Induction of Human Fetal Hemoglobin Expression by Selective Inhibitors of Histone Deacetylase 1 and 2 (HDAC1/2)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3259-3259
Author(s):  
Jeffrey R Shearstone ◽  
John H van Duzer ◽  
James E Bradner ◽  
Ralph Mazitschek ◽  
Simon S Jones ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Clinical Trials ◽  
Dna Methyltransferase ◽  
Fetal Hemoglobin ◽  
Beta Thalassemia ◽  
Selective Inhibitors ◽  
Genetic Ablation ◽  
Histone Deacetylase 1 ◽  
Hbf Induction ◽  
Equity Ownership

Abstract Abstract 3259 Introduction: Fetal hemoglobin (HbF) induction is an established therapeutic strategy in sickle cell disease (SCD), and could also be effective in treating beta-thalassemia (bT). The only drug with proven efficacy in SCD is hydroxyurea, which is cytotoxic, poorly tolerated, and only reduces the frequency and severity of SCD crises in a subset of patients. For bT there are no approved drug treatments and, for the most severe forms of bT, patients require repeated blood transfusions for life and chelation therapy to reduce iron overload. Fetal (γ) globin expression is silenced in adults partly through the action of a complex containing BCL11A and HDAC1/2. Genetic ablation and chemical inhibition of HDAC1 or HDAC2, but not HDAC3, resulted in the induction of γ-globin synthesis in adult bone marrow derived CD34+ erythroid cells (Bradner, Proc Natl Acad Sci 2010). While a variety of non-specific HDAC inhibitors have been used successfully to induce HbF, further clinical development has been limited by their variable efficacy and concerns over off-target side-effects observed in small clinical trials, potentially due to inhibition of HDAC3. Therefore, development of selective and potent HDAC1/2 inhibitors leading to HbF induction represents a refined and targeted therapeutic approach for the treatment of SCD and bT. Results: Recently, selective inhibitors for HDAC1 and 2 have been described for compounds containing a benzamide zinc chelating group (Witter, Bioorg Med Chem Lett 2008). Acetylon Pharmaceuticals has generated a library of structurally distinct compounds containing a modified benzamide biasing element. We have screened these molecules in an HDAC biochemical assay platform and found IC50 values for HDAC1 and 2 ranging from 5 to 10 nM and 10 to 30 nM, respectively, with approximately 10- to 100-fold selectivity over inhibition of HDAC3. These compounds had good oral bioavailability in rat, with exposure (AUC) exceeding 5000 hr*ng/mL following an oral dose of 5 mg/kg. Furthermore, half-life was found to range between 4 to 8 hours. In cultured human CD34+ bone marrow cells undergoing erythroid differentiation, these compounds induced a dose dependent increase in fetal hemoglobin expression, with a 5-fold induction observed at 1 μM. The γ-globin induction we observe is comparable to MS-275, a non-selective HDAC1/2/3 inhibitor, and decitabine, a DNA methyltransferase inhibitor which has been in clinical trials to induce HbF in SCD. The absolute levels of adult β-globin remained unchanged. These results were confirmed using flow cytometry, where a 2- to 4-fold increase in the number of cells expressing HbF protein was observed. Conclusion: These results suggest that inhibition of HDAC1 and 2 is sufficient to induce fetal globin expression. Our selective HDAC1/2 inhibitors have highly favorable oral pharmacokinetic profiles suitable for further development towards the treatment of SCD and bT. Disclosures: Shearstone: Acetylon Pharmaceuticals, Inc.: Employment. van Duzer:Acetylon Pharmaceuticals, Inc.: Employment. Bradner:Acetylon Pharmaceuticals, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Mazitschek:Acetylon Pharmaceuticals, Inc. : Consultancy, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc.: Employment. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment.

scholarly journals Pharmacological Inhibition of Histone Deacetylases 1 and 2 (HDAC1/2) Induces Fetal Hemoglobin (HbF) through Activation of Gata2

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 335-335
Author(s):  
Jeffrey R Shearstone ◽  
Olga Golonzhka ◽  
Apurva Chonkar ◽  
Matthew Jarpe
Keyword(s):  
Bone Marrow Cells ◽  
Fetal Hemoglobin ◽  
Fold Increase ◽  
Erythroid Differentiation ◽  
Beta Globin ◽  
Employment Equity ◽  
Regulatory Regions ◽  
Genetic Ablation ◽  
Equity Ownership ◽  
Highly Correlated

Abstract Induction of HbF is an established therapeutic strategy for the treatment of sickle cell disease, and could also be effective in treating beta-thalassemia. Genetic ablation of HDAC1 or HDAC2, but not HDAC3, results in the induction of the fetal beta-like globin gene (HbG) transcript (Bradner JE, Proc Natl Acad Sci, 2010). We have previously shown that selective chemical inhibitors of HDAC1/2 elicit a dose and time dependent induction of HbG mRNA and HbF protein in cultured human CD34+ bone marrow cells undergoing erythroid differentiation (Shearstone JS, ASH Annual Meeting Abstracts, 2012). In this work, we have utilized our proof of concept molecule ACY-957, a selective inhibitor of HDAC1/2, to discover a novel role for Gata2 in the activation of HbG. To identify genes affected by HDAC1/2 inhibition, CD34+ bone marrow cells undergoing erythroid differentiation were treated with ACY-957 or vehicle, followed by mRNA expression profiling. Among the genes differentially regulated by both pharmacological inhibition and genetic ablation of HDAC1/2 were Bcl11a and Sox6, known HbG repressors, and Gata2, a potential HbG activator. Quantitative real time PCR (QRT-PCR) time course experiments confirmed that ACY-957 treatment leads to a 2-fold and 10-fold decrease in Bcl11A and Sox6, respectively, and an 8-fold increase in Gata2 mRNA. Unlike Bcl11a and Sox6, Gata2 induction by ACY-957 was highly correlated with HbG induction, suggesting a possible role for this transcription factor in the direct activation of HbG. To investigate this possibility, lentiviral infection was utilized to overexpress full length Gata2 transcript in differentiating primary erythroblasts. After 5 days of differentiation, Gata2 overexpression resulted in a 2.5-fold increase in HbG mRNA, while the level of the major adult beta-like globin chain (HbB) mRNA was unaffected. HbG mRNA remained elevated by Gata2 overexpression at day 7 of differentiation, while HbB was reduced by 1.6-fold. Gata2 overexpression appeared to have minimal effect on cell differentiation, as determined by the cell surface markers CD71 and GlycophorinA, a finding consistent with observations in ACY-957 treated cells with elevated Gata2. Furthermore, lentiviral delivery of short hairpin RNA (shRNA) targeting Gata2, attenuated HbG induction by ACY-957. These data suggest that elevated levels of Gata2 resulting from HDAC1/2 inhibition is sufficient to induce HbG at early stages of erythroid cell differentiation. To understand how HDAC1/2 inhibition drives Gata2 activation, chromatin immunoprecipitation coupled with either next generation sequencing (ChIP-seq) or QRT-PCR was performed in ACY-957 and vehicle treated cells. HDAC1 and HDAC2 were present throughout the Gata2 gene body and promoter regions, and HDAC1/2 binding levels were highly correlated, suggesting co-occupancy of these enzymes at this locus. ACY-957 treatment led to elevated histone acetylation at previously described Gata2 gene regulatory regions (Bresnick et. al. 2010, J Biol Chem). Specifically, the -1.8 kb and -2.8 kb regulatory regions showed a 6-fold increase in histone H3K9 and H2BK5 acetylation, while the +9.5 kb and -3.9 kb regions showed a 3-fold increase. The Gata2 protein showed increased binding at these regulatory regions in response to ACY-957 treatment, with a maximum increase of 3-fold at the -1.8 kb region. This finding is consistent with the known positive autoregulation of the Gata2 gene. Taken together, these data suggest that selective inhibition of HDAC1/2 leads to elevated Gata2 through acetylation-induced activation of a positive autoregulatory loop. The tight temporal correlation between Gata2 and HbG activation following HDAC1/2 inhibition argues that Gata2 may affect the beta-globin locus directly. ChIP-seq data across the 70-kb beta-globin locus demonstrated that ACY-957 treatment altered Gata2 binding only at a single region, lying within the promoter for delta globin. This region is suspected in playing a role in switching from fetal to adult globin during development, as naturally occurring deletions of this region are associated with elevated fetal hemoglobin in adults (Sankaran et. al. 2011, NEJM). Whether the change in GATA2 binding to this region is responsible for the increased expression of HbG in cells treated with HDAC1/2-selective inhibitors is under investigation. Disclosures Shearstone: Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Golonzhka:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Chonkar:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership.

scholarly journals Enhanced fetal hemoglobin production by phenylacetate and 4- phenylbutyrate in erythroid precursors derived from normal donors and patients with sickle cell anemia and beta-thalassemia

Blood ◽  
1993 ◽  
Vol 82 (7) ◽  
pp. 2203-2209 ◽  
Author(s):  
E Fibach ◽  
P Prasanna ◽  
GP Rodgers ◽  
D Samid
Keyword(s):  
Sickle Cell ◽  
Clinical Symptoms ◽  
Fetal Hemoglobin ◽  
Underlying Disease ◽  
Beta Thalassemia ◽  
Globin Mrna ◽  
Two Phase ◽  
Erythroid Progenitor ◽  
Patient Response ◽  
Hbf Induction

Abstract In both sickle cell (SS) anemia and beta-thalassemia (beta-thal), an increase in fetal hemoglobin (HbF) ameliorates the clinical symptoms of the underlying disease. Several pharmacologic agents have been used to elevate HbF levels in adults; however, concerns regarding adverse effects of the prevailing drugs raise an urgent need for other agents capable of stimulating HbF production. We show here that sodium phenylacetate (NaPA) and its precursor, sodium 4-phenylbutyrate (NaPB), can enhance HbF production in cultured erythroid progenitor derived from normal donors and patients with SS anemia or beta-thal, when used at pharmacologic concentrations. Treatment resulted in (1) reduced cell proliferation, (2) elevated hemoglobin (Hb) content per cell (mean cellular Hb [MCH]), and (3) an increased proportion of HbF produced, associated with elevated levels of gamma-globin mRNA. Moreover, the active phenyl-fatty acids, with NaPA as a prototype, potentiated HbF induction by other drugs of clinical interest, including hydroxyurea (HU), sodium butyrate, and 5-azacytidine (5AzaC). Efficacy could be further enhanced by introducing chlorine substituents at the phenyl ring to increase drug lipophilicity. Our findings indicate that NaPA and NaPB, both already proven safe and effective in treatment of children with urea cycle disorders, might benefit also patients with severe hemoglobinopathies. The two-phase liquid culture procedure used in this study should prove valuable in further studies exploring the mechanisms of HbF induction by these agents, and might provide an assay to predict patient response in the clinical setting.

Amplicon-sequencing of BCL11a-edited stem cells used to treat beta thalassemia and sickle cell disease suffices to demonstrate that CRISPR is a chromosome shredder

2019 ◽  
Author(s):  
Sandeep Chakraborty
Keyword(s):  
Clinical Trials ◽  
Target Gene ◽  
Fetal Hemoglobin ◽  
Amplicon Sequencing ◽  
Beta Thalassemia ◽  
Conservative Estimate ◽  
Cell Disease ◽  
High Confidence ◽  
Gene Space ◽  
Cell Study

The quanta of ’genome vandalism’ CRISPR does is easily observed by amplicon sequencing of the target gene. The algorithm chooses reads that match the targeted gene GENE, but also another gene (OFFGENE) - meaning there has been a translocation/integration. This translocation/integration is possible if and only if OFFGENE has also been edited - an off-target. Larger the number of reads with GENE-OFFGENE, larger the confidence. For example, the stem-cell study, which edits BCL11a to increase fetal hemoglobin, has 111 BCL11a-RNA45SN5 reads [1]. Analysis of the edit location in RNA45SN5 shows 8 mismatches with the gRNA, which falls within the permissible limits [2]. There are 20 such genes which have integrated with the BCL11a gene with high confidence. This is a very conservative estimate for two reasons. First, amplicon-sequencing only looks at GENE. If two OFFGENE’s were integrating, the data would not show that. Secondly, I have only looked the gene space - so only 2% of the genome. Since we find 20 genes integrating with BCL11a after such constraints, it is fair to assume that CRISPR is literally shredding the chromosome - a possible reason for significant fatalities in clinical trials [3].

Efficacy and Safety of Eltrombopag in Elderly Patients with Chronic Immune Thrombocytopenia: Analysis of Five Clinical Trials,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3294-3294 ◽  
Author(s):  
Harold J. Olney ◽  
Ingrid Pabinger ◽  
Bhabita Mayer ◽  
Kalpana Bakshi ◽  
Christine K Bailey ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Clinical Trials ◽  
Adverse Events ◽  
Immune Thrombocytopenia ◽  
Age Groups ◽  
Efficacy And Safety ◽  
Employment Equity ◽  
Fatal Bleeding ◽  
Fatal Hemorrhage ◽  
Equity Ownership

Abstract Abstract 3294 Introduction: Patients (pts) with immune thrombocytopenia (ITP) with platelet counts persistently <30,000/μL are at risk for severe and even fatal bleeding events; this risk increases with age. Risk for major non-fatal hemorrhage has been estimated at 3% per year for pts <40 years and 71% per year for pts >60. The 5-year risk of fatal hemorrhage has been estimated at 2% for pts <40 years and 48% for pts >60 (Cohen 2000). Eltrombopag, an oral, nonpeptide thrombopoietin receptor agonist approved for the treatment of chronic ITP, stimulates bone marrow progenitor cells, promoting differentiation and proliferation of megakaryocytes and platelet production. No data have been reported on the safety and efficacy of eltrombopag specifically in elderly pts. Aim: To assess efficacy and safety of eltrombopag in pts ≥65 years. Methods: A retrospective analysis by age was completed for 446 adult chronic ITP pts receiving eltrombopag in 5 clinical trials: two 6-week and one 6-month placebo-controlled studies (TRA100773A/B, RAISE); an open-label study with pts treated intermittently in 3 cycles of up to 6 weeks (REPEAT); and an ongoing extension study (EXTEND) of 299 pts who completed a prior eltrombopag trial. Bleeding was assessed prospectively using the WHO Bleeding Scale and using the NCI Common Terminology Criteria for Adverse Events (CTCAE) v3.0. Results: Pts receiving eltrombopag in each trial aged 18–49, 50–64, and ≥65 are shown in the Table. In the 6-week trials, response was defined as platelets ≥50,000/μL at day 43. In TRA100773A (eltrombopag 50 mg), response was achieved by 67% of pts aged 18–49 and 50–64, and 100% in pts ≥65. In TRA100773B, response was achieved by 50% of pts 18–49, 64% of pts 50–64, and 74% of pts ≥65. In RAISE, at any given visit, 40–53% of pts aged 18–49, 40–61% aged 50–64, and 42–75% ≥65 responded (platelets >50,000/μL and <400,000/μL). In REPEAT, response (platelets ≥50,000/μL and ≥2x baseline) in all 3 cycles was achieved by 67%, 72%, and 83% of pts who responded in Cycle 1, in the 18–49, 50–64, and ≥65 groups. In EXTEND, 84%, 94%, and 86% of pts aged 18–49, 50–64, and ≥65 had a response (platelets ≥50,000/μL); 15, 18, and 6 of these pts had platelets ≥50,000/μL at baseline. In EXTEND, 66%, 73%, and 78% of pts aged 18–49, 50–64, and ≥65 responded for >50% of assessments and 43%, 48%, and 53% for >75% of assessments. Across all 5 studies, pts aged 18–49 (n=220), 50–64 (n=148), and ≥65 (n=78) were treated with eltrombopag for a median (range) of 399 (5–1255), 484.5 (5–1302), and 244.5 (2–1267) days; median daily dose was 53 mg for pts 18–49 and 50 mg each for pts 50–64 and ≥65. The most common adverse events (AEs) (headache, nasopharyngitis, upper respiratory tract infection, and diarrhea) across all 5 studies were reported in similar proportions of pts across age groups. Fatigue, arthralgia, constipation, and cataracts were more common in elderly pts (11%, 10%, 5%, and 4% for pts 18–49 vs 19% each for pts ≥65). Thromboembolic events were reported in 4 (2%), 5 (3%), and 7 (9%) pts aged 18–49, 50–64, and ≥65. No age specific trend toward arterial vs venous events was observed. Proportions of liver enzymes elevation and bone marrow reticulin grade ≥2 were similar across age groups. Bleeding serious AEs (SAEs) were reported in 7% of pts aged 18–49 and 50–64, and 3% of pts ≥65. The most common bleeding SAEs were GI and CNS events, with no apparent difference in proportion of pts among age groups. Two pts experienced a fatal bleeding event (GI and CNS hemorrhages) 55 and 107 days after stopping eltrombopag; both were 18–49 and never responded to eltrombopag. Conclusion: No significant difference in the safety or efficacy profile of eltrombopag was observed for elderly versus younger pts, although elderly pts seemed to exhibit slightly more robust responses and slightly more nonhemorrhagic AEs (including thrombosis), which are not unexpected in an elderly population. Disclosures: Olney: GlaxoSmithKline: Consultancy, Membership on an entity's Board of Directors or advisory committees. Pabinger:GlaxoSmithKline: Research Funding, Speakers Bureau. Mayer:GlaxoSmithKline: Employment, Equity Ownership. Bakshi:GlaxoSmithKline: Employment. Bailey:GlaxoSmithKline: Employment, Equity Ownership. Brainsky:GlaxoSmithKline: Employment, Equity Ownership.

Fetal Hemoglobin Expression Is Differentially Affected by Inhibition of the Proposed Dred Complex Constituents, LSD1 and DNMT1

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3263-3263 ◽  
Author(s):  
Tara L Arvedson ◽  
Lynn Tran ◽  
Sandra L Ross ◽  
Sean Yoder ◽  
Alexandra Hertz ◽  
...  
Keyword(s):  
Fetal Hemoglobin ◽  
Erythroid Differentiation ◽  
Beta Thalassemia ◽  
Cell Disease ◽  
Hematopoietic Progenitor ◽  
Gamma Globin ◽  
Hbf Induction ◽  
Effect Of Treatment ◽  
Globin Promoter

Abstract Abstract 3263 Introduction Sickle cell disease and beta thalassemia are disorders caused by mutations in adult hemoglobin (HbA) or defects in HbA expression. A potential therapeutic solution is reactivation of fetal hemoglobin (HbF) expression. Although HbF, comprising two alpha and two gamma globin chains, is the primary form of hemoglobin expressed in utero, gamma globin expression is silenced in adults. One proposed mechanism of gamma globin silencing involves binding of the direct repeat erythroid definitive (DRED) repressor complex to sequences in the gamma globin promoter. The DRED complex is reported to include the orphan nuclear hormone receptors TR2 and TR4, lysine specific demethylase (LSD1) and DNA methyltransferase (DNMT1). As both LSD1 and DNMT1 are epigenetic modifiers, gamma globin repression is proposed to be mediated by LSD1- and DNMT1-induced epigenetic changes. To investigate the role of DNMT1 and LSD1 in HbF silencing, HbF expression was evaluated in an erythroid differentiation model where hematopoietic progenitor cells were treated with either DNMT1 or LSD1 small molecule inhibitors or siRNA. Methods Human hematopoietic progenitor cells from healthy donors were induced to become erythroid using a two step protocol including erythropoietin, SCF, IL-3 and hydrocortisone for days 1–7 and erythropoietin and SCF for days 8–14. Cultures were treated with a range of concentrations of either tranylcypromine or S2101 (LSD1 inhibitors) or 5-azacytidine (DNMT1 inhibitor) and compared to HbF-inducing, positive control small molecules pomalidomide and lenalidomide. Cultures were also treated with LSD1 siRNAs and compared to controls. The effect of treatment on gamma, beta and alpha globin transcription was determined by qRT-PCR. The effect of treatment on HbA and HbF levels was determined by ELISA, HPLC, flow cytometry and imaging. Differentiation was characterized by morphology and flow-based detection of CD34 and glycophorin. Effects on viability were characterized by ViCell and flow cytometry. Results Treatment with a concentration range of 5-azacytidine increased the rate of red blood cell differentiation as measured by daily changes in CD34 and glycophorin and hemoglobinization. Quantitative ELISA demonstrated that HbF expression increased two-fold. In contrast, LSD1 inhibition reduced both the rate of proliferation and differentiation of erythroid progenitors. Consistent with impaired differentiation, both beta globin transcription and HbA expression were reduced by up to 84% (qRT-PCR) and 65% (quantitative ELISA), respectively. No increase in gamma globin transcription or HbF expression was observed in response to LSD1 inhibition. Control cultures differentiated as expected: after 14 days of treatment the majority of vehicle-, lenalidomide- or pomalidomide-treated cells were glycophorin-positive and enucleation was readily apparent. Both lenalidomide and pomalidomide treatment induced a two-fold increase in HbF expression, as previously reported. Conclusions Although both LSD1 and DNMT1 are reported to be components of the DRED complex and are proposed to be jointly responsible for epigenetically modifying the gamma globin promoter to silence HbF expression, inhibition of the two proteins had different outcomes on HbF expression. DNMT1 inhibition upregulated HbF expression to a similar extent as pomalidomide (currently in Phase 1 clinical trials for HbF induction), whereas LSD1 inhibition impaired erythroid differentiation and hemoglobinization. These results suggest that the mechanism of gamma globin silencing and the proposed role of the DRED complex require further evaluation. Furthermore, this work also suggests that LSD1 inhibition is not a therapeutic strategy for HbF induction in patients with sickle cell disease or beta thalassemia. Disclosures: Arvedson: Amgen: Employment. Tran:Amgen: Employment. Ross:Amgen: Employment. Yoder:Amgen: Employment. Hertz:Amgen: Employment. Hale:Amgen: Employment. Eschelbach:Amgen: Employment. Dineen:Amgen: Employment. Matyas:Amgen: Employment. Hartley:Amgen: Employment. Morgenstern:Amgen: Employment. Winters:Amgen: Employment. Cindy:Amgen: Employment. Molineux:Amgen: Employment. Coxon:Amgen: Employment.

Targeted Therapeutic Strategies for Fetal Hemoglobin Induction

Hematology ◽  
2011 ◽  
Vol 2011 (1) ◽  
pp. 459-465 ◽  
Author(s):  
Vijay G. Sankaran
Keyword(s):  
Fetal Hemoglobin ◽  
Therapeutic Strategies ◽  
Cell Disease ◽  
Therapeutic Approaches ◽  
Histone Deacetylase 1 ◽  
Effective Strategies ◽  
Limited Success ◽  
Hbf Induction ◽  
Adult Hemoglobin ◽  
Insight Into

Abstract Increased levels of fetal hemoglobin (HbF) can ameliorate the severity of the β-hemoglobin disorders, sickle cell disease (SCD) and β-thalassemia, which are major sources of morbidity and mortality worldwide. As a result, there has been a longstanding interest in developing therapeutic approaches for inducing HbF. For more than 3 decades, the majority of HbF inducers developed were based on empiric observations and have had limited success. Recently, human genetic approaches have provided insight into previously unappreciated regulators of the fetal-to-adult hemoglobin switch and HbF silencing, revealing molecular targets to induce HbF. This article reviews these developments and discusses how molecules including BCL11A, KLF1, MYB, SOX6, miRNAs 15a and 16-1, and histone deacetylase 1 and 2 (HDAC1/2) could be important targets for HbF induction in humans. The current understanding of how these molecules function and the benefits and drawbacks of each of these potential therapeutic targets are also examined. The identification of these regulators of HbF expression is extremely promising and suggests that rationally designed approaches targeting the very mechanisms mediating this switching process could lead to better, less toxic, and more effective strategies for HbF induction.

scholarly journals IMR-261, a Novel Oral Nrf2 Activator, Induces Fetal Hemoglobin in Human Erythroblasts, Reduces VOCs, and Ameliorates Ineffective Erythropoiesis in Experimental Mouse Models of Sickle Cell Disease and Beta-Thalassemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 853-853
Author(s):  
Thiago Trovati Maciel ◽  
Caroline Carvalho ◽  
Rachel Rignault ◽  
Biree Andemariam ◽  
Betty S. Pace ◽  
...  
Keyword(s):  
Research Funding ◽  
Fetal Hemoglobin ◽  
Fold Change ◽  
Beta Thalassemia ◽  
Human Monocytes ◽  
Ineffective Erythropoiesis ◽  
Antioxidant Genes ◽  
F Cells ◽  
Hbf Induction

Abstract Background Sickle cell disease (SCD) is an autosomal recessive disorder where mutated hemoglobin (HbS) polymerizes and can lead to irreversible red blood cell (RBC) sickling and painful vaso-occlusive crisis (VOC). The RBC sickling is amplified by inflammation, resulting in tissue and organ damage. The transcription factor Nuclear factor erythroid 2-related factor 2 (Nrf2) coordinates the expression of antioxidant genes in response to oxidative stress, regulates inflammation, inhibits the NFkB pathway, and induces fetal hemoglobin (HbF), making it an attractive target in SCD and beta-thalassemia. IMR-261 is a novel oral activator of Nrf2 and has been tested in Phase 2 clinical trials (previously as CXA-10). Methods & Results CD14+ human monocytes were exposed to IMR-261 at 3µM and 10µM for 3 hours, to determine via quantitative PCR (qPCR) its ability to induce expression of antioxidant genes. IMR-261 at 10 µM significantly increased (p&lt;0.05) the expression of Nrf2-dependent genes (p&lt;0.05), including HMOX1, HSPA1A, HSP90, GCLM, SOD1 and TXNRD1. Human monocytes were treated with lipopolysaccharide (LPS) to test the ability of IMR-261 to block inflammatory genes with a NFkB target dataset. IMR-261 significantly inhibited (p&lt;0.05) LPS-induced expression of IL-1-beta, TNF-alpha and IL-6 in human monocytes. To test the effects of IMR-261 on HbF induction, human erythroblasts were derived from CD34+ blood marrow progenitor cells sourced from healthy or SCD subjects. IMR-261 induced expression of the gamma-globin gene (4.0-fold change at 3µM and 7.18-fold change at 6 µM). This was accompanied by increased %F-cells (2.8-fold change at 3µM and 3.0-fold change at 6 µM). IMR-261 was also tested in the Townes HbSS mouse model of SCD to assess the potential for HbF induction. Mice were dosed with IMR-261 at 12.5 mg/kg or 37.5 mg/kg BID for 4 weeks (N=4-8/group). After 4 weeks of treatment, IMR-261 at 12.5 mg/kg and 37.5 mg/kg resulted in a significant increase in HbF relative to control, and 37.5 mg/kg resulted in a significant increase in %F-cells relative to control (Table 1, p&lt;0.05). In addition, both doses of IMR-261 led to significant increases in RBC counts and total hemoglobin (Hb) (Table 1, p&lt;0.05). IMR-261 at 37.5 mg/kg also significantly decreased (p&lt;0.05) both reticulocyte counts and spleen cellularity. The ability of IMR-261 to reduce VOCs was assessed in separate Townes HbSS mice after the administration of TNF-alpha (0.5 µg/mice i.p.). IMR-261 was dosed at 37.5 mg/kg BID for 5 days before triggering VOCs. RBCs were stained with Ter-119 antibodies on spleen and liver of mice. Compared to controls, IMR-261 significantly reduced the presence of RBC on occluded vessels. This was coupled with a reduction of P-selectin (3109±97 Mean Fluorescence Units [MFI] in vehicle-treated vs. 1974±379 MFI in IMR-261 group, p&lt;0.05) and L-selectin (375±20 MFI in vehicle-treated vs. 242±60 MFI in IMR-261 group, p&lt;0.05). IMR-261 also reduced select hemolysis biomarkers: bilirubin (11.2±0.3 mg/dL in vehicle-treated vs. 8.4±0.7 mg/dL in IMR-261 group, (p&lt;0.05) and free-heme (325±52 µM in vehicle-treated vs. 203±51 µM in IMR-261 group, p&lt;0.05). A beta-thalassemia experimental model Hbb th1/th1 was tested to evaluate whether IMR-261 could improve ineffective erythropoiesis seen in beta-thalassemia. IMR-261 treatment at 37.5 mg/kg BID significantly increased hemoglobin levels, RBC counts and hematocrit (p&lt;0.05), with significant reductions observed in reticulocytes (p&lt;0.05). flow cytometry analysis (CD71/Ter119) showed that IMR-261 significantly decreased late basophilic and polychromatic erythroblasts (Ery.B) and increased orthochromatic erythroblasts and reticulocytes (Ery.C) cell numbers in the spleen (p&lt;0.05). Conclusions IMR-261 activates Nrf2-dependent antioxidant genes and inhibits NFkB-induced pro-inflammatory genes in human monocytes. In human erythroblasts, IMR-261 significantly increased HbF and %F-cells. In vivo SCD models show that IMR-261 significantly induced HbF and %F-cells, improved hemolytic markers, and decreased VOCs. IMR-261 also increased Hb and improved ineffective erythropoiesis in a beta-thalassemia in-vivo model. Together these data suggest that IMR-261 is a promising, novel, oral therapy that warrants clinical testing in SCD and beta-thalassemia. Figure 1 Figure 1. Disclosures Maciel: Imara Inc.: Research Funding. Carvalho: Imara Inc.: Research Funding. Rignault: Imara Inc.: Research Funding. Pace: Imara Inc.: Consultancy. OCain: Imara Inc.: Current Employment, Current equity holder in publicly-traded company. Ballal: Imara Inc.: Current Employment, Current equity holder in publicly-traded company.

Genotype of Beta Thalassemia Patients As Predictors for Prognosis & Management without Blood Transfusion,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3199-3199
Author(s):  
Saqib Hussain Ansari ◽  
Tahir S Shamsi ◽  
Mohammed Tahir Khan ◽  
Muneera Bohray ◽  
Tasneem Farzana ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Transplantation ◽  
Blood Transfusion ◽  
Marrow Transplantation ◽  
Response Rate ◽  
Fetal Hemoglobin ◽  
Beta Thalassemia ◽  
Genetic Mutations ◽  
Blood Transfusions ◽  
Blood Diseases

Abstract Abstract 3199 Introduction: Packed red blood cell (PRC) transfusion with iron chelation, despite their undesirable effects, has been the mainstay of treatment for patients with beta-thalassaemia major. In the recent past introduction of oral medications that augment Hemoglobin F (HbF) has opened new horizons in the management & prognosis of these patients sparing many of them from the hardtimes of blood transfusions & related adversities. Methods: On the basis of our previous studies evaluating the safety & efficacy of Hydroxyurea (HU) in beta thalassemia patients which is an oral HbF augmentation agent, at 10–15 mg/kg/day was used on 238 patients for 24 months under the guidelines of Helsinki's declaration. Response was measured by using transfusion requirement prior to 6 months period of enrollment in the study as control. Patients were finally divided into 3 groups on the basis of response, Group 1 consisted of Complete Responders, group 2 were partial responders & group 3 were non-responders. Group1 patients needed regular blood transfusions prior to HU therapy while after 24 months they never required transfusion as they maintained their mean Hb at ≥7gm/dL. Partial responders substantially decreased their need for transfusion (less than 50 %), while Non-responders had no decrease in their need for transfusions. All patients' genetic mutation profiles were investigated upon. Results: Most common genetic mutations observed were IVS1-5 (48%) either homozygous or heterozygous, Fr 8–9 (11.8%), IVS1-1 (10.5%), Cd 30 (7.6 %), Fr 41–42 (6.3%), Cap+1 (3.3%) & Del-619 (8%). Homozygous Xmn-polymorphism was found in 20% & heterozygous Xmn polymorphism was observed in 26% of the patients, while rest 54% had no Xmn polymorphism. Astounding results were observed when these mutations were correlated with response of HU. Response rate was found to be most closely related with gene mutations IVS1-1 (68% Responders), Cd-30 (56% Responders), Cap+1 (38% Responders)IVS1-5 (37% Responders). Xmn polymorphism was found to be significantly associated with Response rate 73% (p-value 0.00). Discussion: It was observed from our study that certain genetic mutations in beta-thalassemia & presence of Xmn polymorohism responds exceptionally well to oral HbF augmentation agents, sparing the patients from the adversities of blood transfusion. Most notably presence of IVS1-1 spared 68% of the patients from blood transfusions while Xmn polymorphism spared 73% of them. It is recommended, therefore, that a new classification be made on the basis of genetic profile of beta thalassemia patients for better treatment & prognosis preventing unnecessary blood transfusions. Disclosures: Ansari: National Institute of Blood Diseases & Bone Marrow Transplantation: Consultancy. Off Label Use: We used Hydroxyurea or hydroxy carbamide. Hydroxycarbamide increases the concentration of fetal hemoglobin. The precise mechanism of action is not yet clear, but it appears that hydroxycarbamide increases nitric oxide levels, causing soluble guanylyl cyclase activation with a resultant rise in cyclic GMP, and the activation of gammaglobin synthesis necessary for fetal hemoglobin. Shamsi:National Institute of Blood Diseases & Bone Marrow Transplantation: Consultancy. Bohray:National Institute of Blood Diseases & Bone Marrow Transplantation: Consultancy. Farzana:National Institute of Blood Diseases: Employment. Erum:National Institute of Blood Diseases: Employment.

scholarly journals Enhanced fetal hemoglobin production by phenylacetate and 4- phenylbutyrate in erythroid precursors derived from normal donors and patients with sickle cell anemia and beta-thalassemia

Blood ◽  
1993 ◽  
Vol 82 (7) ◽  
pp. 2203-2209 ◽  
Author(s):  
E Fibach ◽  
P Prasanna ◽  
GP Rodgers ◽  
D Samid
Keyword(s):  
Sickle Cell ◽  
Clinical Symptoms ◽  
Fetal Hemoglobin ◽  
Underlying Disease ◽  
Beta Thalassemia ◽  
Globin Mrna ◽  
Two Phase ◽  
Erythroid Progenitor ◽  
Patient Response ◽  
Hbf Induction

In both sickle cell (SS) anemia and beta-thalassemia (beta-thal), an increase in fetal hemoglobin (HbF) ameliorates the clinical symptoms of the underlying disease. Several pharmacologic agents have been used to elevate HbF levels in adults; however, concerns regarding adverse effects of the prevailing drugs raise an urgent need for other agents capable of stimulating HbF production. We show here that sodium phenylacetate (NaPA) and its precursor, sodium 4-phenylbutyrate (NaPB), can enhance HbF production in cultured erythroid progenitor derived from normal donors and patients with SS anemia or beta-thal, when used at pharmacologic concentrations. Treatment resulted in (1) reduced cell proliferation, (2) elevated hemoglobin (Hb) content per cell (mean cellular Hb [MCH]), and (3) an increased proportion of HbF produced, associated with elevated levels of gamma-globin mRNA. Moreover, the active phenyl-fatty acids, with NaPA as a prototype, potentiated HbF induction by other drugs of clinical interest, including hydroxyurea (HU), sodium butyrate, and 5-azacytidine (5AzaC). Efficacy could be further enhanced by introducing chlorine substituents at the phenyl ring to increase drug lipophilicity. Our findings indicate that NaPA and NaPB, both already proven safe and effective in treatment of children with urea cycle disorders, might benefit also patients with severe hemoglobinopathies. The two-phase liquid culture procedure used in this study should prove valuable in further studies exploring the mechanisms of HbF induction by these agents, and might provide an assay to predict patient response in the clinical setting.

scholarly journals 8a, a New Acridine Antiproliferative and Pro-Apoptotic Agent Targeting HDAC1/DNMT1

2021 ◽  
Vol 22 (11) ◽  
pp. 5516
Author(s):  
Qiting Zhang ◽  
Ziyan Wang ◽  
Xinyuan Chen ◽  
Haoxiang Qiu ◽  
Yifan Gu ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Dna Methyltransferase ◽  
Hdac Inhibitors ◽  
Mitochondrial Pathway ◽  
Epigenetic Therapy ◽  
Protein Interaction Data ◽  
Dependent Manner ◽  
Histone Deacetylase 1 ◽  
Histiocytic Lymphoma ◽  
Dose Dependent

Epigenetic therapy using histone deacetylase (HDAC) inhibitors has become an attractive project in new drug development. However, DNA methylation and histone acetylation are important epigenetic ways to regulate the occurrence and development of leukemia. Given previous studies, N-(2-aminophenyl)benzamide acridine (8a), as a histone deacetylase 1 (HDAC1) inhibitor, induces apoptosis and shows significant anti-proliferative activity against histiocytic lymphoma U937 cells. HDAC1 plays a role in the nucleus, which we confirmed by finding that 8a entered the nucleus. Subsequently, we verified that 8a mainly passes through the endogenous (mitochondrial) pathway to induce cell apoptosis. From the protein interaction data, we found that 8a also affected the expression of DNA methyltransferase 1 (DNMT1). Therefore, an experiment was performed to assess the binding of 8a to DNMT1 at the molecular and cellular levels. We found that the binding strength of 8a to DNMT1 enhanced in a dose-dependent manner. Additionally, 8a inhibits the expression of DNMT1 mRNA and its protein. These findings suggested that the anti-proliferative and pro-apoptotic activities of 8a against leukemia cells were achieved by targeting HDAC1 and DNMT1.

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