HbF induction 3rd Pan-European Conference on Haemoglobinopathies & Rare Anaemias

1. HbF induction 3rd Pan-European Conference on Haemoglobinopathies & Rare Anaemias Limassol, 24 – 26 October 2012 Mehran Karimi Shiraz University of Medical Sciences, Shiraz, Iran

2. Agenda • HbF structure • Regulation of HbF production • HbF inducers • Conclusions

3. 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

4. hemoglobin switching during development

5. 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

6. 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

7. 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

8. HbF switching

9. Molecular regulation of the fetal-to-adult hemoglobin switch

10. 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

11. Fetal hemoglobin levels vs. morbidity in TI R2=0.933 R2=0.825 Musallam KM, Sankaran VG, Cappellini MD, et al. Blood. 2012;119:364-7. NRBC = nucleated red blood cell; NTBI = non-transferrin-bound iron.

12. 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)

13. Molecular targets for HbF induction Hematology, 2011

14. 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

15. 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

16. 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

17. Hypomethylating agents • Decitabine (5-aza-2́-deoxycytidine): demethylate and reactivate expression of the gamma globingene • 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

18. 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

19. 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

20. 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

21. 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

22. 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

23. 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 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 Blood, 2003,Int.J.Hematology 2012

24. 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

25. 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

26. 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

27. Conclusions (1) • An ideal Hb F inducers should have three characteristics: • orally active and tolerable • not inhibit erythropoiesis • 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

28. 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

29. THANKS FOR YOUR ATTENTION karimim@sums.ac.ir