 and cMyc binding, which was abolished by mutation of the E-box sequence (CACGTG) in the FCD core. To build on the KU812 cell data, we performed μChIP assay using primary erythroid progenitors generated from adult CD34+ stem cells (Zhu et al., Haematologica 2017). In our system, the γ-globin to β-globin gene switch occurred on day 4 in culture. Initially, we measured levels of acetylated histone H3 (AcH3), H3K4Me1, and TFIID binding in the β-globin locus control region hypersensitivity site 2 (HS2) and FCD. As expected, high AcH3 and H3K4Me1 marks occurred in HS2 consistent with its enhancer function. Similar patterns of histone marks and binding of TFIID, NFE2 and BHLHE40 occurred in HS2 and FCD region. It is known that HS2 participates in DNA looping to accomplish developmentally regulated globin gene expression during hemoglobin switching therefore chromosome conformation capture (3C) assay was performed in primary erythroid progenitors to determine if the FCD facilitates changes in chromatin structure. We observed strong long-range interactions between the FCD and HS2 with 4.4-fold maximal chromatin enrichment on day 5; likewise the FCD showed interactions with Aγ-globin (4.7-fold) and Gγ-globin (3.5-fold) by day 3 before switching occurred. Of note, low level interactions occurred between the FCD and β-globin gene at all time points tested. Collectively, these data support the FCD as a unidirectional enhancer element involved in γ-globin regulation during erythropoiesis. To further define mechanisms of FCD function, the effects of CrisprCas9-mediated deletions in the FCD on globin gene expression will be explored. Disclosures No relevant conflicts of interest to declare.

Experimental Biology and Medicine, Aug 31, 2019

Sickle cell disease (SCD) and b-thalassemia are inherited blood disorders caused by genetic defec... more Sickle cell disease (SCD) and b-thalassemia are inherited blood disorders caused by genetic defects in the b-globin gene on chromosome 11, producing severe disease in people worldwide. Induction of fetal hemoglobin consisting of two a-globin and two c-globin chains ameliorates the clinical symptoms of both disorders. In the present study, we investigated the ability of d-aminolevulinate (ALA), the heme precursor, to activate c-globin gene expression as well as its effects on cellular functions in erythroid cell systems. We demonstrated that ALA induced c-globin expression at both the transcriptional and protein levels in the KU812 erythroid cell line. Using inhibitors targeting two enzymes in the heme biosynthesis pathway, we showed that cellular heme biosynthesis was involved in ALA-mediated c-globin activation. Moreover, the transcription factor NRF2 (nuclear factor [erythroidderived 2]-like 2), a critical regulator of the cellular antioxidant response, was activated by ALA and contributed to mechanisms of c-globin activation; ALA did not affect cell proliferation and was not toxic to cells. Subsequent studies demonstrated ALA-induced c-globin activation in erythroid progenitors generated from normal human CD34 þ stem cells. These data support future study to explore the potential of stimulating intracellular heme biosynthesis by ALA or similar compounds as a novel therapeutic strategy for treating SCD and b-thalassemia.

Blood, Nov 16, 2006

The design and evaluation of therapies for sickle cell disease rely on our understanding of the s... more The design and evaluation of therapies for sickle cell disease rely on our understanding of the sequential expression of the β-like globin genes during development. Drug inducers that reactivate γ-globin transcription after birth ameliorate symptoms in sickle cell patients. Therefore, the objective of our study is to identify trans-factors involved in γ-gene activation during the γ- to β-globin switch recapitulated in erythropoiesis. We previously established the one-phase liquid culture system using buffy coat mononuclear cells grown in erythropoietin (Epo; 4U/mL), stem cell factor (50ng/ml) and Interleukin-3 (IL-3; 10ng/ml). We observed high γ-globin transcription on day 7, equal γ- and β-globin mRNA levels by day 20 followed by high β-globin expression on day 28. Cell surface markers were measured by quantitative PCR (qPCR) and correlated with globin gene expression. During lineage commitment and erythroid maturation CD34+ cells decreased while the erythroid markers CD71, glycophorin A and Epo receptor increased 12 to 144-fold, followed by a decrease on day 28. We concluded that the γ- to β-globin switch was recapitulated in our system. Subsequent studies were performed with microarray and gene profiling by principle component analysis (PCA) to characterize proteins involved in switching. The Discover Chip from ArrayIt (Sunnyvale, CA) containing 380 human genes was used. cDNA from day 0/7, 7/21 and 21/28 were labeled with Cy3 and Cy5 respectively and used for standard chip hybridization. Fluorescence data was acquired using the Axon scanner and GenePixPro 6.0 software. By day 7, 88 genes were silenced and 68 activated; at day 28 an additional 38 genes were silenced and 47 activated. PCA defined the four major gene profiles consistent with developmental globin gene regulation. Profile 1 genes including IL-3, IL-9, heat shock protein 1a and neurofibromin were activated greater than 2-fold on day 7 suggesting positive regulators of γ-globin; trans-activators were not identified. Profile 2 genes were highly expressed around the switch on day 20. GATA-1, guanine nucleotide binding protein, ZF protein 132 and ERK1 expression peaked during this time. This data is consistent with a γ-globin silencer role previously demonstrated for ERK. Profile 3 genes also peaked on day 20 but continued to be highly expressed during β-globin transcription; EKLF, IL-8, Ras, Fos, and histone H3 were identified in this group. Experimental data support EKLF binding in the locus control region to compete with γ-globin expression. Finally Profile 4 genes increased steadily and peaked on day 28 consistent with positive β-globin regulators including NF-E2, ATF-2, IL-12, and thymosin β10. The microarray data were confirmed by qPCR in 90% of the genes tested. Our findings support the one-phase erythroid liquid culture system as a model for globin switching. We will expand gene profiling efforts using the Agilent 44K chip and PCA to confirm this model.

Blood, Nov 16, 2004

Erythroid differentiation recapitulates the temporal switch from γ to β-globin gene expression th... more Erythroid differentiation recapitulates the temporal switch from γ to β-globin gene expression that occurs during fetal development. We and others have demonstrated a p38 mitogen activated protein kinase (MAPK)-dependent mechanism for γ-globin activation by fetal hemoglobin inducers in the setting of permissive chromatin structure. Furthermore, by electrophoretic mobility shift assay we confirmed that CREB and ATF-2 bind the CRE at nucleotide −1222 in the Gγ-promoter (G-CRE). A naturally occurring C→G mutation in the CRE occurs is associated with the Benin haplotype, producing lower fetal hemoglobin levels in sickle cell patients. To understand the functional relevance of the G-CRE we used a luciferase reporter system established with promoter mutants at nucleotides −1500, −1350 and −1180 to produce the respective reporters -1500Gγluc, −1350Gγluc and −1180Gγluc. Transient transfections were performed in K562 cells by electroporation. In the −1180Gγluc reporter there was a loss of the 15 to 20-fold activation induced by 2mM butyrate and 0.5μM trichostatin in the −1500Gγluc reporter . The loss of trans-activation when the G-CRE was deleted in the −1180 mutant confirms functional relevance for this element. Trans-activation studies were also completed with a wtCREB (pCMV-CREB) and dominant negative, pKCREB expression vector. The latter forms an inactive heterodimer with CREB by blocking the DNA binding domain. To determine the direct trans-activation potential of wtCREB, co-transfection studies were completed with 10 to 40μg of either pCMV-CREB or pKCREB alone. We observed a dose-dependent 1.5 to 11-fold increase in luciferase activity with wtCREB compare to a robust 18 to 100-fold Gγ-promoter induction by pkCREB. This ability of both vectors to activate was reversed when the G-CRE was deleted. Interestingly, when an equal concentration of both vectors was co-transfected, a 40-fold increase in promoter activity was observed. Additional studies with pkCREB and butyrate or trichostatin inductions produced a similar synergistic effect. Collectively, the findings suggest that other transcription factors with greater trans-activation potential bind the G-CRE when CREB is inactivated by its dominant negative protein. We will test the two most likely candidate transcription factors, cJun and ATF-2 which bind the G-CRE and are downstream effectors of p38 MAPK signaling. Transfection experiments with cJun confirmed a dose-dependent 80-fold activation of −1500Gγluc. Interestingly, when cJun was combined with wtCREB, γ-promoter trans-activation dropped 60-fold supporting two possible interactions between cJun and CREB: 1) competitive binding to the G-CRE or 2) the formation of an inactive or low activity heterodimer between the two proteins. Additional studies with site-directed mutants of the G-CRE will be performed to ferret out the DNA-protein interactions in this region, thereby providing gene-based approaches for fetal hemoglobin induction.

Blood, Nov 16, 2004

We previously demonstrated that signal transducer and activator of transcription 3β (Stat3β) acts... more 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.

Cellular & Molecular Biology Letters, 1998

Blood, Nov 16, 2004

Enhancing γ-globin expression is an effective therapeutic strategy for sickle cell disease. Fetal... more Enhancing γ-globin expression is an effective therapeutic strategy for sickle cell disease. Fetal hemoglobin (HbF) inducers such as sodium butyrate (NaB), trichostatin A (TSA) and hydroxyurea (HU) have been studied extensively to ascertain molecular and cell signaling mechanisms for γ-gene activation. Reactive oxygen species (ROS) including nitric oxide, superoxide, hydroxyl radicals and hydrogen peroxide stimulate signaling through the cyclic guanosine monophosphate (cGMP) and p38 MAPK pathways. Indirect evidence supports HU as a nitric oxide donor thus providing a mechanism for HbF induction via cGMP activation. Recently, we and others demonstrated a role for p38 signaling in γ-gene induction by HU as well as NaB, TSA and apicidin. However, the ability of HbF inducers to stimulate ROS formation as a mechanism for γ-globin activation via cGMP or p38 MAPK signaling has not been determined. To clarify this mechanism we measured ROS levels using 2′,7′-dichlorofluorescin diacetate which emits fluorescence at a 529 nm wavelength when activated by hydrogen peroxide. We correlated ROS levels with p38 phosphorylation and γ-globin mRNA levels measured by western blot and real-time PCR respectively. Studies were also completed in the absence and presence of myxothiazol, a known inhibitor of ROS formation to confirm our findings. The positive controls menadione and hemin increased ROS levels 4-fold and 3-fold with a concomitant 6.5-fold and 4-fold increase in γ-globin mRNA respectively. Pretreatment with myxothiazol inhibited maximal HbF induction by both agents. By contrast, NaB and TSA did not increase ROS levels in K562 cells therefore they activate γ-globin by a ROS-independent, p38 MAPK-dependent mechanism. We concluded that ROS play a major role in the mechanism for HbF induction by hemin and the vitamin K derivative, menadione. Investigations were completed which confirmed the ability of HU to stimulate ROS formation as well. Studies are underway to determine the role of hydrogen peroxide and/or nitric oxide in HbF induction by HU via cGMP/p38 MAPK cell signaling.

Blood, Dec 3, 2015

Sickle cell disease (SCD) affects &gt;100,000 Americans and millions more worldwide. Symptoms... more Sickle cell disease (SCD) affects &gt;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..

Experimental Biology and Medicine, Jul 26, 2020

NRF2 is the master regulator for the cellular oxidative stress response and regulates γ-globin ge... more NRF2 is the master regulator for the cellular oxidative stress response and regulates γ-globin gene expression in human erythroid progenitors and sickle cell disease mice. To explore NRF2 function, we established a human β-globin locus yeast artificial chromosome transgenic/NRF2 knockout (β-YAC/NRF2−/−) mouse model. NRF2 loss reduced γ-globin gene expression during erythropoiesis and abolished the ability of dimethyl fumarate, an NRF2 activator, to enhance γ-globin transcription. We observed decreased H3K4Me1 and H3K4Me3 chromatin marks and association of TATA-binding protein and RNA polymerase II at the β-locus control region (LCR) and γ-globin gene promoters in β-YAC/NRF2−/− mice. As a result, long-range chromatin interaction between the LCR DNase I hypersensitive sites and γ-globin gene was decreased, while interaction with the β-globin was not affected. Further, NRF2 loss silenced the expression of DNA methylcytosine dioxygenases TET1, TET2, and TET3 and inhibited γ-globin gene DNA hydroxymethylation. Subsequently, protein-protein interaction between NRF2 and TET3 was demonstrated. These data support the ability of NRF2 to mediate γ-globin gene regulation through epigenetic DNA and histone modifications.Impact statementSickle cell disease is an inherited hemoglobin disorder that affects over 100,000 people in the United States causing high morbidity and early mortality. Although new treatments were recently approved by the FDA, only one drug Hydroxyurea induces fetal hemoglobin expression to inhibit sickle hemoglobin polymerization in red blood cells. Our laboratory previously demonstrated the ability of the NRF2 activator, dimethyl fumarate to induce fetal hemoglobin in the sickle cell mouse model. In this study, we investigated molecular mechanisms of γ-globin gene activation by NRF2. We observed the ability of NRF2 to modulate chromatin structure in the human β-like globin gene locus of β-YAC transgenic mice during development. Furthermore, an NRF2/TET3 interaction regulates γ-globin gene DNA methylation. These findings provide potential new molecular targets for small molecule drug developed for treating sickle cell disease.

Hematology-oncology Clinics of North America, Apr 1, 2014

Fetal globin (gamma globin; HBG) is normally expressed during fetal life and prevents the clinica... more Fetal globin (gamma globin; HBG) is normally expressed during fetal life and prevents the clinical manifestations of beta hemoglobinopathies before birth. HBG genes are normally integrated in hematopoietic stem cells in all humans, and are at least partially amenable to reactivation. Inducing expression of fetal globin (HBG) gene expression to 60% to 70% of alpha globin synthesis produces a β-thalassemia trait phenotype, and reduces anemia. Tailoring combinations of therapeutics to patient subsets characterized for quantitative trait loci which modulate basal fetal hemoglobin and erythroid cell survival should provide effective amelioration of clinical symptoms in β-thalassemia and sickle cell disease.

Blood, Aug 1, 1996

We describe an in vivo approach, in transgenic mice, aimed to identify promotar elements responsi... more We describe an in vivo approach, in transgenic mice, aimed to identify promotar elements responsible for the induction of y globin expression by butyrate. Transgenic lines carrying human ' y gene promoter truncations at position-141,-201,-382, and-730 'y were treated with a amino butyric acid (aABA), and effects on y globin expression were ana

Experimental Hematology, Nov 1, 2003

Objective. Our goal was to determine the role of p38 mitogen-activated protein kinase (MAPK) sign... more Objective. Our goal was to determine the role of p38 mitogen-activated protein kinase (MAPK) signaling in fetal hemoglobin (HbF) induction. Two histone deacetylase inhibitors (HDAIs), sodium butyrate (NB), and trichostatin (TSA) and hemin were analyzed. In addition, the effect of direct activation of p38 MAPK on g-globin gene activity was studied. Method. Primary erythroid progenitors derived from peripheral blood mononuclear cell and K562 erythroleukemia cells were analyzed. Cells were grown in NB, TSA, hemin, or anisomycin either alone or in the presence of the p38 MAPK inhibitor SB203580. The effects of the various treatments on g-globin RNA, HbF, and phosphorylated p38 MAPK levels were measured by RNase protection assay, alkaline denaturation, and Western blot analysis, respectively. A K562 stable line overexpressing constitutively active p38 MAPK was established using MAPK kinase kinase 3 (MKK3) and MKK6, the immediate upstream activators of p38. The direct effect of p38 MAPK overexpression on g-globin mRNA synthesis was analyzed. Results. NB and TSA activated p38 MAPK and increased g-globin mRNA levels in K562 cells and primary erythroid progenitors. Pretreatment with SB203580 blocked p38 MAPK and gglobin gene activation. In contrast, no change in p38 activity was observed with hemin inductions. Direct activation of p38 by anisomycin or constitutive overexpression also increased g-globin mRNA in the absence of HbF inducers in wild-type K562 cells and in the MKK stable lines. Conclusion. This study supports a novel role for p38 MAPK in g-globin regulation in human erythroid progenitors. Ć 2003 International Society for Experimental Hematology. Published by Elsevier Inc. Recent evidence has emerged demonstrating the important role of signaling pathways in mediating the effects of druginduced fetal hemoglobin (HbF) production. The JAK-STAT (Janus kinase-signal transducers and activators of transcription) pathways transmit the cell surface signals initiated by many growth factors, which regulate cellular proliferation and differentiation [1]. Sodium butyrate (NB)-induced erythroid maturation in mouse erythroleukemia cells is associated with rapid phosphorylation of Jak2 and Stat5 [2]. Mitogen-activated protein kinases (MAPKs) are key regulatory enzymes that transduce external signals into a complex array of intracellular responses [3-5]. The MAPK family consists of extracellular signal-related kinases (ERK1,

Blood, Nov 16, 2005

The design and evaluation of therapies for sickle cell disease (SCD) rely on our understanding of... more The design and evaluation of therapies for sickle cell disease (SCD) rely on our understanding of hemoglobin accumulation during erythropoiesis and sequential globin gene expression (ε → Gγ → Aγ → δ → β) during development. To gain insights into globin gene switching, we completed time course micorarray analyses of erythroid progenitors to identify trans-factors involved in γ gene activation. Studies were completed to map the pattern of γ and β globin gene expression in progenitors grown from normal peripheral blood mononuclear cells. We compared cells grown in a 2-phase (phase 1, d0-6: SCF, IL-3, IL-6, and GM-CSF and phase 2, d7-25: SCF and EPO) vs. 1-phase (d0-34: SCF, IL-3, and EPO) liquid culture system. From day 0 to 34 in either system cell viability remained &amp;amp;amp;gt;99%. Total RNA was isolated using Trizol and column cleanup (Qiagen). Globin mRNA levels were measured at 2–3 day intervals by quantitative PCR (qPCR). In the 2-phase system γ-globin mRNA&amp;amp;amp;gt;β-globin mRNA up to d14, 4 days of approximately equal expression then β mRNA &amp;amp;amp;gt; γ mRNA by d20. By contrast, in 1-phase studies there was a rapid switch around d20(see graph). We speculate that this difference may be due to the early addition of EPO on d0 therefore we continued our detailed analysis in this system. To confirm that our in vitro system recapitulates in vivo gene expression patterns, we completed studies to ascertain Gγ - vs. Aγ globin mRNA levels. The normalized Gγ:Aγ ratio decreased from ~3:1 on d7 to ~1:1 by d34; These findings were confirmed using two sets of Gγ and Aγ globin primers. We concluded that the 1-phase system recapitulated normal γ/β globin switching and that gene profiling studies to identify the trans-factor involved in switching mechanisms were feasible. We used Discover oligo chips (ArrayIt, Sunnyvale, CA) containing 380 human genes selected from 30 major functional groups including hematopoiesis. To aide interpretation of chip data, cell populations were rated morphologically using Giemsa stained cytospin preps. From d16 on we observed an increase in late erythroid progenitors (normoblasts) from 1% to 71% by d31. After verifying RNA quality by gel inspection of ribosomal molecules, we prepared Cy3 and Cy5 probes for early and late time-point RNA samples respectively. Chip analysis was performed at several time points but d0/21, d7/21, and d21/28 were most informative. Based on Axon GenePixPro 6.0 and Acuity 4.0 software analysis we found the following genes with &amp;amp;amp;gt;1.5-fold change in expression profile (shown as down-regulated/up-regulated genes): d0/21: 33/73, d7/21: 13/25, and d21/28:35/26. Principal component analysis (PCA), hierarchical clusters and self organizing maps were constructed. Gene profiles were correlated with the γ/β switching curve using d7 (γ &amp;amp;amp;gt;β), d21 (γ ~ β), and d28 (γ &amp;amp;amp;lt;β) data. Hematopoietic dataset analysis at d21 revealed 4 candidate γ-globin gene activators including v-myb, upsteam binding transfactor -RNApol1 and 2 zinc finger proteins. Analysis of a d28 dataset revealed 12 proteins involved in γ-globin gene silencing including IL-3, SCF, MAPKKK3, v-raf-1, ATF-2, and glucocorticoid receptor DNA binding factor 1 among others. Gene expression profiles will be validated using qPCR and promising candidates will be tested by forced expression in transient and stable reporter systems. Figure Figure

Blood, Apr 28, 2022

Excessive intravascular release of lysed cellular contents from damaged red blood cells (RBCs) in... more Excessive intravascular release of lysed cellular contents from damaged red blood cells (RBCs) in patients with sickle cell anemia (SCA) can activate the inflammasome, a multiprotein oligomer promoting maturation and secretion of proinflammatory cytokines, including interleukin-1β (IL-1β). We hypothesized that IL-1β blockade by canakinumab in patients with SCA would reduce markers of inflammation and clinical disease activity. In this randomized, double-blind, multicenter phase 2a study, patients aged 8 to 20 years with SCA (HbSS or HbSβ0-thalassemia), history of acute pain episodes, and elevated high-sensitivity C-reactive protein &gt;1.0 mg/L at screening were randomized 1:1 to received 6 monthly treatments with 300 mg subcutaneous canakinumab or placebo. Measured outcomes at baseline and weeks 4, 8, 12, 16, 20, and 24 included electronic patient-reported outcomes, hospitalization rate, and adverse events (AEs) and serious AEs (SAEs). All but 1 of the 49 enrolled patients were receiving stable background hydroxyurea therapy. Although the primary objective (prespecified reduction of pain) was not met, compared with patients in the placebo arm, patients treated with canakinumab had reductions in markers of inflammation, occurrence of SCA-related AEs and SAEs, and number and duration of hospitalizations as well as trends for improvement in pain intensity, fatigue, and absences from school or work. Post hoc analysis revealed treatment effects on weight, restricted to pediatric patients. Canakinumab was well tolerated with no treatment-related SAEs and no new safety signal. These findings demonstrate that the inflammation associated with SCA can be reduced by selective IL-1β blockade by canakinumab with potential for therapeutic benefits. This trial was registered at www.clinicaltrials.gov as #NCT02961218.

Cellular & Molecular Biology Letters, 1998

Journal of clinical and translational science, 2023

Although mentoring is critical for career advancement, underrepresented minority (URM) faculty of... more Although mentoring is critical for career advancement, underrepresented minority (URM) faculty often lack access to mentoring opportunities. We sought to evaluate the impact of peer mentoring on career development success of URM early career faculty in the National Heart Lung and Blood Institute-sponsored, Programs to Increase Diversity Among Individuals Engaged in Health-Related Research-Functional and Translational Genomics of Blood Disorders (PRIDE-FTG). The outcome of peer mentoring was evaluated using the Mentoring Competency Assessment (MCA), a brief open-ended qualitative survey, and a semi-structured exit interview. Surveys were completed at baseline (Time 1), 6 months, and at the end of PRIDE-FTG participation (Time 2). The following results were obtained. Between Time 1 and Time 2, mentees' self-assessment scores increased for the MCA (p < 0.01) with significant increases in effective communication (p < 0.001), aligning expectations (p < 0.05), assessing understanding (p < 0.01), and addressing diversity (p < 0.002). Mentees rated their peer mentors higher in the MCA with significant differences noted for promoting development (p < 0.027). These data suggest that PRIDE-FTG peer mentoring approaches successfully improved MCA competencies among URM junior faculty participants with faculty ranking peer mentors higher than themselves. Among URM faculty, peer mentoring initiatives should be investigated as a key strategy to support early career scholar development.

Blood Cells Molecules and Diseases, Mar 1, 2007

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Blood, Dec 8, 2017

Abstract The hemoglobinopathies including sickle cell disease (SCD) and β-thalassemia are the mos... more Abstract The hemoglobinopathies including sickle cell disease (SCD) and β-thalassemia are the most common genetic disorders in the world producing significant morbidity and mortality. Drug-mediated fetal hemoglobin (HbF) induction ameliorates the clinical severity of SCD and improves long-term survival. To define mechanisms of γ-globin regulation, previously we demonstrated the role of the Gγ-globin cAMP response element, in drug-mediated HbF induction (Sangerman et al., Blood 2006). Subsequently, data generated by the ENCODE project led us to investigate a region 4 kb upstream of Gγ-globin, which harbors a DNase hypersensitive site and enrichment of the histone 3 lysine 4, mono-methylated (H3K4Me1) enhancer mark, that we named the Fetal Chromatin Domain (FCD). Recently, a synthetic zinc finger DNA-binding domain targeting this region was shown to reduce γ-globin gene expression in K562 cells (Shen et al., Blood 128:320, 2016). Initially, we performed multispecies sequence alignments which revealed the FCD is located in a genomic region shared by Old World monkeys (humans and Baboons) with a delayed fetal to adult globin switch that occurs after birth. By contrast, the FCD region is absent in New World monkeys where the switch occurs during the fetal developmental stage. These findings support involvement of the FCD region in γ-globin regulation during hemoglobin switching. Subsequent studies were performed to define the functional role of the FCD during erythropoiesis. ENCODE long RNA-sequencing assay detected PolyA RNA species throughout the FCD region in K562 cells. Based on these findings, we designed two small-interference RNA molecules targeting the core FCD (siFCD). Both siFCD molecules were transfected into KU812 cells and individual γ-globin gene expression measured using RT-qPCR with gene-specific primers. The level of Gγ-globin mRNA increased by 64%, while Aγ-globin transcription was reduced 38%. These results suggest the FCD facilitates differential regulation of the γ-globin genes. To further characterize FCD function, a series of γ-globin promoter luciferase reporters were constructed with the sense or antisense 500-bp core FCD region, cloned downstream of the luciferase gene. In KU812 stable cell lines, the sense FCD construct enhanced Aγ-globin promoter activity 10-fold, whereas the antisense construct repressed promoter activity. By contrast, the FCD in both orientations silenced Gγ-globin promoter activity. These results support unidirectional gene-specific enhancer properties of the FCD. Subsequent pulldown assay with a 34-bp biotinylated FCD probe confirmed NFE2, BHLHE40 and cMyc binding, which was abolished by mutation of the E-box sequence (CACGTG) in the FCD core. To build on the KU812 cell data, we performed μChIP assay using primary erythroid progenitors generated from adult CD34+ stem cells (Zhu et al., Haematologica 2017). In our system, the γ-globin to β-globin gene switch occurred on day 4 in culture. Initially, we measured levels of acetylated histone H3 (AcH3), H3K4Me1, and TFIID binding in the β-globin locus control region hypersensitivity site 2 (HS2) and FCD. As expected, high AcH3 and H3K4Me1 marks occurred in HS2 consistent with its enhancer function. Similar patterns of histone marks and binding of TFIID, NFE2 and BHLHE40 occurred in HS2 and FCD region. It is known that HS2 participates in DNA looping to accomplish developmentally regulated globin gene expression during hemoglobin switching therefore chromosome conformation capture (3C) assay was performed in primary erythroid progenitors to determine if the FCD facilitates changes in chromatin structure. We observed strong long-range interactions between the FCD and HS2 with 4.4-fold maximal chromatin enrichment on day 5; likewise the FCD showed interactions with Aγ-globin (4.7-fold) and Gγ-globin (3.5-fold) by day 3 before switching occurred. Of note, low level interactions occurred between the FCD and β-globin gene at all time points tested. Collectively, these data support the FCD as a unidirectional enhancer element involved in γ-globin regulation during erythropoiesis. To further define mechanisms of FCD function, the effects of CrisprCas9-mediated deletions in the FCD on globin gene expression will be explored. Disclosures No relevant conflicts of interest to declare.

Experimental Biology and Medicine, Aug 31, 2019

Sickle cell disease (SCD) and b-thalassemia are inherited blood disorders caused by genetic defec... more Sickle cell disease (SCD) and b-thalassemia are inherited blood disorders caused by genetic defects in the b-globin gene on chromosome 11, producing severe disease in people worldwide. Induction of fetal hemoglobin consisting of two a-globin and two c-globin chains ameliorates the clinical symptoms of both disorders. In the present study, we investigated the ability of d-aminolevulinate (ALA), the heme precursor, to activate c-globin gene expression as well as its effects on cellular functions in erythroid cell systems. We demonstrated that ALA induced c-globin expression at both the transcriptional and protein levels in the KU812 erythroid cell line. Using inhibitors targeting two enzymes in the heme biosynthesis pathway, we showed that cellular heme biosynthesis was involved in ALA-mediated c-globin activation. Moreover, the transcription factor NRF2 (nuclear factor [erythroidderived 2]-like 2), a critical regulator of the cellular antioxidant response, was activated by ALA and contributed to mechanisms of c-globin activation; ALA did not affect cell proliferation and was not toxic to cells. Subsequent studies demonstrated ALA-induced c-globin activation in erythroid progenitors generated from normal human CD34 þ stem cells. These data support future study to explore the potential of stimulating intracellular heme biosynthesis by ALA or similar compounds as a novel therapeutic strategy for treating SCD and b-thalassemia.

Blood, Nov 16, 2006

The design and evaluation of therapies for sickle cell disease rely on our understanding of the s... more The design and evaluation of therapies for sickle cell disease rely on our understanding of the sequential expression of the β-like globin genes during development. Drug inducers that reactivate γ-globin transcription after birth ameliorate symptoms in sickle cell patients. Therefore, the objective of our study is to identify trans-factors involved in γ-gene activation during the γ- to β-globin switch recapitulated in erythropoiesis. We previously established the one-phase liquid culture system using buffy coat mononuclear cells grown in erythropoietin (Epo; 4U/mL), stem cell factor (50ng/ml) and Interleukin-3 (IL-3; 10ng/ml). We observed high γ-globin transcription on day 7, equal γ- and β-globin mRNA levels by day 20 followed by high β-globin expression on day 28. Cell surface markers were measured by quantitative PCR (qPCR) and correlated with globin gene expression. During lineage commitment and erythroid maturation CD34+ cells decreased while the erythroid markers CD71, glycophorin A and Epo receptor increased 12 to 144-fold, followed by a decrease on day 28. We concluded that the γ- to β-globin switch was recapitulated in our system. Subsequent studies were performed with microarray and gene profiling by principle component analysis (PCA) to characterize proteins involved in switching. The Discover Chip from ArrayIt (Sunnyvale, CA) containing 380 human genes was used. cDNA from day 0/7, 7/21 and 21/28 were labeled with Cy3 and Cy5 respectively and used for standard chip hybridization. Fluorescence data was acquired using the Axon scanner and GenePixPro 6.0 software. By day 7, 88 genes were silenced and 68 activated; at day 28 an additional 38 genes were silenced and 47 activated. PCA defined the four major gene profiles consistent with developmental globin gene regulation. Profile 1 genes including IL-3, IL-9, heat shock protein 1a and neurofibromin were activated greater than 2-fold on day 7 suggesting positive regulators of γ-globin; trans-activators were not identified. Profile 2 genes were highly expressed around the switch on day 20. GATA-1, guanine nucleotide binding protein, ZF protein 132 and ERK1 expression peaked during this time. This data is consistent with a γ-globin silencer role previously demonstrated for ERK. Profile 3 genes also peaked on day 20 but continued to be highly expressed during β-globin transcription; EKLF, IL-8, Ras, Fos, and histone H3 were identified in this group. Experimental data support EKLF binding in the locus control region to compete with γ-globin expression. Finally Profile 4 genes increased steadily and peaked on day 28 consistent with positive β-globin regulators including NF-E2, ATF-2, IL-12, and thymosin β10. The microarray data were confirmed by qPCR in 90% of the genes tested. Our findings support the one-phase erythroid liquid culture system as a model for globin switching. We will expand gene profiling efforts using the Agilent 44K chip and PCA to confirm this model.

Blood, Nov 16, 2004

Erythroid differentiation recapitulates the temporal switch from γ to β-globin gene expression th... more Erythroid differentiation recapitulates the temporal switch from γ to β-globin gene expression that occurs during fetal development. We and others have demonstrated a p38 mitogen activated protein kinase (MAPK)-dependent mechanism for γ-globin activation by fetal hemoglobin inducers in the setting of permissive chromatin structure. Furthermore, by electrophoretic mobility shift assay we confirmed that CREB and ATF-2 bind the CRE at nucleotide −1222 in the Gγ-promoter (G-CRE). A naturally occurring C→G mutation in the CRE occurs is associated with the Benin haplotype, producing lower fetal hemoglobin levels in sickle cell patients. To understand the functional relevance of the G-CRE we used a luciferase reporter system established with promoter mutants at nucleotides −1500, −1350 and −1180 to produce the respective reporters -1500Gγluc, −1350Gγluc and −1180Gγluc. Transient transfections were performed in K562 cells by electroporation. In the −1180Gγluc reporter there was a loss of the 15 to 20-fold activation induced by 2mM butyrate and 0.5μM trichostatin in the −1500Gγluc reporter . The loss of trans-activation when the G-CRE was deleted in the −1180 mutant confirms functional relevance for this element. Trans-activation studies were also completed with a wtCREB (pCMV-CREB) and dominant negative, pKCREB expression vector. The latter forms an inactive heterodimer with CREB by blocking the DNA binding domain. To determine the direct trans-activation potential of wtCREB, co-transfection studies were completed with 10 to 40μg of either pCMV-CREB or pKCREB alone. We observed a dose-dependent 1.5 to 11-fold increase in luciferase activity with wtCREB compare to a robust 18 to 100-fold Gγ-promoter induction by pkCREB. This ability of both vectors to activate was reversed when the G-CRE was deleted. Interestingly, when an equal concentration of both vectors was co-transfected, a 40-fold increase in promoter activity was observed. Additional studies with pkCREB and butyrate or trichostatin inductions produced a similar synergistic effect. Collectively, the findings suggest that other transcription factors with greater trans-activation potential bind the G-CRE when CREB is inactivated by its dominant negative protein. We will test the two most likely candidate transcription factors, cJun and ATF-2 which bind the G-CRE and are downstream effectors of p38 MAPK signaling. Transfection experiments with cJun confirmed a dose-dependent 80-fold activation of −1500Gγluc. Interestingly, when cJun was combined with wtCREB, γ-promoter trans-activation dropped 60-fold supporting two possible interactions between cJun and CREB: 1) competitive binding to the G-CRE or 2) the formation of an inactive or low activity heterodimer between the two proteins. Additional studies with site-directed mutants of the G-CRE will be performed to ferret out the DNA-protein interactions in this region, thereby providing gene-based approaches for fetal hemoglobin induction.

Blood, Nov 16, 2004

We previously demonstrated that signal transducer and activator of transcription 3β (Stat3β) acts... more 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.

Cellular & Molecular Biology Letters, 1998

Blood, Nov 16, 2004

Enhancing γ-globin expression is an effective therapeutic strategy for sickle cell disease. Fetal... more Enhancing γ-globin expression is an effective therapeutic strategy for sickle cell disease. Fetal hemoglobin (HbF) inducers such as sodium butyrate (NaB), trichostatin A (TSA) and hydroxyurea (HU) have been studied extensively to ascertain molecular and cell signaling mechanisms for γ-gene activation. Reactive oxygen species (ROS) including nitric oxide, superoxide, hydroxyl radicals and hydrogen peroxide stimulate signaling through the cyclic guanosine monophosphate (cGMP) and p38 MAPK pathways. Indirect evidence supports HU as a nitric oxide donor thus providing a mechanism for HbF induction via cGMP activation. Recently, we and others demonstrated a role for p38 signaling in γ-gene induction by HU as well as NaB, TSA and apicidin. However, the ability of HbF inducers to stimulate ROS formation as a mechanism for γ-globin activation via cGMP or p38 MAPK signaling has not been determined. To clarify this mechanism we measured ROS levels using 2′,7′-dichlorofluorescin diacetate which emits fluorescence at a 529 nm wavelength when activated by hydrogen peroxide. We correlated ROS levels with p38 phosphorylation and γ-globin mRNA levels measured by western blot and real-time PCR respectively. Studies were also completed in the absence and presence of myxothiazol, a known inhibitor of ROS formation to confirm our findings. The positive controls menadione and hemin increased ROS levels 4-fold and 3-fold with a concomitant 6.5-fold and 4-fold increase in γ-globin mRNA respectively. Pretreatment with myxothiazol inhibited maximal HbF induction by both agents. By contrast, NaB and TSA did not increase ROS levels in K562 cells therefore they activate γ-globin by a ROS-independent, p38 MAPK-dependent mechanism. We concluded that ROS play a major role in the mechanism for HbF induction by hemin and the vitamin K derivative, menadione. Investigations were completed which confirmed the ability of HU to stimulate ROS formation as well. Studies are underway to determine the role of hydrogen peroxide and/or nitric oxide in HbF induction by HU via cGMP/p38 MAPK cell signaling.

Blood, Dec 3, 2015

Sickle cell disease (SCD) affects &gt;100,000 Americans and millions more worldwide. Symptoms... more Sickle cell disease (SCD) affects &gt;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..

Experimental Biology and Medicine, Jul 26, 2020

NRF2 is the master regulator for the cellular oxidative stress response and regulates γ-globin ge... more NRF2 is the master regulator for the cellular oxidative stress response and regulates γ-globin gene expression in human erythroid progenitors and sickle cell disease mice. To explore NRF2 function, we established a human β-globin locus yeast artificial chromosome transgenic/NRF2 knockout (β-YAC/NRF2−/−) mouse model. NRF2 loss reduced γ-globin gene expression during erythropoiesis and abolished the ability of dimethyl fumarate, an NRF2 activator, to enhance γ-globin transcription. We observed decreased H3K4Me1 and H3K4Me3 chromatin marks and association of TATA-binding protein and RNA polymerase II at the β-locus control region (LCR) and γ-globin gene promoters in β-YAC/NRF2−/− mice. As a result, long-range chromatin interaction between the LCR DNase I hypersensitive sites and γ-globin gene was decreased, while interaction with the β-globin was not affected. Further, NRF2 loss silenced the expression of DNA methylcytosine dioxygenases TET1, TET2, and TET3 and inhibited γ-globin gene DNA hydroxymethylation. Subsequently, protein-protein interaction between NRF2 and TET3 was demonstrated. These data support the ability of NRF2 to mediate γ-globin gene regulation through epigenetic DNA and histone modifications.Impact statementSickle cell disease is an inherited hemoglobin disorder that affects over 100,000 people in the United States causing high morbidity and early mortality. Although new treatments were recently approved by the FDA, only one drug Hydroxyurea induces fetal hemoglobin expression to inhibit sickle hemoglobin polymerization in red blood cells. Our laboratory previously demonstrated the ability of the NRF2 activator, dimethyl fumarate to induce fetal hemoglobin in the sickle cell mouse model. In this study, we investigated molecular mechanisms of γ-globin gene activation by NRF2. We observed the ability of NRF2 to modulate chromatin structure in the human β-like globin gene locus of β-YAC transgenic mice during development. Furthermore, an NRF2/TET3 interaction regulates γ-globin gene DNA methylation. These findings provide potential new molecular targets for small molecule drug developed for treating sickle cell disease.

Hematology-oncology Clinics of North America, Apr 1, 2014

Fetal globin (gamma globin; HBG) is normally expressed during fetal life and prevents the clinica... more Fetal globin (gamma globin; HBG) is normally expressed during fetal life and prevents the clinical manifestations of beta hemoglobinopathies before birth. HBG genes are normally integrated in hematopoietic stem cells in all humans, and are at least partially amenable to reactivation. Inducing expression of fetal globin (HBG) gene expression to 60% to 70% of alpha globin synthesis produces a β-thalassemia trait phenotype, and reduces anemia. Tailoring combinations of therapeutics to patient subsets characterized for quantitative trait loci which modulate basal fetal hemoglobin and erythroid cell survival should provide effective amelioration of clinical symptoms in β-thalassemia and sickle cell disease.

Blood, Aug 1, 1996

We describe an in vivo approach, in transgenic mice, aimed to identify promotar elements responsi... more We describe an in vivo approach, in transgenic mice, aimed to identify promotar elements responsible for the induction of y globin expression by butyrate. Transgenic lines carrying human ' y gene promoter truncations at position-141,-201,-382, and-730 'y were treated with a amino butyric acid (aABA), and effects on y globin expression were ana

Experimental Hematology, Nov 1, 2003

Objective. Our goal was to determine the role of p38 mitogen-activated protein kinase (MAPK) sign... more Objective. Our goal was to determine the role of p38 mitogen-activated protein kinase (MAPK) signaling in fetal hemoglobin (HbF) induction. Two histone deacetylase inhibitors (HDAIs), sodium butyrate (NB), and trichostatin (TSA) and hemin were analyzed. In addition, the effect of direct activation of p38 MAPK on g-globin gene activity was studied. Method. Primary erythroid progenitors derived from peripheral blood mononuclear cell and K562 erythroleukemia cells were analyzed. Cells were grown in NB, TSA, hemin, or anisomycin either alone or in the presence of the p38 MAPK inhibitor SB203580. The effects of the various treatments on g-globin RNA, HbF, and phosphorylated p38 MAPK levels were measured by RNase protection assay, alkaline denaturation, and Western blot analysis, respectively. A K562 stable line overexpressing constitutively active p38 MAPK was established using MAPK kinase kinase 3 (MKK3) and MKK6, the immediate upstream activators of p38. The direct effect of p38 MAPK overexpression on g-globin mRNA synthesis was analyzed. Results. NB and TSA activated p38 MAPK and increased g-globin mRNA levels in K562 cells and primary erythroid progenitors. Pretreatment with SB203580 blocked p38 MAPK and gglobin gene activation. In contrast, no change in p38 activity was observed with hemin inductions. Direct activation of p38 by anisomycin or constitutive overexpression also increased g-globin mRNA in the absence of HbF inducers in wild-type K562 cells and in the MKK stable lines. Conclusion. This study supports a novel role for p38 MAPK in g-globin regulation in human erythroid progenitors. Ć 2003 International Society for Experimental Hematology. Published by Elsevier Inc. Recent evidence has emerged demonstrating the important role of signaling pathways in mediating the effects of druginduced fetal hemoglobin (HbF) production. The JAK-STAT (Janus kinase-signal transducers and activators of transcription) pathways transmit the cell surface signals initiated by many growth factors, which regulate cellular proliferation and differentiation [1]. Sodium butyrate (NB)-induced erythroid maturation in mouse erythroleukemia cells is associated with rapid phosphorylation of Jak2 and Stat5 [2]. Mitogen-activated protein kinases (MAPKs) are key regulatory enzymes that transduce external signals into a complex array of intracellular responses [3-5]. The MAPK family consists of extracellular signal-related kinases (ERK1,

Blood, Nov 16, 2005

The design and evaluation of therapies for sickle cell disease (SCD) rely on our understanding of... more The design and evaluation of therapies for sickle cell disease (SCD) rely on our understanding of hemoglobin accumulation during erythropoiesis and sequential globin gene expression (ε → Gγ → Aγ → δ → β) during development. To gain insights into globin gene switching, we completed time course micorarray analyses of erythroid progenitors to identify trans-factors involved in γ gene activation. Studies were completed to map the pattern of γ and β globin gene expression in progenitors grown from normal peripheral blood mononuclear cells. We compared cells grown in a 2-phase (phase 1, d0-6: SCF, IL-3, IL-6, and GM-CSF and phase 2, d7-25: SCF and EPO) vs. 1-phase (d0-34: SCF, IL-3, and EPO) liquid culture system. From day 0 to 34 in either system cell viability remained &amp;amp;amp;gt;99%. Total RNA was isolated using Trizol and column cleanup (Qiagen). Globin mRNA levels were measured at 2–3 day intervals by quantitative PCR (qPCR). In the 2-phase system γ-globin mRNA&amp;amp;amp;gt;β-globin mRNA up to d14, 4 days of approximately equal expression then β mRNA &amp;amp;amp;gt; γ mRNA by d20. By contrast, in 1-phase studies there was a rapid switch around d20(see graph). We speculate that this difference may be due to the early addition of EPO on d0 therefore we continued our detailed analysis in this system. To confirm that our in vitro system recapitulates in vivo gene expression patterns, we completed studies to ascertain Gγ - vs. Aγ globin mRNA levels. The normalized Gγ:Aγ ratio decreased from ~3:1 on d7 to ~1:1 by d34; These findings were confirmed using two sets of Gγ and Aγ globin primers. We concluded that the 1-phase system recapitulated normal γ/β globin switching and that gene profiling studies to identify the trans-factor involved in switching mechanisms were feasible. We used Discover oligo chips (ArrayIt, Sunnyvale, CA) containing 380 human genes selected from 30 major functional groups including hematopoiesis. To aide interpretation of chip data, cell populations were rated morphologically using Giemsa stained cytospin preps. From d16 on we observed an increase in late erythroid progenitors (normoblasts) from 1% to 71% by d31. After verifying RNA quality by gel inspection of ribosomal molecules, we prepared Cy3 and Cy5 probes for early and late time-point RNA samples respectively. Chip analysis was performed at several time points but d0/21, d7/21, and d21/28 were most informative. Based on Axon GenePixPro 6.0 and Acuity 4.0 software analysis we found the following genes with &amp;amp;amp;gt;1.5-fold change in expression profile (shown as down-regulated/up-regulated genes): d0/21: 33/73, d7/21: 13/25, and d21/28:35/26. Principal component analysis (PCA), hierarchical clusters and self organizing maps were constructed. Gene profiles were correlated with the γ/β switching curve using d7 (γ &amp;amp;amp;gt;β), d21 (γ ~ β), and d28 (γ &amp;amp;amp;lt;β) data. Hematopoietic dataset analysis at d21 revealed 4 candidate γ-globin gene activators including v-myb, upsteam binding transfactor -RNApol1 and 2 zinc finger proteins. Analysis of a d28 dataset revealed 12 proteins involved in γ-globin gene silencing including IL-3, SCF, MAPKKK3, v-raf-1, ATF-2, and glucocorticoid receptor DNA binding factor 1 among others. Gene expression profiles will be validated using qPCR and promising candidates will be tested by forced expression in transient and stable reporter systems. Figure Figure