Jose Sangerman’s research while affiliated with Boston University and other places
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Induction of fetal hemoglobin (HbF) expression ameliorates the clinical severity and prolong survival in persons with sickle cell disease (SCD). Hydroxyurea (HU) is the only FDA-approved HbF inducer however, additional therapeutics that produce an additive effect in SCD are needed. To this end, development of potent Class I histone deacetylase inhibitors (HDACi) for HbF induction represents a rational molecularly targeted approach. In studies here, we evaluated CT-101, a novel Class I-restricted HDACi, a Largazole derivative, for pharmacodynamics, cytotoxicity, and targeted epigenetic effects. In SCD derived erythroid progenitors, CT-101 induced HbF expression with additive activity in combination with HU. CT-101 preferentially activated γ-globin gene transcription, increased acetylated histone H3 levels and conferred an open chromatin conformation in the γ-globin promoter. These data indicate CT-101 represents a strong potential candidate as a molecularly targeted inducer of HbF.
Increased expression of developmentally silenced fetal globin (HBG) reduces the clinical severity of β-hemoglobinopathies. Benserazide has a relatively benign safety profile having been approved for 50 years in Europe and Canada for Parkinson's disease treatment. Benserazide was shown to activate HBG gene transcription, in a high throughput screen and subsequent studies confirmed fetal hemoglobin (HbF) induction in erythroid progenitors from hemoglobinopathy patients, transgenic mice containing the entire human β-globin gene (β-YAC) and anemic baboons. The goal of this study is to evaluate efficacies and plasma exposure profiles of benserazide racemate and its enantiomers to select the chemical form for clinical development. Intermittent treatment with all forms of benserazide in β-YAC mice significantly increased proportions of red blood cells expressing HbF and HbF protein per cell with similar pharmacokinetic profiles and with no cytopenia. These data contribute to the regulatory justification for development of the benserazide racemate. Additionally, dose ranges and frequencies required for HbF induction using racemic benserazide were explored. Orally administered escalating doses of benserazide in an anemic baboon induced γ-globin mRNA up to 13-fold and establish an intermittent dose regimen for clinical studies as a therapeutic candidate for potential treatment of β-hemoglobinopathies.
High-level fetal (γ) globin expression ameliorates clinical severity of the beta (β) hemoglobinopathies, and safe, orally-bioavailable γ-globin inducing agents would benefit many patients. We adapted a LCR-γ-globin promoter-GFP reporter assay to a high-throughput robotic system to evaluate five diverse chemical libraries for this activity. Multiple structurally- and functionally-diverse compounds were identified which activate the γ-globin gene promoter at nanomolar concentrations, including some therapeutics approved for other conditions. Three candidates with established safety profiles were further evaluated in erythroid progenitors, anemic baboons and transgenic mice, with significant induction of γ-globin expression observed in vivo. A lead candidate, Benserazide, emerged which demonstrated > 20-fold induction of γ-globin mRNA expression in anemic baboons and increased F-cell proportions by 3.5-fold in transgenic mice. Benserazide has been used chronically to inhibit amino acid decarboxylase to enhance plasma levels of L-dopa. These studies confirm the utility of high-throughput screening and identify previously unrecognized fetal globin inducing candidates which can be developed expediently for treatment of hemoglobinopathies.
Sickle cell disease (SCD) is the most common monogenic disorder, afflicting millions worldwide, and causing hemolytic anemia and chronic organ damage from vaso-occlusion. Fetal hemoglobin (HbF) is an endogenous type of hemoglobin present in all humans during development, which is normally suppressed in infancy. Biochemical and clinical studies have shown that increased synthesis of HbF inhibits sickle hemoglobin (HbS) polymerization and reduces clinical severity. Concerted efforts have been made to induce the synthesis of HbF in adult erythroid cells with chemical inducers of HbF and through disruption of transcription factors in repressor complexes. As wide variability in individual responses to drug candidates have been observed in clinical trials, consistently effective HbF inducers are highly desired. We previously identified that Lysine-specific histone demethylase 1 (LSD1) is involved in the regulation of the fetal γ-globin genes, and inhibition of LSD1 using either RNAi or by the momoamine oxidase inhibitor tranylcypromine (TC) in primary human erythroid progenitor cells induces HbF to therapeutic levels. However, TC treatment has potentially problematic side effects, and at high concentrations decreases adult b-globin mRNAs and impairs erythroid maturation. We have now investigated another LSD1 inhibitor, RN-1, which is a cell-permeable TC analog that acts as a potent, irreversible inhibitor of LSD1 with a lower IC50 than TC. We investigated in vivo effects of RN-1 on γ-globin gene expression and erythroid physiology in a transgenic mouse model of SCD which expresses human α- and sickle β-globin, and has many genetic, hematologic, and pathophysiological features found in SCD patients, including irreversibly sickled RBCs, hemolytic anemia, high reticulocyte counts, hepatosplenomegaly and organ pathology. We found a robust increase in human fetal γ-globin (15-fold) and murine embryonic εY- and βH1-globin mRNAs (36 and 54-fold) and 4-fold increases in human HbF in SCD mice following repeated RN-1 treatment (at 10 μg/g body weight) within 4 weeks. Further, irreversibly sickled RBCs were significantly reduced, and RBC lifespan increased markedly in RN-1-treated SCD mice, leading to significantly decrease pathophysiologic indicators (hemolysis, splenomegaly, and organ necrosis) compared to untreated SCD mice. To begin to evaluate potential effects of RN-1 on erythroid progenitor cells from patients with SCD, peripheral blood from 5 adult SCD patients was cultured with RN-1 (0.07 to 0.25 μM) in a 2-phase progenitor assay, with mRNA analyzed on day 12 and F-reticulocytes on day 13-14 of the erythroid differentiation phase. RN-1 treated progenitors demonstrated a mean 3.4-fold higher g-globin mRNA (p=0.04) and 5% higher absolute F-reticulocytes than were observed in untreated progenitors from the same subject, with responses occurring in 5/5 subjects' assays. These preclinical studies provide additional evidence that modulating LSD-1 activity is a promising approach to inducing HbF expression as a mechanism to reduce clinical severity of SCD.
Disclaimer: "Research reported in this publication was supported by the NHLBI under Award Number P50HL118006. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health"
R01 DK052962 10A1
R42-HL-110727
Disclosures
No relevant conflicts of interest to declare.
Pharmacologic augmentation of γ-globin expression sufficient to reduce anemia and clinical severity in patients with diverse hemoglobinopathies has been challenging. In studies here, representative molecules from four chemical classes, representing several distinct primary mechanisms of action, were investigated for effects on γ-globin transcriptional repressors, including components of the NuRD complex (LSD1 and HDACs 2–3), and the downstream repressor BCL11A, in erythroid progenitors from hemoglobinopathy patients. Two HDAC inhibitors (MS-275 and SB939), a short-chain fatty acid derivative (sodium dimethylbutyrate [SDMB]), and an agent identified in high-throughput screening, Benserazide, were studied. These therapeutics induced γ-globin mRNA in progenitors above same subject controls up to 20-fold, and increased F-reticulocytes up to 20%. Cellular protein levels of BCL11A, LSD-1, and KLF1 were suppressed by the compounds. Chromatin immunoprecipitation assays demonstrated a 3.6-fold reduction in LSD1 and HDAC3 occupancy in the γ-globin gene promoter with Benserazide exposure, 3-fold reduction in LSD-1 and HDAC2 occupancy in the γ-globin gene promoter with SDMB exposure, while markers of gene activation (histone H3K9 acetylation and H3K4 demethylation), were enriched 5.7-fold. These findings identify clinical-stage oral therapeutics which inhibit or displace major co-repressors of γ-globin gene transcription and may suggest a rationale for combination therapy to produce enhanced efficacy.
Induction of gamma globin expression to certain target levels reduces clinical severity in sickle cell disease, globin imbalance, and anemia in the beta thalassemias. However, achieving high level expression in diverse patients with variable baseline HbF levels is clinically challenging. To determine whether different therapeutics have complimentary molecular actions that could be combined for higher efficacy, we evaluated a panel of oral fetal globin-inducing therapeutics in clinical use or trials for effects on transcriptional suppressors of HBG, including components of the NuRD complex, LSD1 and HDACs 1-3, and the downstream repressor BCL11A, in erythroid progenitors cultured from sickle cell and beta thalassemia patients. Using chromatin immunoprecipitation assays, immunoblot, and qRT-PCR, we investigated two pan-HDAC inhibitors, MS-275 and SB939, a short chain fatty acid derivative without global histone deacetylation effects, sodium dimethylbutyrate (ST20), and PB-04, a PK-enhancing drug identified through high throughput screening (HTS), which has a long-standing benign clinical safety profile. These therapeutics induce fetal globin expression by 1.5 to 12-fold in different individuals’ progentiors over untreated controls and are active in 60-90% of patients’ progenitors. BCL11A mRNA and protein were suppressed by 3-7 fold and 5-10 fold, respectively, over untreated cells from the same subject by all agents tested. PB-04 treatment decreased LSD1 mRNA by 5-fold. MS-275 and ST20 suppressed KLF-1 mRNA by 3- and 2.5-fold respectively. ChIP analyses demonstrated that PB-04 reduced LSD1 and HDAC-3 occupancy on the gamma globin gene promoter by 4-fold and 3-fold, respectively, coincident with gamma globin induction; ST20 decreased HDAC-2 and LSD1 binding by 6- and 2-fold respectively. Further, histone marks associated with gene activation were enriched at the gamma-globin promoter by treatment with PB-04, including acetylated Histone H3K9 (AcH3K9), the target of HDAC3, and dimethylated H3K4 (H3K4me2), a target of LSD1. Collectively, these results identify oral clinical stage therapeutics which disrupt two major mechanisms of transcriptional repression. The findings provide a basis for targeting therapies in individuals and combining pharmacologic agents which induce HbF expression through complimentary molecular mechanisms to provide high level induction.
Disclosures
Faller: Phoenicia BioSciences: Employment, Membership on an entity's Board of Directors or advisory committees. Perrine:Phoenicia BioSciences: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
High-level expression of fetal (Υ) globin reduces clinical complications in sickle cell disease and this is achieved with hydroxyurea (HU) in young children. However, non-cytotoxic high-potency therapeutics, particularly which can be utilized in combination with HU, are needed for most adolescent and adult patients who have continued serious clinical events. We have identified additional pharmaceutical candidates which induce HbF without cytotoxicity, using a Υ-globin gene promoter linked to GFP for robotic high-throughput screening, and screening five diverse chemical libraries. From a library of US and EU drugs which are approved for treatment of other medical conditions, a small panel of approved therapeutics were found to induce Υ globin expression, and have benign safety profiles, are orally active, and are suitable for long-term use. Three orally active candidates were evaluated in anemic baboons, and two, DLT and PB-04, induced Υ-globin mRNA by 15- to 33-fold over baseline levels. In 3/3 beta-globin locus YAC transgenic mice, one candidate (PB-04; 20 mg/kg) given by intra-peritoneal (IP) injections (for experimental feasibility) 3 times/ week for 5 wks significantly increased F-cells from 0.1 to 9%, 0.4 to 18%, and 0.13 to 12% respectively; and mean fluorescence intensity (MFI) increased by 10- to 33-fold. Responses were observed within one week. In hydroxyurea treated mice (100 mg/kg; IP, 5 days/ wk) F-cells increased from 0.3 to 2.3% on average (p<0.05), and MFI increased by 6- to 7-fold, while water vehicle did not increase F-cells significantly. PB-04 has been used clinically for decades as an excipient solely to prolong the half-life of another pharmaceutical, and is suitable for repurposing. In ChIP assays in K562 cells, PB-04 treatment demonstrated dual actions of displacing HDAC3 by 20-fold and LSD-1 by 3-fold from the Υ globin gene promoter.
To investigate potential effects of genetic modifiers of HbF on responses to different HbF inducer classes, erythroid progenitors from 40 sickle and beta thalassemic subjects were sub-genotyped for 3 major quantitative trait loci (QTL) (Bcl-11A, HMIP, Xmn-I) and cultured +/- 7 HbF inducers which are in clinical trials or approved. Most HbF inducers, including decitabine and butyrate used as positive controls, are active in 50-70% of progenitors, with differential Υ-globin mRNA responses observed. Only 10% of progenitors did not respond to any inducing agent. Most progenitors with the Xmn-1 variant responded with higher Υ globin transcription to all inducers. Sodium dimethylbutyrate (HQK-1001) and decitabine, produced 6-fold overall mean induction. PB-04 produced 9-fold mean induction above untreated control levels from the same subject. HDAC inhibitors (Butyrate, MS-275) which suppress Bcl-11A expression, demonstrate higher activity in progenitors from subjects without an underlying SNP in Bcl-11A. Another HDAC inhibitor, SB939, produced responses in 80% of progenitors. 25-30% of subjects’ progenitors exhibit high induction, 12-to 40-fold above untreated controls, to dimethylbutyrate, PB-04, decitabine, and an HDAC1/2 inhibitor 14F, suggesting a “high responder genotype” of which only half had a recognized favorable QTL. Taken together, these in vitro and in vivo studies identify a mini-pipeline of clinical-stage HbF-inducing therapeutics, with both epigenetic and targeted molecular actions, which can be investigated clinically to develop tailored therapeutics and therapeutic combinations for high-level induction of HbF in subgenotyped hemoglobinopathy patients.
Seven new and established inducers have been evaluated in erythroid progenitors cultured from sub-genotyped hemoglobinopathy patients, 3 new drugs (PB-04, DLT, RSV) and two HDAC inhibitors (MS-275 and SB939), induced 3- to 40-fold higher Υ-globin mRNA above untreated control levels.
Disclosures
Faller: Phoenicia BioSciences, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Perrine:Phoenicia BioSciences, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Research Funding.
In addition to being a part of the metabolic fatty acid fuel cycle, butyrate is also capable of inducing growth arrest in a variety of normal cell types and senescence-like phenotypes in gynecological cancer cells, inhibiting DNA synthesis and cell growth in colonic tumor cell lines, suppressing hTERT mRNA expression and telomerase activity in human prostate cancer cells, and inducing stem cell differentiation and apoptosis by DNA fragmentation. It regulates gene expression by inhibiting histone deacetylases (HDACs), enhances memory recovery and formation in mice, stimulates neurogenesis in the ischemic brain, promotes osteoblast formation, selectively blocks cell replication in transformed cells (compared to healthy cells), and can prevent and treat diet-induced obesity and insulin resistance in mouse models of obesity, as well as stimulate fetal hemoglobin expression in individuals with hematologic diseases such as the thalassemias and sickle-cell disease, in addition to a multitude of other biochemical effects in vivo. However, efforts to exploit the potential of butyrate in the clinical treatment of cancer and other medical disorders are thwarted by its poor pharmacological properties (short half-life and first-pass hepatic clearance) and the multigram doses needed to achieve therapeutic concentrations in vivo. Herein, we review some of the methods used to overcome these difficulties with an emphasis on HDAC inhibition.
... We previously conducted in vitro studies using primary erythroid progenitors derived from adults with SCD to develop a novel Class I HDAC inhibitor, CT-101 [25]. Treatment with CT-101 was not toxic to erythroid cells with normal growth and maturation, and induction of HbF expression; moreover, an additive effect was observed when CT-101 was combined with HU. ...
... In addition, benserazide, which is currently used in combination with levo-dopa for the treatment of Parkinson's disease, demonstrated a good induction of HbF. A phase I trial of benserazide using patients with NTDT is ongoing (NCT04432623) [164,169,170]. Figure 4 provides the most important elements of CTZ, Sirolimus, IMR-687, and benserazide. [164,169,170]. ...
... Binding of Simvastatin and tBHQ with KLF-1 mRNA in the proposed model leads to γ-globin gene expression induction, which agrees with results in Macari et al. (2013). Drug treatments of erythroid progenitors cultured from Sickle Cell Anemia and β-thalassemia patients revealed that MS-275 and ST-20 decrease the level of KLF-1 mRNA by 3-and 2.5-fold, respectively Dai et al. (2014). The simulation results at 100 pt show that the kinetic parameter of T30 and T31 should be considered as (0.3, 0.4, 0.5) and (0.35, 0.37, 0.40) fuzzy numbers to suppress KFL-1 mRNA level by 3-and 2.5-fold, respectively. ...
... Although the molecular mechanism of action of HQK-1001 is not fully elucidated, similar short-chain fatty acid derivatives were found to reactivate the g-globin gene by inducing a dissociation of histone deacetylase class 3 (HDAC-3) and its adaptor protein nuclear receptor corepressor (NCoR) from the g-globin gene promoter, with coincident recruitment of RNA polymerase II to the g-globin gene promoter [3]. HQK-1001 was also found to displace HDAC-2 in chromatin immunoprecipitation assays and to suppress BCL-11A protein levels in treated erythroid progenitors [4]. In addition, HQK-1001 enhances erythropoiesis and decreases erythroid apoptosis by phosphorylation and activation of STAT-5, c-cis, and Bcl-xL [5][6][7]. ...
... It has been used for a different activity in combination with Ldopa to enhance its half-life for the treatment of Parkinson's disease for > 40 years in Europe and Canada. 123,124 It was discovered in a high throughput screen of clinical-stage drugs to have high inducing activity compared to other active and non-cyotoxic candidates. 104,105 Benserazide profoundly suppressed or displaced several repressors of the fetal globin gene promoter in patients' erythroid progenitors and induced γ globin mRNA and protein, F-reticulocytes, F-cells, and total Hb in nonhuman primates and transgenic mice. ...
... The FA pathway may coordinate the activity of HR, MMR and NER proteins in ICL repair [124, 125]. Several FA proteins are known to bind to DNA containing ICLs either directly or via nonerythroid α spectrin (also called SPTAN1) [126]. This protein also associates with ICLs in telomeres in the S phase and is required for their maintenance [127]. ...
... Although tractable for high-throughput compound screening, these synthetic gene constructs may not address essential endogenous DNA elements that tightly regulate and control γ-globin gene expression in erythroid cells. Further, until recently, many cell-based screens for HbF-inducers have been conducted in human immortalized myelogenous leukemia K562 cells [25,26] or murine erythroleukemia MEL cells [27], which are poor surrogates for normal erythroid cells and thus are not optimal systems for studying hemoglobin switching and regulation. In addition, the use of flow cytometry assays (FACS) or ELISA-based readouts for HbF quantitation have constrained their widespread application due to time-consuming and expensive assay protocols that are poorly adaptable to large-scale industrial drug discovery screening campaigns. ...
... 101,103,104 Interesting new molecular mechanisms were identified with these candidates. 112 One involved EKLF (erythroid Kruppel-like factor) which was previously associated with activating the β-globin gene in erythroid cells specifically. A next generation molecule identified in molecular modeling, RB7, was found to have a novel action of recruiting EKLF and a remodeling complex BRG1 from the β to the γglobin gene promoter, inducing high level γ globin expression. ...
... Butyrate and propionate are SCFAs that play essential roles in fatty acid metabolism and synthesis. 70,71 Butyrate serves as a cell energy source and acts as a histone deacetylase (HDAC) inhibitor, influencing gene expression related to lipid metabolism and inflammation. 72 Propionate participates in gluconeogenesis and contributes to the maintenance of metabolic balance. ...
... There is growing appreciation for the multifunctional nature of spectrin family members beyond providing structural support for the cell membrane. For example, spectrins support longrange cellular communication involving, in part, coordination of signaling nanodomains for ion channels [42,[53][54][55][56][57][58][59][60]. More recently, it has been discovered that spectrins modulate gene expression to control remodeling of cell function in response to chronic stress stimuli, although the precise mechanisms remain to be determined [61][62][63]. ...