scholarly journals Domain-focused CRISPR screen identifies HRI as a fetal hemoglobin regulator in human erythroid cells

Science ◽  
2018 ◽  
Vol 361 (6399) ◽  
pp. 285-290 ◽  
Author(s):  
Jeremy D. Grevet ◽  
Xianjiang Lan ◽  
Nicole Hamagami ◽  
Christopher R. Edwards ◽  
Laavanya Sankaranarayanan ◽  
...  
Keyword(s):  
Fetal Hemoglobin ◽  
Protein Translation ◽  
Kinase Domain ◽  
Erythroid Cells ◽  
Cell Disease ◽  
Potential Therapeutic Target ◽  
Protein Kinase Domain ◽  
Guide Rna ◽  
Specific Manner ◽  
Crispr Screen

Increasing fetal hemoglobin (HbF) levels in adult red blood cells provides clinical benefit to patients with sickle cell disease and some forms of β-thalassemia. To identify potentially druggable HbF regulators in adult human erythroid cells, we employed a protein kinase domain–focused CRISPR-Cas9–based genetic screen with a newly optimized single-guide RNA scaffold. The screen uncovered the heme-regulated inhibitor HRI (also known as EIF2AK1), an erythroid-specific kinase that controls protein translation, as an HbF repressor. HRI depletion markedly increased HbF production in a specific manner and reduced sickling in cultured erythroid cells. Diminished expression of the HbF repressor BCL11A accounted in large part for the effects of HRI depletion. Taken together, these results suggest HRI as a potential therapeutic target for hemoglobinopathies.

Human Fetal Hemoglobin Expression Is Regulated by the Developmental Stage-Specific Repressor BCL11A

Science ◽  
2008 ◽  
Vol 322 (5909) ◽  
pp. 1839-1842 ◽  
Author(s):  
Vijay G. Sankaran ◽  
Tobias F. Menne ◽  
Jian Xu ◽  
Thomas E. Akie ◽  
Guillaume Lettre ◽  
...  
Keyword(s):  
Association Studies ◽  
Globin Gene ◽  
Genetic Association Studies ◽  
Fetal Hemoglobin ◽  
Sequence Variants ◽  
Erythroid Cells ◽  
Cell Disease ◽  
Direct Role ◽  
Globin Gene Regulation

Differences in the amount of fetal hemoglobin (HbF) that persists into adulthood affect the severity of sickle cell disease and the β-thalassemia syndromes. Genetic association studies have identified sequence variants in the gene BCL11A that influence HbF levels. Here, we examine BCL11A as a potential regulator of HbF expression. The high-HbF BCL11A genotype is associated with reduced BCL11A expression. Moreover, abundant expression of full-length forms of BCL11A is developmentally restricted to adult erythroid cells. Down-regulation of BCL11A expression in primary adult erythroid cells leads to robust HbF expression. Consistent with a direct role of BCL11A in globin gene regulation, we find that BCL11A occupies several discrete sites in the β-globin gene cluster. BCL11A emerges as a therapeutic target for reactivation of HbF in β-hemoglobin disorders.

Additive Effect of Butyrate and Hemin on Fetal Hemoglobin Induction in Erythroid Cells from Sickle Cell Disease and Beta-Thalassemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1224-1224
Author(s):  
Hassana Fathallah ◽  
Ali Taher ◽  
Ali Bazarbachi ◽  
George F. Atweh
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Histone Deacetylases ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Beta Thalassemia ◽  
Erythroid Cells ◽  
Cell Disease ◽  
Globin Mrna ◽  
In Erythroid Cells

Abstract High levels of fetal hemoglobin (HbF) are known to ameliorate the pathophysiology of β-globin disorders. The objective of this study is twofold: the first is to evaluate the efficacy of hemin as an inducer of HbF in erythroid cells from patients with sickle cell disease (SCD) and β-thalassemia (β-thal); the second is to determine if the combination of butyrate and hemin can induce higher levels of expression of HbF than either agent alone. BFU-E derived cells from the peripheral blood of two patients with homozygous SCD, three patients with β-thal, one patient with sickle β-thalassemia (S/β-thal) and one normal individual (AA) were cultured in the absence (control) or presence of butyrate (B), hemin (H) or butyrate and hemin (B+H). As expected, the levels of γ-globin mRNA [expressed as % γ/(β+γ)] increased upon butyrate exposure in progenitor-derived erythroid cells from SS and S/β-thal patients, and to a lesser extent in patients with β-thal (P = 0.01). In contrast, butyrate did not increase γ-globin expression in BFU-E derived colonies from the AA individual. Moreover, hemin exposure increased the γ/(β+γ) ratio in all subjects (P = 0.02). These findings confirm that hemin can be an effective HbF inducing agent in SCD and β-thal. Although the mechanism of induction of HbF by hemin is not known, unlike butyrate, hemin is clearly not a direct inhibitor of histone deacetylases and is likely to induce HbF by a different mechanism of action. Thus, we investigated the effect of the combination of hemin and butyrate on γ-globin gene expression. Interestingly, the combination of butyrate and hemin resulted in additive increases in the γ/(β+γ) ratios in all patients compared to butyrate alone (P = 0.03) or hemin alone (P = 0.01) (Table I). Just as importantly, exposure to both drugs resulted in a decrease in the α/(β+γ) mRNA imbalance in β-thal, which is the predominant pathophysiological feature of this disorder. In conclusion, combination therapy consisting of butyrate and hemin, which are two agents with different mechanisms of action and different toxicity profiles, may provide a more effective way of inducing HbF in patients with SCD and β-thal. Table I mRNA SCD β-Thal S/β-Thal AA n 2 3 1 1 %γ/(β+γ) Control 36 42 26 7.1 B 45 50 41 6.9 H 55 55 52 15 B+H 60 61 59 13 α/(β+γ) Control 3.1 8.9 1.8 1.9 B 2.0 7.7 2.9 1.7 H 3.0 7.5 1.7 1.0 B+H 2.9 6.4 2.2 1.3

scholarly journals In Vivo Characterization of Ftx-6058, a Novel Small Molecular Fetal Hemoglobin Inducer for Sickle Cell Disease

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 26-27
Author(s):  
Keqiang Xie ◽  
Mark Roth ◽  
Ivan Efremov ◽  
Serena Silver ◽  
Lucienne Ronco ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Fetal Hemoglobin ◽  
Erythroid Cells ◽  
Cell Disease ◽  
Publicly Traded ◽  
F Cells ◽  
Biological Target

Sickle cell disease (SCD) results from genetic mutation in the β-globin gene encoding a subunit of the adult form of hemoglobin (HbA), leading to red blood cell (RBC) deformation and disease pathology. It has been demonstrated that reactivation of the fetal ortholog of the hemoglobin beta subunit, HBγ (also referred to as HBG proteins), can prevent or reduce disease-related pathophysiology. In SCD, the presence of HBG protein in hemoglobin tetramers prevents sickle hemoglobin polymerization under deoxygenated conditions and therefore may be of therapeutic benefit in SCD. FTX 6058, a novel orally bioavailable small molecule, is in development for the treatment of sickle cell disease (SCD) by Fulcrum Therapeutics. FTX-6058 was demonstrated to inhibit the novel biological target and elevate the expression of HBγ, resulting in induction of fetal hemoglobin (HbF) tetramer in differentiated human primary CD34+ cells. The in vivo target engagement (TE) and pharmacologic effects of FTX-6058 were characterized in wild-type CD-1 mice and humanized Townes SCD mice, with TE also confirmed in non-human primates. In CD-1 mice, once-daily (QD) FTX-6058 oral administration induced TE in a time- and dose-dependent manner and most markedly in erythroid lineage (Ter119+) cells derived from bone marrow, the putative therapeutic compartment, and increased transcript levels of Hbb-bh1, a murine embryonic hemoglobin surrogate for human HBG gene. Steady state TE in circulating monocytes, following repeated QD FTX-6058 administration, correlated well with that in bone marrow-derived erythroid cells, suggesting peripheral monocytes as a suitable surrogate for assessing erythroid TE activity in Fulcrum's Phase 1 study. In non-human primate cynomolgus monkeys, QD oral dosing of FTX-6058 as early as for 7 days induced robust and comparable TE in bone marrow derived CD34+ erythroid progenitors and circulating monocytes, further supporting the use of monocytes to assess TE in bone marrow. Mouse data also provided evidence of the reversibility of TE effects once dosing is stopped. In repeat-dose studies in the humanized Townes SCD mouse model, FTX-6058 was superior to standard of care hydroxyurea as measured by human HBG1 transgene induction and increased %F-cells and HBG1 protein levels. Furthermore, the induction of %F cells was sustainable for several days after dosing cessation. These in vivo studies have demonstrated that FTX-6058 engages its novel biological target in multiple preclinical models and induces HbF expression at plasma concentrations likely to b e readily achievable in clinic, and peripheral monocytes is a suitable surrogate for assessing TE in bone marrow erythroid cells, which could be beneficial to patients with SCD. Disclosures Xie: Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Roth:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Efremov:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Silver:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Ronco:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Thompson:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Stickland:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Moxham:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Wallace:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company.

scholarly journals Control of Human Hemoglobin Switching By LIN28B-Mediated Regulation of BCL11A Translation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 412-412
Author(s):  
Anindita Basak ◽  
Mathias Munschauer ◽  
Jacob C. Ulirsch ◽  
Kara E. Montbleau ◽  
Christina R. Hartigan ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Protein Expression ◽  
Sickle Cell ◽  
Developmental Regulation ◽  
Fetal Hemoglobin ◽  
Globin Genes ◽  
Human Hemoglobin ◽  
Erythroid Cells ◽  
Cell Disease ◽  
Hemoglobin Switching

Abstract Both sickle cell disease and β-thalassemia are major sources of morbidity and mortality worldwide. Continued production of the β-like γ-globin genes that form fetal hemoglobin after infancy has been shown to ameliorate the severity of these disorders. As a result, there has been considerable interest in understanding the underlying regulation of the physiologic fetal-to-adult hemoglobin switch in humans to be able to better manipulate this process for therapeutic purposes. To date, only a single factor, BCL11A, has been identified as being involved in the developmental regulation of human hemoglobin switching. BCL11A is a direct transcriptional repressor of the γ-globin genes. Moreover, BCL11A is expressed in a developmental stage-specific manner to regulate human hemoglobin switching. However, despite extensive studies, the mechanisms that act upstream to regulate BCL11A expression and thereby control hemoglobin switching have remained elusive. To gain further insights, we have directly explored the developmental regulation of BCL11A expression at various stages of human erythropoiesis. We find that BCL11A is regulated at the level of mRNA translation during development. While BCL11A mRNA is comparably expressed in a similar manner at all developmental stages in erythroid cells, robust protein expression only occurs in adult erythroid cells. Importantly, we demonstrate that at the earlier stages of development, the observed reduction in protein expression is attributable to decreased synthesis and not increased degradation of BCL11A through direct assessment of both protein synthesis and degradation rates in primary erythroid cells from various stages of human development. Interestingly, while BCL11A protein is not well synthesized at these earlier stages of development, we find that its mRNA curiously continues to be associated with ribosomes in a comparable manner between newborn and adult erythroid precursors using polysome fractionation of stage-matched erythroid cells. Through use of an unbiased proteomic analysis approach involving RNA affinity purification of the 18S ribosomal RNA in erythroid cells, we demonstrate that the RNA-binding protein LIN28B, which is developmentally expressed in a reciprocal pattern to BCL11A, directly interacts with ribosomes. We additionally show that the observed suppression of BCL11A protein translation is mediated by LIN28B. Using a newly developed RNA isolation and crosslinking immunoprecipitation approach coupled to massively parallel sequencing we mapped the direct interaction of LIN28B with BCL11A mRNA at nucleotide resolution. Through a number of functional assays in both newborn and adult erythroid cells, we additionally demonstrate that this alteration of BCL11A translation is entirely independent of the role of LIN28B in the biogenesis of let-7 microRNAs. Finally, we show that BCL11A is the major functional target in LIN28B-mediated fetal hemoglobin induction through functional complementation experiments in primary human erythroid cells. Our results reveal a previously unappreciated regulatory mechanism underlying human hemoglobin switching. Moreover, our findings highlight opportunities for developing improved treatments for sickle cell disease and β-thalassemia by understanding the upstream regulation of human hemoglobin switching. Disclosures No relevant conflicts of interest to declare.

scholarly journals 3,3′,5-Triiodothyroacetic acid (TRIAC) induces embryonic ζ-globin expression via thyroid hormone receptor α

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Huiqiao Chen ◽  
Zixuan Wang ◽  
Shanhe Yu ◽  
Xiao Han ◽  
Yun Deng ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Hormone Receptor ◽  
Thyroid Hormone Receptor ◽  
Globin Gene ◽  
Erythroid Cells ◽  
Cell Disease ◽  
Potent Inducer ◽  
Thyroid Hormone Receptor Α

AbstractThe human ζ-globin gene (HBZ) is transcribed in primitive erythroid cells only during the embryonic stages of development. Reactivation of this embryonic globin synthesis would likely alleviate symptoms both in α-thalassemia and sickle-cell disease. However, the molecular mechanisms controlling ζ-globin expression have remained largely undefined. Moreover, the pharmacologic agent capable of inducing ζ-globin production is currently unavailable. Here, we show that TRIAC, a bioactive thyroid hormone metabolite, significantly induced ζ-globin gene expression during zebrafish embryogenesis. The induction of ζ-globin expression by TRIAC was also observed in human K562 erythroleukemia cell line and primary erythroid cells. Thyroid hormone receptor α (THRA) deficiency abolished the ζ-globin-inducing effect of TRIAC. Furthermore, THRA could directly bind to the distal enhancer regulatory element to regulate ζ-globin expression. Our study provides the first evidence that TRIAC acts as a potent inducer of ζ-globin expression, which might serve as a new potential therapeutic option for patients with severe α-thalassemia or sickle-cell disease.

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

Blood ◽  
1994 ◽  
Vol 84 (9) ◽  
pp. 3198-3204 ◽  
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.

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

2008 ◽  
Vol 105 (33) ◽  
pp. 11869-11874 ◽  
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

In silico profiling and structural insights of missense mutations in RET protein kinase domain by molecular dynamics and docking approach

2014 ◽  
Vol 10 (3) ◽  
pp. 421-436 ◽  
Author(s):  
C. George Priya Doss ◽  
B. Rajith ◽  
Chiranjib Chakraboty ◽  
V. Balaji ◽  
R. Magesh ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Protein Kinase ◽  
In Silico ◽  
Kinase Domain ◽  
Missense Mutations ◽  
Protein Kinase Domain ◽  
Docking Approach ◽  
Structural Insights

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

Hematology ◽  
2013 ◽  
Vol 2013 (1) ◽  
pp. 362-369 ◽  
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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