Epigenetic alterations and microRNAs

Abstract

The term epigenetics refers to the heritable changes in gene expression that do not represent changes in DNA sequence. DNA methylation and histone modification are the best studied epigenetic mechanisms. However, microRNAs, which affect gene expression at the posttranscriptional level, should be considered as members of the epigenetic machinery too. Myelodysplastic syndromes (MDS) are clone disorders of the hematopoietic stem cell with increased risk of leukemic transformation. Over the years, increased number of studies indicates the role of epigenetic mechanisms, including microRNAs, in MDS pathogenesis and prognosis. Indeed, epigenetic therapy with demethylating agents has already been applied to MDS. In this review we summarize current knowledge on the role of epigenetic alterations in MDS pathogenesis and treatment.

Keywords: :

• MDS
• DNA methylation
• histone modification
• microRNAs
• epigenetics
• DNMTs
• polycomb system

Introduction

Epigenetics refer to the heritable changes in gene expression that are not reflected in changes in DNA sequence. The term was first used in the 1940s by Conrad Waddington, who defined epigenetics as “the causal interactions between genes and their products, which bring the phenotype into being.”¹ Several definitions of epigenetics have been proposed since then. For example, Holliday R. defined epigenetics as “changes in gene expression that are not due to alteration in DNA sequence.”² Recently, another definition was proposed by Adrian Bird “the structural adaptation of chromosomal regions so as to register, signal or perpetuate altered activity states.”³ The epigenetic machinery comprises DNA methylation, histone modification and RNA interference. RNA interference refers mostly to the recently discovered microRNAs. microRNAs are small, non-coding RNAs that regulate gene expression at the posttranscriptional level repressing the translation of their mRNA target without affecting the DNA sequence of the targeted gene.

Myelodysplastic syndromes (MDS) are heterogeneous disorders of hematopoietic stem cell associated with ineffective hematopoiesis and increased risk of transformation to acute myeloid leukemia (AML). The precise pathophysiological mechanisms of MDS remain unclear. However, it seems that the pathways involved in the pathogenesis of this disease are genetic alterations, repression of apoptosis and deregulation of the microenvironment.⁴ Emerging data support the role of the aforementioned epigenetic mechanisms in MDS pathogenesis and prognosis. Indeed, several studies, discussed below, provide evidence that key molecules involved in DNA methylation, histone modification and microRNA regulation are implicated in MDS.

The aim of this review is to summarize the role of epigenetics with respect to pathogenesis, prognosis and treatment of MDS.

DNA Methylation

DNA methylation is the best studied epigenetic mechanism. It involves the addition of a methyl group at the 5′ carbon of a cytosine of a 5′-CpG-3′ dinucleotide by DNA methyltransferases (DNMTs).⁵ Most of the CpG dinucleotides in mammals are methylated. However, regions in genome with high frequency of CpG dinucleotides, called CpG islands, reside in or near promoters of housekeeping genes and are unmethylated.^6,7 DNA methylation is transmitted through mitosis and controls gene function by inhibiting DNA transcription. The latter is achieved by two ways. First, DNA methylation prevents transcription factors from binding to gene promoter and, second, methyl-CpG groups bind proteins, namely methyl-CpG-binding domain proteins (MeCPs), which, in turn, recruit histone-modifying proteins leading to chromatin inactivation.⁸

Several alterations have been reported in MDS regarding DNA methylation, such as aberrant genome methylation and impaired function of proteins that regulate methylation (Table 1). Aberrant methylation of selected genes, such as p15INK4B, has been reported by several groups.^9,10 However, it seems that altered methylation in MDS is not restricted to unique genes but affects the whole genome. Widespread promoter hypermethylation in MDS and secondary AML has been reported by the recent study of Figueroa et al. According to this study, bone marrow cells from patients with MDS and secondary AML exhibit an extensive pattern of promoter hypermethylation that differs from normal cells and de novo AML and is not limited to cancer-associated genes but affects almost the whole genome. The extension of this phenomenon suggests that general mechanisms that normally control DNA methylation are deregulated in MDS. Of note, members of WNT and MAPK pathways as well as CDKN2A genes were found to be hypermethylated in this study.¹¹ However, WNT-antagonist genes (sFRP1, sFRP2, sFRP4, sFRP5 and DKK1) are found methylated too in another study. As a result WNT-pathway is activated and WNT downstream genes TCF1 and LEF1 are upregulated.¹⁹ A possible explanation of these contradictory phenomena is that the relevant methylation status of WNT-pathway genes and WNT-antagonists determines the level of WNT activation. In accordance to the study of Figueroa et al., another group reported the aberrant methylation of CDKN2A gene in MDS and AML. In this cohort, CDKN2B was hypermethylated too while both genes exhibited higher levels of methylation in AML compared with MDS. Interestingly, aberrant methylation of the aforementioned genes was associated with disease progression.¹²

Table 1. The effect of gene methylation in MDS

Gene	Alteration	Result	Reference
p15INK4B	hypermethylation	Inactivation	9,10
WNT pathway	hypermethylation	inactivation	11
MAPK pathway	hypermethylation	inactivation	11
CDKN2A	hypermethylation	inactivation	11
CDKN2B	hypermethylation	inactivation	12
DOCK4	hypermethylation	Invasion of cancer cells	13
SOX7	hypermethylation	No inhibition of WNT pathway	14
Dab2	hypermethylation	Advanced MDS	15
DNMT3A	Somatic mutations (missence, nonsence, frameshift)	Aberrant DNA methylation	16,17
TET-2	Somatic mutations (missence, nonsence, frameshift)	DNA hypomethylation	18
IDH	Heterozygous mutations	Poor-prognosis MDS	37-40

Global methylation analysis of total leukocytes form peripheral blood revealed different methylation status between MDS patients and normal controls. These results were consistent with the ones reported by previous studies in bone marrow cells. Of note, aberrant hypermethylation occurred mainly outside CpG islands. Among the most affected genes was DOCK4 (Dedicator of cytokinesis 4) an activator of G-proteins, whose mutations have been associated with invasion of cancer cells.¹³ However, the fact that total leukocytes from peripheral blood contain normal and dysplastic cells should always be taken into consideration when evaluating these results.

During the last few years, several studies of gene methylation in MDS have been published. SOX7 (sex determining region Y-box) gene, which codes for an inhibitor of WNT pathway, is found to be methylated in its CpG island in 58.1% of MDS patients and the percentage of patients with hypermethylation of SOX7 gene was higher among patients with advanced disease. Interestingly, the percentage of methylation of CpG island seems to represent a predictive factor of MDS prognosis.¹⁴ In addition, methylation of Dab2 (human disabled - 2) gene is associated with MDS pathogenesis, since it is methylated in 50.6% of MDS patients and its hypermethylation is mostly associated with advanced stages of disease.¹⁵ Another gene whose methylation is implicated in MDS pathogenesis is the erythroid transcription factor GATA1, whose upregulation is necessary for erythroid and megakaryocytic development. Methylation of GATA1 in MDS patients inhibits upregulation of the gene and leads to ineffective erythropoiesis.²⁰ Finally, the promoter of p73, a tumor suppressor gene and homolog of p53 gene, is hypermethylated in MDS and is negatively associated with patients’ prognosis.²¹

As previously mentioned, DNA methylation is catalyzed by methyltransferases (DNMTs). There are three DNMTs in mammals, DNMT3A and B that catalyze de novo methylation and DNMT1 that is associated with maintenance of methylation. 5′-methylcytocine is converted to 5′-hydroxymethylcytocine by Ten Eleven Translocation (TET) proteins (TET1, 2 and 3), whose action depends on aKG and Fe²⁺. 5′-hydroxymethylcytocine (5-hmC) is short lived and is followed by demethylation of cytocine. Of note, isocitrate dehydrogenase 1 (IDH1) converts isocitrate to aKG which is necessary for TET2 function.²² As a result, proper function of DNMTs, TET proteins and IDH1 protein are necessary for normal DNA methylation.

Mutations in DNMT3A were first reported in AML^23,24 and were recently identified in advanced stages MDS patients (RAEB I and II).¹⁶ Additionally, Walter et al. found 13 heterozygous, missence, nonsense and frame shift somatic mutations in DNMT3A in 8% of patients. Four of them occurred in R882 amino acid which residues in methyltransferase domain of DNMT3A and has been associated with impaired function of the enzyme since it reduces its ability to bind DNA. These mutations seem to occur at early stages of MDS course and are associated with worse overall survival (OS) and event free survival (EFS). However, multivariate analysis could not be performed due to the small number of patients.¹⁷ Another study confirmed these results and also found that global methylation status did not differ between mutated and wild type patients.²⁵ The result of DNMT3A mutations in DNA methylation and the mechanism by which they affect gene expression remain unclear. Further studies will clarify this subject. Apart from somatic mutations of DNMT3A, overexpression of DNMT1, 3A and B in MDS has also been reported, particularly in refractory anemia (RA) and refractory anemia with excess of blasts (RAEB), which is consistent with the hypermethylation described in MDS.²⁶

Patients with MDS and AML and myeloproliferative neoplasms (MPN) exhibit uniparental disomy (UPD) on chromosome 4q24. The deleted region on chromosome 4q24 contains the aforementioned gene, TET2. Deletions or missence, nonsence and frameshift mutations of TET2 are observed in MDS, MPN and AML. The defects occur at early stages of MDS pathogenesis, which is deduced from the fact that mutations and deletions of TET2 are observed in CD34+ cells.^27-30 Homozygous and hemizygous mutations as well as heterozygous and compound heterozygous mutations occur in patients with MDS/MPN and affect conserved regions of TET2 protein and the N-terminal domain.¹⁸ The clinical impact of TET2 mutations has not been clarified yet. It seems that mutations are associated with impaired TET2 catalytic activity leading to low levels of 5-hmC. In spite of the fact that the most expected result of such a modification is DNA hypermethylation, low levels of 5-hmC are associated with DNA hypomethylation.³¹ However, hypermethylation of DNA has also been reported.¹¹ Of note, the effect of TET2 mutations on patients’ prognosis is still a contradictory issue. There are a few studies supporting the favorable effect of TET2 mutations on OS and response rate to azacitidine^29,32,33 while Smith et al. did not found any correlation between TET2 mutations and OS.³⁴ As reported by the authors, these contradictory results may be attributed to differences in study design and source of data. Further studies will illuminate this issue.

IDH1 and 2 are enzymes that participate in the citric acid cycle and catalyze the oxidative decarboxylation of isocitrate producing a-ketoglutarate in the cytoplasm and peroxisomes respectively. IDH1 is located in cytoplasm and peroxisomes and IDH2 in mitochondria. IDH1 mutations were first found in gliomas and were then discovered in AML patients by whole genome sequencing.³⁵ Most mutations affect highly conserved arginine residues: IDH1-R132, IDH2-R172 and IDH2-R140. These heterozygous mutations are found to inhibit the catalytic activity of IDH1 in gliomas resulting in loss-of function of IDH1, reduction in aKG and activation of HIF-a pathway.³⁶ However, the fact that only one gene is mutated raised the possibility that these mutations may result in gain of function, not simply in loss of function. This is the reason why Dang et al. performed metabolic studies and showed that IDH1 mutations result in the production of 2-hydroxyglutarate (2-HG), which is an onco-metabolite and its accumulation is associated with progression of gliomas.³⁷ Very little is known about these rare, albeit important, mutations in MDS. Most of the studies published so far agree that IDH1 and 2 mutations are rare in MDS (3–5 and 12% in one study), are associated with normal or good risk karyotype and represent and independent factor of poor prognosis and AML transformation.^38-41 Apart from the aforementioned mutations, a new nonsense mutation of IDH1 has been recently discovered; R100X, which results in a truncated molecule, without enzymatic activities.⁴⁰ It is worth noting that IDH1 and 2 mutations result in DNA hypermethylation in AML and are mutually exclusive with TET2 mutations.⁴² Thus, IDH1 and 2 mutations may represent a potential prognostic marker as well as therapeutic target in MDS treatment.

Finally, methylation of imprinted genes seems to play a role in MDS. The term genomic imprinting refers to the preferential expression of a gene depending on whether they come from the maternal or paternal DNA. As a result, the inherited genes are unequal expressed and the genetic locus acts as haploid although it is diploid. A mechanism by which this phenomenon takes place is DNA methylation. This is a normal mechanism, however aberrant methylation of imprinted genes have been associated with myeloid malignancies. Thus, the imprinted genes MEG3 (maternally expressed gene 3) and SNRPN (small nuclear ribonucleoprotein polypeptide N) are hypermethylated in MDS and AML. MEG3 methylation is also associated with worse overall survival suggesting that it is a potential prognostic factor.⁴³

Histone Modification

Histones are proteins that organize the DNA of a eukaryotic cell into structural units called nucleosomes. There are two classes of histone proteins: the core histones H2A, H2B, H3 and H4 and the linker histone H1. Each nucleosome is an octamer consisting of two pairs of the core histone proteins and 147 base pairs of DNA wrapped around them. H3 and H4 histones have tails of amino acids that comprise mostly lysine and arginine. Epigenetic modifications of these amino acids (acetylation, methylation, phosphorylation, ubiquitylation, sumoylation, ADP-ribosylation, deimination, proline isomerization) are associated with compaction or relaxation of nucleosomes resulting in repression or activation of gene translation, respectively.

Histone methylation and acetylation are the best-studied modifications. Acetylation is mediated by histone acetylases (HAT) and results in activation of gene transcription while deacetylation by histone deacetylases (HDAC), prevents transcription. HATs are divided into group A, which are located in the nucleus, and group B, located in the cytoplasm. HDACs are divided into three classes. Class I HDACs are located in the nucleus and participate in transcription. Class II HDACs can be found in the nucleus and in the cytoplasm and mediate protein modifications, and Class III are associated with cell cycle.⁴

The effect of methylation depends on the residue modified and the degree of modification and could be associated with activation or repression of gene transcription. Trimethylation of lysine 27 of H3 (H3K27me3) by the polycomb complex PRC2 results in transcription inactivation. Similarly, di- and tri- methylation of lysine 9 of H3 (H3K9me2/3) leads to gene silencing. In contrast, trimethylation of lysine 4 (H3K4me3) and lysine 6 of H3 (H3K6me3) are marks of active gene translation. Thus, histone modification determines the “on” or “off” status of DNA, which is necessary for the accurate control of gene transcription. In conclusion, it should be mentioned that the two important epigenetic mechanisms, DNA methylation and histone modification, are related to each other, since histone modification directs DNA methylation in early development and DNA methylation represents a template for the histone modification.⁴⁴

Alteration of histone modification has been associated with MDS pathogenesis and prognosis (Table 2). Genes that regulate histone modification, like Polycomb group (PcG) proteins, are mutated in MDS. PcG acts in two complexes namely PRC1 and 2 that are responsible for histone modification and gene silencing. PRC1 comprises three proteins: RING1A, RING1B and BMI1 while PRC2 contains four core proteins namely EZH2, EED, SUZ12 and RbAp46/48. EZH2, located in 7q36.1, is the catalytic unit of PRC2 and mediates methylation of H3K27 resulting in gene silencing. Missense, nonsense, donor-splice-site and frameshift mutations of EZH2 have been discovered in MDS patients. Hemizygous mutations were found in patients with microdeletions in 7q and homozygous mutations in 7q acquired UPD.^45,47,48 These mutations contribute to loss-of-function and decreased H3K27 methylation suggesting that EZH2 acts as tumor suppressor. However, recent studies support the oncogenic role of EZH2 in myeloid malignancies.^46,49 Finally, EZH2 mutations are associated with decreased overall survival but more studies are required to clarify the role of these mutations in MDS prognosis.⁴⁸

Table 2. Histone modification in MDS

Gene	Alteration	Result	Reference
EZH2	Loss of function mutations (missence, donor-splice-site, frameshift)	Oncogenesis, ↓overall survival	45,47-49
SUZ1,2 EED	Mutations	Compromised PCR2 function	50 51
ASXL1	Loss of function mutations	Compromised PCR2 function	53
BMI1	Mutations	Poor prognosis MDS	56,57

SUZ12 (suppressor of zeste 12 homolog), member of PRC2 complex is necessary for cell proliferation in vivo and in vitro and EZH2 activity in vivo.⁵⁰ Mutation in the SUZ12 gene as well as in the EED gene (also member of PRC2 complex) has been recently discovered in MDS and have been associated with compromised PRC2 function.⁵¹

The ASXL1 gene is located on 20q11 chromosome and codes for a nuclear protein that is a member of PRC2 complex and regulates gene expression by repressing transcription of some genes while enhancing the transcription of others. Mutations of ASXL1 have been reported in 11.4–20.7% of MDS patients.⁵² The vast majority of these mutations are loss-of-function frameshift and nonsense mutations and result in compromised function of PRC2 regarding H3K27 methylation, similarly to the effect of aforementioned EZH2 mutations.⁵³ Finally, combined frameshift and point mutations as well as frameshift mutations of ASXL alone represent an independent prognostic marker in MDS.⁵⁴

BMI1 protein belongs to PRC1 complex and mediates ubiquitylation of H2AK119 leading to PRC2 recruitment.⁵⁵ It is responsible for CD34+ cells’ self – renewal and has been associated with poor prognosis MDS (RAEB and RAEB-T) and disease progression to AML.^56,57 Finally, overexpression of EZH, RING1 and BMI1in MDS has been recently reported and has been associated with poor prognosis too.⁵⁸ It is worth noting that combined mutations of the previously mentioned genes: ASLX1, IDH1, IDH2, TET2 and WT1, have been recently reported, highlighting the complexity of the pathways involved in MDS pathogenesis and progression.⁵⁹

Finally, it should be mentioned that the chromatin remodeling factor ATRX was found to be mutated in α thalassaemia associated MDS (ATMDS). ATRX is a nuclear protein that regulates gene expression through interaction with the HP1 components of heterochromatin. Its normal function is important for α-globin expression, while the hematological phenotype of patients with ATMDS is attributed to ATRX mutations.⁶⁰

The Role of microRNAs in MDS and Epigenetic Machinery

microRNAs are small non-coding RNAs that regulate gene expression at the posttranscriptional level by inhibiting protein translation or destabilizing target transcripts. They are transcribed as long, capped and polyadenylated primary precursors (pri-microRNA) by RNA polymerase II.^61,62 Pri-microRNAs are then cleaved by Drosha (a nuclear RNase III) into a hairpin shaped precursor that is called pre-microRNA and consists of 70–100 nucleotides.⁶³ Drosha creates a complex with the dsRNA-binding protein, DGCR8 (DiGeorge syndrome chromosomal region 8),^64-66 which, unlike Drosha, can stably interact with pri-microRNAs.⁶⁷ In that way, Drosha is stabilized. Pre-microRNAs are exported to the cytoplasm by exportin 5.⁶⁸ At the cytoplasm, the RNase III Dicer in association with the dsRNA-binding protein TRBP (transactivation-responsive RNA-binding protein) and PACT (in humans), removes the terminal loop of pre-microRNAs and creates a 19–24 nucleotide microRNA duplex.^69-71 The protein complex RISC (RNA-induced silencing complex) binds to the aforementioned duplex. The mature microRNA is determined by the strand of the complex that remains stably associated with RISC. Then, the mature microRNA drives RISC to the target mRNA.^72,73 microRNAs bind to the 3′-untranslated regions (UTRs) of mRNAs targets by their “seed” region (nucleotides 2 to 8 counted from their 5′ end) through perfect (in plants) or imperfect (in mammals) base-pairing.⁷⁴

microRNAs have been associated with almost every normal cell function as well as with normal hematopoiesis and hematologic malignancies including MDS.⁷⁵ These small non-coding RNAs hold a leading role in the epigenetic machinery acting in a bidirectional way: They target key molecules of the epigenetic reactions but are susceptible to epigenetic regulation themselves. However, the available data on this interesting, albeit confusing, subject are still very few. The results of different studies overlap or even oppose to each other suggesting that there is a long way to the understanding of microRNA function in MDS.

Expression of microRNAs in MDS

As shown in Tables 3 and 4, many studies using different techniques have tried to shed light on the role of microRNAs in MDS pathogenesis and prognosis, the exact pathways are not yet understood.⁸² Interestingly, the expression profile of microRNAs was found different between MDS patients and normal controls. Indeed, microarray and RT-PCR analysis of microRNA expression in mononuclear cells (MNC) of patients suffering from low risk MDS identified 13 microRNAs differentially expressed between patients and controls.⁷⁶ This was also true when Pons et al. compared the expression of microRNAs in MNC from PB and BM of patients and controls using RT-PCR. Twelve microRNAs were differentially expressed between these two groups.⁸⁰ Furthermore, expression analysis of microRNAs with Taqman arrays and RT-PCR showed differential expression between patients with 5q- syndrome and controls.⁷⁷ A recent study identified another microRNA whose expression differs between patients and controls. MiR-21 is overexpressed in bone marrow samples of MDS patients compared with normal controls and directly targets SMAD-7, by binding to its 3′-UTR. SMAD-7 is a negative regulator of TGF-β receptor-I kinase. Thus, SMAD-7 inhibition by miR-21 leads to the activation of TGF-β pathway and ineffective hematopoiesis and especially anemia and dysplasia. Indeed, miR-21 inhibition resulted in SMAD elevation in vivo and stimulated erythropoiesis in vitro.⁸¹ Finally, miR-150 has been found upregulated in patients with del (5q) MDS compared with controls and miR-221 was found decreased in sAML compared with other subgroups of MDS.⁷⁸

Table 3. Downregulated microRNAs in MDS patients

microRNA	MDS subtype	Method	Reference
miR-10b, miR-103, miR-126, miR-140, miR-150, miR-342, miR-365, miR-378, miR-483, miR-558, miR-632, miR-636, miR-639	Low risk MDS	Agilent miRNA microarray and RT-PCR	76
miR-128b, miR-213, miR-95, miR-520c	5q- syndrome	Taqman miRNA microarray and RT-PCR	77
miR-221	sAML	RT-PCR	78
miR-196a*, miR-423–5p, miR-525–5p, miR-507, miR-583, miR-940, miR-1284, miR-1305, HS_122.1	All subtypes	Illumina miRNA microarray	79
miR-124, miR-206, miR-146a, miR-150, miR326, let-7e, mir-197, mir-875–5p	All subtypes	miRNA microarray-OSU3 and RT-PCR	85

Table 4. Upregulated microRNAs in MDS

microRNAs	MDS subtype	Method	Reference
miR-17–3p, miR-17–5p, miR-18a, miR-10a, miR-10b, miR-15a, miR-16, miR-222, miR-155, miR-181a, miR-21, miR-126	All subtypes	RT-PCR	80
miR-10a, miR-10b, miR-125a, miR-126, miR-130a, miR-148a, miR-151, miR-199a, miR-199b, miR-24, miR-29c, miR-335, miR-34a, miR-486, miR-99b	5q- syndrome	Taqman microRNA microarray and RT-PCR	77
miR-21	RCMD and RAEB	RT-PCR	81
miR-150	MDS-del(5q), MDS with low risk karyotype	RT-PCR	78
miR-299–3p, miR-299–5p, miR-323–3p, miR-329, miR-370, miR-409–3p, miR-431, miR-432, miR-494, miR-654–5p, miR-665, HS_40, HS_206	All subtypes	Illumina array	79
miR-222, miR-10a, miR-196a, miR-320, miR-100	All subtypes	miRNA microarray-OSU3 and RT-PCR	85

In addition, microRNA expression differs between MDS subgroups and is associated with disease prognosis. Hussein et al. used low-density RT–PCR based array and found different microRNA signature between patients with normal karyotype and those with chromosomal alteration.⁸³ Furthermore, Merkerova et al. used illumina array platform and demonstrated that 22 microRNAs distinguished between patients and controls and MDS subtypes.⁷⁹ Our group found that mir-17–5p and mir-20a were under expressed in high risk MDS, compared with low risk MDS patients and that let-7a was under expressed in patients with intermediate or high-risk karyotype. Additionally, mir-17–5p and mir-20a expression was associated with increased overall survival of MDS patients suggesting that these two microRNAs are favorable prognostic factors.⁸⁴ Finally, Sokol et al. focused on the prognostic value of microRNA expression in MDS and found 10 microRNAs that were associated with International Prognostic Scoring System (IPSS) and that patients with high mir-181 expression had worse overall survival than patients with lower expression.⁸⁵

microRNAs control epigenetic machinery in MDS

microRNAs that affect the epigenetic machinery are called epi-microRNAs. The first study reporting the existence of epi-microRNAs was performed by Fabbri et al., who found that mir-29 family downregulates DNMT3A and B levels in lung cancer cells and its enforced expression results in DNA hypomethylation (through DNMT3A and B inhibition) and tumor-suppressor genes re-expression.⁸⁶ The same group confirmed the role of mir-29b in DNMT1, DNMT3A and B downregulation in AML cells. They illustrated that mir-29b downregulates DNMT3A and B by binding to the 3′- UTR of their mRNA and DNMT1 by inhibiting the transcription factor Sp1 (specific protein 1). In addition, enforced expression of mir-29b reduced global DNA methylation while its overexpression resulted in upregulation of the tumor suppressor genes ESR1 and p15INK4b.⁸⁷ These studies illuminated the role of mir-29 family in lung cancer and AML and paved the way for the possible therapeutic use of mir-29. Despite the paucity of data for the role of mir-29 family in MDS, we can hypothesize that, similarly to AML, overexpression of DNMTs and global promoter hypermethylation (including p15INK4b promoter) occurring in MDS could be associated with mir-29 function. Therefore, studies of the role of mir-29 in MDS pathogenesis and prognosis would be of great interest.

Besides their role in posttranscriptional regulation of gene expression, microRNAs seem to act as regulators of gene translation and hematopoietic stem cell differentiation too. Mir-223, which regulates human granulopoiesis by targeting the nuclear factor I-A (NFA-I)⁸⁸ localizes in the nucleus, forms a complex with PcG proteins and binds NFA-I promoter. This results in NFA-I silencing, which favors granulopoiesis. On the other hand, NFA-I activation leads to erythroid differentiation.⁸⁹ This is a novel pathway of direct control of hematopoietic stem cell differentiation by a microRNA suggesting that deregulation of mir-223 could contribute to impaired granulopoiesis and possibly disease pathogenesis. Indeed, in AML, AML-ETO fusion gene binds to mir-223 promoter, recruits a complex consisting of DNMTs, HDAC and MCPS and results in its silencing. This modification is associated with impaired myeloid differentiation, which characterizes AML blast cells.⁹⁰

Epigenetic regulation of microRNA expression in MDS

Apart from their role in regulation of gene expression, microRNAs are susceptible to epigenetic control themselves. microRNA expression depends on their genomic location (microRNAs associated with cancer are located in genomic fragile regions), transcription regulation (i.e., promoter hyper/hypomethylation, transcription factors, etc.) and posttranscriptional events including maturation process.

A recent study demonstrated that Dicer and Drosha, two ribonucleases necessary for microRNA maturation, are found downregulated in mesenchymal stromal cells (MSC) from BM of MDS patients compared with normal controls and MNC from MDS patients. The same study found global microRNA downregulation (including the hematopoiesis related mir-155, 181 and 222), which shows that the maturation mechanism of microRNAs in these cells is impaired. Additionally, the authors note that mir-155, 181 and 222 target Dicer and Drosha, which highlights the complexity of the pathways involved in MDS pathogenesis.⁹¹ To our knowledge, this is the first study that brings evidence of decreased levels of Dicer and Drosha in BM MSC from MDS patients and addresses the role of the microenvironment in MDS.

Another microRNA affected by the epigenetic machinery in MDS is miR-124. It is transcribed in three regions of the genome as miR-124a-1 (8p), miR-124a-2 (8q) and miR-124a-3 (20q). In the study of Castoro et al., miR-124a-1 was methylated in the majority of MDS (78%) and AML (95%) patients and was associated with worse overall survival while miR-124a-3 was methylated in 47% of MDS and 65% of AML patients. Treatment with the DNA methylation inhibitor decitabine (DAC) resulted in a significant decrease in methylation status of miR-124a-1 and 3 in patients who responded to treatment. On the other hand, non-responders had no significant de-methylation of this microRNA. Methylation levels were measured at day 5, 12 and 30 after DAC treatment. In addition, patients who responded to DAC showed a significant increase in miR-124 expression at day 5 while patients who did not respond to treatment had no significant increase in miR-124 expression. Finally, miR-124 re-expression was associated with the repression of its target mRNA, CDK6, at day 5.⁹² This study provides a representative example of the effects of DNA methylation and epigenetic therapies on microRNA expression. Of interest, these results were confirmed by a recent study according to which, miR-124 is downregulated in MDS patients and is related with the level of its promoter methylation.⁹³

The role of the aforementioned miR-124 in MDS pathogenesis and its relationship with its regulator EVI1 was later studied by another group. EVI1 is a transcription factor that regulates the expression of many genes responsible for cell cycle, including SMAD3 (a member of TGF-β pathway) and generates a model of MDS when expressed in murine bone marrow. Its activation in MDS patients is associated with megakaryocytic and erythroid dysplasia, refractory anemia and bone marrow failure. Dickstein at al created an MDS model by expressing EVI1 in murine bone marrow and studied the role of this transcription factor in MDS pathogenesis. One of the major pathways resulting in MDS phenotype in this study was miR-124 silencing by CpG methylation of its promoter.⁹⁴ These data, which are summarized in Figure 1, demonstrate an example of epigenetic alteration of microRNAs in MDS pathogenesis.

Figure 1. Epigenetic alteration of microRNAs and MDS pathogenesis Mir-124, which is hypermethylated and under-expressed in MDS patients, participates in MDS pathogenesis. It down-regulates the cyclin dependent kinase 6 (CDK6), which in turn phosphorylates and inactivates the tumor-suppressor protein Rb. EVI1 represses miR-124 expression by promoter methylation. It also down-regulates the expression of SMAD3, a member of TGF-β pathway that regulates cell differentiation, proliferation and apoptosis. Mir-124 activity can be restored by the treatment with the DNA methylation inhibitor, Decitabine. The TGF-β pathway is also regulated by mir-21 which down-regulates the expression of SMAD7, which in turn is a negative regulator of TGF-β receptor.

MiR-10a expression is induced by the transcription factor TWIST-1, which is upregulated in CD34+ cells of MDS, interacts with the DNA-binding domain of p53 and leads to resistance in apoptosis. In addition, TWIST-1 binds to the promoter of miR-10a/b and induces its expression. A recent study found that mir-10a/b is overexpressed in CD34+ cells from BM of MDS patients compared with healthy controls correlating with TWIST-1 levels. Furthermore, miR-10a/b, TWIST-1, NF-kB and p53 form a pathway which control sensitivity to apoptosis in MDS patients.⁹⁵ In summary, transcriptional regulation of mir-10a/b seems to be important for MDS pathogenesis and constitutes a candidate pathway for target therapy.

Epigenetic Therapy of MDS

The term epigenetic therapy refers to treatment of a disease by targeting epigenetic pathways. As illustrated above, the role of epigenetic mechanisms in MDS pathogenesis and prognosis has received very much attention. The observation that DNA methylation, histone modification and microRNA interference play a critical role in MDS raised hope that key molecules of these pathways could represent new therapeutic targets or even therapeutic agents. The first epigenetic drugs for MDS treatment, 5-azacytidine (azacitidine) and 5-aza-2’-deoxycytidine (decitabine), have already been used in MDS patients with promising results.

Targeting DNA methylation

The central epigenetic pathway (DNA methylation) has already been targeted by the aforementioned hypomethylating agents azacitidine and decitabine. Both drugs were synthesized in the 1960s and represent cytocine analogs in which the carbon at position 5 of the ring is replaced by N. Azacitidine incorporates first into RNA and then into DNA and binds irreversibly DNMTs causing its degradation. As a result, when DNA replicates, the newly synthesized DNA strands remain unmethylated. The anti-neoplastic effect of azacitidine is a dose-dependent: At low doses it inhibits DNMTs while in high doses is cytotoxic. It was established in the treatment of high-risk MDS with a randomized, open-label phase III study of azacitidine compared with conventional regimens, which proved the survival benefit of patients receiving azacitidine. Nowadays, azacitidine is used for the treatment of intermediate II and high risk IPSS MDS.⁹⁶ Decitabine, on the other hand, incorporates directly into DNA and inhibits DNMTs action in a similar way as azacitidine. It is also indicated for MDS and AML treatment in the US

Although azacitidine has proved its demethylating effect in vivo and vitro, questions were raised about the exact mechanism of action in MDS. According to many scientists the belief that demethylation itself is sufficient for the anti-tumor effect of azacitidine and decitabine is just simplicity of what really takes place into the cell. Indeed, there are still no data proving a direct relationship between DNA hypomethylation and disease response. On the other hand, azacitidine is administered in low doses in which the drug exhibits hypomethylating function.⁹⁷ Whether DNA hypomethylation is the only mechanism of action or a part of a complicated pathway still remains an open question. Finally, it should be mentioned that several clinical trials that study the combination of azacitidine with other agents like lenalidomide, valproic acid etc or even oral administration of azacitidine have been already launched aiming to change the current treatment approach of MDS.

Targeting histone modification

Targeting histone modification currently means targeting histone deacetylases. As discussed above, histone modifications are involved in MDS pathogenesis so the use of histone deacetylace inhibitors (HDACi) in MDS treatment seems rational. HDACi induce differentiation, cell cycle arrest and expression of p21^cip/waf1.^98,99 However, the exact mechanism of their action has not been clarified yet. The application of HDACi as therapeutic agents of hematological malignancies is still under clinical research. A phase II study of belinostat (PXD101) for the treatment of MDS stopped at the first stage of enrollment.¹⁰⁰ However, there are several phase I and II clinical trials of HDACi with or without azacitidine and chemotherapy regiments for the treatment of MDS and AML. In addition, it has been showed that valproic acid targets HDAC and in combination with all-trans retinoic acid can induce differentiation of malignant cells.^101,102 Thus, the possibility that new therapeutic agents will change the current treatment of MDS cannot be excluded.

Conclusions and Future Directions

In this review we have summarized the current knowledge in epigenetic alterations, including microRNAs, in MDS. Growing evidence supports the role of aberrant DNA methylation, histone modification and microRNA regulation in MDS pathogenesis and prognosis and helps decipher the complicated pathways that are involved in this disease. The greatest offer of these data so far is the development of epigenetic therapies. The most illustrative example is azacitidine, which is the only agent associated with increased overall survival in MDS patients. However, the development of drugs that target histone modification is expected to change thoroughly the therapeutic approach in MDS.

In conclusion, the current knowledge on epigenetic alterations in MDS seems to be only the tip of the iceberg since many important issues remain unclear. Further studies are required that will elucidate the exact role of epigenetic alterations in MDS as well as the role of microRNAs in epigenetic machinery.

Acknowledgments

The authors acknowledge the help of Dr. A. Pouliakis who designed Figure 1.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.