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Epigenetic mechanisms such as DNA methylation and histone modifications drive stable, clonally propagated changes in genes expression and can therefore serve as molecular mediators of pathway dysfunction in neoplasia. MDS is characterized by frequent epigenetic abnormalities, including the hypermethylation of genes that control proliferation, adhesion, and other characteristic features of this leukemia. Aberrant DNA hypermethylation is associated with a poor prognosis in MDS that can be accounted for by more rapid progression to AML. In turn, treatment with drugs that modify epigenetic pathways (DNA methylation and histone deacetylation inhibitors) induce durable remissions and prolong life in MDS, offering some hope and direction in the future management of this deadly disease.
The myelodysplastic syndromes are a group of diverse and heterogeneous syndromes characterized by clonal proliferation, bone marrow failure and an increased risk of development of AML95. The natural course of the disease ranges from slow progression to rapid evolution into AML. There are several classification systems for MDS such as the International Prognostic Scoring System (IPSS)30, the WHO classification10, or more recent clinical schemes53 but none captures the full heterogeneity of the disease. Heterogeneity and progression are likely related to molecular changes that drive the disease phenotype and biology, but these remain incompletely understood. Insights into the pathogenesis of cancer has come from studying genetic changes in neoplastic cells38;76. In MDS, several cytogenetic abnormalities have been identified that characterize subgroups of patients such as deletions of chromosome 5 and/or 7 in patients with poor prognosis, isolated deletion of 5q or trisomy 8. Mutations of several genes also characterize subsets of cases, including RAS, TET2, RUNX1 and others. Despite these advances, the molecular causes of MDS and its peculiar clinical features remain poorly understood. Epigenetic changes have been recognized in the past decade as major drivers of the malignant phenotype48. Epigenetics refers to the study of clonally inherited changes in gene expression without accompanying genetic changes. There are three general molecular mechanisms carrying epigenetic information – DNA methylation, histone modifications and RNA interference15;43. In cancer, there has been particular recent interest in the involvement of aberrant DNA methylation and histone modifications in gene silencing that then mediate altered physiology9;49. They may be particularly relevant to MDS pathogenesis given that the disease responds quite well to drugs that affect DNA methylation40, a major epigenetic modulator. In this article, recent progress on MDS epigenetics and epigenetic-based therapies will be reviewed.
DNA methylation is a covalent modification of cytosines resulting in the formation of 5-methyl-cytosine (5mC), a base that changes the interactions between protein and DNA. CpG methylation can occur anywhere in the genome, but is particularly relevant when it involves CpG rich regions called CpG islands. In turn, these can be present in about half of human gene promoters. CpG island methylation is associated with absent transcription from the involved promoter, and this silencing is stably transmitted through mitosis, thus insuring clonal inheritance12. This association between CpG island methylation and absent transcription is most striking when one considers the two physiological conditions where this process was described, X-inactivation34 and imprinting7. In both cases, one of the two copies of the involved genes is transcriptionally silent in association with promoter methylation, despite continued normal expression of the unmethylated allele. Evidence for a direct role of methylation in maintaining the silenced state came from studies where methylation was relieved via pharmacologic47 or genetic66 reduction in DNA methyltransferase (DNMT) activity. In most such studies, bi-allelic expression could be restored, in association with decreased methylation of the affected promoters.
The mechanism whereby CpG island methylation suppresses gene transcription has been partially elucidated recently (Fig. 1), at least in vitro51. Methylated CpG islands form excellent binding sites for methylated-DNA binding proteins (often with transcriptional repression properties), such as MeCp2. MeCp2 binding is followed by the recruitment of a protein complex that includes histone deacetylases (HDAC), and eventually leads to a closed chromatin configuration. This closed chromatin configuration results in exclusion of transcription factors, thus insuring allele-specific inactivation. Methylation-related epigenetic silencing has also been found to be associated with histone H3 lysine 9 (H3K9) methylation43. Evidence suggests that H3K9 methylation is a critical modification that is associated with closed chromatin at DNA methylation sites, and it was proposed that a cascade of events follows DNA methylation (MeCP2 binding, H3K9 deacetylation, H3K9 methylation) and ensures transcriptional suppression22;59;74 (Fig. 1). Separately, DNMT1 can directly suppress transcription (without DNA methylation) through interactions with histone deacetylases25;89. H3K9 methylation itself appears to set-up a silencing loop by attracting more DNA methylation97, and may sometimes precede hypermethylation5.
There are complex changes in DNA methylation in cancer. For the most part, these changes involve simultaneous global demethylation, increased expression of DNMTs and de-novo methylation at previously unmethylated CpG islands. Demethylation was first discovered by studying overall 5-methy-cytosine (5mC) content in tumors, and appears to involve primarily satellite DNA, repetitive sequences, and CpG sites located in introns23;44. The cause of this demethylation remains unclear, although it could be related to alterations in proliferation or cell-cycle control29. The functional consequences of hypomethylation are not entirely clear, but there is mounting evidence that gene-specific hypomethylation can cause increased expression of various genes that could contribute to the neoplastic phenotype21. An increased mutation rate was demonstrated in cells in which severe hypomethylation (>75%) was achieved by homozygous deletion of DNMT116, but it is not clear whether this degree of hypomethylation is ever achieved in neoplasms107.
Increased enzymatic DNMT activity is a property of nearly all transformed cells55. Increased mRNA levels for DNMT1, DNMT3a and DNMT3b have also been described in some neoplasms including leukemias19;41;85, and these three DNA-methyltransferase genes probably account for the observed increase in activity. The causes and functional significance of these increases remain unclear. DNMTs appear to be cell-cycle regulated96, and it has been argued that DNMT levels do in fact reflect the physiologic state of increased proliferation in neoplasia63. On the other hand, DNMT1 has been reported to increase following oncogene activation6;88, and it is possible that its levels in neoplasia reflect the various molecular defects seen in tumors. The functional significance of increased DNMT activity is also poorly defined. In several systems, increased DNMT activity has been found to be transforming6;106, but it is not clear whether this is due to increased CpG island methylation and tumor-suppressor gene silencing, or due to direct effects on the cell-cycle. The facts that, in primary tumors and cell lines, no correlation was found between DNMT activity and gene silencing67;100, and that DNMT-related transformation is reversible when the oncogenic stimulus is removed6 support the latter possibility. Nevertheless, simultaneous inhibition of multiple DNMTs does inhibit cancer cell growth82, making them potential therapeutic targets.
In parallel to global hypomethylation and increased DNMT activity, there also are distinct and frequent localized increases in methylation, often involving CpG islands9;49. Because CpG island methylation is associated with repressed transcription that is stably inherited through mitosis, this de-novo methylation in transformed cells has been proposed to serve as an alternate mechanism for inactivating tumor-suppressor genes48. Indeed, several genes have now been shown to be transcriptionally silent in neoplasia, in association with CpG island methylation, including in leukemias. The most convincing evidence for CpG island methylation as a true alternative to mutations in neoplasia came from studies of RB190, p1673, VHL36 and MLH152. For each of these genes, the tumor-spectrum of methylation events is virtually the same as that for mutations, and there are described cases where one allele of the gene is inactivated by methylation while the other is mutated, suggesting an equivalent growth advantage for each event in neoplasia. For example, in colorectal cancer, the HCT116 cell line carries one mutated unmethylated p16 allele, while the second allele is unmutated but densely hypermethylated and transcriptionally silent73. Moreover, the MLH1 mismatch repair gene is unmutated but densely methylated in the mismatch repair deficient cell line RKO, and nearly normal levels of mismatch repair can be restored by inhibiting DNMT activity37.
Histones are small proteins that form a core around which DNA is wrapped, forming nucleosomes. Nucleosomes are the basic in-vivo structural unit of DNA, and consist of 8 histone molecules (2 each of Histones H2A, H2B, H3 and H4) around which a loop of DNA is wrapped56. While H2A and H2B are thought to play primarily a structural role, it has become apparent that Histones H3 and H4 and key integrators of a variety of signals that regulate gene transcription43;62;83. In particular, these two histone proteins have histone “tails” or strings of amino acids that protrude outside of the basic nucleosomal structure and make contact with DNA. Specific post-translational modifications of the amino acids in these histone tails (e.g. methylation, acetylation, phosphorylation, ubiquination, sumoylation) interact with other proteins to create nucleosomes that are relaxed and promote transcription, or nucleosomes that are closed, exclude transcription factors and result in gene silencing. These histone modifications occur relatively dynamically and are mediated by specific histone modifying proteins. Targeting of these histone modifiers to specific gene promoters in-turn is achieved by transcriptional activator/co-activator complexes in response to physiologic stimuli. Thus, histone modifications form a “code” that integrates gene activation/inactivation/silencing signals, such that the transcriptional activity of a given promoter can be predicted by looking at the specific histone modifications43.
Currently, the best understood histone modifications are acetylation and methylation of specific residues, although it is clear that other modifications also play a role in the process43. Acetylation of specific residues on histone H3 and H4 is typically associated with active gene transcription32. Methylation of specific residues can be associated with either activation of transcription (H3K4) or silencing (H3K9, H3K27)62. The different modifications also significantly interact. Thus, H3K4 methylation promotes H3K9 acetylation. Moreover, H3K9 can be either acetylated or methylated (but not both), and the switch from acetylation to methylation at this residue appears to be a key element of silencing across many organisms. H3K9 promotes silencing by recruiting HP1 and a silencing complex that changes chromatin structure locally and results in exclusion of transcription factors. An added complexity to the process was demonstrated when residues were shown to have several possible methylation states77;84;92. For H3K9, mono and dimethylation are associated with euchromatin silencing, and trimethylation is associated with pericentric heterochromatin silencing. The H3K9 methylation/silencing switch appears conserved across evolution and is active in mammalian cells62. H3K27 methylation appears distinct from H3K9 methylation; H3K27 trimethylation has been reported to occur early in X-chromosome inactivation87, and leads to silencing by recruitment of polycomb group (PCG) proteins75. By contrast H3K9 trimethylation is primarily a mark of heterochromatin, and leads to silencing by recruitment of HP1 proteins62. Thus, these two processes, which are mediated by different enzymes, are potentially functionally distinct. There is now considerable interest in the involvement of the histone code in stemness and differentiation. A bivalent chromatin domain has been described in embryonic stem cells11, whereby some unexpressed genes have co-existence of activating (H3K4Me) and silencing marks (H3K27Me). Upon differentiation, this bivalency resolves either into the activated or the suppressed state.
While histone modifications are relatively stable, histones are eventually degraded, and the nucleosome structure is substantially altered during DNA synthesis28. Moreover, histone acetylation and methylation can be directly reversed by a large number of histone deacetylases and demethylases. Thus, for histone modifications to retain epigenetic memory, some form of targeting has to be operative. DNA methylation is one such form of targeting; so is persistent expression of transcription factors. It has also been argued that the histone modifications themselves could be a form of epigenetic memory in the absence of DNA methylation or other proteins to direct the process33.
Histone modifications have been implicated in the neoplastic process in a variety of ways. Indirectly, a number of genes that are altered in cancer affect gene expression via recruitment of histone modifying enzymes and resulting changes in gene expression. For example, the PML-RAR gene translocation in acute promyelocytic leukemia recruits HDAC to inhibit the expression of target genes, thus contributing to malignant transformation72. More directly, several histone-modifying enzymes are molecularly altered in cancer. For example, the histone acetyltransferase CBP is mutated in some acute leukemias64, the MLL gene, an H3K4 methylase is rearranged in a significant portion of acute leukemias3, the RIZ1 gene, a putative histone methylase is silenced in some cancers18 and EZH2, a H3K27 tri-methylase is over-expressed in various malignancies103 which also exhibit de-novo H3K27triM mediated silencing58. Another mechanism modifying the histone cancer code in cancer is aberrant promoter methylation35. Such DNA methylation may lead to silencing via recruitment of histone modifying enzymes that ultimately alter the histone code in favor of gene silencing.
Most studies of epigenetics in MDS have focused on DNA methylation so far. Several genes have been shown to be transcriptionally silenced in association with promoter DNA methylation in this disease (Table 1). These include genes involved in cell-cycle regulation (CDKN2A), apoptosis (DAPK1, RIL), adhesion and motility (CDH1, CDH13) and others. Separately, some of these genes clearly have minimal functional impact on the disease, not being expressed in normal hematopoietic cells. MDS cases often show hypermethylation of several genes simultaneously93. Thus, hypermethylation can be viewed in a similar way as mismatch repair deficiency and microsatellite instability in cancer: many loci are affected simultaneously, a few of which likely have functional consequences.
In MDS, CDKN2B (P15) has been the most extensively studied gene. CDKN2B was reported to be methylated in 30-80% of the cases, with the variability being likely due to different methods of measurement, as well as inclusion of different types of MDS. Thus, CDKN2B methylation has been reported to be very frequent in therapy related MDS, as well as in CMML, in RAEB-T or AML arising from MDS4;17;81;99;101. CDKN2B methylation in MDS has also been associated with older age, deletions of 5q and 7q, and a poor prognosis4;17;81;99;101. Interestingly, when present, CDKN2B methylation in MDS has been shown to affect multiple lineages from clonogenic cells to circulating mononuclear cells2. In a mouse model, loss of CDKN2B was associated with enhanced myeloid progenitor and reduced erythroid progenitor formation86, suggesting that its inactivation plays a functional role in MDS.
In a recent study focusing on quantitative analysis of the methylation status of 10 separate genes, a hypermethylator phenotype was identified that marks a subset of cases with MDS93. This phenomenon, first described in colon cancer100, results in the simultaneous inactivation of multiple genes by an unknown mechanism. In MDS, this form of intense hypermethylation is associated with rapid progression to AML and a shortened survival in multivariate analyses93. This explains in part why the methylation of so many genes has been reported to be prognostic in MDS (Table 1): In all likelihood, all these studies of individual genes are pointing to a common subset of cases affected by the hypermethylator phenotype.
Whole genome scans for DNA methylation are beginning to reveal the complexity of the disease at an epigenetic level24;45. Hundreds of genes are frequently hypermethylated in MDS, with evidence for enrichment of WNT pathway genes24. As indicated by studies of individual genes, hypermethylation across the genome is associated with poor prognostic features and transformation to AML. Intriguingly, there was a distinct methylation pattern in AMD and related AMLs compared to de-novo AML, pointing to distinct pathogenic mechanisms24. Thus, a consensus has emerged that DNA methylation is abnormal early on in MDS, and that progression of the disease is associated with accumulation of additional epigenetic events.
By contrast to DNA methylation, detailed studies of histone modifications in MDS remain to be described. A few cases of AML have chromosomal translocations involving MLL, a known epigenetic modifier. Recently, mutations in the polycomb related gene ASXL1 were described in about 10% of cases27, and mutations in the TET2 gene were found in about 25% of cases. TET2 is related to TET1, a gene which converts 5-methylcytosine to 5-hydroxymethylcytosine, and thus may be involved in regulation of DNA methylation. It is not known whether MDS cases with abnormalities in MLL, ASXL1 or TET2 have characteristic epigenetic patterns.
Treatment of cancer by targeting epigenetic pathways has been referred to as epigenetic therapy20. The principle of this approach is to reverse pathologic gene expression changes in malignant cells, which is presumed to elicit a therapeutic effect that culminates in tumor responses. The specific therapeutic effect induced by these agents is still a matter of debate, and there are data favoring many differing pathways as affected by this therapy, including differentiation, senescence, apoptosis, immune recognition etc.40 In addition, many epigenetic drugs show dose-dependent cytotoxicity8;80, which could also be a factor in responses. The therapeutic index of this approach lies, in principle, on the fact that cancer cells are more dependent on continued silencing tumor-suppressor genes and like molecules than normal cells. There have been concerns that epigenetic therapy could have dramatic effects on normal cells through reactivating, for example, imprinted genes, and may even lead to cancer formation. In-vivo, however, these fears have not been substantiated in studies so far, with no evidence of unusual side-effects, new chromosomal defects or secondary malignancies107. It is likely that the doses of drugs that elicit such effects are not achievable in-vivo.
Targeting epigenetics at the present time essentially equates to targeting silencing pathways in cancer and their potential interactions. A central pathway (Fig. 1) involves DNA methylation, methyl-binding proteins, histone deacetylation, histone (H3K9) methylation and eventual binding of a silencing protein complex that, itself, may trigger more methylation57. This “silencing loop” is stable, as evidenced by imprinted genes and the inactive X-chromosome, which remain turned off for decades in adult cells. There is also a distinct silencing mechanism in development and cancer that involves the PCG complexes PRC1 and PRC2, resulting in H3K27me3 modification. These complexes also involve histone deacetylases. Each step in these silencing cascades is a potential target for therapeutic intervention. Combining inhibition of multiple epigenetic targets will likely be synergistic, as demonstrated for DNA methylation and HDAC inhibition14. There are two epigenetic targets for which drugs are available in clinical trials – DNA methylation and histone deacetylation.
5-Azacytidine (azacitidine) and 5-aza-2′-deoxycytidine (DAC, decitabine) are two hypomethylating cytosine analogues with activity in leukemia65;91. Both drugs were synthesized in the 1960’s as cytosine analogues, and were shown in the early 1980’s to be potent DNA methylation inhibitors and in-vitro differentiation inducers50. DNA methylation inhibition is related to the shared modified structure of the cytosine ring with a C to N substitution at the 5 position, resulting in trapping and eventual degradation of DNA methyltransferases. Azacitidine incorporates into RNA and, after intracellular conversion to DAC, incorporates into DNA and inhibits DNA methylation. Unlike azacitidine, DAC does not incorporate into RNA and is directly incorporated into DNA, resulting in 10 fold higher demethylating activity at equimolar concentrations in-vitro50. Both drugs have activity in MDS demonstrated in randomized studies65;70;94, and both are now approved by the United States Food and Drug Administration. A feature of both azacitidine and DAC studies in MDS has been delayed clearance of blasts, delayed myelosuppression, slow responses (median number of cycles to best response was >3) and eventual cytogenetics responses. A similar phenomenon has been seen with CML patients treated with DAC54. These studies have suggested that the mechanism of action of these drugs is not cytotoxicity, although the exact mechanism of achieving complete remissions is not known.
There are a variety of Histone deacetylase inhibitors (HDACi) in clinical trials currently71. These include drugs discovered through activity in the NCI-60 cell panel (romidepsin), drugs discovered through in-vitro differentiation screens (vorinostat), or drugs discovered to be HDACi serendipitously (Valproic Acid78). For all these drugs, in-vitro inhibition of HDAC activity was demonstrated, as well as gene reactivation and induction of apoptosis. Once again, the mechanism of downstream action of these drugs is unclear, with recent data pointing towards DNA damage and effects on reactive oxygen species102 (in addition to gene expression activation). Clinically, several of these drugs are in phase I/II studies, with activity demonstrated for depsipeptide and SAHA in lymphomas79, and both drugs are now approved for the treatment of cutaneous T-cell lymphoma. Less data is available in MDS and AML, though anecdotal responses have been reported26;61.
Drugs targeting other epigenetic pathways are currently in development or pre-clinical studies. There is particular interest in drugs that can inhibit the activity of various histone methyltransferases60;98, as these could work independently of (and complement) DNA methylation and histone deacetylation inhibitors. It is likely that several drugs targeting epigenetic pathways will enter clinical trials in MDS in the next few years.
Epigenetic pathways mediate cancer-specific gene expression abnormalities in multiple genes involved in determining the neoplastic phenotype. MDS is characterized by multiple epigenetic abnormalities that can help determine prognosis and risk of progression to AML. Drugs that target epigenetic pathways have changed the natural history of MDS and offer some hope that the disease could be treated successfully in the next decade.
Relevant work in the author’s laboratory is supported by National Institutes of Health grants CA100632, CA098006, and CA121104. JPI is an American Cancer Society Clinical Research professor supported by a generous gift from the F. M. Kirby Foundation.
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