ALBERT

Description: The erythroid Kruppel-like factor, EKLF, is an essential transcription factor for mammalian β-type globin gene switching, and specifically activates transcription of the adult β-globin gene through binding of its zinc finger domain to the β-globin promoter. We report now that EKLF is also required for activation of the γ-globin gene by short-chain fatty acid (SCFA) derivatives. We found that specific knockdown of EKLF levels by siRNA prevents SCFA induced-expression of an integrated γ-globin promoter in a stably-expressed mLCRβprRluc AγprFluc cassette, and prevents induction of the endogenous γ-globin gene in primary human erythroid progenitors. In chromatin immunoprecipitation (ChIP) assays, EKLF was found to be actively recruited to the endogenous γ-globin gene promoter with exposure of human erythroid progenitors, and hematopoietic cell lines, to SCFA derivatives. The human SWI/WNF complex is a ubiquitous multimeric complex that regulates gene expression by remodeling nucleosomal structure in an ATP-dependent manner. We found that the SWI/SNF complex chromatin-modifying core ATPase BRG1 is also required for γ-globin gene induction by SCFA derivatives. Furthermore, BRG1 is actively recruited to the endogenous γ-globin promoter of human erythroid progenitors with exposure to SCFA derivatives, and this recruitment is dependent upon the presence of EKLF. These findings all demonstrate that EKLF, and the co-activator BRG1, previously demonstrated to be required for definitive or adult erythropoietic patterns of globin gene expression, are co-opted by SCFA derivatives to activate the fetal globin genes. Recently. we also identified a γ-globin-specific repressor complex, consisting of NCoR and HDAC3, which is displaced from the proximal γ-globin promoter by exposure to SCFA derivatives prior to activation of transcription (Blood, 108:3179–86, 2006). Collectively, these studies identify critical activating and repressing cofactors regulating γ-globin gene expression, and provide new targets for therapeutic interventions.

Description: Abstract 4280 Beta thalassemia intermedia syndromes are serious conditions for which there is no satisfactory therapy to correct the underlying globin chain imbalance. Some agents that induce fetal globin gene expression have ameliorated anemia in thalassemia patients by reducing the imbalance in alpha: non-alpha globin synthesis, but none have been broadly accepted or are currently approved by regulatory authorities. HQK-1001 is an oral agent that targets the fetal globin gene promoter, thereby increasing fetal hemoglobin (HbF) expression. It has been well tolerated in single dose and multiple dose escalation clinical studies in healthy volunteers. We now report the results of a randomized, double blind, placebo-controlled, multiple ascending dose Phase I/II trial in 21 adult patients with beta thalassemia intermedia (BTI), including 14 with HbE/ß0 thalassemia and 7 with ß+/ß0 thalassemia (including 12 different beta globin gene mutations). Study medication was taken as a single daily dose for 8 weeks. Four ascending dose levels (10, 20, 30, and 40 mg/kg/day) were sequentially evaluated in 4 dose level cohorts after the preceding dose and schedule were determined safe by an independent and unblinded Safety Monitoring Committee. HQK-1001 was well-tolerated. Adverse events in treated subjects included headache, upper respiratory infection and nausea, but the rates of such events were not markedly different than those observed in the placebo-treated subjects. The 20 mg/kg dose was associated with a 10% mean increase above baseline in HbF, (p〈 0.001). Total hemoglobin (Hgb) increased by a mean of 1.1 gram/dL in 3 of 6 treated BTI patients with Mediterranean mutations. F-cells increased over the study period with maximal increases often observed 2 weeks following therapy. Doses higher than 20 mg/kg were not associated with the same magnitude of pharmacodynamic effects. These observations indicate that HQK-1001 is well-tolerated at doses associated with favorable pharmacodynamic effects on Hgb and HbF. These findings with brief treatment provide a rationale for conducting larger and longer studies in BTI patients. Disclosures: Fuchareon: HemaQuest Pharmaceuticals, Inc: Honoraria, Research Funding. Inati:HemaQuest Pharmaceuticals, Inc: Honoraria, Research Funding. Boosalis:HemaQuest Pharmaceuticals, Inc: Equity Ownership, Research Funding. Thein:HemaQuest Pharmaceuticals, Inc: Research Funding. Wallis:HemaQuest Pharmaceuticals: Consultancy, Equity Ownership. Bobbitt:HemaQuest Pharmaceuticals, Inc: Employment, Equity Ownership, Patents & Royalties. Thomson:HemaQuest Pharmaceuticals: Employment, Equity Ownership. Johnson:HemaQuest Pharmaceuticals: Employment, Equity Ownership. Berenson:HemaQuest Pharmaceuticals, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Perrine:HemaQuest Pharmaceuticals, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Description: Abstract 251 Beta thalassemia intermedia (BTI) syndromes are characterized by globin chain imbalance, ineffective erythropoiesis, hemolytic anemia, and have no therapeutic approved for the underlying pathology. Higher fetal globin (HbF) expression can compensate for beta globin deficiency, reduce globin chain imbalance, and ameliorate phenotype within the same genotypes. HQK-1001 (HemaQuest Pharmaceuticals, San Diego, CA) is an orally bioavailable, non-cytotoxic, short-chain fatty acid derivative which induces fetal globin expression in multiple experimental models. In thalassemic erythroid progenitors in vitro, HQK-1001 prolongs STAT-5 phosphorylation and increases expression of the pro-survival protein Bcl-xL. In a Phase I/II dose-escalation trial in BTI, HQK-1001 administered at 10, 20, 30, and 40 mg/kg/day for 8 weeks was well–tolerated, demonstrated a t½ of 9–11 hours, and treatment resulted in an increase in HbF and total hemoglobin (Hgb), with best results observed at 20 mg/kg. This open-label Phase 2 trial evaluated HQK-1001 administered orally at 20 mg/kg once daily for 26 weeks in adult patients with HbE-β0 thalassemia (NCT01609595). Ten subjects were enrolled, ages 20–36 years, including 7/10 female and 5/10 splenectomized subjects. The beta globin molecular mutations of the subjects, in addition to Codon 26 (G-A), included Codon 41/42 (-TTCT), Codon 17 (A-T), Codon 110 (T-C), and IVS I-I (G-T). At the time of this analysis, 9 of 10 subjects have completed at least 20 weeks of dosing. HbF has increased in all subjects above baseline levels by a mean of 10% (range 4.3% to 20.9%) and total HbF has increased by a mean of 1.07 g/dL, (range 0.52–2.38 g/dL, with positive changes in total HbF observed in 9/10 subjects. Total Hgb has increased by 〉0.5 g/dL above baseline (mean of 1.27 g/dL, range 0.7 to 2.2 g/dL) in 3 subjects after 3 to 5 months of dosing. Treatment was well-tolerated. There have been no severe drug-related adverse events, no serious adverse events, and no clinically relevant laboratory abnormalities. One subject developed transient proteinuria and edema without changes in renal function 3 weeks following an episode of pharyngitis. This ongoing trial demonstrates consistent induction of fetal globin expression by HQK-1001 at a well-tolerated dose level in subjects with HbE-beta thalassemia. Disclosures: Fucharoen: HemaQuest Pharmaceuticals: Research Funding. Perrine:HemaQuest Pharmaceuticals: Equity Ownership, Patents & Royalties.

Description: 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.

Description: 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

Description: Orally bioactive compounds that induce γ globin gene expression at tolerable doses are needed for optimal treatment of the β-hemoglobinopathies. Short-chain fatty acids (SCFAs) of 2 to 6 carbons in length induce γ globin expression in animal models, and butyrate, phenylbutyrate, and valproate induce γ globin in human patients. The usefulness of these compounds, however, is limited by requirements for large doses because of their rapid metabolism and their tendency to inhibit cell proliferation, which limits the pool of erythroid progenitors in which γ globin can be induced. Selected short-chain fatty acid derivatives (SCFADs) were recently found to induce γ globin and to stimulate the proliferation of hematopoietic cells in vitro. These SCFADs are now evaluated in vivo in nonanemic transgenic mice containing the human β globin gene locus and in anemic phlebotomized baboons. In mice treated with a SCFAD once daily for 5 days, γ globin mRNA increased 2-fold, reticulocytes increased 3- to 7-fold, and hematocrit levels increased by 27%. Administration of 3 SCFADs in anemic baboons increased F-reticulocytes 2- to 15-fold over baseline and increased total hemoglobin levels by 1 to 2 g/dL per week despite ongoing significant daily phlebotomy. Pharmacokinetic studies demonstrated 90% oral bioavailability of 2 SCFADs, and targeted plasma levels were maintained for several hours after single oral doses equivalent to 10% to 20% of doses required for butyrate. These findings identify SCFADs that stimulate γ globin gene expression and erythropoiesis in vivo, activities that are synergistically beneficial for treatment of the β hemoglobinopathies and useful for the oral treatment of other anemias.

Description: The mechanisms by which pharmacologic agents stimulate γ-globin gene expression in β-globin disorders has not been fully established at the molecular level. In studies described here, nucleated erythroblasts were isolated from patients with β-globin disorders before and with butyrate therapy, and globin biosynthesis, mRNA, and protein-DNA interactions were examined. Expression of γ-globin mRNA increased twofold to sixfold above baseline with butyrate therapy in 7 of 8 patients studied. A 15% to 50% increase in γ-globin protein synthetic levels above baseline γ globin ratios and a relative decrease in β-globin biosynthesis were observed in responsive patients. Extensive new in vivo footprints were detected in erythroblasts of responsive patients in four regions of the γ-globin gene promoter, designated butyrate-response elements gamma 1-4 (BRE-G1-4). Electrophoretic mobility shift assays using BRE-G1 sequences as a probe demonstrated that new binding of two erythroid-specific proteins and one ubiquitous protein, CP2, occurred with treatment in the responsive patients and did not occur in the nonresponder. The BRE-G1 sequence conferred butyrate inducibility in reporter gene assays. These in vivo protein-DNA interactions in human erythroblasts in which γ-globin gene expression is being altered strongly suggest that nuclear protein binding, including CP2, to the BRE-G1 region of the γ-globin gene promoter mediates butyrate activity on γ-globin gene expression. © 1998 by The American Society of Hematology.

Description: High-level γ-globin gene expression for definitive therapy of beta globin disorders likely requires γ-globin gene promoter activation, dissociation of repressor complexes, and/or local chromatin modification. To define the molecular mechanisms employed by high-potency γ-globin inducers, the effects of butyrate on transcriptional co-regulatory protein recruitment to the human γ- and β-globin gene promoters were defined and compared to those of novel SCFAD γ-globin inducers recently identified through computational modeling. One particularly potent SCFAD inducer, RB7, which has no inhibitory effect on class I HDAC enzymes (HDAC1, HDAC2, and HDAC3), demonstrated 5-fold induction, (2.5 to 3-fold greater γ-globin gene-inducing activity than butyrate), in a transcriptional reporter assay which detects only strong γ-globin gene inducers. Chromatin immunoprecipitation (ChIP) assays performed on the integrated human gamma and beta globin gene promoters demonstrated that RB7, butyrate, and other transcriptionally-active SCFADs, induced dissociation of HDAC-3, and its adaptor protein NCoR, from the γ-globin gene promoter, with no such effect on the β-globin promoter, coincident with recruitment of RNA Polymerase II to the γ-globin promoter and initiation of γ-globin transcription. In a reciprocal fashion, as HDAC-3 dissociated, the chromatin-modifying ATPases Brg-1 and Brm were recruited specifically to the γ-globin gene promoter in response to the active SCFADs. The ability of the novel SCFADs to induce dissociation of HDAC-3 (but not HDAC-1 or -2) from the γ-globin promoter was proportional to the degree of γ-globin promoter induction by these agents. Knockdown of HDAC-3 by siRNA induced transcription of γ-globin specifically, demonstrating the functional importance of the activity of SCFADS in dissociating HDAC-3 from the γ-globin gene promoter. These studies demonstrate new dynamic alterations in transcriptional regulatory complexes specifically-associated with high-level activation of γ-globin and also identify essential molecular targets for future therapeutic interventions.

Description: Induction of EBV lytic-phase gene expression, combined with exposure to an antiherpes viral drug, represents a promising targeted therapeutic approach to EBV-associated lymphomas. Short-chain fatty acids or certain chemotherapeutics have been used to induce EBV lytic-phase gene expression in cultured cells and mouse models, but these studies generally have not translated into clinical application. The recent success of a clinical trial with the pan-histone deacetylase (pan-HDAC) inhibitor arginine butyrate and the antiherpes viral drug ganciclovir in the treatment of EBV lymphomas prompted us to investigate the potential of several HDAC inhibitors, including some new, highly potent compounds, to sensitize EBV+ human lymphoma cells to antiviral agents in vitro. Our study included short-chain fatty acids (sodium butyrate and valproic acid); hydroxamic acids (oxamflatin, Scriptaid, suberoyl anilide hydroxamic acid, panobinostat [LBH589], and belinostat [PXD101]); the benzamide MS275; the cyclic tetrapeptide apicidin; and the recently discovered HDAC inhibitor largazole. With the exception of suberoyl anilide hydroxamic acid and PXD101, all of the other HDAC inhibitors effectively sensitized EBV+ lymphoma cells to ganciclovir. LBH589, MS275, and largazole were effective at nanomolar concentrations and were 104 to 105 times more potent than butyrate. The effectiveness and potency of these HDAC inhibitors make them potentially applicable as sensitizers to antivirals for the treatment of EBV-associated lymphomas.