Fetal Globin Gene Expression Research Articles

Targeting a Novel Epigenetic Silencing Mechanism to Efficiently Upregulate Fetal Globin Gene Expression

Abstract 352L3MBTL1 is a Polycomb group protein, commonly deleted in patients with myeloid disorders associated with the 20q- chromosomal abnormality. After crystallizing the MBT repeat domain, we demonstrated that L3MBTL1 compacts chromatin by binding mono- and di-methylated lysine residues in histones H1 (H1K26) and H4 (H4K20), ultimately leading to gene repression. Despite its role in affecting the chromatin structure, the role of L3MBTL1 in hematopoiesis has remained largely unknown.We recently demonstrated that lack of L3MBTL1 accelerates the erythroid differentiation of human hematopoietic stem cells and here we reveal that L3MBTL1 represses the expression of the fetal gamma globin gene. We lentivirally expressed shRNAs targeting L3MBTL1 in human cord blood (CB) CD34+ cells and in K562 erythroleukemia cells, and consistently observed upregulation of gamma globin gene expression, while beta globin gene expression decreased. Remarkably, we observed similar findings in human embryonic stem (hES) cells, where knock-down of L3MBTL1 triggered a BMP4-like spontaneous differentiation. Given the potential impact of therapeutically increasing fetal hemoglobin expression in patients with hemoglobinopathies, we targeted L3MBTL1 in induced pluripotent stem (iPS) cells derived from patients with β-thalassemia. The gene expression profile of L3MBTL1-KD normal and thalassemic iPS cells indicated clear activation of fetal hemoglobin (HbF) expression, activation of BMP4 signaling and upregulation of specific smad5 target genes (e.g. EKLF, HHEX, ID2/3). We generated and utilized a model of “stress erythropoiesis” in L3MBTL1 KO mice and observed in vivo BMP4-mediated expansion of spleen immature erythroid progenitors, as indicated by increased spleen weight and splenic BFU-E colonies in KO mice compared to controls.We also examined K562 cells, human CB CD34+ cells and hES cells, using chromatin immunoprecipitation assays, and found that L3MBTL1 directly associates with the human β-globin locus, occupying discrete regions within the human β-globin cluster. Furthermore, L3MBTL1 colocalized with H4K20me within the Locus Control Region (LCR), a primary attachment site for chromatin modifiers. We observed clearance of L3MBTL1 and its associated histone marks (H4K20me1/2) from the LCR upon treatment with hemin, erythropoietin or TGFβ, three agents that potently induce erythroid differentiation. This suggests that this polycomb repressor complex responds to cytokine signaling.In summary, we have identified a novel epigenetic regulatory mechanism to control fetal globin gene expression; the Polycomb protein L3MBTL1 regulates BMP4 signaling and the chromatin structure of globin genes. Targeting this regulatory system represents a means to efficiently increase HbF in a human model of β-thalassemia (i.e. with the use of patient-derived iPS cells) and to potentially ameliorate hematological and clinical symptoms of patients with red cell disorders. Disclosures:No relevant conflicts of interest to declare.

Correction of Sickle Cell Disease in Adult Mice by Interference with Fetal Hemoglobin Silencing

Persistence of human fetal hemoglobin (HbF, α(2)γ(2)) in adults lessens the severity of sickle cell disease (SCD) and the β-thalassemias. Here, we show that the repressor BCL11A is required in vivo for silencing of γ-globin expression in adult animals, yet dispensable for red cell production. BCL11A serves as a barrier to HbF reactivation by known HbF inducing agents. In a proof-of-principle test of BCL11A as a potential therapeutic target, we demonstrate that inactivation of BCL11A in SCD transgenic mice corrects the hematologic and pathologic defects associated with SCD through high-level pancellular HbF induction. Thus, interference with HbF silencing by manipulation of a single target protein is sufficient to reverse SCD.

The PcG Protein L3MBTL1 Transcriptionally Represses Human Embryonic and Fetal Globin Genes: a Novel Prospect for HbF Activation

AbstractAbstract 1014L3MBTL1 is the human homolog of the Drosophila Polycomb Group tumor suppressor gene, lethal(3)malignant brain tumor. We demonstrated that human L3MBTL1 functions as a transcriptional repressor and after crystallizing the MBT repeat domain determined that L3MBTL1 compacts chromatin by binding mono- and di-methylated lysine residues in histones H1 (H1K26) and H4 (H4K20).Despite the known role of L3MBTL1 in affecting chromatin structure, the function of L3MBTL1 in human hematopoiesis has remained largely unknown. We recently demonstrated that L3MBTL1 enforces cell fate decision toward the erythroid lineage and that knockdown of L3MBTL1 accelerates the erythroid differentiation of human hematopoietic stem/progenitor cells, suggesting that its deletion contributes to the pathogenesis of 20q- erythroid malignancies. Consistently with its role in erythropoiesis, here we reveal that L3MBTL1 is a novel transcriptional repressor of fetal globin genes and it may work in concert with BCL11A and EKLF to control globin gene expression.By utilizing RNA interference to reduce L3MBTL1 expression, we have found that knockdown of L3MBTL1 in human cord blood hematopoietic stem/progenitor cells consistently upregulates the expression of the epsilon, gamma, and zeta globin genes, but not the beta globin gene. Similar effects were seen following knockdown of L3MBTL1 in the human erythroleukemia cell line K562, and knockdown of L3MBTL1 in human embryonic stem cells (ESCs) led to the inappropriate expression of fetal and embryonic globin genes (which increases more than 50-fold after the L3MBTL1-KD). These data suggest a role for L3MBTL1 in regulating the globin switch.To investigate the mechanism by which L3MBTL1 silences embryonic and fetal globin gene expression, we used chromatin immunoprecipitation (ChIP) assays to show that L3MBTL1 directly associates with the human β-globin locus. L3MBTL1 occupies several discrete regions within the human β-globin cluster and colocalizes with H4K20me within the Locus Control Region (LCR), a primary attachment site for chromatin modifiers. As confirmation, we found that treatment of K562 cells with hemin, which broadly increases H3K9 acetylation over the β-globin locus and activates the transcription of globin genes, leads to decreases in expression of the repressive H4K20me2 methylmark and L3MBTL1 to the beta-globin cluster.Given the recent identification of the repressor of gamma globin gene expression, BCL11A, we investigated a potential relationship between L3MBTL1 and BCL11A. We found that knockdown of L3MBTL1 led to downregulation of BCL11A mRNA. Accordingly, we have also found that overexpression of L3MBTL1 is associated with an upregulation of BCL11A mRNA, suggesting that L3MBTL1 and BCL11A may function cooperatively to silence globin gene expression. Knockdown of L3MBTL1 also upregulated EKLF mRNA levels which could relate to the decreased BCL11A expression.In summary our data demonstrate that knock-down of L3MBTL1 upregulates embryonic and fetal globin genes in cell contexts where they are usually silenced, indicating the functional importance of this Polycomb protein for repressing the globin gene locus. The clearance of L3MBTL1 and its associated histone mark (H4K20me2) during treatments that induce potent transcriptional activation of globin genes suggest that repression induced by L3MBTL1 is dynamic and may be involved in the fetal-to-adult globin switch. L3MBTL1 therefore emerges as a novel transcriptional repressor of fetal globin genes whose expression may be coordinated with that of BCL11A and EKLF. Understanding the role of L3MBTL1 and the H4K20 methylmark in globin gene switching offers the prospect of the targeted activation of HbF in erythroid cells of patients with hemoglobin disorders. Disclosures:No relevant conflicts of interest to declare.

Structural and Functional Characterization of Csda Protein Complexes Involved In Modulation of Fetal Globin Gene Expression

AbstractAbstract 2071Impaired switching from fetal hemoglobin (HbF) to adult globin gene expression leads to hereditary persistence of fetal hemoglobin (HPFH) in adult life. This is of prime interest because elevated HbF levels ameliorate beta-thalassemia and sickle cell anemia. Fetal hemoglobin levels are regulated by complex mechanisms involving factors linked or not to the beta-globin gene locus. Recently, we reported an inverse relationship between Ggamma-globin gene (HBG2) and Cold Shock Domain Protein A (CSDA) expression levels. Based on mRNA differential display analysis, RNA interference and over-expression studies in K562 and primary erythroid cells we postulated that CSDA could contribute to regulate HBG2 expression. The putative mechanism by which CSDA modulates HBG2 expression was investigated in K562 cells by gene reporter assays on wild-type and mutant constructs of the HBG2 promoter region suspected to bind CSDA, providing experimental evidence that CSDA acts as repressor of HBG2 expression. Furthermore, chromatin immunoprecipitation (ChIP) analysis on K562 cells showed that CSDA interacts in vivo with this promoter region. In this way we were able to demonstrate that CSDA modulates HBG2 expression at least in part at the transcriptional level (Petruzzelli R et al, Br J Haematol 2010). The CSDA gene is located at position 12p13.1 and comprises 10 exons. The C-terminus (exons 6–9) is involved in protein-protein interactions. Alternative splicing of exon 6 results in two main isoforms, namely CSDA isoform a and isoform b, which show different C-terminal domains, potentially able to take part to specific protein complexes. We found that expression levels of CSDA isoform a were reduced in HPFH patients respect to isoform b. These findings suggested that isoform a could be much more involved in repression of HBG2 expression compared to isoform b. To identify putative CSDA interactors, we over-expressed these two FLAG-tagged CSDA isoforms in K562 cells. Western-blot analysis on proteins immunoprecipitated with a FLAG antibody revealed the presence of NF-kB p50 and p65 subunits and histone deacethylase 2 (HDAC2) only in samples co-immunoprecipitated with CSDA isoform a, but not with isoform b (Fig. 1). By ChIP assays with antibodies against p65, p50 and HDAC2, we demonstrated that both the NF-kB p50-p65 heterodimer and HDAC2 interact with the –200 bp region of the HBG2 promoter containing the CSDA binding site (Fig. 2). To examine the role of NF-kB and histone deacetylases on the transcriptional repression of HBG2 expression, we treated K562 cells with the proteasome inhibitor bortezomib which blocks the nuclear traslocation and transcriptional activity of the NF-kB p65-p50 complex or with the histone deacetylase inhibitor trichostatin A (TSA). Quantitative analysis by Real Time PCR showed that HBG2 expression increased following either bortezomib or TSA treatments. Furthermore, by ChIP analysis we were able to demonstrate that knock-down of CSDA abolished these interactions. To investigate if treatment with bortezomib or TSA affects the histone acetylation levels at the -200 bp region of the HBG2 promoter, we performed ChIP assays in K562 cells using an anti-acetyl-H3 antibody. Results indicated that both these drugs induces a considerable increase in H3 acetylation levels at the -200 bp region of the HBG2 promoter (Fig. 3). Taken altogether these data indicate that NF-kB and HDAC2 interact with CSDA to form a multiprotein complex which take part to the regulation of HBG2 expression by modulating local chromatin conformation. Furthermore, our study contributes to better define the role played by CSDA in fetal globin gene expression and shed novel light on the molecular mechanisms involved in globin gene switching. [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

HQK-1001 Is Well Tolerated and Augments Hemoglobin F and Hemoglobin Levels In Patients with Beta Thalassemia Intermedia

AbstractAbstract 4280Beta 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.

Identification of a PRMT5-dependent repressor complex linked to silencing of human fetal globin gene expression

Defining the molecular mechanisms underpinning fetal (gamma) globin gene silencing may provide strategies for reactivation of gamma-gene expression, a major therapeutic objective in patients with beta-thalassemia and sickle cell disease (SCD). We have previously demonstrated that symmetric methylation of histone H4 Arginine 3 (H4R3me2s) by the protein arginine methyltransferase PRMT5 is required for recruitment of the DNA methyltransferase DNMT3A to the gamma-promoter, and subsequent DNA methylation and gene silencing. Here we show in an erythroid cell line, and in primary adult erythroid progenitors that PRMT5 induces additional repressive epigenetic marks at the gamma-promoter through the assembly of a multiprotein repressor complex containing the histone modifying enzymes SUV4-20h1, casein kinase 2alpha (CK2alpha), and components of the nucleosome remodeling and histone deacetylation complex. Expression of a mutant form of PRMT5 lacking methyltransferase activity or shRNA-mediated knockdown of SUV4-20h1 resulted in loss of complex binding to the gamma-promoter, reversal of both histone and DNA repressive epigenetic marks, and increased gamma-gene expression. The repressive H4K20me3 mark induced by SUV4-20h1 is enriched on the gamma-promoter in erythroid progenitors from adult bone marrow compared with cord blood, suggesting developmental specificity. These studies define coordinated epigenetic events linked to fetal globin gene silencing, and provide potential therapeutic targets for the treatment of beta-thalassemia and SCD.

Fetal globin gene inducers: novel agents and new potential

Inducing expression of endogenous fetal globin (gamma-globin) gene expression to 60-70% of alpha globin synthesis produces beta-thalassemia trait globin synthetic ratios and can reduce anemia to a mild level. Several classes of therapeutics have induced gamma-globin expression in beta-thalassemia patients and subsequently raised total hemoglobin levels, demonstrating proof-of-concept of the approach. Butyrate treatment eliminated transfusion requirements in formerly transfusion-dependent patients with treatment for as long as seven years. However, prior generation inducers were not readily applicable for widespread use. Currently, a novel oral dual-action therapeutic, sodium 2,2-dimethylbutyrate, is in clinical trials, an oral decitabine formulation is under development, and agents with complementary mechanisms of action can be applied in combined regimens. Identification of three major genetic trait loci which modulate clinical severity provides avenues for developing tailored regimens. These refinements offer renewed potential to apply fetal globin induction as a treatment approach in patient-friendly regimens that can be used worldwide.

Short Chain Fatty Acids and p38 in the Induction of Embryonic/Fetal Globin Genes During Definitive Erythropoiesis.

Abstract 460Augmentation of fetal hemoglogin (HbF) is therapeutically desirable in β –globin gene disorders, such as sickle cell anemia and β-thalassemia. Short chain fatty acids (SCFAs) can augment embryonic/fetal β-type globin gene expression in vivo and in vitro during adult definitive erythropoiesis (EryD), but the underlying molecular mechanisms are not well understood. p38 signaling, triggered by stress erythropoiesis, has been implicated in the induction of fetal globin gene expression. Here, we examined p38 signaling and its effects using a murine primary cell model of EryD, isolated from fetal liver. This model overcomes some limitations of previously used transformed or long-term primary cell models, and has afforded complementary data. E14.5 erythroid fetal cells (eFLCs) were examined at harvest and after culture in insulin and erythropoietin (‘ins/EPO') with or without the SCFA butyrate, and in the presence or absence of p38 inhibitors SB 203580, SB 202190, PD 169316 at concentrations of 0-50 μM for up to 72 hours. Western blots showed that p38 phosphorylation was constitutive at harvest and preserved in cultures with ins/EPO, but was diminished by half after concurrent culture in PD169316. Embryonic globin gene expression, ((βH1+εY)/(βH1+εY+βMAJ) × 100), was analyzed by real-time PCR, normalized to 18S expression. Embryonic globin gene induction, relative to maximal expression at 72 hours in butyrate with ins/EPO, was blocked in a dose dependent manner by all inhibitors of p38 signaling, at 10-30% of maximal induction when cultured in 50 mM of any inhibitor. Primary transcripts from the adult β-globin gene, βmaj, were likewise diminished by p38 inhibition, with a median level of 8.3% and 4.0% of maximal transcript in ins/EPO and butyrate & ins/EPO respectively after culture for 72 hours in 50 μM PD 169316 (n=3, p<.01 relative to non-inhibited cultures). Erythroid differentiation, analyzed by FACS quantitation of CD71 and TER119 co-immunostaining, was delayed by p38 inhibitors, with 22-38% of ins/EPO +/- SCFA-treated cells comprised of orthochromatophilic cells after 72 hours, compared with 4-8% of parallely treated cells to which p38 inhibitors had been added. To further explore stress erythropoesis, cell divisions were analyzed using carboxyfluorescein succinimidyl ester (a fluorescent cytoplasmic dye that is diluted with each cell division) as monitored by FACS. Cell divisions were modestly slowed, but not blocked, by SCFAs. A median of 60% of ins/EPO-, vs. 29% of butyrate & ins/EPO-, treated cells had undergone 2 cell divisions at 48 hours (n=2, p<.05), but by 72 hours a median of 56% and 51% of treated cells, respectively, had completed 2 divisions (n=2, p=n.s.). In contrast, cell division was perturbed by p38 inhibitors, with <26% of eFLCs having undergone a 2nd division by 72 hours. Our studies suggest that p38 signaling is required for, but is not specific to, the SCFA-mediated induction of embryonic/fetal globin gene expression. We do not find that p38 phosphorylation is stimulated by SCFAs in short-term primary EryD. The central role that p38 signaling plays in the induction of embryonic and fetal globin gene expression, as manifested by lost induction with p38 inhibition, may instead reflect p38's constitutive function in cell division and differentiation. Disclosures:No relevant conflicts of interest to declare.

HQK-1001 Has Additive HbF-Inducing Activity in Combination with Hydroxyurea and Decitabine.

Abstract 977Several classes of HbF inducers, including chemotherapeutic agents, ESAs, and short chain fatty acids (SCFADs) have demonstrated efficacy in inducing HbF in beta hemoglobinopathy and thalassemia patients, but broad clinical application of agents except Hydroxyurea (HU) has been limited by requirements for parenteral administration, high doses and toxicity concerns for long-term use. Yet, combination therapy is likely necessary to achieve the high fetal globin expression levels necessary to ameliorate anemia in the severe beta thalassemias. HQK-1001 is an oral, non-mutagenic SCFAD, which induces fetal globin gene expression through displacement of a repressor complex from the gamma globin gene promoter, and has shown a favorable safety and PK profile with a T1/2 of 11 hours with once daily oral doses from 5-20 mg/kg in normal human volunteers. To investigate whether HQK-1001 has additive activity with other fetal globin inducing agents with different mechanisms of action, dose-escalation studies of HQK-1001 were performed in baboons, and PK profiles and fetal globin mRNA were compared to treatment with HU. Fetal globin mRNA increased with escalating doses of HQK-1001 from 50-150 mg/kg, reaching peak effects at an AUClast of 1267 h.micrograms/ml, comparable to a human equivalent dose of 20 mg/kg (AUClast = 1174-2205 h. microgram/ml), while a significantly higher AUClast (11,817 h.microgram/ml) was observed at a 200 mg/kg/dose and associated with gamma globin suppression. In baboons treated with sequentially administered Hydroxyurea (HU) (3 days/week), gamma globin mRNA increased by 80-120% above baseline, treatment with HQK-1001 increased γ globin mRNA by 290% above baseline, while the 2-agent combination increased γ globin mRNA by 360%. In Bfu-e cultures comparing HQK-1001 +/− decitabine, F-cells increased above baseline in cultures treated with HQK-1001 alone by 32-71%, with decitabine alone by 66-104%, and with the 2 agents together by 147% above F-cell levels in control cultures. In cultures containing SCF, Bfu-e numbers increased by 25% over controls with both agents added to a peak decitabine conc. of 0.1 microM; erythroid growth declined at higher concentrations of decitabine. These studies together demonstrate higher gamma globin gene induction with HQK-1001 in combination with HU and with decitabine, and with reduced cytotoxicity with added HQK-1001. Dose-escalating Phase 2 clinical trials of HQK-1001 have begun in patients with beta thalassemia. Disclosures:Perrine:HemaQuest Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Faller:HemaQuest Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Boosalis:HemaQuest Pharmaceuticals: Equity Ownership. Thomson:HemaQuest Pharmaceuticals: Employment, Equity Ownership. Berenson:hemaQuest Pharmaceuticals: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Humanized Mouse Model of Cooley's Anemia

A novel humanized mouse model of Cooley's Anemia (CA) was generated by targeted gene replacement in embryonic stem (ES) cells. Because the mouse does not have a true fetal hemoglobin, a delayed switching human gamma to beta(0) globin gene cassette (gammabeta(0)) was inserted directly into the murine beta globin locus replacing both adult mouse beta globin genes. The inserted human beta(0) globin allele has a mutation in the splice donor site that produces the same aberrant transcripts in mice as described in human cells. No functional human beta globin polypeptide chains are produced. Heterozygous gammabeta(0) mice suffer from microcytic anemia. Unlike previously described animal models of beta thalassemia major, homozygous gammabeta(0) mice switch from mouse embryonic globin chains to human fetal gamma globin during fetal life. When bred with human alpha globin knockin mice, homozygous CA mice survive solely upon human fetal hemoglobin at birth. This preclinical animal model of CA can be utilized to study the regulation of globin gene expression, synthesis, and switching; the reactivation of human fetal globin gene expression; and the testing of genetic and cell-based therapies for the correction of thalassemia.

A Novel Model of Short Chain Fatty Acid (SCFA)- Mediated up-Regulation of Embryonic/Fetal Globin Genes during Definitive Erythropoiesis.

Persistence of fetal hemoglobin can ameliorate adult beta (β)-globin gene disorders such as sickle cell disease and β-thalassemia. Short chain fatty acids (SCFAs) up-regulate embryonic/fetal globin gene expression in vitro and in vivo and have great therapeutic potential. We first studied the SCFA-responsiveness of embryonic and fetal globin gene expression during short-term culture in a murine primary cell model. Pooled erythroid fetal liver cells (eFLCs) from wild-type (wt) and human β-globin gene locus-containing mice were examined after treatment with SCFAs. Cells were cultured in basal media alone or in basal media plus propionate (5mm), butyrate (1mM), and/or insulin (2 U/mL) and erythropoietin (10 mg/mL, ‘ins/EPO‘). Murine embryonic β-type globin gene expression, ((βH1+εY)/( βH1+εY+βMAJ)x100) was markedly increased at 72 hours in SCFA-treated wt eFLCs, from 1.7±1.2% at baseline to, at 72 hours, 4.88±2.21% in propionate, 5.40±3.39% in butyrate, 19.48±8.30% in butyrate & ins/EPO, compared with ins/EPO-only-treated wt eFLCs, at 0.3±0.3% (n ≥ 3 for each, p<.05). Human fetal γ-globin gene expression was also up-regulated in human transgenic eFLCs, at 29.0±5.0% in butyrate & ins/EPO vs. 3.07±1.08 in ins/EPO only (n=2, p<.05). PCR analysis of FACS-sorted individual eFLCs after culture confirmed that both ins/EPO- and butyrate & ins/EPO-treated cells express detectable α-globin (n=15 for both, p= n.s.). However, only butyrate & ins/EPO-treated cells also express detectable embryonic βH1 globin (11/15, p<.005). Apoptosis was not increased in SCFA-treated eFLCs, with or without ins/EPO. Erythroid differentiation was analyzed by FACS quantitation of CD71 and TER119 co-immunostaining. At 72 hours, 21.5±3.5% of ins/EPO- vs. 65.5±9.9% of butyrate-only- and 77.5±4.0% of propionateonly-treated erythroid cells were highly differentiated (p<.005). Cytospins confirmed a marked increase in erythroid differentiation in SCFA-treated eFLCs.We also investigated SCFA-associated phosphorylation of signaling molecules, notably STAT5 and p38, which had been described in other models of erythropoiesis. Western blotting of protein extracts from cultured eFLCs showed phosphorylation of STAT5 in ins/EPO- and butyrate & ins/EPO-, but not in butyrate-only-, treated cells. However, p38 was constitutively phosphorylated in all of these conditions. Inhibitors of p38 kinase activity, SB 203580 (5–100 μM) or PD 169316 (5–300 μM), added to each condition throughout culture, prevented SCFA-mediated induction of embryonic globin gene expression in a concentration dependent manner. Erythroid differentiation was also blocked by p38 inhibition. Our data suggest that SCFAs augment both pancellular embryonic globin gene expression and erythroid differentiation. However, in our model, p38 phosphorylation and kinase activity are not unique to SCFA treatment in eFLCs; p38-associated erythroid differentiation, common to eFLCs in all culture conditions tested, may be an essential precursor for additional, as yet uncharacterized, molecular triggers for SCFA-mediated reactivation of embryonic/fetal globin gene expression in eFLCs.

Phase 1 Clinical Testing of HQK-1001, a Novel Oral Fetal Globin Gene Inducer

Development of non-cytotoxic, orally-active therapeutics, which induce fetal globin production, would be beneficial for treatment of beta-thalassemias and hemoglobinopathies. The short-chain fatty acids (SCFA) butyrate and phenylbutyrate are active fetal globin inducers, but their clinical application has been limited by inhibition of erythropoiesis and unfavorable PK profiles. HQK-1001 is a SCFA which stimulates fetal globin gene expression in reporter gene assays, erythroid progenitors, transgenic mice, and anemic baboons. HQK-1001 also stimulates human erythroid progenitor (BFU-E) proliferation by a mean of 30% above controls, and increased red blood cell counts in phlebotomized baboons. HQK-1001 has demonstrated a wide safety margin in pre-clinical toxicology testing, with no significant adverse effects in sub-chronic (6-month) studies and negative mutagenicity testing. Based on these pre-clinical data, a placebo-controlled, randomized, blinded, single-dose escalating Phase 1 trial was conducted to evaluate HQK-1001 at 4 dose levels ranging from 2 to 20 mg/kg in 32 normal volunteers. The drug was welltolerated, with no significant adverse events. Pharmacokinetic analyses showed low intersubject variability, a linear increase in Cmax and AUC with increasing dose, and T½ of 11 hours, indicating suitability for once-daily dosing. Administration under fed conditions demonstrated equivalent AUC to fasting conditions. In a subsequent 14-day Phase 1 dosing study in 41 normal subjects, the drug was also well-tolerated, with no significant adverse effects at the studied dose levels (5, 10 and 15 mg/kg). F-reticulocytes increased in subjects treated with 5 to 10 mg/kg doses. Absolute reticulocytes increased significantly above baseline (p <0.02 – 0.002) following 14-day dosing at all dose levels, while no significant change occurred in placebo controls. These first-in-human clinical studies demonstrating safety of HQK-1001 in 73 normal volunteer subjects and an improved pharmacologic profile over prior SCFAs indicate that HQK-1001 merits further clinical investigation in anemic patients with beta-globin gene disorders.

Delayed decline of γ‐globin expression in infant age associated with the presence of Gγ−158 (C→T) polymorphism

Persistent production of fetal hemoglobin (HbF) in adult has ameliorative effects on hemoglobinopathies and great efforts are currently made to achieve an exhaustive understanding of the molecular mechanisms of the switching in globin gene expression. One of the factors reported to be associated with the expression of fetal globin genes is the Xmn I Ggamma-158 polymorphism, although it is still unclear if it is involved in this mechanism either by itself or in strong linkage disequilibrium with other loci. Here, we report a novel effect of the Xmn I Ggamma-158 site that was found associated with a significant delayed decline of HbF production in infant age. The prolonged decay trend was enhanced when the Ggamma-158 C-->T substitution was co-inherited with a beta-thalassemic trait. Our observations reinforce the hypothesis that this region plays an important role in the expression of the gamma-globin genes and give new insights on the intriguing and still poorly understood mechanisms of globin gene expression switching.

Endogenous Elevations of Short Chain Fatty Acids (SCFAs) Can Up-Regulate Embryonic Globin Gene Expression.

Persistence of fetal hemoglobin can ameliorate adult beta (β)-globin gene disorders. Since SCFAs can affect embryonic and fetal globin gene expression, we examined their role during development. Murine globin gene expression, β-type (embryonic βH1, and epsilon-y, εY, and adult βmajor), and alpha (α)-type (embryonic zeta, ζ, >α, adult α), were compared between wildtype (wt) and transgenic mice, in which a key enzyme for SCFA metabolism, PCCA, had been knocked out (PCCA−/−, (Miyazaki et al, 2001). E10.5 PCCA−/− yolk sac (n= 9), showed increased α, βH1 and ζ gene expression, at respectively 2-, 2.6- and 1.6-fold relative to wt (n=13, p<.05), and εY gene expression, at 1.7-fold (p=0.07). The embryonic-to-adult globin gene switch was modestly delayed in yolk sacs from E12.5 PCCA−/− (n=9) vs. wt (n=4) and E 14.5 PCCA−/− (n=6) vs. wt (n=6). % embryonic β-type globin gene expression (% βH1 and εY of total β globin) was 77±6 PCCA−/− and 74±3 wt at E12.5, p=n.s., and 42±13 PCCA−/− and 21±3 wt at E14.5, p<.05; % emvbryonic α-type expression (% ζ of total α) was 32±3 PCCA−/−, 25±1wt at E12.5, p<.05 and 7±2 PCCA−/− and 4±1 wt at E14.5, p<.05). Embryonic globin gene expression in E 12.5 and 14.5 fetal livers was not different between PCCA−/− and wt embryos. Cultures of pooled E14.5 wt fetal liver cells (FLCs, n=4 separate experiments), however, suggested that embryonic globin genes can be activated in FLCs. The percent of total β-type globin gene expression that was embryonic after culture with butyrate (1mM) was 11.6±2.6%, with propionate (2.5 mM) was 3.6±0.2%, and insulin/erythropoietin or basal media was 0.03±0.03% and 0.42±0.26% respectively (p<.05 relative to SCFAs). Dose-response with propionate (n=2 seaparate experiments) suggest inadequate endogenous propionate levels for activation in PCCA −/− fetal liver, as % embryonic β-type globin gene expression rose above basal levels only at concentrations of 1 to 5 mM (2.5 mM maximal) but not at <0.6 mM. We conclude that endogenous SCFAs, at levels achievable in vivo can activate embryonic globin gene expression during development in the murine yolk-sac. However, higher levels than achievable endogenously currently are necessary to produce this effect in murine fetal livers.

Intermediaries of Branched-Chain Amino Acid Metabolism Can Increase Fetal Hemoglobin In Vitro and In Vivo.

Propionic acid and methylmalonic acid, which are intermediaries (BCAA-I) of branched-chain amino acid (leucine, isoleucine, valine) metabolism, can up-regulate HbF during definitive erythropoiesis [italics]in vitro[/italics] and [italics]in vivo [/italics](Little 1995). We tested ‘proximal' (prior to thiolester formation with Coenzyme A) BCAA-Is, which would be elevated in maple syrup urine disease (MSUD), and ‘distal', which would be elevated in Isovaleric Acidemia (IVA) Propionic Acidemia (PA) or Methylmalonic Acidemia (MMA), for their ability to simultaneously stimulate adult erythroid differentiation and embryonic globin gene expression in an adult erythroid cell line, murine erythroleukemia (MEL) cells after 96 hours in culture. Compounds and maximal concentrations (X) tested were: α-keto isocaproic acid, α−keto β−methylvaleric acid, and α−keto isovaleric acid, all at 50 mM, which are elevated in ‘proximal' MSUD; isobutyric acid (IB), isovaleric acid (IV), methylmalonic acid, all at 50 mM, and propionate (5mM), all of which are elevated in ‘distal' perturbations of BCAA metabolism, plus butyrate (2mM), which has been used pharmacologically to increase HbF (Atweh et al, 1999), and DMSO (adult erythroid differentiating agent in MELs). Concentration ranges were, relative to maximum, X, [X], [0.5X], [0.2X], [0.1X] and [0.01X]. RNA was analyzed by real-time PCR for murine adult alpha (α) and adult beta-major (βmaj) globin gene expression and embryonic α-type zeta (ζ) and β-type beta-H1 (βH1) and epsilon-y (εY). Only four BCAA-I compounds, plus DMSO, induced adult α−globin gene expression in MEL cells (by >100-fold, relative to uninduced). These compounds, but not DMSO, also augmented β−type embryonic globin gene expression ((βH1+εY)/(β−total))*100, as shown: propionate (5mM) 24%, butyrate (1 mM) 8% (2mM) 10%, IBA (25 mM) 5%, IVA (5 mM) 9% and (10mM) 13%. Samples from 10 individuals with ‘proximal' abnormalities in BCAA-I (n=4, median age 5.5 years) due to MSUD and ‘distal' abnormalities in BCAA-I (n=6, median age 6 years) due to IVA, PA, and MMA were examined by HPLC for % HbF. All patients were under good metabolic control at evaluation; mean Hgb (g/dL) was 13.1±0.9 in ‘proximal' (MSUD) patients, and 12.2±1.6 in patients with distal perturbations in BCAA metabolism (p=n.s.). HbF was 0.9±0.3% (one beta-thalassemia excluded) and 0.2±0.4%(p<.01) in proximal and distal BCAA-I defects, respectively. Gamma (γ)-globin gene composition was examined by HPLC-mass spectromety in 3 patients with proximal BCAA-I and 5 with distal BCAA-I. No γ chains were detectable in patients with MSUD, and only Gγ chains, at >5% of total β−type chains, were present in 3 of 5 patients with distal BCAA-I (p<.05). Our data show that, as predicted by MEL cells [italics]in vitro[/italics], a subset of metabolic intermediaries can up-regulate fetal globin gene expression in non-anemic patients with endogenous elevations in BCAA-I due to inborn metabolic errors. Gγ, which predominates during normal fetal life, may be most affected by these compounds.

DNA methyltransferase as targets for cancer therapy

Methylation of DNA at 5-position of cytosine, catalyzed by DNA methyltransferases, is the predominant epigenetic modification in mammals. Aberrations in methylation play a causal role in a variety of diseases, including cancer. Recent studies have established that like mutation, methylation-mediated gene silencing often leads to tumorigenesis. Paradoxically, genome-wide DNA hypomethylation may also play a causal role in carcinogenesis by inducing chromosomal instability and spurious gene expression. Since methylation does not alter DNA base sequence, much attention has been focused recently on developing small molecule inhibitors of DNA methyltransferases that can potentially be used as anticancer agents. Vidaza (5-azacytidine), marketed by Pharmion (Boulder, CO, USA), was the first DNA methyltransferase inhibitor approved by the U.S. Food and Drug Administration (FDA) for chemotherapy against myelodysplastic syndrome (MDS), a heterogeneous bone marrow disorder. Recently MGI Pharma Inc. (Bloomington, MN, USA) got FDA approval to market Dacogen (5-aza-2'-deoxycytidine, or decitabine) for treating MDS patients. These drugs were used earlier against certain anemias to induce expression of fetal globin genes. Interest in clinical trials of these drugs as anticancer agents has been renewed only recently because of reversal of methylation-mediated silencing of critical genes in cancer. Clinical trials have shown that both drugs have therapeutic potential against leukemia such as MDS, acute myeloid leukemia, chronic myelogenous leukemia and chronic myelomonocytic leukemia. In contrast, their effectiveness with solid tumors appears to be less promising, which challenges researchers to develop inhibitors with more efficacy and less toxicity. The major hindrance of their usage as anticancer agents is their instability in vivo as well as the toxicity secondary to their excessive incorporation into DNA, which causes cell cycle arrest. Gene expression profiling in cancer cells revealed that antineoplastic property of these drugs is mediated through both methylation-dependent and -independent pathways. Recently, we have shown that treatment of cancer cells with these cytidine analogues also induces proteasomal degradation of DNA methyltransferase 1, the ubiquitously expressed enzyme upregulated in almost all cancer cells. Development of related stable drugs that can facilitate gene activation in cancer cells by enhancing degradation of DNA methyltransferases without being incorporated into DNA would be ideal for chemotherapy. In this monograph we review historical perspective and recent advances on the molecular mechanisms of action and clinical applications of these DNA hypomethylating agents.

The Protein Methyltransferase Prmt5 Links Histone H4 Methylation to Dnmt3a-Dependent DNA Methylation and Transcriptional Silencing of the Fetal Globin Genes.

Elevated levels of fetal hemoglobin ameliorate the severity of sickle cell disease and β-thalassemia, fuelling interest in the mechanisms underpinning the fetal (γ) to adult (β) switch in β-like globin chain subtype. We have previously identified a tripartite protein complex consisting of p22 NF-E4, CP2 and ALY, collectively known as the stage selector protein (SSP) that binds to the proximal γ-promoters, and fosters the preferential expression of the γ-genes in fetal erythroid cells. We have also identified a 14 kDa isoform of the NF-E4 protein that plays a role in γ-gene repression by binding CP2 and sequestering it away from the γ-promoter, resulting in disassembly of the activator SSP complex. Despite the loss of SSP binding, we showed by chromatin immunoprecipitation (ChIP) analysis that p22 NF-E4 remained bound to the γ-promoter in this context. To determine whether p22 NF-E4 could serve as the cornerstone for assembly of a larger repressor complex in this setting, we analyzed the proteins that were co-immunoprecipitated with p22 NF-E4 from K562 cell extract by mass spectrometry. One protein identified was PRMT5, an arginine methyltransferase that has been linked to gene silencing by establishing repressive arginine methyl marks including symmetrical dimethylation of arginine 3 on histone H4 (H4R3me2s). We confirmed the interaction between the two endogenous proteins by direct co-immunoprecipitation, and co-localized p22 NF-E4 and PRMT5 to the γ-globin gene promoters by ChIP. In vitro methylation studies using PRMT5 co-immunoprecipitated with p22 NF-E4 confirmed that histone H4 was the major substrate of the enzyme complex in K562 cells. In accord with this, we demonstrated a marked increase in H4R3me2s at the γ-promoter by ChIP in the setting of enforced expression of wild type PRMT5, accompanied by silencing of γ-gene expression.To determine whether additional factors cooperated with PRMT5 in γ-gene repression, we interrogated PRMT5 containing immunoprecipitates with antisera to a range of candidate proteins. We isolated a large repressor complex containing members of the NuRD complex and the methyl domain-binding proteins (MBD2 and MDB3). We also isolated the DNA methyltransferase 3a (Dnmt3a), a finding of considerable interest in view of the links between γ-gene silencing and methylation of CpG dinucleotides. Using bisulfite DNA sequencing, we demonstrated in K562 cells in which PRMT5 expression had been enforced, an increase in the density of methylated CpG dinucleotides clustered around the transcriptional start site. In contrast, cells transfected with an expression vector stably expressing hairpin short interfering RNAs, which induced a 90% reduction in PRMT5 protein levels, showed complete abrogation of DNA methylation at these CpGs, coincident with a five-fold induction of γ-gene expression. ChIP analysis of the human β-globin locus in βYAC transgenic mice revealed a marked enhancement of H4R3me2s at the γ-promoters in adult erythropoietic cells, and absence of this repressive mark at the γ-promoter in the E12.5 fetal liver. This data establishes a direct link between the PRMT5-induced repressive histone mark H4R3me2s and DNA methylation in developmental regulation of γ-gene expression. It also provides impetus for new strategies aimed at reactivation of fetal globin gene expression.

5-Azacytidine Induction of Human γ-Globin Gene Expression: Experimental Evaluation of Current Models.

5-Azacytidine (5-Aza) was demonstrated to be a potent inducer of human fetal globin gene expression more than 20 years ago. More recently, 5-Aza-2-deoxycytidine has been shown to have similar properties. Since the 1980's there have been two predominant hypotheses to explain the action of these agents. The first is based on the observation that these, and several other active inducing agents, are cytotoxic to differentiating erythroid cells and that drug treatment alters the kinetics of erythroid differentiation. This has been proposed to result in prolonged expression of the γ-globin genes which are normally expressed only early in differentiation. The second is based on the observation that both agents are DNA methyltransferase inhibitors and are presumed to cause demethylation of cellular DNA including the γ-globin gene promoters leading to activation of the genes. These two models lead to specific predictions that we have evaluated using an in vitro erythroid differentiation system. In this system, human adult CD34+ cells are cultured in SCF, Flt3 ligand and IL-3 for 7 days and then switched to Epo for 14 days. This results in an exponential expansion of erythroid cells. As has been described for normal human differentiation, these cells express small amounts of γ-globin mRNA early in differentiation followed by a much larger amount of β-globin mRNA. HPLC at the end of the culture period shows 99% HbA and 1% HbF. Treatment of cultures on a daily basis with 5-Aza starting on day 10 results in dose dependent increases in γ-globin mRNA, Gγ- and Aγ-chain production and HbF. The cytotoxicity model predicts that γ-globin expression will be prolonged to later in differentiation - and this is seen. However, a daily 5-Aza dose of 300 nM, which produces ~80% of the maximal response in γ-globin mRNA and HbF, has no effect on cell growth or differentiation kinetics. This argues against the toxicity model. We next examined the effect of 5-Aza on γ-globin promoter methylation using the bisulfite method. We studied CpGs at −344, −252, −162, −53, −50, +6, +19 and +50 relative to the start site. For untreated controls, all of the sites are nearly 100% methylated at day 1. By day 3, the upstream sites become ~50% methylated except the −53 CpG which was <20%. This pattern persisted at day 10. By day 14 the promoters had become largely remethylated. For cells treated with 5-Aza starting on day 10, there was no change in the levels of methylation seen on days 1,3 and 10, but at day 14 the low levels of upstream methylation persisted - just as γ-globin expression does. However, in both treated and untreated cells, down-stream CpG sites were highly methylated at all time points. This suggests that γ promoter demethylation may be due to a local and not a generalized effect of 5-Aza on cellular DNA methylation. We also made two unexpected observations. At a 300nM dose of 5-Aza, γ-globin mRNA is ~doubled while β-globin mRNA levels are ~halved - indicating that 5-Aza not only induces γ-globin expression also suppresses β-globin. Also despite only a doubling in γ-globin mRNA, there was an ~50-fold increase in HbF, from ~1% to more than 50%, while total per cell Hb levels were unchanged. Neither of these results are easily explained by current models of γ-globin gene induction. Our results raise the possibility that mechanisms beyond cytotoxicity and generalized DNA demethylation may be responsible for pharmacologic induction of γ-globin mRNA and HbF.

Short-chain fatty acids induce γ-globin gene expression by displacement of a HDAC3-NCoR repressor complex

AbstractHigh-level induction of fetal (γ) globin gene expression for therapy of β-hemoglobinopathies likely requires local chromatin modification and dissociation of repressor complexes for γ-globin promoter activation. A novel γ-globin–inducing short-chain fatty acid derivative (SCFAD), RB7, which was identified through computational modeling, produced a 6-fold induction in a reporter assay that detects only strong inducers of the γ-globin gene promoter and in cultured human erythroid progenitors. To elucidate the molecular mechanisms used by high-potency SCFADs, chromatin immunoprecipitation (ChIP) assays performed at the human γ- and β-globin gene promoters in GM979 cells and in erythroid progenitors demonstrate that RB7 and butyrate induce dissociation of HDAC3 (but not HDAC1 or HDAC2) and its adaptor protein NCoR, specifically from the γ-globin gene promoter. A coincident and proportional recruitment of RNA polymerase II to the γ-globin gene promoter was observed with exposure to these γ-globin inducers. Knockdown of HDAC3 by siRNA induced transcription of the γ-globin gene promoter, demonstrating that displacement of HDAC3 from the γ-globin gene promoter by the SCFAD is sufficient to induce γ-globin gene expression. These studies demonstrate new dynamic alterations in transcriptional regulatory complexes associated with SCFAD-induced activation of the γ-globin gene and provide a specific molecular target for potential therapeutic intervention.

Solution Structure and DNA-binding Mode of the Matrix Attachment Region-binding Domain of the Transcription Factor SATB1 That Regulates the T-cell Maturation

SATB1 is a transcriptional regulator controlling the gene expression that is essential in the maturation of the immune T-cell. SATB1 binds to the nuclear matrix attachment regions of DNA, where it recruits histone deacetylase and represses transcription through a local chromatin remodeling. Here we determined the solution structure of the matrix attachment region-binding domain, possessing similarity to the CUT DNA-binding domain, of human SATB1 by NMR spectroscopy. The structure consists of five alpha-helices, in which the N-terminal four are arranged similarly to the four-helix structure of the CUT domain of hepatocyte nuclear factor 6alpha. By an NMR chemical shift perturbation analysis and by surface plasmon resonance analyses of SATB1 mutant proteins, an interface for DNA binding was revealed to be located at the third helix and the surrounding regions. Surface plasmon resonance experiments using groove-specific binding drugs and methylated DNAs indicated that the domain recognizes DNA from the major groove side. These observations suggested that SATB1 possesses a DNA-binding mode similar to that of the POU-specific DNA-binding domain, which is known to share structural similarity to the four-helix CUT domain.