HRI depletion cooperates with pharmacologic inducers to elevate fetal hemoglobin and reduce sickle cell formation

Abstract Increasing fetal hemoglobin (HbF) provides clinical benefit in patients with sickle cell disease (SCD). We recently identified heme-regulated inhibitor (HRI, EIF2AK1), as a novel HbF regulator. Because HRI is an erythroid-specific protein kinase, it presents a potential target for pharmacologic intervention. We found that maximal HbF induction required >80% to 85% HRI depletion. Because it remains unclear whether this degree of HRI inhibition can be achieved pharmacologically, we explored whether HRI knockdown can be combined with pharmacologic HbF inducers to achieve greater HbF production and minimize potential adverse effects associated with treatments. Strongly cooperative HbF induction was observed when HRI depletion was combined with exposure to pomalidomide or the EHMT1/2 inhibitor UNC0638, but not to hydroxyurea. Mechanistically, reduction in the levels of the HbF repressor BCL11A reflected the cooperativity of HRI loss and pomalidomide treatment, whereas UNC0638 did not modulate BCL11A levels. In conjunction with HRI loss, pomalidomide maintained its HbF-inducing activity at 10-fold lower concentrations, in which condition there were minimal observed detrimental effects on erythroid cell maturation and viability, as well as fewer alterations in the erythroid transcriptome. When tested in cells from patients with SCD, combining HRI depletion with pomalidomide or UNC0638 achieved up to 50% to 60% HbF and 45% to 50% HbF, respectively, as measured by high-performance liquid chromatography, and markedly counteracted cell sickling. In summary, this study provides a foundation for the exploration of combining future small-molecule HRI inhibitors with additional pharmacologic HbF inducers to maximize HbF production and preserve erythroid cell functionality for the treatment of SCD and other hemoglobinopathies.

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Heme-Regulated Inhibitor (HRI) Loss and Pharmacologic Treatments Cooperate to Strongly Elevate Fetal Hemoglobin and Reduce Sickle Cell Formation

Blood ◽

10.1182/blood-2018-99-117436 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 721-721

Author(s):

Scott A. Peslak ◽

Jeremy D. Grevet ◽

Xianjiang Lan ◽

Osheiza Abdulmalik ◽

Junwei Shi ◽

...

Keyword(s):

Protein Kinase ◽

Sickle Cell ◽

Cell Formation ◽

Fetal Hemoglobin ◽

Cell Maturation ◽

Erythroid Cell ◽

Cd34 Cells ◽

Hbf Induction ◽

Pharmacologic Treatments

Abstract Increasing fetal hemoglobin (HbF) provides significant clinical benefit for patients with sickle cell disease (SCD) and is a critical goal that is being pursued with a variety of pharmacologic strategies. Hydroxyurea (HU) is currently the only FDA-approved drug aimed at raising HbF for SCD patients but is limited in its efficacy. Recent work in our laboratory utilizing a protein kinase-domain focused CRISPR-Cas9 based genetic screen identified heme-regulated inhibitor (HRI), an erythroid-specific protein kinase, as a novel HbF regulator (Grevet et al., Science 361:6399, 285-290). Depleting HRI in a human erythroid cell line and primary human erythroid cultures significantly raised HbF levels. Besides HU, several compounds have been recently identified as in vitro HbF inducers, including pomalidomide, a third-generation imide, and UNC0638, an EHMT 1/2 histone methyltransferase inhibitor. While all these approaches induce HbF in vitro, it remains to be seen whether these would achieve sufficient levels of HbF induction in patients when used as single agents. Although the specific mechanisms by which HRI, HU, pomalidomide and UNC0638 regulate HbF remain to be fully resolved, we hypothesized that they might work in distinct pathways, and that by combining them might improve effect size and diminish potential adverse effects on erythroid cell maturation and viability. We utilized a three-stage in vitro culture system that recapitulates normal erythropoiesis by terminal differentiation of adult CD34+ hematopoietic cells. Primary human CD34+ cells were treated with select pharmacologic inducers of HbF, including hydroxyurea, pomalidomide, or UNC0638, in combination with shRNA lentiviral knockdown of HRI. HbF levels were assessed by RT-qPCR, Western blot, flow cytometry, and cation-exchange HPLC. We find that knockdown of HRI leads to significant HbF induction as expected. We further observed an inverse correlation between remaining HRI levels and HbF induction with maximal effect size requiring 85%-90% HRI depletion. HU displayed comparatively little activity and failed to increase the effects of HRI knockdown. However, treatment with pharmacologic levels of pomalidomide or UNC0638 combined with HRI depletion each showed greater than additive effects in HbF with levels reaching 25-30% HbF for UNC0638-treated and 30-40% HbF for pomalidomide-treated combinations, suggesting underlying cooperativity of HbF induction. RT-qPCR and Western blot analyses suggest that the diminished expression of the HbF repressor BCL11A accounts in large part for HbF induction in HRI knockdown samples, particularly when combined with pomalidomide treatment in which Bcl11A depletion was greater than 90%, while other HbF repressors such as LRF were unchanged. To determine whether HbF induction results in reduced sickle cell formation, we performed combination HRI depletion and HbF pharmacologic induction in primary CD34+ cells derived from sickle cell patients. HRI depletion in primary SCD cells showed a marked increase in HbF from baseline levels; in addition, significant cooperativity with pomalidomide and UNC0638 was observed, achieving 45-50% HbF for UNC0638-treated and 50-60% HbF for pomalidomide-treated drug combinations with no apparent detrimental effects on erythroid differentiation or maturation. Importantly, combination of HRI knockdown and HbF pharmacologic treatments markedly reduced in vitro sickling as measured by low-oxygen sickling assays, suggesting significant amelioration of the sickle cell phenotype in vitro. Overall, we find that the combination of HbF pharmacologic induction and shRNA-mediated HRI inhibition results in significant cooperative upregulation of HbF levels in both normal and sickle cell-derived primary human cells without impairing red cell maturation. We are currently exploring additional potential synergies with HbF regulators to determine the optimal modalities to maximize HbF induction. While no effective and specific HRI inhibitors are currently available, our work suggests that future small molecule inhibition of HRI may be combined with other pharmacotherapies to achieve significant, clinically meaningful HbF induction for the treatment of SCD and other hemoglobinopathies. Disclosures No relevant conflicts of interest to declare.

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Large-Scale Drug Screen Identifies FDA-Approved Drugs for Repurposing in Sickle-Cell Disease

Journal of Clinical Medicine ◽

10.3390/jcm9072276 ◽

2020 ◽

Vol 9 (7) ◽

pp. 2276

Author(s):

Matthew Cannon ◽

Hannah Phillips ◽

Sidney Smith ◽

Katie Williams ◽

Lindsey Brinton ◽

...

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Large Scale ◽

Treatment Options ◽

Fetal Hemoglobin ◽

Erythroid Cell ◽

Drug Screen ◽

Cell Disease ◽

Approved Drugs ◽

Fda Approved Drugs

Sickle-cell disease (SCD) is a debilitating hematological disorder with very few approved treatment options. Therapeutic reactivation of fetal hemoglobin (HbF) is one of the most pursued methods for ameliorating the systemic manifestations of SCD. Despite this, very few pharmacological agents have advanced to clinical trials or marketing for use. In this study, we report the development of an HbF in situ intracellular immunoblot assay coupled to a high-throughput drug screen to identify Food and Drug Administration (FDA) approved drugs that can be repurposed clinically for treatment of SCD. Using this assay we evaluated the National Institute of Health (NIH) Clinical Collection (NCC), a publicly available library of 725 small molecules, and found nine candidates that can significantly re-express HbF in erythroid cell lines as well as primary erythroblasts derived from SCD patients. Furthermore, we show the strong effects on HbF expression of these candidates to occur with minimal cytotoxicity in 7 of the 9 drugs. Given these data and their proven history of use for other indications, we hypothesize that several of these candidate drugs warrant further investigation for use in SCD.

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Heme-Regulated Inhibitor (HRI) Depletion Cooperates with Pharmacologic Inducers to Strongly Elevate Fetal Hemoglobin and Reduce Sickle Cell Formation

Blood ◽

10.1182/blood-2019-125664 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3557-3557

Author(s):

Scott A. Peslak ◽

Xianjiang Lan ◽

Eugene Khandros ◽

Peng Huang ◽

Jennifer A. Yano ◽

...

Keyword(s):

Sickle Cell ◽

Research Funding ◽

Fetal Hemoglobin ◽

Cell Maturation ◽

Erythroid Cell ◽

Dose Titration ◽

Cell Phenotype ◽

Erythroid Cells ◽

Hbf Induction

Increasing fetal hemoglobin (HbF) provides significant clinical benefit for patients with sickle cell disease (SCD) and is a critical goal that is being pursued with a variety of pharmacologic strategies. Hydroxyurea (HU) is currently the only FDA-approved drug aimed at raising HbF but is limited in its efficacy. Recent work in our laboratory identified heme-regulated inhibitor (HRI), an erythroid-specific protein kinase, as a novel HbF regulator (Grevet et al., Science 2018). Depleting HRI in primary human erythroid cells significantly raised HbF levels; however, maximal HbF induction by HRI knockdown required at least 80-85% HRI depletion. It is currently unknown whether this degree of HRI inhibition can be achieved pharmacologically. Besides HU, several compounds have been recently identified as in vitro HbF inducers, including pomalidomide, a third-generation imide, and UNC0638, an EHMT 1/2 histone methyltransferase inhibitor. We therefore set out to test whether combining HRI depletion with pharmacologic HbF inducers would cooperatively increase HbF levels and diminish potential adverse effects on erythroid cell maturation and viability. We performed three-stage in vitro culture of human CD34+ cells treated with select pharmacologic inducers of HbF, including HU, pomalidomide, or UNC0638, in combination with shRNA knockdown of HRI. HbF levels were assessed by RT-qPCR, Western blot, flow cytometry, and HPLC. We found that treatment with HU displayed comparatively little activity and failed to increase effects of HRI knockdown. However, treatment with UNC0638 combined with HRI depletion showed greater than additive effects on HbF with levels reaching 25-30% HbF, while HRI depletion combined with pomalidomide treatment showed the highest levels of cooperativity, reaching 30-40% HbF. Combination of HRI depletion and HbF pharmacologic inducers showed minimal effects on the erythroid transcriptome by RNA-Seq and did not significantly impair erythroid maturation. Intriguingly, dose-titration experiments indicated that HRI knockdown sensitizes erythroid cells to low doses of pomalidomide, maintaining HbF levels of greater than 40% in HRI-depleted samples despite a ten-fold decrease in pomalidomide concentration. Diminished expression of the HbF repressor BCL11A accounted in large part for HbF induction in HRI knockdown samples, particularly when combined with pomalidomide treatment in which BCL11A depletion exceeded 90%, while other HbF repressors such as LRF were unchanged. We will present RNASeq analyses aimed at elucidating mechanisms of HRI cooperativity. Finally, we found that HRI depletion in SCD patient-derived cells showed significant cooperativity with pomalidomide and UNC0638, achieving 45-50% HbF for UNC0638-treated and 50-60% HbF for pomalidomide-treated drug combinations. Importantly, combination of HRI knockdown and HbF pharmacologic treatments markedly reduced in vitro sickling as measured by low-oxygen sickling assays, suggesting significant amelioration of the sickle cell phenotype. In sum, we find that dual targeting of HbF induction via HRI inhibition and pharmacologic inducers results in successful cooperative upregulation of HbF levels in both normal and SCD primary human cells without impairing red cell maturation. Furthermore, our data suggest that dose titration of HbF inducers combined with HRI depletion could maximize HbF induction while potentially reducing off-target effects. Moreover, HRI is an attractive target for HbF induction as it is expressed in an erythroid-specific manner. While no specific HRI inhibitors are currently available, our work suggests that future small molecule inhibitors of HRI may be combined with other pharmacotherapies to achieve significant, clinically meaningful HbF induction for the treatment of SCD and other hemoglobinopathies. Disclosures Abdulmalik: The Children's Hospital of Philadelphia: Patents & Royalties: Provisional Patent. Blobel:Bioverativ: Research Funding; Pfizer: Research Funding.

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Salubrinal induces fetal hemoglobin expression via the stress-signaling pathway in human sickle erythroid progenitors and sickle cell disease mice

10.1101/2021.12.13.472406 ◽

2021 ◽

Author(s):

Betty Pace ◽

Nicole H Lopez ◽

Biaoru Li ◽

Chithra Palani ◽

Umapathy Siddaramappa ◽

...

Keyword(s):

Sickle Cell Disease ◽

Signaling Pathway ◽

Sickle Cell ◽

Fetal Hemoglobin ◽

Stress Signaling ◽

Cell Disease ◽

Erythroid Progenitors ◽

F Cells ◽

Hbf Induction ◽

Stress Signaling Pathway

Sickle cell disease (SCD) is an inherited blood disorder caused by a mutation in the HBB gene leading to hemoglobin S production and polymerization under hypoxia conditions leading to vaso-occlusion, chronic hemolysis, and progressive organ damage. This disease affects ~100,000 people in the United States and millions worldwide. An effective therapy for SCD is fetal hemoglobin (HbF) induction by pharmacologic agents such as hydroxyurea, the only Food and Drug Administration-approved drug for this purpose. Therefore, the goal of our study was to determine whether salubrinal (SAL), a selective protein phosphatase 1 inhibitor, induces HbF expression through the stress-signaling pathway by activation of p-eIF2α and ATF4 trans-activation in the γ-globin gene promoter. Sickle erythroid progenitors treated with 24µM SAL increased F-cells levels 1.4-fold (p=0.021) and produced an 80% decrease in reactive oxygen species. Western blot analysis showed SAL enhanced HbF protein by 1.6-fold (p=0.0441), along with dose-dependent increases of p-eIF2α and ATF4 levels. Subsequent treatment of SCD mice by a single intraperitoneal injection of SAL (5mg/kg) produced peak plasma concentrations at 6 hours. Chronic treatments of SCD mice with SAL mediated a 2.3-fold increase in F-cells (p=0.0013) and decreased sickle erythrocytes supporting in vivo HbF induction.

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Adenosine Base Editing of γ-Globin Promoters Induces Fetal Hemoglobin and Inhibit Erythroid Sickling

Blood ◽

10.1182/blood-2020-141498 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 21-22

Author(s):

Thiyagaraj Mayuranathan ◽

Jonathan S. Yen ◽

Gregory A. Newby ◽

Yu Yao ◽

Shaina N. Porter ◽

...

Keyword(s):

Bone Marrow ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Fetal Hemoglobin ◽

Erythroid Cell ◽

Cell Disease ◽

Private Company ◽

Hematopoietic Stem ◽

Benign Condition ◽

Base Editing

Rare variants in the γ-globin (HBG2 and HBG1) promoters cause sustained postnatal expression of fetal hemoglobin (HbF, α2γ2) in red blood cells (RBCs). This benign condition is termed hereditary persistence of fetal hemoglobin (HPFH). Individuals with HPFH variants are protected from β-hemoglobinopathies including sickle cell disease and β-thalassemia. Our group and others have used CRIPSR/Cas9-mediated non-homologous end joining to generate HPFH-like insertion-deletion (indel) mutations in the γ-globin promoter. However, simultaneous double-stranded breaks (DSBs) in the tandem duplicated γ-globin genes can result in loss or inversion of the intervening genetic material and/or chromosomal rearrangements. More generally, Cas9-associated DSBs can elicit a cytotoxic DNA repair response leading to cell death or evoke p53 loss with malignant transformation. Base editor (BE) proteins represent a promising approach to install precise nucleotide substitutions without DSBs. Adenosine base editors (ABEs), consisting of catalytically impaired Cas9 fused to a modified adenosine deaminase, create targeted A:T-to-G:C mutations. Here we describe the use of ABEs to recapitulate naturally occurring HPFH variants in hematopoietic stem cells (HSCs). We electroporated ABE7.10-single guide (sg) RNA ribonucleoprotein (RNP) complex into mobilized peripheral blood CD34+ hematopoietic stem and progenitor cells (HSPCs) to recreate 3 different HPFH variants in the HBG1/2 promoters (-198 T>C, -175 T>C and -113 A>G). Measured editing frequency was maximal on day 10 after electroporation and transferred to erythroid differentiation media. 20% editing efficiency was observed for the -198 site, 58% for -175 and 50% for -113. Indel frequencies were <2% at each of the three sites, reflecting a low rate of DSBs. Fetal hemoglobin levels in erythroid cells generated in vitro from A base-edited CD34+ HSPCs were 26±4% (-198 T>C), 60±10% (-175 T>C), and 42±7% (-113 A>G) versus14±2% in unedited control cells. Base editing at the -175 site in sickle cell disease (SCD) donor CD34+ HSPCs resulted in the induction of HbF to 55% in erythroid progeny compared to 6% in controls. After exposure to hypoxia (2% oxygen), reticulocytes generated from -175 T>C-edited CD34+ HSPCs exhibited sickling rates of 24%, compared to 52% in controls. Thus, creation of this variant, which generates a de novo binding site for the transcriptional activator TAL1, reactivates erythroid cell HbF to levels that inhibit sickle hemoglobin polymerization and cell sickling. To assess base editing in HSCs, we used ABE RNP to modify the -175 site in SCD donor CD34+ HSPCs, followed by transplantation into NBSGW mice. The editing frequency in CD34+ HSPCs before transplantation was ~30% and declined to approximately 20% in bone marrow-repopulating donor cells at 16 weeks post-transplantation. Editing frequencies were similar in CD34+ donor cell-derived myeloid, erythroid, and B cells, indicating that hematopoietic differentiation was not altered. Bone marrow erythroblasts derived from base-edited and control CD34+ HSPCs exhibited similar maturation profiles and enucleation. Erythroblasts generated in vivo from SCD patient HSPCs exhibited 32±2% HbF compared to unedited controls (4±1%) (n=4, P>0.0001). Our studies provide proof of concept that adenosine base editors can be used therapeutically for β-hemoglobinopathies. Specifically, generation of the -175 T>C HPFH mutation in patient HSCs followed by autologous transplantation represents a new therapeutic approach for SCD and β-thalassemia. Disclosures Yen: Beam Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Sharma:Spotlight Therapeutics: Consultancy; Magenta Therapeutics: Other: Research Collaboration; CRISPR Therapeutics, Vertex Pharmaceuticals, Novartis: Other: Clinical Trial PI. Liu:Pairwise Plants: Consultancy, Patents & Royalties; Editas Medicine: Consultancy, Patents & Royalties; Beam Therapeutics: Consultancy, Patents & Royalties; Prime Medicine: Consultancy, Patents & Royalties. Weiss:Beam Therapeuticcs: Consultancy, Current equity holder in private company; Esperion Therapeutics: Consultancy, Current equity holder in private company; Novartis: Consultancy, Current equity holder in private company; Cellarity Inc.: Consultancy, Current equity holder in private company; Rubius Inc.: Consultancy, Current equity holder in private company.

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LSD1 Inhibition Induces Fetal Hemoglobin Expression and Provides a Novel Therapeutic Approach to Sickle Cell Disease

Blood ◽

10.1182/blood.v128.22.2472.2472 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2472-2472

Author(s):

Sang Hyun Lee ◽

Maxim Soloviev ◽

Yan Zhang ◽

Valerie Roman ◽

Gengjie Yang ◽

...

Keyword(s):

Gene Expression ◽

Sickle Cell ◽

Therapeutic Approach ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Epigenetic Mechanisms ◽

Cell Disease ◽

F Cells ◽

Hbf Induction ◽

Novel Therapeutic

Abstract Sickle cell disease (SCD) is an autosomal recessive genetic disorder caused by a point mutation in the human β-globin gene. Patients harboring this mutation can exhibit long-chain polymers of hemoglobin and sickle-shaped red blood cells, and suffer from severe medical manifestations including hemolysis and vaso-occlusive crises. Multiple preclinical, clinical and epidemiologic studies have shown that the levels of unmutated fetal hemoglobin (HbF encoded by the γ-globin gene) correlate with less severe disease, validating HbF induction as a therapeutic approach in SCD. Treatment with hydroxyurea (HU), the only approved therapy for SCD, results in a variable induction of HbF and significant improvement in the frequency of pain crises. However, a significant percentage of patients treated with HU fail to exhibit durable benefit, necessitating the need for alternative therapeutic agents. The human γ-globin gene is repressed in the post-natal period by epigenetic mechanisms, and therefore may lend itself to pharmacological intervention aimed at derepressing gene expression. One of the most important of these epigenetic mechanisms is catalyzed by lysine-specific demethylase 1 (LSD1), a histone demethylase that removes mono-/dimethyl marks from the lysine 4 and 9 residues of histone H3 through an FAD-directed redox process. Here, we report the characterization of selective, potent, and orally bioavailable LSD1 inhibitors from two classes - FAD-directed inhibitors that achieve inhibitory activity through formation of covalent FAD-adducts and non-FAD-directed, reversible inhibitors - and demonstrate their ability to induce γ-globin gene expression in murine and primate preclinical models. In the Towne's SCD mouse model, oral administration of LSD1 inhibitors significantly increased HbF+ cell (F cell) production. Concurrent with the increase in F cells, sickle cell numbers, reticulocyte counts, and bilirubin levels were all markedly reduced, indicating an amelioration of several pathophysiological features of SCD. FAD- and non-FAD-directed LSD1 inhibitors were more effective than HU in increasing F cells production, and the combination of HU and suboptimal doses of LSD1 inhibitors resulted in a greater induction of F cells and more pronounced reductions in reticulocyte counts and bilirubin levels. In addition to the humanized SCD model, HbF induction in response to LSD1 inhibitor treatment was evaluated in non-anemic cynomolgus monkeys. Oral administration of LSD1 inhibitors significantly induced F cells and HbF in a dose-dependent manner and over a sustained period (>50 days) following the discontinuation of treatment. The percentage of induced F cells in total RBCs was linearly correlated with the percentage of HbF protein induced by LSD1 inhibition. Taken together, these results support the potential utility of LSD1 inhibition as a novel therapeutic approach to increase HbF production. Disclosures Lee: Incyte Corporation: Employment, Other: Stock. Soloviev:Incyte Corporation: Employment, Other: Stock. Zhang:Incyte Corporation: Employment, Other: Stock. Roman:Incyte Corporation: Employment, Other: Stock. Yang:Incyte Corporation: Employment, Other: Stock. Bowman:Incyte Corporation: Employment, Other: Stock. Burke:Incyte Corporation: Employment, Other: Stock. Margulis:Incyte Corporation: Employment, Other: Stock. O'Connor:Incyte Corporation: Employment, Other: Stock. Yang:Incyte Corporation: Employment, Other: Stock. Wu:Incyte Corporation: Employment, Other: Stock. Wynn:Incyte Corporation: Employment, Other: Stock. Burn:Incyte Corporation: Employment, Other: Stock. Shuey:Incyte Corporation: Employment, Other: stock. Diamond:Incyte Corporation: Employment, Other: Stock. Yao:Incyte Corporation: Employment, Other: Stock. Hollis:Incyte Corporation: Employment, Other: Stock. Yeleswaram:Incyte Corporation: Employment, Other: Stocks. Roberts:Incyte Corporation: Employment, Other: Stock. Huber:Incyte Corporation: Employment, Other: Stock. Scherle:Incyte Corporation: Employment, Other: Stock. Ruggeri:Incyte Corporation: Employment, Other: Stock.

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Fetal hemoglobin induction by acetate, a product of butyrate catabolism [see comments]

Blood ◽

10.1182/blood.v84.9.3198.3198 ◽

1994 ◽

Vol 84 (9) ◽

pp. 3198-3204 ◽

Cited By ~ 32

Author(s):

G Stamatoyannopoulos ◽

CA Blau ◽

B Nakamoto ◽

B Josephson ◽

Q Li ◽

...

Keyword(s):

Fatty Acids ◽

Sickle Cell Disease ◽

Umbilical Cord Blood ◽

Sodium Acetate ◽

Fetal Hemoglobin ◽

Cell Disease ◽

Gamma Globin ◽

Hbf Induction ◽

Globin Synthesis

Abstract Butyrate induces fetal hemoglobin (HbF) synthesis in cultures of erythroid progenitors, in primates, and in man. The mechanism by which this compound stimulates gamma-globin synthesis is unknown. In the course of butyrate catabolism, beta oxidation by mitochondrial enzymes results in the formation of two acetate molecules from each molecule of butyrate. Studies were performed to determine whether acetate itself induces HbF synthesis. In erythroid burst-forming unit (BFU-E) cultures from normal persons, and individuals with sickle cell disease and umbilical-cord blood, dose-dependent increases in gamma-globin protein and gamma mRNA were consistently observed in response to increasing acetate concentrations. In BFU-E cultures from normal adults and patients with sickle cell disease, the ratio of gamma/gamma + beta mRNA increased twofold to fivefold in response to acetate, whereas the percentage of BFU-E progeny staining with an anti-gamma monoclonal antibody (MoAb) increased approximately twofold. Acetate-induced increases in gamma-gene expression were also noted in the progeny of umbilical cord blood BFU-E, although the magnitude of change in response to acetate was less because of a higher baseline of gamma- chain production. The effect of acetate on HbF induction in vivo was evaluated using transgenic mouse and primate models. A transgenic mouse bearing a 2.5-kb mu locus control region (mu LCR) cassette linked to a 3.3-kb A gamma gene displayed a near twofold increase in gamma mRNA during a 10-day infusion of sodium acetate at a dose of 1.5 g/kg/d. Sodium acetate administration in baboons, in doses ranging from 1.5 to 6 g/kg/d by continuous intravenous infusion, also resulted in the stimulation of gamma-globin synthesis, with the percentage of HbF- containing reticulocytes (F reticulocytes) approaching 30%. Surprisingly, a dose-response effect of acetate on HbF induction was not observed in the baboons, and HbF induction was not sustained with prolonged acetate administration. These results suggest that both two- carbon fatty acids (acetate) and four-carbon fatty acids (butyrate) stimulate synthesis of HbF in vivo.

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DNA polymorphisms at the BCL11A, HBS1L-MYB, and -globin loci associate with fetal hemoglobin levels and pain crises in sickle cell disease

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0804799105 ◽

2008 ◽

Vol 105 (33) ◽

pp. 11869-11874 ◽

Cited By ~ 326

Author(s):

G. Lettre ◽

V. G. Sankaran ◽

M. A. C. Bezerra ◽

A. S. Araujo ◽

M. Uda ◽

...

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Fetal Hemoglobin ◽

Dna Polymorphisms ◽

Cell Disease

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Vaso-occlusion in sickle cell disease: pathophysiology and novel targeted therapies

Hematology ◽

10.1182/asheducation-2013.1.362 ◽

2013 ◽

Vol 2013 (1) ◽

pp. 362-369 ◽

Cited By ~ 33

Author(s):

Deepa Manwani ◽

Paul S. Frenette

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Targeted Therapies ◽

Therapeutic Intervention ◽

Fetal Hemoglobin ◽

Cell Disease ◽

The Past ◽

Symptomatic Management

Abstract Recurrent and unpredictable episodes of vaso-occlusion are the hallmark of sickle cell disease. Symptomatic management and prevention of these events using the fetal hemoglobin–reactivating agent hydroxyurea are currently the mainstay of treatment. Discoveries over the past 2 decades have highlighted the important contributions of various cellular and soluble participants in the vaso-occlusive cascade. The role of these elements and the opportunities for therapeutic intervention are summarized in this review.

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Hydroxyurea Treatment for Sickle Cell Disease

The Scientific World JOURNAL ◽

10.1100/tsw.2002.295 ◽

2002 ◽

Vol 2 ◽

pp. 1706-1728 ◽

Cited By ~ 13

Author(s):

Martin H. Steinberg

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Fetal Hemoglobin ◽

Hemoglobin Concentration ◽

Pharmacologic Therapy ◽

Acute Chest Syndrome ◽

Cell Disease ◽

Globin Chains ◽

Hydroxyurea Treatment ◽

Erythroid Precursors

High fetal hemoglobin (HbF) levels inhibit the polymerization of sickle hemoglobin (HbS) and reduce the complications of sickle cell disease. Pharmacologic agents that can reverse the switch from γ- to β-chain synthesis — γ-globin chains characterize HbF, and sickle β-globin chains are present in HbS — or selectively increase the proportion of adult erythroid precursors that maintain the ability to produce HbF are therapeutically useful. Hydroxyurea promotes HbF production by perturbing the maturation of erythroid precursors. This treatment increases the total hemoglobin concentration, reduces the vaso-occlusive complications of pain and acute chest syndrome, and attenuates mortality in adults. It is a promising beginning for pharmacologic therapy of sickle cell disease. Still, its effects are inconsistent, trials in infants and children are ongoing, and its ultimate value — and peril — when started early in life are still unknown.

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