HbF inducers

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HbF induction
3rd Pan-European Conference on
Haemoglobinopathies & Rare Anaemias
Limassol, 24 – 26 October 2012
Mehran Karimi
Shiraz University of Medical Sciences,
Shiraz, Iran
Agenda
● HbF structure
● Regulation of HbF production
● HbF inducers
● Conclusions
Fetal Hb
● 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
hemoglobin switching during development
HbF structure
● 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
Nat Rev Genet, 2001
HbF structure
● 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
Br J Haematol, 2008
Regulation of HbF production
● 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)
Human Molecular Genetics, 2009
HbF switching
Molecular regulation of the fetal-to-adult hemoglobin
switch
HbF induction
● 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
Ann Hematol, 2009
Fetal hemoglobin levels vs.
morbidity in TI
R2=0.933
R2=0.825
Variable
beta (95% CI)
p-value
Variable
beta (95% CI)
p-value
Fetal hemoglobin level (%)
-0.050 (-0.059 to –0.042)
<0.001
Log fetal hemoglobin level (%)
-3.401 (-3.915 to -2.886)
<0.001
Age (years)
0.000 (-0.010 to 0.009)
0.937
Age (years)
-0.003 (-0.001 to 0.006)
0.560
Splenectomy
-0.026 (-0.345 to 0.292)
0.868
Splenectomy
0.039 (-0.266 to 0.343)
0.800
Total hemoglobin level (g/l)
-0.288 (-0.522 to -0.055)
0.017
Total hemoglobin level (g/l)
-0.053 (-0.255 to 0.148)
0.596
NRBC count (×106/l)
0.002 (0.002 to 0.003)
<0.001
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)
0.695
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)
0.136
Serum ferritin level (µg/l)
0.000 (0.000 to 0.000)
0.516
NTBI level (µmol/l)
0.005 (-0.073 to 0.083)
0.121
NTBI level (µmol/l)
0.012 (-0.061 to 0.085)
0.747
NRBC = nucleated red blood cell; NTBI = non-transferrin-bound iron.
Musallam KM, Sankaran VG, Cappellini MD, et al. Blood. 2012;119:364-7.
HbF inducers
● 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)
Molecular targets for HbF
induction
Hematology, 2011
HbF inducers
● 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
Hypomethylating agents
● 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
Exp Hematol, 2007
Hypomethylating agents
● 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)
Blood, 2008 Br J Haematol, 2004
Hypomethylating agents
• 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
Blood, 2008 Br J Haematol, 2004
Histone deacetylase
inhibitors
● 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
Butyrate
● 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
Blood, 1998
Butyrate
● 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.
Blood, 1999 J Clin Invest, 2007
Hydroxyurea
● 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
JAMA, 2003 Blood, 1997
Hydroxyurea
● 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 transfusiondependent 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
Blood, 2003,Int.J.Hematology 2012
Stem cell factor (SCF)
● 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
Blood, 2008
Erythropoietin (EPO)
• 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
Blood, 2008
Other compounds
● 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
J Clin Invest, 2008 and blood review 2012
Conclusions (1)
● 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 (2)
● 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
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