DNA Methylation Inhibitors: Retrospective and Perspective View

Introduction

DNA methylation is a hallmark in current cell biology research and constitutes one of the most important epigenetic alterations that contributes to both diseases and normal biologic development. DNA methylation contributes to vital biologic processes such as chromosome stability, genome integrity, embryogenesis, cell differentiation and growth (1, 2). Apart from DNA methylation, other epigenetic mechanisms like histone modifications, miRNA and long non-coding RNAs (lncRNAs) also regulate gene expression (3, 4).

DNA methylation is a covalent attachment of a methyl group at the C-5 position of cytosine forming 5-methyl cytosine (5mC) in the cytosine-guanosine (CpG) dinucleotide sequence that are frequently located in the promoter regions of CpG-rich gene sequences known as CpG islands. In mammalian genomic DNA, about 5% of cytosine exists as 5mC. There are approximately 28 million CpG sites in the entire genome and approximately 70% of 5mC is located within these CpG sequences. Non-CpG methylation, such as methylation of CpA and CpT, is very uncommon and exists only under restricted conditions. Majority of CpG islands are 500 – 1000 base pairs (bp) in length and commonly associated with housekeeping genes and tissue-specific genes, which suggest that DNA methylation in CpG sites has noticeable consequences on gene expression (5). CpG DNA methylation near gene promoters has direct consequences on chromatin structure by recruiting methyl binding proteins and histone deacetylases (HDACs) to repress transcription. Moreover, methylated DNA can prevent the binding of methylation-sensitive transcription factors such as E2F and CREB leading to blockade of transcription (6–8).

As discussed so far, DNA methylation is a vital mechanism in normal cell development and plays important role in the regulation of gene expression. On the other hand, aberrant DNA methylation can contribute to disease conditions like inflammatory diseases, precancerous lesions and cancer (9 – 11). Recent studies have demonstrated that aberrant DNA methylation caused by alterations in DNMT activity is a hallmark in the initiation and development of various cancers through mechanisms like inactivation of gene transcription, repression of gene transcription or by affecting chromatin structure and function (12, 13). It is well documented that methylated DNA is closely associated with closed inactive heterochromatin (14, 15). On the Contrary, CpG islands in the promoter region of tumor suppressor genes (TSGs) are unmethylated and associated with open euchromatin conformation (12, 16).

DNA methyltransferase (DNMT) enzymes

DNMT enzymes catalyze the transfer of methyl group from S-adenosyl methionine (SAM), which acts as a universal donor of methyl group to cytosine DNA (17). The DNMT family is comprised of five isoforms: DNMT1, DNMT2, DNMT3a, DNMT3b and DNMT3L. Reversal of DNA methylation is catalyzed by the Ten-Eleven Translocation (TET) enzymes family and can also be mediated by a passive mechanism due to loss of function mutations in the DNMT enzymes. The role of DNMT and TET enzymes in DNA methylation is summarized in Figure 1.

Figure 1. DNA methylation and demethylation regulation.

Maintenance and de novo DNA methylation are catalyzed by DNMT1 and DNMT3a/3b, respectively. Loss of function (LOF) mutation of DNMT enzymes induces passive demethylation. TET enzymes catalyze active demethylation by sequential oxidation reactions that leads to the formation of sequential intermediate products like 5hmc, 5-formylcytosine and finally 5-carboxylcytosine, which is decarboxylated to form cytosine. C, mC, and hmC indicate cytosine, methylcytosine, and 5 hydroxylmethylcytosine, respectively.

DNMT1 is responsible for the maintenance of methylated DNA by regenerating fully methylated DNA state during cell division (18). DNMT2 and DNMT3L are catalytically inactive and the former one is in fact, an RNA methyl transferase that mostly methylates transfer RNAs (tRNAs) (19). DNMT3L is a DNA methyl transferase-like protein that modulates DNMT3a and DNMT3b to synergize their catalytic activities. The main de novo methylation enzymes are DNMT3a and DNMT3b and are mainly involved in the development of embryonic stem cells, where they target unmethylated CpG sequences and establish new DNA methylation patterns. Despite their similar catalytic activities, DNMT3a and DNMT3b exhibit distinctive and site-specific activity (20, 21). For instance, DNMT3a exerts higher methylation activity than DNMT3b on naked DNA. Moreover, DNMT3b significantly methylates DNA in the core region of nucleosomes while DNMT3a hardly does this. The reason for these differences is not clear, but it is speculated that the expression pattern of DNMTs at different times during embryonic development is contributing to such site-specific catalytic activities (22). Table 1 summarizes the regulatory function of the enzymes involved in DNA methylation/demethylation and the different tumor types caused by their dysregulation.

Table 1.

Key Regulatory Factors and their Functions in DNA Methylation/Demethylation

Regulator	Function	Cancer type	Biological processes involved
DNMT1	Maintenance DNA methyltransferase	Acute myeloid leukemia (AML) pituitary adenoma, colorectal cancer, pancreatic cancer, gastric cancer, lung cancer, thyroid cancer	Cell apoptosis, cell proliferation
DNMT3a	De novo DNA methyltransferase	Vulvar squamous cell carcinoma, Pituitary adenoma, AML	Cell proliferation and invasion
DNMT3b	De novo DNA methyltransferase	Lung cancer, hepatocellular carcinoma, ovarian cancer, breast cancer	Cell proliferation and invasion
DNMT3L	Cofactor in De novo DNA methyltransferase
TET1	DNA demethylation via hydroxylation of methylcytosine	AML, breast cancer, ovarian cancer, lung cancer, renal cancer	Cell migration and invasion
TET2	DNA demethylation via hydroxylation of methylcytosine	AML Chronic myelomonocytic leukemia Lymphomas Myeloproliferative neoplasms	Cell proliferation, colony formation and metastasis
TET3	DNA demethylation via hydroxylation of methylcytosine	Renal cell carcinoma, head and neck cancer, ovarian cancer, breast cancer	Embryonic development
MBD1	Methyl CpG binding protein	Pancreatic cancer, prostate cancer	Cell proliferation, invasion and metastasis
MBD2	Methyl CpG binding protein	Lung cancer, colon cancer, breast cancer, prostate cancer	Cell invasion and metastasis

While DNA methylation is a stable and highly conserved epigenetic modification in many organisms, DNA demethylation and subsequent depletion of 5mC are also key factors in many biological processes. For example, DNA demethylation is an important step towards pluripotent functionality of the primordial germ cells (PGCs) (23, 24). Furthermore, disruption of normal DNA demethylation is considered to be associated with oncogenesis.

DNA methylation and genomic instability

DNA methylation is directly involved in modulating the genomic integrity (25). While hypermethylation in the CpG promoter regions can silence TSGs and affect key cellular processes such as DNA repair, genome-wide hypomethylation is responsible for increased genomic instability (26–28). CpG-poor regions normally tend to undergo hypomethylation during tumorigenesis, resulting in global decrease in DNA methylation as observed in several cancers such as colon adenocarcinoma, prostate cancer and chronic lymphocytic leukemia (29–32). Extensive hypomethylation in intergenic and intragenic regions was observed in studies with breast tumor cell lines such as MCF-7 and MDAMB-231 and was associated with fragile sites and chromosomal breakpoints. These findings suggests that DNA hypomethylation contributes to genomic instability (4).

On the other hand, promoter hypermethylation of TSGs is another contributing factor to genomic instability. The common targets for such methylation are DNA repair genes and cell cycle regulatory genes that are closely associated with genomic instability. For example, epigenetic silencing of MLH1 gene involved in DNA repair may cause an ultimate downstream genetic mutation that leads to genome-wide instability (33).

Current Approaches in Developing DNA methylation Inhibitors

Global alterations in epigenetic modifications are one of the hallmarks of carcinogenesis. The three main epigenetic alterations involved in initiation and development of tumors are aberrant DNA methylation, histone modifications and aberrant expressions of miRNA (34). In this review, we will focus mainly on DNA methylation inhibitors and discuss the different classes of DNMT inhibitors under development, relevant mechanisms involved, potential challenges and future directions. Several approaches may be considered in developing drugs for reversing aberrant DNA methylation like inhibition of the universal methyl group donor SAM, downregulation of methyl-CpG binding domain (MBD) proteins and inhibition of DNMT enzymes (35–38). The expected outcome of the first two approaches is still questionable as they also interfere with other biological processes resulting in substantial non-specific undesirable effects. Taking these limitations into consideration, inhibition of DNMT enzymes is still considered the most preferred approach in developing DNA demethylating agents. DNMT inhibitors can be broadly classified into two groups, the nucleoside analogs and the non-nucleoside analogs (5, 17, 35). The nucleoside analogs include cytidine and S-adenosyl-L-homocysteine (SAH) derivatives, while the non-nucleoside inhibitors are mostly natural products and small molecules (39).

Nucleoside Analogs as DNA methylation Inhibitors

The rationale for developing nucleoside analogs as drug candidates is that they possess a modified cytosine ring that can be transformed into nucleotides and incorporated into newly synthesized DNA or RNA (Figure 2). Consequently, DNMT enzymes bind irreversibly to the analogs with subsequent depletion of the DNMT pool and inhibition of DNA methylation. 5-azacytidine (5AC) and decitabine (5-aza-2’-deoxycytidine, DAC) are the prototype of the nucleoside derivatives and were approved by the US Food and Drug Administration (FDA) in 2004 and 2006 for the treatment of myelodysplastic syndrome (MDS), respectively (17, 40 – 42). Historically, these drugs were being used as antimetabolites for cancer chemotherapy since 1960s and their DNA methylation reversal effect was identified in the early 1980s to recognize them as potential DNA hypomethylating agents that induce gene expression and differentiation of tumor cells. Both drugs are administered as injections and recommended for the treatment of MDS and chronic myelomonocytic leukemia (CMML) (5, 43). Recently an oral tablet formulation of 5AC was FDA-approved. Decitabine is also effective against sickle cell anemia and beta-thalassemia (44). Both 5AC and decitabine undergo phosphorylation prior to incorporation into DNA but 5AC additionally incorporates into RNA, which generates the main difference between the two drugs. Previous animal studies demonstrated teratogenic and abnormal embryonic development associated with the use of 5AC but not with its analog 6-azacytidine signifying that these side effects are linked to DNA methylation reversal as 6-azacytidine lacks a hypomethylating effect (45, 46). There are no current data on the use of 5AC in pregnant women to confirm the animal studies and consequently pregnant women should be advised of the potential risk to the fetus.

Figure 2. Nucleoside analogs DNA demethylating agents.

The chemical structures of cytosine derivatives and the cytosine analogs.

The compromised oral bioavailability and excessive cytotoxic effects of 5AC and decitabine created increased demand for developing new nucleoside analogs such as 6-thioguanine (6-TG), zebularine and guadecitabine (SGI-110). 6-TG is the third nucleoside analog antimetabolite approved by the FDA for the treatment of AML. Upon incorporation into DNA, 6-TG entraps DNMT1 via covalent attachment with subsequent degradation of DNMT1 (47 – 52). Zebularine which was originally developed as a cytidine deaminase (CDA) inhibitor, is another stable nucleoside analog and was the first orally active DNMT inhibitor with minimum reported toxicity (53 – 55). Zebularine reactivated silenced p16 gene through complete inhibition of DNMT1 and partial inhibition of DNMT3a and DNMT3b in human T24 bladder carcinoma cells (55, 56). Unfortunately, its rapid and extensive metabolism limited its further evaluation in clinical trials (57). Guadecitabine is a dinucleotide molecule composed of decitabine linked to deoxyguanosine via phosphodiester bond where decitabine is the active moiety that confers the DNA hypomethylating effect (58, 59). Guadecitabine shows several advantages over other decitabine derivatives such as improved aqueous stability and resistance to CDA degradation with consequent prolonged in vivo duration of action after subcutaneous administration. It has demonstrated promising outcome in the treatment of AML and MDS both in Phase I and Phase II clinical trials (60, 61). Guadecitabine has also been tested in solid tumors and showed promising response both in vivo and in vitro (62).

Non-nucleoside Analogs as DNA methylation Inhibitors:

The nucleoside analogs developed and commercialized so far have shown numerous obstacles such as low pharmacokinetic profile, chemical instability, undesirable toxicity and lack of selectivity. Direct inhibition of DNMT enzymes with small molecules that do not incorporate into DNA is expected to eradicate most of the drawbacks associated with the nucleoside analogs (35). In the last two decades, several non-nucleoside small molecule drugs demonstrated DNA hypomethylating activity (Figure 3). The non-nucleoside DNA demethylating agents can be classified into three different classes based on their mechanism of action: DNA binders, SAM antagonists and agents with unknown or multiple mechanisms.

Figure 3. Non-nucleoside small molecules DNMT inhibitors.

All the drugs in this group are derived from natural origin except RG108.

Although the non-nucleoside analogs exert their demethylating effects without being incorporated into DNA, they do bind to DNA and compete for the DNA binding sites of DNMTs (63). Shikonin, a naturally occurring naphthoquinone derivative extracted from purple gromwell herb has been reported to show antitumor activity in human thyroid cancer (64 – 67). Moreover, psammaplin, a group of natural products isolated from the marine sponge pseudoceratina purpurea, have been reported to inhibit both DNMTs and HDAC with mild toxicity (68). Isofitsularin-3 is another alkaloidal natural product isolated from Aplysina aerophoba (a Mediterranean Sea sponge) demonstrated moderate inhibitory effect against DNMT1 (69). Polyphenols in green tea are strong antioxidants and also demonstrated antitumor activity through epigenetic mechanisms (70 – 72). Epigallocatechin-3-gallate (EGCG) is the major polyphenol component in green tea and is a potent inhibitor of the enzyme catechol-O-methyltransferase (COMT), which shares structural similarity to DNMT enzymes (73, 74). Indeed, EGCG reversibly demethylated multiple TSGs including hMLH1, p16 and Rb in colon, esophageal and prostate cancer cell lines (75).

Procainamide and hydralazine are FDA-approved for the treatment of cardiovascular diseases and have shown DNA demethylating activity in cloned T-cell lines (76 –78). Procainamide reversed GSTP1 hypermethylation in LNCaP human prostate cancer cells both in vitro and in xenograft tumors in athymic nude mice (79). Both hydralazine and procainamide have demonstrated substantial demethylation activity through binding to GC-rich regions and demonstrated promising antitumor activity in breast cancer (80, 81). Unfortunately, the demethylating effect of both drugs require high doses that far exceeds the safe clinical dose.

Developing DNMT inhibitors targeting the catalytic pocket of DNMTs is another alternative strategy to reverse DNA methylation. The non-nucleoside DNMT inhibitor RG108 (N-phthaloyl-L-tryptophan 1) selectively inhibits DNMT1 and has been credited as the first small molecule non-nucleoside DNMT inhibitor that shows demethylating activity in cancer cells (82). Docking studies with DNMT1 revealed that RG108 binds to the SAM binding site on DNMT1 with its indole moiety. In vitro experiments in HCT116 colon cancer cells lines showed that RG108 reactivates the expression of TSGs such as p16, TIMP3 and SFRP1 via promoter demethylation (82). RG108 was not cytotoxic to HCT116 cells even at high concentrations. On the contrary to previous reports, the activity of RG108 was compared to the nucleoside analog decitabine using DNA pyrosequencing of three CpG sites within LINE-1 sequence in leukemia cells (83). High concentrations of RG108 did not reverse methylation of any of the three CpG sites. Minor decrease in methylation (3–5% decrease only) was observed after using irrelevant pharmacologic concentrations. Decitabine was used as a positive control in these experiments and induced 25% decrease in methylation at a concentration that was 100 times less than RG108 concentration, demonstrating the huge difference in activity between the two compounds.

Curcumin is a polyphenolic natural compound isolated from turmeric that has been known for its anti-inflammatory and anticancer properties. Despite its poor bioavailability and rapid metabolism, epigenetic changes induced by curcumin have been evidenced by inhibition of both HDAC and HAT enzymes in cancer cells (84). Several studies have shown that curcumin and its stable analog dimethoxycurcumin (DMC) induce DNA hypomethylation in leukemia cells (85). On the other hand, other reports showed that curcumin has a weak and partial activity. The partial activity indicates the preferential demethylation of partially-methylated over not fully-methylated CpG sites. A previous study investigating the DNA methylation reversal activity of curcumin and DMC in leukemia cells was concordant with the weak and partial demethylating activity of both compounds (85). Interestingly, induction of gene expression of promoter-methylated genes like CDKN2B and CDH-1 was observed after treatment with DMC but not curcumin. The mechanism of induction involved an increase in H3K36me3 and histone acetylation without reversing DNA methylation. The combination of DMC with the DNMT inhibitor DAC was investigated to detect possible synergy between the two compounds. The combination enhanced the reexpression of epigenetically silenced genes like CDH-1 with consequent increase in protein expression. Chromatin analysis of the promoter region of CDH-1 showed significant increase in H3K27 acetylation upon using the combination versus the single agents. The data highlight the potential for combining curcumin derivatives with nucleoside DNMT inhibitors to maximize their impact on gene expression. In spite of the extensive research conducted on curcumin and its analogs, none of them is currently FDA-approved for any clinical indication. Future clinical research utilizing curcumin was criticized and expected to further produce controversial results because curcumin is considered a pan-assay interference compound (PAIN).

Antisense Oligonucleosides as DNA Demethylating Agents:

It is well documented that both nucleoside and non-nucleoside analogs exhibit several nonspecific cytotoxic effects because of their non-selective binding to multiple targets. Another approach to eradicate this potential drawback might be the direct and specific inhibition of DNMTs using antisense oligonucleotides or small interfering RNA (siRNA) (35). siRNA or antisense oligonucleotide mediated degradation of DNMTs has been explored in developing DNA demethylating agents. Knocking down of DNMT1 induced re-expression of the p16 and RASSF1 genes in colorectal and renal cells (86). DNMT1 Knock down approach was tested in a clinical trial using antisense molecules such as MG98 in the subsequent years. MG98 is a second generation phosphorothioate antisense oligodeoxynucleotide that demonstrated effective DNA methylation reversal in different models of cancer. Unfortunately, the results of a phase I study were not encouraging (87, 88). Additionally, MG98 inhibited DNMT1 translation in nude mice xenograft models without any antitumor activity (87). Recently, a stem-loop structured aptamer was capable of displacing the hemimethylated DNA duplex in DNMT1. However, this aptamer could not inhibit DNMT3a or DNMT3b-mediated de novo methylation or even bind to them (89). Overall, the development of antisense oligonucleotide and siRNA as DNA demethylating agents is challenging because of their chemical instability, delivery and off-target effects. Indeed, no single approach can eradicate all the existing drawbacks of DNA methylation inhibitors. Another approach is to disrupt protein-protein interaction between DNMTs and their protein counterparts rather than targeting their catalytic pockets (90). However, the approach was not selective.

DNA Methylation Inhibitors and Combination Therapy:

Currently, there is no single drug that can bypass the existing drawbacks of DNA methylation inhibitors. The combination of DNA methylation inhibitors with other antitumor drugs like chemotherapy, HDAC inhibitors and immunomodulatory derivatives like thalidomide and lenalidomide has been in practice for many years. Concomitant administration of DNMT inhibitors and the above agents has been practiced to harness their additive or synergistic effect on gene reexpression and cytotoxicity. Another advantage of the combination approach is to overcome drug resistance, a major obstacle in the success of chemotherapy. For instance, silencing of TSGs by DNA methylation may disrupt apoptosis with consequent resistance development to chemotherapy (35). Decitabine used in combination with the HDAC inhibitor belinostat efficiently restored the expression of TSGs, while decitabine as a single agent partially restored TSGs expression (91). Mammalian target of rapamycin (mTOR) plays a critical role in cell growth and is often altered in cancer. The combination of rapamycin with decitabine effectively eliminated the downstream effect of mTOR activation in colorectal cancer cell lines (5). Similarly, simultaneous administration of 5AC and doxorubicin elicited synergistic cytotoxic effects in myeloma cells (92). Sequential administration of decitabine followed by cytarabine also demonstrated synergistic cytotoxicity in leukemia cells. Sequential administration of decitabine followed by the HDAC inhibitor TSA demonstrated substantial re-expression of promoter-methylated genes, while TSA as a single agent was less effective (93).

Dual inhibition of DNMTs and HDACs by a single molecule is an attractive approach (94). Recently, several hydroxamic acid derivatives have been reported as multifunctional inhibitors dually acting on DNMTs and HDACs. Treatment of human H937 cells with these molecules demonstrated enhanced H3K9 and H4K8 acetylation levels and induced p16 CpG island demethylation leading to increased p16 expression and cell apoptosis. C02S is a novel compound that inhibits both DNMTs and HDACs and was effective against breast cancer cells (95). CM-272 is another potent dual inhibitor of G9a (a histone methyl transferase) and DNMTs that demonstrated promising outcome in hematological malignancies (96).

Conclusion

Epigenetic events are an integral part of normal biological processes as well as in tumorigenesis. Accordingly, epigenome-targeted therapy is an encouraging approach for cancer treatment. However, potential concerns and challenges exist. The selectivity of the drug candidates is the major concern. As epigenetic events are randomly distributed in normal cells and cancer cells, some cancers are sensitive to certain epigenetic alterations while the normal cells are usually capable of adjusting with those changes. The second potential concern is the range of indications. Because of the biological and pathological differences between hematologic malignancies and solid tumors, DNMT inhibitors demonstrated promising results in hematological malignancies, but not in solid tumors (17). A large number of DNMT inhibitors candidates are in the developmental pipeline and some of them are currently tested in clinical trials. The success of these candidates will have a positive impact on the strategies for cancer treatment. Considering the achievements observed so far, DNA methylation-targeted epigenetic therapy is a promising strategy for the treatment of cancer.