HbF induction3rd Pan-European Conference on
Haemoglobinopathies & Rare AnaemiasLimassol, 24 – 26 October 2012
Mehran KarimiShiraz University of Medical Sciences,
Shiraz, Iran
● HbF structure
● Regulation of HbF production
● HbF inducers
● Conclusions
Agenda
● Hemoglobin F is the major Hb of the fetus and the newborn infant
● HbF levels reach the adult levels of <1% of the total Hb from about 18–24 months of age and its replaced by adult Hb (HbA)
● HbF is able to bind oxygen with greater affinity than HbA
Fetal Hb
hemoglobin switching during development
● It consists of two α-globin changes and two γ-globin changes (α2γ2)
● Alpha globin gene is located on chromosome 16 and gamma globin gene is located on chromosome 11 as a part of the human β-globin locus
● The human β-globin locus consisting of about 100 kb and is composed of five functional genes: ɛ, Gγ, Aγ, δ, and β
HbF structure
Nat Rev Genet, 2001
● The two α-chains composed of 142 and two γ-chains, composed of 147-amino-acid residues
● γ-chains are the results of two γ-globin genes which differ by one amino acid at position 136, having either alanine (Aγ) or glycine (Gγ)
● In adult erythrocytes, HbF is composed by about 40% Gγ and 60% Aγ, while in the newborn by about 70% Gγ and 30% Aγ
● There is no functional deference between two types of HbF
HbF structure
Br J Haematol, 2008
● Adult production of Hb F is regulated by multiple genetic loci including (5 QTL):
The HMIP locus at chromosome 6q23 The bcl11A locus on chromosome 2 Xp22.2 region of the X chromosome 8q region on chromosome 8 11p15 region on chromosome 11 (including the
Xmn1- HBG1 site in the β-globin locus)
Regulation of HbF production
Human Molecular Genetics, 2009
HbF switching
Molecular regulation of the fetal-to-adult hemoglobin switch
● Experimental and clinical data revealed that increased HbF levels can ameliorate the severity of sickle cell disease and β-thalassemia
● So there has been a longstanding interest in developing therapeutic approaches for inducing HbF
● The increase in HbF in response to HbF inducers varies among patients with β-thalassemia due to individual genetic determinants
HbF induction
Ann Hematol, 2009
Fetal hemoglobin levels vs. morbidity in TI
R2=0.825 R2=0.933
Variable beta (95% CI) p-value
Fetal hemoglobin level (%) -0.050 (-0.059 to –0.042) <0.001
Age (years) 0.000 (-0.010 to 0.009) 0.937
Splenectomy -0.026 (-0.345 to 0.292) 0.868
Total hemoglobin level (g/l) -0.288 (-0.522 to -0.055) 0.017
NRBC count (×106/l) 0.002 (0.002 to 0.003) <0.001
GDF-15 level (pg/ml) 0.000 (0.000 to 0.000) 0.695
Serum ferritin level (µg/l) 0.000 (0.000 to 0.000) 0.136
NTBI level (µmol/l) 0.005 (-0.073 to 0.083) 0.121
Variable beta (95% CI) p-value
Log fetal hemoglobin level (%) -3.401 (-3.915 to -2.886) <0.001
Age (years) -0.003 (-0.001 to 0.006) 0.560
Splenectomy 0.039 (-0.266 to 0.343) 0.800
Total hemoglobin level (g/l) -0.053 (-0.255 to 0.148) 0.596
NRBC count (×106/l) 0.000 (0.000 to 0.000) 0.850
GDF-15 level (pg/ml) 0.000 (0.000 to 0.000) 0.261
Serum ferritin level (µg/l) 0.000 (0.000 to 0.000) 0.516
NTBI level (µmol/l) 0.012 (-0.061 to 0.085) 0.747
Musallam KM, Sankaran VG, Cappellini MD, et al. Blood. 2012;119:364-7. NRBC = nucleated red blood cell; NTBI = non-transferrin-bound iron.
● HbF inducers should have some effect on molecule targets to induce HbF including:
BCL11A (B-cell lymphoma/leukemia 11A) EKLF1 (Erythroid Kruppel-like factor 1) cMYB (transcription factor) SOX6 (transcription factor) miRNAs 15a and 16-1 Histone deacetylase 1 and 2 (HDAC1/2)
HbF inducers
Molecular targets for HbF induction
Hematology, 2011
● The HbF inducers can be grouped in several classes based on their chemical structures and mechanisms of action including
Hypomethylating agents (eg; 5-azacytidine and decitibine)
Short Chain Fatty acids: Histone deacetylase inhibitors (eg; sodium butyrate)
Chemotherapeutic agents (eg; hydroxyurea) Stem cell factor and erythropoietin Others
HbF inducers
● DNA methylation is one of gene silencing mechanisms
● Genes with heavily methylated promoter regions cannot be transcribed and are effectively silenced
● Analysis of γ- and β-globin promoter methylation showed that, in general, promoter methylation and globin gene expression are inversely related
● In fetal erythroid progenitors, the γ-globin promoter is hypomethylated and becomes progressively more methylated as erythroid differentiation progresses and is hypermethylated in the mature erythroid progeny
Hypomethylating agents
Exp Hematol, 2007
● 5-Azacytidine (5-Aza) was the first agent able to reactivate HbF synthesis in humans used for the treatment of β-thalassemia and sickle cell disease
● Although the mechanism through which 5-Aza stimulates HbF synthesis is unclear but one hypothesis is based on the capacity of 5-Aza to inhibit DNA methyltransferase enzymes, resulting in inhibition of DNA methylation
● Increased in Hb levels was seen 2.5 gr/dl (1.5-4)
Hypomethylating agents
Blood, 2008Br J Haematol, 2004
Hypomethylating agents
Blood, 2008Br J Haematol, 2004
• Decitabine (5-aza-2́-deoxycytidine): demethylate and reactivate expression of the gamma globin gene
• Dosage:0.2 mg/kg /sc two times per week
• It can increase hemoglobin levels about 1.6 gr/dl
● Gene expression is controlled by alterations in chromatin structure produced by acetylation and deacetylation of histone tails
● Histone deacetylase (HDAC) enzymes determine deacetylation of histone tails, causing chromatin condensation and repression of transcription
● Many experimental evidences have indicated that inhibition of the activity of HDAC causes an increased HbF synthesis
Hematology, 2004
Histone deacetylase inhibitors
● Several HDAC inhibitors, such as sodium butyrate, adipicin, scriptaid and trichostatin A (TSA), have been shown to induce HbF synthesis in vitro
● Some studies have been carried out using valproic acid, a compound related to short fatty acid derivatives, reported increased HbF levels
Histone deacetylase inhibitors
● Butyrate was shown to promote cell differentiation and to enhance globin gene expression to cause higher HbF levels
● The mechanism through which butyrate stimulates HbF synthesis remains unclear but since butyrate is an inhibitor of histone deacetylase, it was proposed that butyrate increases HbF levels by enhancing the transcription rate of the γ-globin gene via changes in histone acetylation at the levels of critical promoter regions
Butyrate
Blood, 1998
● Studies showed that not all sickle cell disease patients responded to butyrate treatment: Particularly, those exhibiting >2% HbF baseline levels responded to treatment, while those with lower HbF levels were resistant to this treatment
● Butyrate was also assayed in β-thalassemia patients
● Na phenyl butyrate: increase Hb levels 2gr/dl(1-2.5)
● Arginine butyrate ± EPO: increase Hb levels 2.7gr/dl (1-5). It is one of the most effective compound
● New generations of SCFADs: Na 2,2-dimethylbutyrate and α-methylhydrocinnamate.
Butyrate
Blood, 1999J Clin Invest, 2007
● Hydroxyurea is a well-known drug used for treatment of myeloproliferative disorders
● Initial studies carried out in 1984 in sickle cell anemia patients showed that HU could stimulate HbF production
● As a consequence placebo-controlled study, HU was approved by the FDA for the treatment of sickle cell disease patients
● Hydroxyurea response in β-thalassemia has been extensively studied and, according to the majority of the reports, its administration was safe and able to significantly decrease transfusion requirements
Hydroxyurea
JAMA, 2003 Blood, 1997
● More recent studies have confirmed in various types of β-thalassemia major and β-thalassemia intermedia patients a significant clinical benefit following hydroxyurea therapy, with elimination of transfusion requirements in a significant proportion of transfusion-dependent β-thalassemic children
● Hydroxyurea seems to have a good role in the treatment of thalassemia especially TI, but resistance to this drug is observed in some cases
● It can increase Hb levels 0.6-2.7gr/dl
Hydroxyurea
Blood, 2003,Int.J.Hematology 2012
● Because of pronounced effects of SCF on erythropoiesis, it seemed important to evaluate a possible effect of this cytokine on HbF synthesis
● The majority of these studies have been carried out by investigating in vitro the effect of SCF on HbF synthesis in cultures of adult erythroid cells
● The effect of SCF was also investigated in cultures of β-thalassemic erythroid cells
● It induces and expansion of erythropoiesis and the reactivation of HbF synthesis
● There is strong evidence of synergy effect of SCF with Epo
Stem cell factor (SCF)
Blood, 2008
• It promotes red blood cell survival and increased Hb levels.
• It can use alone or combine by hydroxyurea
• EPO, Darbopoietin: increase Hb levels 1.6-2gr/dl
• EPO is expensive
Erythropoietin (EPO)
Blood, 2008
● Thalidomide, Lenalidomide and pomalidomide are other components that induces increased expression of the γ-globin gene and HbF production
● when combined with hydroxyurea, pomalidomide and, to a lesser extent, lenalidomide were found to have synergistic effects on HbF up-regulation
● Rapamycin (immunosuppressant drug) and Resveratrol ( antioxidant) both can increase Hb F production, natural products, not associated with cytotoxicity or cell growth inhibition
Other compounds
J Clin Invest, 2008 and blood review 2012
● An ideal Hb F inducers should have three characteristics:
1. orally active and tolerable
2. not inhibit erythropoiesis
3. Not be mutagenic
● The experimental and clinical observations support the idea that agents enhancing HbF synthesis represent a rational approach for the treatment of β-thalassemia and it is expected to be critical for developing countries with low economic status
Conclusions (1)
● Multiple modalities approach to induce Hb F have relatively satisfactory with different variation response
● However it is important to note that therapy of hemoglobinopathies based on the administration of agents stimulating HbF synthesis cannot be curative and needs a chronic administration
● Therefore, better agents that more effective targeted therapies to induce higher levels of HbF are needed to develop in future
Conclusions (2)