Targeted Therapeutic Strategies for Fetal HemoglobinInduction

Vijay G. Sankaran1-3

1Children’s Hospital Boston, Harvard Medical School, Boston, MA; 2Whitehead Institute for Biomedical

Research, Cambridge, MA; and 3Broad Institute, Cambridge, MA

Increased levels of fetal hemoglobin (HbF) can ameliorate the severity of the �-hemoglobin disorders, sickle celldisease (SCD) and �-thalassemia, which are major sources of morbidity and mortality worldwide. As a result, there hasbeen a longstanding interest in developing therapeutic approaches for inducing HbF. For more than 3 decades, themajority of HbF inducers developed were based on empiric observations and have had limited success. Recently,human genetic approaches have provided insight into previously unappreciated regulators of the fetal-to-adulthemoglobin switch and HbF silencing, revealing molecular targets to induce HbF. This article reviews thesedevelopments and discusses how molecules including BCL11A, KLF1, MYB, SOX6, miRNAs 15a and 16-1, and histonedeacetylase 1 and 2 (HDAC1/2) could be important targets for HbF induction in humans. The current understanding ofhow these molecules function and the benefits and drawbacks of each of these potential therapeutic targets are alsoexamined. The identification of these regulators of HbF expression is extremely promising and suggests that rationallydesigned approaches targeting the very mechanisms mediating this switching process could lead to better, less toxic,and more effective strategies for HbF induction.

IntroductionThe �-hemoglobin disorders, sickle cell disease (SCD) and �-thalassemia, are major causes of morbidity and mortality world-wide,1 as the most common genetic disorders in the world. With theepidemiological transition that many developing countries areundergoing, resulting in significant decreases in mortality frominfectious and nutritional causes, there will likely be an increase inthe prevalence of these diseases in the ensuing years. In SCD, asingle base substitution causes a missense mutation of a valine forglutamic acid at amino acid 6 of the �-globin protein chain, whichleads to a propensity of the sickle hemoglobin to polymerize. Thisresults in deformation of RBCs containing this hemoglobin, whichcan block small blood vessels, leading to impaired oxygen deliveryto tissues. This can cause significant pain crises, respiratorycomplications, and organ damage. In �-thalassemia, insufficientproduction of the �-globin molecule results in an excess of unpaired�-globin chains that can precipitate within erythroid precursors. Theprecipitation of these free �-globin chains impairs the maturation ofthese precursors, leading to their death and thereby causing ineffec-tive production of RBCs. Significant anemia results and theconsequent expansion of erythroid precursors can lead to secondaryproblems in bones and other organs.

Whereas the only cures for these disorders involve BM transplanta-tion2 and gene or cellular therapy,3 there are numerous challengesassociated with these treatments and therefore they have significantlimitations for widespread use, particularly in the developingworld.4 These curative therapies still remain largely experimental,although important advances in these approaches are being made.5

Currently, the major treatment for SCD and �-thalassemia involvessymptomatic care and transfusion of RBCs as clinically necessary.However, the use of regular transfusion therapy can lead tosignificant problems, particularly iron overload, which can evenoccur when the strictest iron-chelation regimens are used.6

Treating the ��-hemoglobinopathies through HbFinductionA series of important natural observations demonstrated that theseverity of SCD and �-thalassemia could be ameliorated viaincreased production of fetal hemoglobin (HbF), which is composedof the �-like globin chain, �-globin, and the adult �-globinmolecule. In normal infants, the predominant �-like globin beforebirth is �-globin, and there is a switch to adult �-globin expressionin the months after birth, a process known as the fetal-to-adulthemoglobin switch (Figure 1).7,8 Children with SCD were noted tobe asymptomatic until after infancy, which was postulated to beattributable to elevated HbF levels.9 This notion was substantiatedby observations of patients with compound heterozygosity for SCDand hereditary persistence of HbF mutations, who were largelyasymptomatic.10 Moreover, certain populations of patients withSCD, such as those from parts of Saudi Arabia and India, hadunusually high levels of HbF associated with a less symptomaticclinical course.10-12 These observations were confirmed with largerepidemiological studies showing that increased HbF levels cansignificantly ameliorate the clinical severity of SCD.13-15 Similarobservations were made in patients with �-thalassemia. Clinicalobservations in rare �-thalassemia patients with highly elevatedlevels of HbF showed that these increased levels resulted in a milderclinical course, and infants with �-thalassemia only begin to showsymptoms after the expression of HbF declines in the months afterbirth.10,16 Larger epidemiological studies of thalassemia populationshave confirmed these findings.17-19

The human globin genes were among the first genes to be cloned,and soon thereafter the structure of the entire �-globin cluster wasmapped and sequenced. Around this time, it was observed that thesilenced �-globin genes in the �-globin cluster underwent DNAmethylation in adult erythroid cells, whereas this was not the case inerythroid cells from embryonic life.4 Given these observations,

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preclinical trials in primates of the DNA-hypomethylating agent5-azacytidine were subsequently performed and demonstrated suc-cess at inducing HbF.4,20 This approach was then tested in patientswith �-thalassemia and SCD.21 However, there was concern overthe long-term consequences of a potentially mutagenic compoundsuch as 5-azacytidine. Around this time, it was suggested that the

effect of 5-azacytidine on HbF induction might relate, at least inpart, to its ability to act as an S-phase cell-cycle inhibitor, rather thanthrough alteration of DNA methylation at the �-globin genepromoters.4 Studies in primates demonstrated that numerousS-phase inhibitors, including hydroxyurea, were all highly effectiveas inducers of HbF. This then led to small clinical trials of

Figure 1. Molecular regulation of the fetal-to-adult hemoglobin switch. The human �-globin gene locus, which is located on chromosome 11, has aseries of 5 genes that are developmentally expressed at distinct stages of human ontogeny (panel A bottom). The �-globin gene is restricted to thetransiently expressed embryonic primitive lineage of RBC produced in the first several weeks of gestation. The �-globin genes encode the genes thatuniquely form HbF, which is the predominant hemoglobin during the course of gestation. There is a switch to expression of the adult �-globin genearound the time of birth (B). B-cell lymphoma/leukemia 11A (BCL11A) and its protein partners, including GATA binding protein 1 (GATA1), zinc fingerprotein multi-type 1 (ZFPM1 or FOG1), and the nucleosome remodeling and deacetylase (NuRD) complex, bind to sequences within the globin locus(panel A tan ovals) and repress the expression of the �-globin genes. Kruppel-like factor 1 (KLF1) interacts with this process by positively regulating theexpression of BCL11A (green arrow in panel A) and also by directly binding to and promoting transcription of the adult �-globin gene. It is unknownwhether KLF1 affects HbF expression through more global effects on erythropoiesis (panel A top), which may also be targeted by other factorsassociated with HbF (eg, MYB). HSs indicates DNase I–hypersensitive sites; LCR, locus control region. (Reprinted with permission from Sankaranand Nathan.7)

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hydroxyurea, which had the safest side-effect profile, in SCDpatients in whom a robust HbF inductive response was demon-strated.22 The use of hydroxyurea was then examined in largerclinical trials that led to its US Food and Drug Administrationapproval for use in adult patients with SCD.23,24 Unfortunately,hydroxyurea is only effective to some extent in a subset of patientswith SCD. There has also been limited success with the use ofhydroxyurea in �-thalassemia, which may be attributable to therequirement for much more HbF to achieve globin chain balance inthis disease and to the inability to escalate hydroxyurea dosing as aresult of cytopenias.

In the mid-1980s, it was shown that infants of diabetic mothers havea delayed fetal-to-adult hemoglobin switch.25,26 Because it wasknown that hydroxybutyrate is elevated in these diabetic mothers, aseries of experiments was performed to determine whether butyrateor other similar short-chain fatty acids may be effective as inducersof HbF. Whereas initial trials showed promise,27,28 these therapieswere not widely effective in larger clinical trials.4,29 It should benoted nonetheless that a subset of patients did show responses inthese trials.4,29 However, additional concerns were raised overpossible toxic effects and the difficulty in delivery of thesemedications, which limited further clinical trials and drug-development efforts. It has been suggested that these short-chainfatty acids are likely to induce HbF through the inhibition of histonedeacetylases (HDACs).30 A variety of HDAC inhibitors can bepotent inducers of HbF in vitro, and so it is possible that thesecompounds may show clinical efficacy in vivo.31,32

Identifying molecular targets for HbF inductionMolecules that regulate the fetal-to-adult hemoglobin switch andhelp to maintain HbF silencing in humans have been long soughtgiven the possibility of using this information to develop targetedtherapeutic approaches for HbF induction.7,32 Whereas numerousregulators of erythropoiesis and globin gene regulation had beenidentified, none of the previously examined molecules appeared tobe specific regulators of the fetal-to-adult switch. Recent insight intothis process has come from studies using human genetic approaches.By taking advantage of natural variation in HbF levels in humans,genome-wide association (GWA) studies were performed to findcommon genetic variants associated with variations in HbF levels.The initial GWA studies of HbF were performed in nonanemicindividuals. Two separate studies were performed around the sametime and showed association of variants at 3 major genomic lociwith HbF levels.33,34 This included the �-globin locus on chromo-some 11, a region between the genes HBS1L and MYB onchromosome 6, and a region within the BCL11A gene on chromo-some 2. These findings were replicated in patients with SCD and�-thalassemia, and the ameliorating effect of these loci upon thesediseases was also demonstrated in multiple other studies.18,19,33,35

Regulation of HbF expression by BCL11A and SOX6The role of BCL11A was described previously for B-lymphocyteproduction, but no studies had explored whether this gene had a rolein erythropoiesis. As a result, the role of this gene in human globingene regulation and erythropoiesis was assessed soon after theinitial association studies.36 BCL11A protein levels appeared to becorrelated with the developmental stage of expression, such thatprimitive and fetal liver erythroid cells that expressed high levels of�-globin had low or absent expression of the full-length forms ofBCL11A, whereas shorter variant forms of the protein were noted inthese cells. This result suggested that this gene product may act as a

repressor of the �-globin genes. To directly test this, knock-down ofBCL11A using siRNA approaches was performed in primary adulterythroid progenitors, and �-globin expression could be induced.The degree of �-globin induction appeared to be linearly related tothe extent of knock-down of BCL11A. Interestingly, major perturba-tions in erythropoiesis did not appear to occur despite the robust�-globin induction seen. Follow-up studies have demonstratedsimilar results when knocking down the expression of BCL11Ausing siRNAs.37-39 It was also shown that BCL11A directly interactswith chromatin at the human �-globin locus in primary erythroidcells and that it appeared to act as part of a complex with thetranscription factor GATA-1 and the NuRD chromatin remodelingand repressor complex (Figure 1).36 The NuRD complex includesHDAC1 and HDAC2, which have been suggested to be the criticalHDACs necessary for HbF silencing (Table 1).40

Whereas hemoglobin switching in mouse models, even thoseharboring a human �-globin locus transgene, appears to divergefrom the normal ontogeny of globin expression seen in humans, anevolutionarily conserved role of BCL11A in globin gene silencingand switching was demonstrated in mice.41 Specifically, transgenicmice with the human �-globin locus express the �-globin gene in amanner similar to the endogenous mouse embryonic globin genes,although there is some variation in the expression between theseglobin genes.42 Mice lacking BCL11A appear to have normalerythropoiesis, but fail to silence the embryonic globin genes indefinitive erythroid cells, allowing persistent expression of �-globinwhen the intact human �-globin locus is present.41 This studyreinforces the importance of BCL11A as a critical mediator ofglobin switching in mammals.

The exact mechanism by which BCL11A silences �-globin expres-sion remains unclear. A recent study suggests that this may bemediated through both interactions with transcription factors suchas SOX6 that bind chromatin at the proximal �-globin promotersand through long-range interactions with a variety of regionsthroughout the �-globin gene cluster.43 When BCL11A is absent,the conformation of the �-globin locus changes such that theupstream enhancer known as the locus control region is juxtaposedwith the transcriptionally activated �-globin genes. A similarphenomenon occurs when cells are treated with HDAC inhibitorsthat induce �-globin gene expression.40 However, it is unclearwhether these conformational alterations are directly mediated byBCL11A or if they occur secondary to the HbF-inductive effect ofBCL11A (or HDAC) inhibition. Nonetheless, these findings stronglysupport the notion that BCL11A has a direct role in silencing�-globin expression within the �-globin locus. Therefore, BCL11Ais likely to be an important therapeutic target. The fact that it caninduce HbF without resulting in a major perturbation in erythropoi-esis is very promising, although it is known to have importanteffects in other lineages such as B lymphocytes, stressing theimportance of in vivo modeling and analysis as part of ongoingefforts to target BCL11A for HbF induction (Table 1). Strategiesaimed at targeting BCL11A may rely on altering its expression levelor activity. SOX6 may also be a potentially promising target forHbF induction,43 although it is known to be necessary for normalerythropoiesis (Table 1).44 Interestingly, however, a patient has beenreported with a heterozygous disruption of SOX6 who did not haveelevated HbF levels, suggesting that the SOX6 gene may have amechanism for dosage compensation or that there may be aparticular threshold necessary for reducing the expression of thisgene to have robust HbF induction.45

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Silencing of HbF by KLF1Using independent and complementary approaches, two studieshave demonstrated that the expression of BCL11A appears to becontrolled by the erythroid-specific transcription factor KLF1. Inone study, a mouse with a hypomorphic allele of KLF1 hadelevations of embryonic globin expression, and transgenic micewith the human �-globin locus showed persistent expression of�-globin.39 As a result, the investigators used siRNA approach tofind out whether the same regulation could occur in primary humanerythroid cells and demonstrate a similar connection. They wereable to demonstrate that this effect occurs through both direct effectsof KLF1 at the �-globin locus, but also via indirect effects mediatedthrough reduced expression of BCL11A upon KLF1 knock-down.This finding demonstrated that KLF1 was a direct positive transcrip-tional regulator of BCL11A expression. A different study examinedthe genetic basis of an unlinked form of hereditary persistence ofHbF that was found to be due to a KLF1 missense mutation in thisfamily.38 Using primary cells from these patients and from unaf-fected controls, the investigators of that study were able todemonstrate that the effect seen was in part attributable to asilencing effect of KLF1 on BCL11A.

One area of ongoing uncertainty relates to the human phenotypesseen in various cases of KLF1 mutations. Whereas in the initialreport, all recipients of the missense mutation had elevations in HbFexpression, it should be noted that this actually varied between 3%and 19% of total hemoglobin levels.7 In addition, other reports ofheterozygous KLF1 mutations in humans either show concomitant

disruption of erythropoiesis or little effect on HbF expression.46,47

The basis of this variation remains to be determined and will beimportant to better understand the mechanisms by which KLF1 acts,both directly and indirectly, to affect HbF expression (Figure 1).This will be critical for any future work attempting to target KLF1for HbF induction, particularly if the untoward effects of suchinhibition on erythroid differentiation are to be avoided. However,given the specificity of KLF1 within the erythroid lineage, and ifdisruption of erythropoiesis could be avoided while allowing forHbF expression, it may be a very promising therapeutic target toinduce HbF (Table 1).

The role of MYB and miRNAs 15a/16-1 in HbF regulationThe GWA studies also demonstrated strong associations of variantsin the intergenic region between the genes HBS1L and MYB withHbF levels. The exact mechanism by which these variants result inelevated HbF levels has remained unclear, although it has beensuggested that this may occur through an effect on the expression orfunction of MYB.7,48 Overexpression of MYB in K562 cells reduced�-globin expression, and primary erythroid cultures with higherHbF expression had lower levels of MYB expression.49 More directevidence lending further support to a connection between MYB andHbF expression has recently been reported. Following up on aclassic clinical observation that patients with a trisomy of chromo-some 13 have elevations of HbF, and using partial trisomy cases tomap this trait, it was shown that a gene in 13q14 appears to beresponsible for mediating this effect.50 Using an integrative genomicapproach, 2 co-transcribed miRNAs, 15a and 16-1, appeared to be

Table 1. A sampling of molecular targets for HbF induction

TargetTarget alterationsfor HbF induction Benefits Drawbacks/limitations

BCL11A 2 ● Robust HbF induction seen in a variety ofexperimental settings with no perturbation oferythropoiesis

● Robust effects of putative regulatory commongenetic variants on clinical severity inhemoglobinopathies

● Required in B lymphocytes and other celltypes, so toxicity or untoward effects oftargeting approaches in vivo may be seen

● Transcription factors can be difficult to targetwith small molecules

SOX6 2 ● HbF induction seen after knock-down in primaryhuman cells

● May affect erythropoiesis and be necessary inother cell types

● Transcription factors can be difficult to targetwith small molecules

● Heterozygous mutation in patient occurswithout HbF elevation

KLF1 2 ● Specific to the erythroid lineage● Supported by humans with heterozygous

mutations and elevated HbF levels

● Knock-down may affect erythropoiesis● Transcription factors can be difficult to target

with small molecules● Variable phenotype in humans with

heterozygous mutationsMYB 2 ● Relative specificity in hematopoiesis

● Robust effects of putative regulatory commongenetic variants on clinical severity inhemoglobinopathies

● Interfering with expression or function mayaffect erythropoiesis

● Transcription factors can be difficult to targetwith small molecules

● Limited mechanistic knowledge of function inglobin gene regulation

miRNA-15a/16–1 1 ● Patients with trisomy 13 have elevations of HbFlevels and increased expression of these miRNAswithout other perturbations in erythropoiesis

● Limited knowledge of the physiological role ofthese miRNAs in erythropoiesis and other celltypes

● miRNAs target a variety of molecules, whichmay lead to unpredictable side effects

HDAC1/2 2 ● Robust drugs targeting these molecules arealready available and in clinical trials

● Newer HDAC inhibitors can be very specific

● These HDACs play a wide variety of roles in anumber of tissues and toxicity could be amajor problem before HbF-inductive effectsare seen

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top candidates as mediators of this trait. This was supported by thefinding that slight overexpression of these miRNAs in primary adulterythroid cells could result in elevations of �-globin expression.Consistent with prior studies,51 these miRNAs appeared to potentlytarget and down-regulate MYB protein expression. Finally, siRNA-mediated knock-down of MYB could result in a similar effect,suggesting a direct connection between the effect of these miRNAson HbF silencing and their effect on MYB expression. This findingalso supports the idea that the intergenic variants between HBS1Land MYB may act to effect MYB expression.48 Sequencing of MYBhas revealed rare coding variants within this gene that appear to beassociated with elevated HbF levels in patients with SCD, providingfurther support for a role of MYB in modulating HbF expression.52

Numerous studies have shown that MYB is necessary for normalerythropoiesis, although hypomorphic mouse alleles of this genehave subtle effects on erythropoiesis.53 Moreover, such in vivostudies demonstrate the specificity of MYB in hematopoietic celltypes. If the function of MYB could be partially disrupted andtargeted in an erythroid-specific manner, then it may be an attractivetherapeutic target (Table 1). The exact physiological role ofmiRNA-15a and miRNA-16-1 in erythropoiesis remains to beelucidated; however, it is possible that these miRNAs may beattractive targets for HbF induction. Support comes from the findingthat trisomy 13 patients have relatively normal erythropoiesis whilehaving persistent HbF expression.50 Much more work is necessaryto elucidate the mechanisms through which these factors affect HbFexpression, which will be important in strategies for HbF induction.

Other factors involved in HbF regulationIn addition to the aforementioned molecules, a few other factorshave been suggested to play a role in HbF expression, althoughsupport from human genetic studies is not currently present for theseother factors. One recent study described the role of the Friend ofPRMT1 (FOP1) protein in HbF silencing.54 Knock-down of thisfactor in primary human adult erythroid cells resulted in robustelevations of HbF levels. The mechanism through which this factorcarries out this activity remains unclear and further work will benecessary to more carefully investigate this observation. In addition,other factors such as the orphan nuclear hormone receptors TR2,TR4, and COUP-TFII have been suggested to play a role in HbFsilencing.8 Further work to examine the role of these factors in HbFregulation will be needed to better understand how these moleculesregulate this process.

As mentioned above, the HDACs are promising targets and arerecruited by and interact with a variety of transcription factorsinvolved in globin gene regulation, such as BCL11A.8 Other DNA-and histone-modifying enzymes have been suggested to play a rolein globin gene regulation and thus are worthwhile considerations aspossible targets for HbF induction. For example, there is support fora role of the DNA methyltransferase DNMT1 in silencing HbF.55

Because 5-azacytidine and its derivative, decitabine, which bothinduce HbF in vivo, target DNMT1, it is possible that futurestrategies targeting this pathway may lead to more effectivetherapies.

Approaches and future directionsWhereas the recent findings described above have uncoveredpromising molecular targets for therapeutic efforts aimed at HbFinduction, a great deal of further work is necessary before thesefindings can be translated to therapeutic advances. The question of

how best to develop such therapies is of great interest in this field.One immediately applicable approach would be to use genetherapeutic approaches to deliver siRNAs that target the moleculesdescribed above. With the exception of miRNAs 15a and 16-1, theother therapeutic targets discussed here all appear to have HbF-inductive effects when their expression is reduced (Table 1).Preliminary preclinical findings support such approaches.37 Inaddition to or in place of delivery of a nonmutated globin gene, suchsiRNA molecules could be extremely useful. These gene therapymethods are promising and could be targeted to the hematopoieticsystem alone. Given recent advances in this field,56 it is possible thatsuch approaches could enter the clinical arena in the near future.Such gene therapy trials that are performed on a relatively smallscale may help to reveal which therapeutic targets would beefficacious in vivo for more broadly applicable small-moleculeapproaches. Another approach would be to introduce exogenousmolecules such as zinc-finger transcription factors, which interferewith the normal activity of endogenous molecules found at the�-globin locus and thus act to induce HbF.37

Small molecules would be ideal as therapies in these globallywidespread diseases. However, nearly all of the molecular targetsthat currently exist are transcriptional regulators of globin geneexpression (Table 1). These molecules have been notoriouslydifficult to target with small molecules, although recent approachessuggest that promising leads could be developed.57 As enzymes, theHDACs are ideal targets, and many specific inhibitors of HDACshave already been developed. Some of these appear to be promisingleads for HbF-inductive therapies and are already in clinical trialsfor other indications such as cancer.58 Other small moleculestargeting epigenetic enzymes, such as DNMT1, may show possiblebenefit as HbF inducers and, in many cases, inhibitors of theseenzymes are already available or are in development. At the presenttime, it is difficult to predict which targets may be best, and, giventhe number of recently identified molecular targets, it is likely thatthe broadest approaches aimed at targeting as many of thesemolecules as possible may eventually yield specific candidates thatcan successfully pass the early stages of evaluation. However, thefact that there are so many recently identified targets lends promiseto this field. In addition, the ongoing basic science studies that areaimed at better understanding the mechanisms by which suchmolecules silence HbF will likely yield additional insights into howthis process can best be modulated.

AcknowledgmentsI apologize to those investigators whose work could not be citedhere because of space limitations. I am grateful to my mentors whohave given me the opportunity to study hemoglobin switching andto the many colleagues I have had the privilege of working with.

DisclosuresConflict-of-interest disclosure: The author declares no competingfinancial interests. Off-label drug use: None disclosed.

CorrespondenceVijay G. Sankaran, Children’s Hospital Boston, 300 LongwoodAvenue, Boston, MA 02115; Phone: (617) 355-6000; e-mail:sankaran@broadinstitute.org.

References1. Weatherall D, Akinyanju O, Fucharoen S, Olivieri N, Mus-

grove P. Inherited Disorders of Hemoglobin. Disease Control

Hematology 2011 463

Priorities in Developing Countries. 2nd ed. New York, NY:Oxford University Press; 2006:663-680.

2. Michlitsch JG, Walters MC. Recent advances in bone marrowtransplantation in hemoglobinopathies. Curr Mol Med. 2008;8:675-689.

3. Persons DA. Hematopoietic stem cell gene transfer for thetreatment of hemoglobin disorders. Hematology Am Soc Hema-tol Educ Program. 2009:690-697.

4. Sankaran VG, Nathan DG. Thalassemia: an overview of50 years of clinical research. Hematol Oncol Clin North Am.2010;24:1005-1020.

5. Hsieh MM, Kang EM, Fitzhugh CD, et al. Allogeneic hemato-poietic stem-cell transplantation for sickle cell disease. N EnglJ Med. 2009;361:2309-2317.

6. Porter JB, Shah FT. Iron overload in thalassemia and relatedconditions: therapeutic goals and assessment of response tochelation therapies. Hematol Oncol Clin North Am. 2010;24:1109-1130.

7. Sankaran VG, Nathan DG. Reversing the hemoglobin switch.N Engl J Med. 2010;363:2258-2260.

8. Wilber A, Nienhuis AW, Persons DA. Transcriptional regula-tion of fetal to adult hemoglobin switching: new therapeuticopportunities. Blood. 2011;117:3945-3953.

9. Watson J. The significance of the paucity of sickle cells innewborn Negro infants. Am J Med Sci. 1948;215:419-423.

10. Weatherall DJ, Clegg JB. The Thalassaemia Syndromes. 4th ed.Malden, MA: Blackwell Science; 2001.

11. Perrine RP, Brown MJ, Clegg JB, Weatherall DJ, May A.Benign sickle-cell anaemia. Lancet. 1972;2:1163-1167.

12. Kar BC, Satapathy RK, Kulozik AE, et al. Sickle cell disease inOrissa State, India. Lancet. 1986;2:1198-1201.

13. Platt OS, Brambilla DJ, Rosse WF, et al. Mortality in sickle celldisease. Life expectancy and risk factors for early death. N EnglJ Med. 1994;330:1639-1644.

14. Platt OS, Thorington BD, Brambilla DJ, et al. Pain in sickle celldisease. Rates and risk factors. N Engl J Med. 1991;325:11-16.

15. Castro O, Brambilla DJ, Thorington B, et al. The acute chestsyndrome in sickle cell disease: incidence and risk factors. TheCooperative Study of Sickle Cell Disease. Blood. 1994;84:643-649.

16. Weatherall DJ. Phenotype-genotype relationships in mono-genic disease: lessons from the thalassaemias. Nat Rev Genet.2001;2:245-255.

17. Premawardhena A, Fisher CA, Olivieri NF, et al. HaemoglobinE beta thalassaemia in Sri Lanka. Lancet. 2005;366:1467-1470.

18. Galanello R, Sanna S, Perseu L, et al. Amelioration ofSardinian beta0 thalassemia by genetic modifiers. Blood. 2009;114:3935-3937.

19. Nuinoon M, Makarasara W, Mushiroda T, et al. A genome-wide association identified the common genetic variants influ-ence disease severity in beta(0)-thalassemia/hemoglobin E.Hum Genet. 2010;127:303-314.

20. DeSimone J, Heller P, Hall L, Zwiers D. 5-Azacytidinestimulates fetal hemoglobin synthesis in anemic baboons. ProcNatl Acad Sci U S A. 1982;79:4428-4431.

21. Ley TJ, DeSimone J, Anagnou NP, et al. 5-azacytidineselectively increases gamma-globin synthesis in a patient withbeta� thalassemia. N Engl J Med. 1982;307:1469-1475.

22. Platt OS, Orkin SH, Dover G, Beardsley GP, Miller B,Nathan DG. Hydroxyurea enhances fetal hemoglobin produc-tion in sickle cell anemia. J Clin Invest. 1984;74:652-656.

23. Platt OS. Hydroxyurea for the treatment of sickle cell anemia.N Engl J Med. 2008;358:1362-1369.

24. Charache S, Terrin ML, Moore RD, et al. Effect of hydroxyureaon the frequency of painful crises in sickle cell anemia.Investigators of the Multicenter Study of Hydroxyurea in SickleCell Anemia. N Engl J Med. 1995;332:1317-1322.

25. Perrine SP, Greene MF, Faller DV. Delay in the fetal globinswitch in infants of diabetic mothers. N Engl J Med. 1985;312:334-338.

26. Bard H, Prosmanne J. Relative rates of fetal hemoglobin andadult hemoglobin synthesis in cord blood of infants of insulin-dependent diabetic mothers. Pediatrics. 1985;75:1143-1147.

27. Perrine SP, Ginder GD, Faller DV, et al. A short-term trial ofbutyrate to stimulate fetal-globin-gene expression in the beta-globin disorders. N Engl J Med. 1993;328:81-86.

28. Perrine SP, Olivieri NF, Faller DV, Vichinsky EP, Dover GJ,Ginder GD. Butyrate derivatives. New agents for stimulatingfetal globin production in the beta-globin disorders. Am JPediatr Hematol Oncol. 1994;16:67-71.

29. Sher GD, Ginder GD, Little J, Yang S, Dover GJ, Olivieri NF.Extended therapy with intravenous arginine butyrate in patientswith beta-hemoglobinopathies. N Engl J Med. 1995;332:1606-1610.

30. Fathallah H, Weinberg RS, Galperin Y, Sutton M, Atweh GF.Role of epigenetic modifications in normal globin gene regula-tion and butyrate-mediated induction of fetal hemoglobin.Blood. 2007;110:3391-3397.

31. Cao H, Stamatoyannopoulos G, Jung M. Induction of humangamma globin gene expression by histone deacetylase inhibi-tors. Blood. 2004;103:701-709.

32. Atweh G, Fathallah H. Pharmacologic induction of fetalhemoglobin production. Hematol Oncol Clin North Am. 2010;24:1131-1144.

33. Uda M, Galanello R, Sanna S, et al. Genome-wide associationstudy shows BCL11A associated with persistent fetal hemoglo-bin and amelioration of the phenotype of beta-thalassemia.Proc Natl Acad Sci U S A. 2008;105:1620-1625.

34. Menzel S, Garner C, Gut I, et al. A QTL influencing F cellproduction maps to a gene encoding a zinc-finger protein onchromosome 2p15. Nat Genet. 2007;39:1197-1199.

35. Lettre G, Sankaran VG, Bezerra MA, et al. DNA polymor-phisms at the BCL11A, HBS1L-MYB, and beta-globin lociassociate with fetal hemoglobin levels and pain crises in sicklecell disease. Proc Natl Acad Sci U S A. 2008;105:11869-11874.

36. Sankaran VG, Menne TF, Xu J, et al. Human fetal hemoglobinexpression is regulated by the developmental stage-specificrepressor BCL11A. Science. 2008;322:1839-1842.

37. Wilber A, Hargrove PW, Kim YS, et al. Therapeutic levels offetal hemoglobin in erythroid progeny of beta-thalassemicCD34� cells after lentiviral vector-mediated gene transfer.Blood. 2011;117:2817-2826.

38. Borg J, Papadopoulos P, Georgitsi M, et al. Haploinsufficiencyfor the erythroid transcription factor KLF1 causes hereditarypersistence of fetal hemoglobin. Nat Genet. 2010;42:801-805.

39. Zhou D, Liu K, Sun CW, Pawlik KM, Townes TM. KLF1regulates BCL11A expression and gamma- to beta-globin geneswitching. Nat Genet. 2010;42:742-744.

40. Bradner JE, Mak R, Tanguturi SK, et al. Chemical geneticstrategy identifies histone deacetylase 1 (HDAC1) and HDAC2as therapeutic targets in sickle cell disease. Proc Natl Acad SciU S A. 2010;107:12617-12622.

41. Sankaran VG, Xu J, Ragoczy T, et al. Developmental andspecies-divergent globin switching are driven by BCL11A.Nature. 2009;460:1093-1097.

42. McGrath KE, Frame JM, Fromm GJ, et al. A transient definitive

464 American Society of Hematology

erythroid lineage with unique regulation of the beta-globinlocus in the mammalian embryo. Blood. 2011;117:4600-4608.

43. Xu J, Sankaran VG, Ni M, et al. Transcriptional silencing of{gamma}-globin by BCL11A involves long-range interactionsand cooperation with SOX6. Genes Dev. 2010;24:783-798.

44. Dumitriu B, Patrick MR, Petschek JP, et al. Sox6 cell-autonomously stimulates erythroid cell survival, proliferation,and terminal maturation and is thereby an important enhancerof definitive erythropoiesis during mouse development. Blood.2006;108:1198-1207.

45. Sankaran VG, Menne J, Heller R. Heterozygous disruption ofhuman SOX6 is insufficient to impair erythropoiesis or silenc-ing of fetal hemoglobin. Blood. 2011;117:4396-4397.

46. Arnaud L, Saison C, Helias V, et al. A dominant mutation in thegene encoding the erythroid transcription factor KLF1 causes acongenital dyserythropoietic anemia. Am J Hum Genet. 2010;87:721-727.

47. Satta S, Perseu L, Moi P, et al. Compound heterozygosity forKLF1 mutations associated with remarkable increase of fetalhemoglobin and red cell protoporphyrin. Haematologica. 2011;96:767-770.

48. Wahlberg K, Jiang J, Rooks H, et al. The HBS1L-MYBintergenic interval associated with elevated HbF levels showscharacteristics of a distal regulatory region in erythroid cells.Blood. 2009;114:1254-1262.

49. Jiang J, Best S, Menzel S, et al. cMYB is involved in theregulation of fetal hemoglobin production in adults. Blood.2006;108:1077-1083.

50. Sankaran VG, Menne TF, Scepanovic D, et al. MicroRNA-15aand -16-1 act via MYB to elevate fetal hemoglobin expression

in human trisomy 13. Proc Natl Acad Sci U S A. 2011;108:1519-1524.

51. Zhao H, Kalota A, Jin S, Gewirtz AM. The c-myb proto-oncogene and microRNA-15a comprise an active autoregula-tory feedback loop in human hematopoietic cells. Blood.2009;113:505-516.

52. Galarneau G, Palmer CD, Sankaran VG, Orkin SH, Hirschhorn JN,Lettre G. Fine-mapping at three loci known to affect fetalhemoglobin levels explains additional genetic variation. NatGenet. 2010;42:1049-1051.

53. Carpinelli MR, Hilton DJ, Metcalf D, et al. Suppressor screenin Mpl-/- mice: c-Myb mutation causes supraphysiologicalproduction of platelets in the absence of thrombopoietinsignaling. Proc Natl Acad Sci U S A. 2004;101:6553-6558.

54. van Dijk TB, Gillemans N, Pourfarzad F, et al. Fetal globinexpression is regulated by Friend of Prmt1. Blood. 2010;116:4349-4352.

55. Banzon V, Ibanez V, Vaitkus K, et al. siDNMT1 increasesgamma-globin expression in chemical inducer of dimerization(CID)-dependent mouse betaYAC bone marrow cells and inbaboon erythroid progenitor cell cultures. Exp Hematol. 2011;39:26-36 e21.

56. Cavazzana-Calvo M, Payen E, Negre O, et al. Transfusionindependence and HMGA2 activation after gene therapy ofhuman beta-thalassaemia. Nature. 2010;467:318-322.

57. Koehler AN. A complex task? Direct modulation of transcrip-tion factors with small molecules. Curr Opin Chem Biol.2010;14:331-340.

58. Boumber Y, Issa JP. Epigenetics in cancer: what’s the future?Oncology (Williston Park). 2011;25:220-226, 228.

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