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Present status of epigenetic drugs in cancer treatment


Present status of epigenetic drugs in cancer treatment

Ranju Kunwor MD*, Yanrong Su PhD, Julia Santucci-Pereira PhD and Jose Russo MD*

1The Irma H Russo MD-Breast Cancer Research Laboratory, Fox Chase Cancer Center-Temple University Health System, Philadelphia, PA 19111, USA

Ranju Kunwor MD: Ranju.Kunwor@fccc.edu; Prof. Jose Russo, MD, FACP: Jose.Russo@fccc.edu


Epigenetic changes such as promoter specific DNA hypermethylation and histone deacetylation cause tumor suppressor genes to become transcriptionally silent and contribute to malignant transformation. DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors can reactivate silenced genes, block cell cycle and induce cell apoptosis which provides rationale for their use in cancer treatment. DNMT and HDAC inhibitors have therefore emerged as an effective strategy against cancer. Epigenetic modifications have a key role in the pathophysiology of many cancers such as myelodysplastic syndrome (MDS), lymphoma, and leukemia and the use of drugs targeting epigenetic changes has become a topic of intense interest in cancer research. The role of transcriptional repression through epigenetic modulation in carcinogenesis has clinically validated the use of inhibitors of DNMT and HDAC. Epigenetic changes can be pharmacologically reversed resulting in gene re-expression.

DNMT inhibitors 5-azacytocine (azacytidine/ AZA) and 2’-deoxy-5-azacytidine (decitabine/ DAC) are cytosine analogues and are currently the most advanced drugs for epigenetic cancer therapy. AZA and DAC have been approved by FDA for the treatment of MDS.   HDAC inhibitors-Vorinostat (SAHA), Romidepsin (depsipeptide FK 219), Belinostat (PXD 101) and LAQ 824/LBH 589 have demonstrated therapeutic benefit in Cutaneous T-cell Lymphoma (CTCL). Both romidepsin and vorinostat are approved by FDA for the treatment of CTCL. In this review, we describe the current status of DNMT inhibitors and HDAC inhibitors usage in cancer treatment and discuss the challenges involved in successful establishment of these novel drugs either alone or in combination therapy for the treatment of various cancers.

Keywords:DNA methyltransferase (DNMT) inhibitor; Histone deacetylase (HDAC) inhibitor; Epigenetics; Cancer


Conrad Waddington coined the term epigenetics (literally meaning ‘over’ or ‘upon’ or ‘beyond’ genetics) in the early 1940s, which  was used to explain why sometimes genetic variation contrast with phenotypic variation; and phenotype is not only a yield of a genotype but a combination of genotype with environmental factors [1] . Epigenetics do explain alternate phenotypes that are not based on the changes in genotype. Epigenetic refers to potentially reversible, heritable changes in gene expressions that occur without changing the DNA primary nucleotide sequence and are generally stably maintained during cell division [2,3] . This is in contrast to genetic mutations which are due to modification in primary nucleotide sequence of a gene and are irreversible. Epigenetic modification includes DNA methylation, covalent histone modification and chromatin remodeling in promoter region of genes [4] . Alteration in gene expression due to modification in DNA methylation and histone acetylation at the promoter region of the genes are two major epigenetic changes and are most widely studied [3-5] .

DNA methylation, DNA methyltransferases and their role in cancer

DNA methylation is the addition of a methyl group to 5th position of the pyrimidine ring of the cytosine [6] . Figure 1 shows the methylation of cytosine to 5-methyl cytosine. In human and other mammalian genomes, methylation occurs only to cytosine that precedes guanosine in DNA sequence, in other words, cytosines that are located 5’ to guanosine and are known as CpG dinucleotides [6,7] . Although the distribution of CpG dinucleotides in a genome is unusually asymmetric, they are usually clustered in the promoter region of the gene and are known as CpG islands [8] . It is in the promoter region where the transcription of DNA to RNA begins. In normal cells, most of the CpG islands are unmethylated which allows gene transcription to take place; allowing transcription of important genes like tumor suppressor genes. Whereas in cancer cells, these CpG islands are heavily methylated impeding transcription [6,8] .

DNA methylation is carried out by DNA methyltransferase (DNMT) enzyme that adds a methyl group to the pyrimidine ring of cytosine, in 5th position to form 5-methyl cytosine, using S-adenosyl methionine (SAM) as a methyl donor [9] . In human and mammals, there are 3 biologically significant DNMT enzymes namely DNMT1, DNMT 3a and DNMT3b [8,10] .  The enzyme DNMT2 has similarities with 5-methyl cytosine methyl transferase of both prokaryotes and eukaryotes, however, this enzyme has been proven to methylate position 38 in aspartic acid tRNA and does not methylate DNA [11,12] . Study of DNMT2 in mouse embryonic stem cells has been shown to cause neither de novo methylation nor maintenance of methylation in proviral DNA [11] . Apart from methylating  tRNA Asp-GTC on cytosine 38 in the anticodon loop, this DNMT2 also methylates  valine tRNA (Val AAC) and Glycine tRNA (Gly GCC). These tRNA methylations protect tRNA against stress induced ribonuclease cleavage [13] .

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Figure 1 Methylation of cytosine to 5-methyl cytosine in CpG dinucleotide is catalysed by DNMT. SAM is a methyl donor. (SAM: S-adenosyl methionine; SAH: S-adenosyl homocysteine; DNMT: DNA methyltransferase)

DNMT1 is the most abundant methyltransferase in mammals and is considered the key one for maintenance of CpG dinucleotide methylation in promoter regions of genome because it preferentially methylates hemi-methylated DNA [10,14] . Therefore, DNMT1 plays an important role in methylation during DNA replication, genomic imprinting and X-chromosome inactivation during embryogenesis [8,15] . DNMT 3a and DNMT3b are responsible for de novo methylation and targets unmethylated DNA. They are classified as de novo methyltransferases even though they have equal preferences for hemi methylated and unmethylated DNA [11,16] .The other enzyme, DNMT3L,  is considered nonfunctional because of its mutated active site and lack of functional catalytic site, but is believed to antagonize DNMT 3a and DNMT3b by competing with their binding sites [17] .DNMT1 is the main enzyme in neoplastic cells for maintaining promoter hypermethylation and transcription repression [17] .  However, studies suggest that the cooperation between both DNMT1 and DNMT3b is required for neoplastic transformation as exemplified in human colon [8] and breast cancer [18] .

DNMTs repress transcription by mechanisms other than promoter hypermethylation. DNMTs recruit histone deacetylases in promoter region causing gene silencing. DNMTs also directly interact with transcription regulatory proteins that demethylate DNA like 5-methylcytosine glycosylase and MBD2b (methyl CpG binding domain isoform 2b). 5-methyl cytosine glycosylase removes methylated cytosine from DNA, and MBD2b demethylates DNA by hydrolyzing 5-methylcytosine to cytosine and methanol [17,19] .

Almost all types of cancer have both hypomethylation and hypermethylation.  In cancer, there is gain of methylation in CpG islands of promoter regions which, in normal cells, are unmethylated. Also, in cancer, CpG dinucleotides outside the promoter region (in coding regions) of the gene are unmethylated where normally the CpG dinucleotide is methylated [19,20] .Abnormal methylation pattern in cancer is explained in figure 2.

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Figure 2 Comparison of DNA methylation pattern in normal and cancer cells. Most of the CpG dinucleotides are present in promoter regions of genome (represented by circles adjacent to transcription site, with in exon 1). Normally, CpG islands in the promoter region of genes are unmethylated (represented by white circles) which allows transcription to occur, whereas, CpG dinucleotides outside promoter region are methylated. In cancer, opposite happens in terms of methylation. CpG islands in the promoter region are methylated inhibiting transcription, and the CpG dinucleotides outside promoter region (in coding regions) of the gene are unmethylated.

Hypermethylation in CpG islands located in promoter regions causes silencing of transcription, including silencing of tumor suppressor genes. Examples of such genes silenced in malignancy include CDKN2A, RB1, hMLH1, BRCA1 and VHL genes. These genes are responsible for various solid (such as breast cancer, ovarian cancer) and hematologic (AML, MDS) malignancies [15,17] .

Global hypomethylation causes over expression of proto-oncogenes, growth factors and genes that leads to cancer cell growth, proliferation, invasion and metastasis. Examples of  malignancies caused by global hypomethylation includes breast, cervical and brain cancers [19] . Hypomethylation can cause increased expression of harmful genes, imprinted genes and repeat elements. Increased proliferation of neoplastic cells due to biallelic expression of IGF2 (there is lack of imprinting of second allele) is an example of hypomethylation leading to loss of imprinting. Malignancies where specific hypomethylation can be seen are hepatocellular cancer, cervical cancer, prostate cancer and B-cell chronic lymphocytic leukemia [19,21] .

Histone acetylation, histone deacetylases and their role in cancer

Histones are the structural proteins responsible for maintaining shape and structure of chromatin. DNA winds around the histone to form nucleosome, a basic unit of DNA packaging in eukaryotes [5] .  A nucleosome have 147 base pairs of DNA wrapped twice around the histone octamer  consisting of four pairs of histone H2A, H2B,H3 and H4 [5,22] . Amino acids on histone tails undergo number of post translational modifications such as acetylation, methylation, phosphorylation, ubiquitylation and sumoylation. These modifications alter chromatin structure from ‘open’ or uncoiled to ‘closed’ or ‘coiled’  form and vice versa [23,24] . Acetylation promotes uncoiling of chromatin and facilitates transcription by allowing the binding of transcription facilitating proteins, whereas removal of acetyl group from histone causes coiling of chromatin inhibiting transcription [23] . Histone deacetylase(HDAC) enzymes remove the acetyl group from histone leading to chromatin coiling and thus inhibits transcription [25] .  Loss of acetylation  of histone H4 at lysine 16 is seen in cancer cells which proves the critical role of HDACs in tumorogenesis [26] .HDACs are divided into 5 classes- class I, class IIA, IIB, III and IV.  Class III is Zn independent whereas rest is Zn dependent [22] .Table 1 shows thesummary of HDAC enzymes [22,26-28] .

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Table 1 Summary of HDAC enzymes

Aberrant expression of HDACs and transcription repression is observed in various types of cancer, such as gastric, breast, pancreatic cancer, hematological malignancies and many more [29] . Studies involving knockout  in yeast and knockout in mammalian cells have shown that class I HDACs are more important for carcinogenesis and have essential role in proliferation and survival [30] . HDAC1 enzyme expression is higher in colon adenocarcinoma, gastric, breast, pancreatic, hepatocellular lung and prostate cancer compare to normal cells. Likewise, HDAC2 expression is higher in colon and lung cancer compared to normal. Knockdown of HDAC2 and use of HDAC inhibitors have arrested growth of colon cancer cell line. High expression of HDAC 1, 2 and 3 is seen in Hodgkin’s lymphoma(HL) [31] , renal cell carcinoma [32] , colorectal cancer [33] and gastric cancer [34] . HDAC4 is overexpressed in breast cancer, waldenstroms macroglobulinemia [35,36] . HDAC4 downregulation is also associated with some cancer. Homozygous deletion of HDAC 4 is seen in melanoma cell lines [37] . Additional work has demonstrated the association of HDAC4 mutation with breast cancer [38] . HDAC 5 and 9 are overexpressed and are valuable markers in medulloblastoma and are associated with poor prognosis [22,26] . Also HDAC 5 aberrant expression in hepatocellular carcinoma(HCC) is proven by successful treatment of preclinical models of HCC with HDAC inhibitor panabinostat [39] .Similarly HDAC9 expression is increased in cervical cancers, medulloblastoma, acute lymphoblastic leukemia (ALL), but HDAC 9 is downregulated in glioblastoma. Increase activity of HDAC is seen in acute promyelocytic leukemia, cutaneous T-cell lymphoma (CTCL) [26,29] .

Expression of HDACs has been related to prognosis of certain cancers [26] . Increased HDAC1 and 3 expressions in breast cancer have been correlated with improved disease free survival [40] .Whereas another study demonstrated that class I HDAC especially HDAC1 overexpression leads to poor prognosis in  breast cancer [41] . Studies have shown that overexpression of class I HDACs especially HDAC1 is associated with poor prognosis in gastric cancer [34] and adenocarcinoma of lung [42] . HDAC8 expression is higher in childhood neuroblastoma and is associated with poor prognosis [43] . On the other hand, lower expression of class II HDACs in non-small cell lung cancer is correlated with poor prognosis [44] .

DNMT inhibitors, their use and rationale

DNMT inhibitors inhibit the epigenetic silencing of gene transcription due to DNMT enzyme mediated covalent modification of the promoter region of genes [45] . These drugs thus act by three ways; (1) preventing methylation (at lower doses of drug), (2) direct incorporation into DNA or RNA after phosphorylation. 2-deoxy-5-azacytidine (Decitabine/DAC) is incorporated into DNA and 5-azacytosine (azacytidine/AZA) is incorporated into RNA which then covalently link with DNMT inhibiting DNA synthesis and protein synthesis respectively. This mechanism is also responsible for cell death at higher doses of drug, and (3) DNA or RNA damage due to structural instability at the site of incorporation [46-48] .

DNMT inhibitors can be divided into two broad categories [49,50] . The first includes the nucleoside inhibitors AZA, DAC, Zebularine and SGI-110. These drugs incorporate into DNA or RNA and act as a suicide inhibitor. These will be described in detail in this review further below. The second includes the non-nucleoside inhibitors EGCG (Epigallocatechin-3-gallate), RG108 and Procaine. They are experimental compounds and have not been thoroughly analyzed systematically yet. EGCG inhibits the enzyme but have no effect on DNA methylation and is extremely cytotoxic due to oxidative stress produced by it. However some trials are going on with EGCG, mainly in preventive aspects of cancer [51] . Procaine is not cytotoxic and has some pro-apoptotic activity at maximum drug concentration. RG 108 is also not cytotoxic but is genotoxic (like AZA and Zebularine) as shown by micronucleus induction assay. RG 108 is the only DNMT inhibitor that can directly inhibit recombinant DNMT in cancer cell lines [49,52] . SGI-110, is a novel second generation DNMT inhibitor being tested for breast, ovarian and hematological malignancies at both pre-clinical and clinical level [53-55] . The clinical trials of SGI-110 are shown in table 5. Clinically, four DNMT inhibitors have been employed- 5-azacytidine (azacytidine/AZA/5-aza-CR), 5-aza-2’deoxycytidine (decitabine/DAC/5-aza-CdR), 1-β-D-arabinofuranosyl-5-azacytocine (fazarabine), dihydro-5-azacytidine (DHAC).  The other agent zebularine is also under studies. A pre-clinical study showed that zebularine induces s-phase cell cycle arrest and apoptosis of two human breast cancer cell lines [56] . AZA and DAC are studied most and have been used in multiple clinical trials effectively [49] . AZA and DAC are FDA approved drugs and are marketed as Vidaza and Dacogen respectively [52] . Some compounds with DNMT inhibiting property that are in use in diseases other than cancer are Hydralazine, Procainamide and Procaine [52] .

Demethylation and reactivation of transcriptionally inactive or silenced genes are observed after treatment with both AZA and zebularine but there is reoccurrence of methylation in dividing cells once drug is stopped [52] . So, continuous delivery of AZA and zebularine may help in maintaining demethylation for longer time although dosage and frequency of drug administration depends on drug bioavailability. Factors like solubility, uptake degradation or metabolism can alter bioavailability.


AZA is one of the major DNMT inhibitors that has been widely studied, and can be considered as a prototype among DNMT inhibitors. Its chemical structure is similar to cytidine, where the 5th position carbon of the pyrimidine ring is replaced by nitrogen as seen in figure 3A. The cellular uptake process of AZA has not been described clearly in molecular level yet [57] . However, nucleoside transporters in human cells may play an important role in cellular uptake of AZA. Also, in vitro experiments show a significant correlation between expression of these nucleoside transport proteins and sensitivity to nucleoside analogues. The four nucleoside transporters in human cells are: (1) Equilibrative uniporters (SLC 29A family), (2) Substrate exchange transporters(SLC 15, SLC 22 family), (3)Concentrative transporters (SLCA28 family), and (4) ATP dependent exporters(ABC family) [57] . AZA is converted into its monophosphate first followed by diphosphate and triphosphate after cellular uptake [57,58] .  It is a ribonucleoside analogue [59] , so 5-azacytidine triphosphate is  incorporated into RNA and thereby exerts its action by inhibiting mRNA translation [59] . Figure 4 summarizes the AZA phosphorylation. A small portion (10-20%) of administered AZA is converted into 5-aza-2’-deoxycytidine diphosphate by the enzyme ribonucleotide reductase; which is then phosphorylated to 5-azadeoxycytidine triphosphate and it is incorporated into DNA [58,60] . Figure 5 shows incorporation of AZA in DNA. This incorporation results in formation of adducts between DNA and DNMT1 [59] , in other words, it traps DNMT1. At lower dose of AZA, these adducts are degraded by proteasomes resulting in restoration of DNA and continued synthesis of DNA in absence of DNMT1 [57,58] . Therefore the aberrant methylation is no longer reproduced in daughter strands after DNA replication. Thus, low dose AZA inhibits DNA methylation by inactivating DNMT1 , the main enzyme responsible for DNA methylation whose expression is high in S-phase of cell cycle [61,62] and cause re-expression of previously silenced gene and restoring normal growth and differentiation of cells [57] .

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Figure 3 Chemical Structures of few of the Epigenetic drugs. (A): Comparison of chemical structure of AZA and DAC with cytidine. In AZA (left), the carbon atom in 5th position of pyrimidine ring is replaced by nitrogen. Structure of DAC is similar to AZA, except there is a removal of one oxygen atom in DAC. (B): Chemical structure of Vorinostat, (C): Chemical structure of Romidepsin

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Figure 4 Schematic representation of AZA phosphorylation after cellular uptake.

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Figure 5 Schematic representation of covalent bond formation between AZA incorporated in DNA and DNMT1 enzyme thus inactivating DNMT. DNMT: DNA methyltransferase

AZA has the strongest demethylation activity compared to other DNMT inhibitor and is very cytotoxic at higher doses. At higher doses, AZA forms very high levels of enzyme DNA adducts and DNA is not able to recover from the adducts resulting in cell lysis [59,63] . Studies have shown demethylation of P53 tumor suppressor gene in patient with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) treated with AZA and DAC; demethylation of Ebstein Barr virus (EBV) genome in EBV associated tumors and demethylation of line elements centromeric satellite repeats and P21 tumor suppressor gene when treated with AZA and DAC [52,64] .       

The only clinical indication of AZA that has been approved by FDA is for treatment of all subtypes of MDS according to French American British (FAB) classification [60,65] . Epigenetic changes resulting in pathogenesis of MDS and AML is well understood, and as these drugs reverse the epigenetic changes associated with them, they both make sense and are useful for the treatment of disease [66] . The FDA approved it on May 19, 2004 as an injectable suspension subcutaneously or intravenously [60] . The drug is marketed as Vidaza by company Celgene [67] .  The approval was based on three clinical studies. Two single arm trials and one randomized controlled phase III trial [68] . That randomized controlled trial (RCT) done in 191 patient with Refractory Anemia(RA) or RA with ringed sideroblast with significant bone marrow suppression showed that the patients treated with AZA showed higher response rates, improved bone marrow response, delayed transformation to AML, improved quality of life, reduced risk of leukemic transformation and improved overall survival compared to the supportive group [68] .  AZA is clearly superior to other drugs tested for MDS like 13-cis-retinoic acid, all-trans-retinoic acid, 1,25-dihydroxy vitamin D, butyrate, cytarabine, hexamethylene bisacetamide and amifostine [68] . None of these other drugs were approved for therapeutic use in MDS before AZA. AZA treatment benefits clearly outweigh risks in MDS patients. AZA is indicated in IPSS (International prognostic scoring system) intermediate-2 or high risk patient 65 or older in age and in patients not suitable for stem cell transplantation [69] .

AZA dosage is 75mg/m2 per day for seven days followed by drug free period of 21 days. The same cycle is repeated after 28 days [58,68] . Treatment should be continued for at least six courses for evaluating the effect of drug on disease. Randomized controlled clinical trial also showed that it takes six cycles to observe an initial response in 90% of the responding patients [68,70] . The response was calculated using International Working group(IWG) criteria as complete response(CR), partial response(PR) and hematological improvement(HI) response [70] . Best response was observed after about eight cycles and lasted for median of 5 cycles [70] . Dose can be adjusted based on toxicity and patient’s response to treatment. As long as the patient shows benefit, the treatment is recommended to continue [58] .

AZA reaches maximum plasma concentration after 30 minutes of subcutaneous injection and after 10 minutes of intravenous (IV) infusion, the time to reach maximum plasma concentration is 11 minutes. The bioavailability after subcutaneous (SC) administration is 89% compared to IV infusion. Plasma half-life is 22 minutes after IV infusion and 41 minutes after SC injection. AZA is rapidly absorbed after SC administration and widely distributed over tissues [60] .Peak plasma concentration in patient treated with AZA is similar to concentration required for invitro DNA demethylation [57] . Primary route of excretion is via urine. AZA is chemically unstable both in aqueous solution and body fluids as they are rapidly hydrolyzed. Also AZA is deaminated by cytidine deaminase to degrade it further [58] , which makes difficult to detect its level after treatment.  However, with the development of a high performance liquid chromatography-electrospray tandem mass spectrometry method, it has become possible to detect AZA and DAC level in human plasma [52] .

Treatment with AZA improves overall survival (OS) in MDS patients, improves quality of life and reduces AML transformation [68] . Response is accessed via IWG criteria [71] and significant response to treatment is seen following 6 weeks of treatment on average [68,70] . HI has better prognostic significance and studies found that AZA treated patient who achieved HI even without CR or PR had better OS. AZA has beneficial response in bone marrow function in International Prognostic scoring system (IPSS) intermediate-2 risk and high risk MDS and Refractory Anemia (RA) and Refractory anemia with ring sideroblast (RARS) [68] . A study done in 282 high risk MDS patients shows a better response to treatment with AZA when bone marrow blast count is <15%, karyotype is normal, and there has been no previous treatment with AZA. AZA treatment in patients with complex karyotype(poor risk karyotype involving chromosome 5 and 17 lesion), transfusion dependency, poor ECOG-PS(Eastern Cooperative Oncology Group performance status) and presence  of >15% blast showed inferior response and shortened OS independently [72] .   The presence of peripheral blast is a poor prognostic factor for low risk MDS as well. Overall survival and prognostic significance is still best predicted by IPSS cytogenetic classification [72,73] .  Mutation in genes that regulates DNA methylation such as TET2 and DNMT 3A and mutation in gene regulating histone acetylation like ASXL1 are considered a good prognostic factor in AZA treated MDS patients. However, the role of genetic factors in OS is yet to be established [58,72] . In therapy related myeloid neoplasms as seen in retrospective review study, AZA has demonstrated comparable response rate(RR) to de novo MDS but with poor overall prognosis [74] .

Common side effects of AZA are nausea, vomiting, diarrhea, anorexia, constipation, neutropenia thrombocytopenia, fever, rigors, arthralgia, headache and dizziness. Myelosuppression (especially decreased neutrophil and platelet numbers) is the most common toxicity and is typically seen in the third week of treatment cycle with most patients recovering until the next treatment cycle. Hematological adverse effects generally improve with subsequent treatment cycles [75,76] . There has been no drug related mortality so far in the clinical setting [60] but one treatment related death was seen in controlled trial done in 191 patients [68] . The RCT showed occurrence of serious adverse effect in about 60% of AZA treated patients and about 36% of patients in observation group [60] .The most common side effects resulting in hospitalization are thrombocytopenia, febrile neutropenia, fever and pneumonia.  The most common side effects resulting in dose reduction or discontinuation are neutropenia, leucopenia and thrombocytopenia.  AZA causes severe myelosuppression in higher doses and this effect has been seen both  in vitro and in vivo [52] . Therefore, a low dose treatment schedules is used for patients as it is better tolerated [75] . FDAs post marketing surveillance also adds tumor lysis syndrome, injection site necrosis and sweet syndrome (acute febrile neutrophilic dermatosis) as rare side effects [69] . AZA is contraindicated in patients with advanced malignant hepatic tumors. Safety in pregnant patient is not yet established. However studies in mice show reproductive toxicity and therefore AZA should not be used in childbearing age groups, pregnant and breastfeeding patients.

The other hematological cancer where use of AZA is widely studied is AML. Low dose AZA, DAC and Cytarabine has been recommended by National comprehensive cancer network, European Leukemia net  and European society for medical oncology for treatment of AML [77] . AZA is European Medicines Agency (EMA) registered for treatment of both MDS and AML. Various studies show that AZA use is beneficial especially in elderly AML patients who cannot tolerate intensive chemotherapy and have poor prognosis with intensive chemotherapy. A retrospective review study done in 671 newly diagnosed AML patients aged 65 or older compared the result of treatment with AZA, DAC and intensive chemotherapy. They demonstrated that patients receiving AZA or DAC have similar survival rates as those receiving intensive chemotherapy [78] . This study shows that treatment of elderly, newly diagnosed AML patient with epigenetic drugs has lower CR and ORR(overall response rate) compared to intensive chemotherapy but are associated with reasonable similar median survival times [78] . Another phase III randomized trial was done in 113 elderly patients with median age of 70 years with AML (WHO defined) and it demonstrated that AZA treatment significantly increases OS compared to conventional care regimens (CCR) in AML patients with low bone marrow blast count. Median OS after a median follow up time of 20.1 months for AZA group was 24.5 months and that of CCR group is 16.0 months with 95% CI. Also, the 2 year OS rate was 50% for the AZA group and 16% for the CCR group [79] . A retrospective study done in 20 WHO defined AML patients, treated with conventional dose outpatient AZA, induced remission of AML. The ORR was 60%, CR 20%, PR 25% and hematological improvement (HI) 15% in that study. The responding patients have median survival of >15 months compared with 2.5 months for non-responding patients (P=0.009) [80] .  Overall survival is again dependent on IPSS and complex karyotypes [81] . These successful clinical trials suggest that AZA can be an alternative treatment in AML patients who are elderly and who cannot undergo intensive chemotherapy for treatment. AZA use is not approved yet by the FDA for AML treatment. However, AZA is approved by EMA in 2008 for treatment of high risk MDS and AML with 20-30% blasts [81] .


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Figure 6 Metabolism of Decitabine (5-AZA-CdR) inside the cell. DAC is converted to its active form by phosphorylation with deoxycytidine kinase (CdR Kinase). This monophosphate form of DAC is converted into di and tri-phosphate form by dCMP kinase and nucleoside diphosphokinase respectively. Cytidine deaminase (CR deaminase) inactivates decitabine by deamination and decitabine monophosphate is inactivated by dCMP deaminase. The triphosphate form (5-AZA-dCTP) is incorporated into DNA with the help of DNA polymerase that forms a covalent bond with enzyme DNMT to inactivate the enzyme.

Similar to AZA, DAC is also a cytidine analogue. Figure 3A shows the chemical structure of DAC and the comparison of its structure with AZA and cytidine . Like AZA, DAC inhibit DNA methylation at low doses by inactivation of DNMT1. It is a deoxyribonucleoside analogue and therefore is incorporated into DNA directly after cellular uptake. For this reason it is less toxic and more specific than AZA. Its cellular uptake  mechanism of action is similar to AZA [50] . Inside the cell, DAC is converted to its active form by phosphorylation with deoxycytidine kinase. This monophosphate form of DAC is converted into di and tri-phosphate form by dCMP kinase and nucleoside diphosphokinase respectively. Cytidine deaminase inactivates decitabine by deamination and decitabine monophosphate is inactivated by dCMP deaminase. Figure 6 shows the intracellular metabolism of DAC. The triphosphate form is incorporated into DNA that forms a covalent bond with enzyme DNMT to inactivate the enzyme [82] .

DAC was approved by the FDA on May 2, 2006 for use in MDS of all FAB subtypes including previously treated or untreated, primary or secondary and IPPS intermediate 1, intermediate-2 and high risk MDS. It is marketed as Dacogen by the company Eisai Inc [83] . This approval was based of multi central randomized clinical trials [84] . Decitabine was found to be clinically effective in MDS treatment improving survival and quality of life. The median time to AML progression or death was 17.5 months for decitabine responders and 9.8 months for non-responders. All decitabine responders became transfusion independent.  Response was determined by using IWG criteria [71,84] .There are two drug regimens approved by the FDA. (1)15mg/m2 by continuous intravenous infusion over 3 hours and repeated every 8 hours for 3 days. This cycle is repeated every 6 weeks [85] . (2)20mg/m2 by continuous intravenous infusion for 1 hour, repeated daily for 5 days [86] . This cycle is repeated every 4 weeks.  For both regimens patient should be treated for at least 4 cycles. The treatment can be longer depending on patient response [84] . In a comparison analysis, there is no significant difference between efficacy of AZA and DAC for treatment of MDS. ORR, OS, event free survival (EFS) and the rate of leukemic transformation in MDS patient is comparable in both the overall and propensity matched cohorts. However, survival was significantly better in propensity score-matched elderly patients (≥65 years), which can be attributed to cytopenia and more frequent infectious episode with DAC [87] . The maximum plasma concentration of DAC after 3 hours of 15mg/m2 intravenous infusion is 64.8 to 77.0 ng/ml reaching the steady state plasma concentration [88] . It is widely distributed in tissue with a volume of distribution of 63 to 89 L/m2. Terminal phase elimination half-life 0.62-0.78 hours [89] .

The most common adverse reaction of DAC is myelosuppression especially neutropenia (87%), thrombocytopenia (85) and anemia (12%). Safety data was evaluated on 164 patients, 83 receiving decitabine and 81 received supportive care. Serious adverse effect was seen in 69% of decitabine patients and 56% of patient receiving supportive care. No direct drug related deaths were observed in the study. All deaths observed were due to MDS related events [90] . The other side effects seen are febrile neutropenia, leukopenia, pyrexia, hyperbilirubinemia and pneumonia [84] .  DAC treatment should not be restarted until all of the following non-hematologic toxicities are resolved: 1) Serum creatinine ≥2mg/dl; 2) SGPT, total bilirubin ≥2 times normal level; 3) Active infection. There are no absolute contraindication of DAC, but should be used cautiously in patient with neutropenia and thrombocytopenia. It is a potential risk to the fetus and should not be used in pregnancy (Category D).

Though not registered by EMA for treatment of MDS, decitabine was registered by EMA in September 2012 for treatment of AML in elderly patients (≥65) who are not eligible for treatment with standard induction chemotherapy. A multicenter, randomized, open label, phase III study done in 485 patient age ≥65 compared low dose decitabine with conventional treatment and supportive care. The study demonstrated improved response rate and improved survival compared to conventional treatment without major differences in safety [77] . Before that, another multicenter, phase II study of DAC in elderly AML patient(mean age 74) demonstrated that dosing schedule of 20mg/m2 daily for 5 days was well tolerated and has a promising response with low treatment related toxicity and mortality [91] .

HDAC inhibitors, their use and rationale

Aberrant HDAC activity has been demonstrated in various cancers like MDS and CTCL, making HDAC inhibitor an important anticancer agent.  HDAC inhibitors that are currently available mainly target class I and class II HDAC enzymes [92] . HDAC enzymes are described in table 1. Only two of them- Romidepsin and Vorinostat are FDA approved drug for treatment of CTCL. HDAC inhibitor causes cancer cell death by five different mechanisms. 1) Acetylation and disruption of client protein activity for heat shock protein. 2) Disturbance in NFKB pathway. 3) Up regulation of extrinsic apoptotic pathway.  4) Activating reactive oxygen species and causing oxidative injury.  5) Generation of pro-apoptotic cell messengers [30,93] . HDAC inhibitors produce a global increase in histone acetylation within hours in both malignant and non-malignant tissue. Acetylated histones causes transcription of genes needed for tumor growth arrest. Acetylation of non-histone proteins such as tissue or development specific proteins (EKLF, GATA-1, ERα,MyoD), oncogenic proteins (c-Myb) and the tumor suppressor (p53) could be of more importance [92] .  The biological effects of HDAC inhibitors are (1) Apoptosis (extrinsic-via Fas, TNF-α, TRAIL upregulation; intrinsic-via upregulation of Bid, Bim, Bad and downregulation of Bcl-2, Bcl-XI, Mcl-1); (2) Mitochondrial injury- due to oxidative stress from inhibition of thioredoxin; (3) Cell cycle arrest- upregulation of CDKN 1A-p21 and stabilization of p53; (4) Inhibition of angiogenesis-decrease in HIF-1A and VEGF expression; (5)Aggresome-proteasome effect of HDAC6 (inhibition of α-tubulin and misfolded protein response). As compared to other HDAC inhibitors, romidepsin has less effect on HDAC6 [94] .Table 2 summarizes the classification of HDAC inhibitors and their current clinical status [30,92,95] .

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Table 2 Classification of HDAC inhibitors


Figure 3B shows the chemical structure of vorinostat. Though the mechanism of action of vorinostat is not yet clear [96] , it is clear that vorinostat is a potent inhibitor of class I and class II HDAC enzymes. Being a histone deacetylase inhibitor, Vorinostat keeps histone, proteins and transcription factor acetylated allowing transcription to occur. Vorinostat induces expression of some genes (p21WAF1, TBP-2, gelsolin, metallothionein 1L, histone H2B) and represses expression of some genes (cyclin D1, ErbB2, thymidylate synthase, importin B). Increased expression of P21WAF1 (cell kinase inhibitor) causes growth arrest of transformed cell [97-99] . Patients with lymphoma have increased activity of transcriptional repressor BCL6 and acetylation of BCL6 can give rise to inhibition of transcription repression providing the rationale for use of HDAC inhibitor in CTCL. A non-transcriptional effect of acetylation is inhibition of mitosis and cell cycle arrest. Vorinostat, by increasing acetylated histones and proteins, disrupts the cell cycle and induces apoptosis by targeting cell cycle check point (G2 check point, which is defective in tumor cell) and a mitotic spindle check point [97] . Vorinostat works by regulating the transcription of genes involved in apoptosis, cell cycle control and differentiation. In vitro studies shows, vorinostat increases malignant T cell apoptosis, G2 arrest and p21WAF1. Also it downregulates VEGF by increasing TSP-1(a potent inhibitor of angiogenesis) in vitro and in skin specimens of 67% of patient treated with vorinostat [100] . Downregulation of VEGF, upregulation of TSP-1 and increased T-cell apoptosis are all responsible for resolving CTCL.

Vorinostat was approved by FDA on October 6, 2006 for use in CTCL in patients who have progressive persistent and recurrent disease on or following two systemic therapies. It is marketed as Zolinza and is available in 100mg capsule form [101] . This approval was based on two phase II studies [102] . An open-label phase IIb multicenter trial conducted on 74 patients who have received at least two prior systemic therapies (at least one of which is bexarotene) to evaluate activity and safety of vorinostat in persistent progressive or recurrent mycosis fungoides or sézary syndrome (CTCL subtypes). The objective response rate (ORR) was 29.7% overall and 29.5% in stage IIb or higher patients. Overall 32% of patient has pruritus relief including 25% of those who did not meet the ORR criteria. Improved pruritus has positive influence in quality of life in CTCL patients. The median duration of response was ≥6.1 months. Thus this study shows that vorinostat was well tolerated and effective as a single agent in CTCL [103] . Another open label, non-randomized phase II trial of oral vorinostat for treatment of refractory CTCL done in 33 patients (13 received vorinostat) who have received and failed prior therapy, demonstrated a response rate of 24%, 8 out of 33 achieved partial response and additional  11 patient have pruritus relief [100] .FDA has approved 400mg once daily dose orally with food. If patient is intolerant to therapy, the dose can be reduced to 300mg once daily or 300mg once daily only for 5 consecutive days each week. Treatment can be continued until progressive disease, unacceptable toxicity, lack of efficacy or patient’s withdrawal of consent [102] .With the oral administration of 400mg of Vorinostat in fasting state, time to maximal concentration (T max) is 1.5 hours and half-life (t1/2) is 1.74 hours. It is eliminated by metabolism via glucorunidation and hydrolysis followed by β-oxidation and less than 1% of administered dose is recovered unchanged in urine [104] .

The most common drug related adverse effects of vorinostat were fatigue, thrombocytopenia, diarrhea and nausea. The most common grade 3 or 4 drug related adverse effect were thrombocytopenia and dehydration [100] . In phase IIb trial in 74 patients, 1 of 3 patients on warfarin at baseline required dose reduction to achieve target prothrombin time/international normalized ratio (PT/INR). One patient had grade 2 QTc prolongations and two had grade 1 QTc prolongation in electrocardiogram (EKG). There were no grade 3 or higher EKG adverse events and none of EKG changes required dose reduction or discontinuation. 28% had grade3/4 adverse effect such as fatigue (5%), pulmonary embolism (5%), thrombocytopenia (5%) and nausea (4%). Median time of onset of grade ¾ adverse effects was 43 days. 11% had serious drug related adverse effect (median time of onset 42 days) such as thromboembolic events, anemia, increase creatinine, death dehydration, gastro-intestinal bleed, ischemic stroke, streptococcal bacteremia, syncope, thrombocytopenia. Three deaths were reported in the study, one due to disease progression, one due to ischemic stroke and one unexplained death [103,105] . Vorinostat is contraindicated in severe hepatic impairment. All patients should be monitored for pulmonary embolism and Deep vein Thrombosis (DVT). Drug related thrombocytopenia and anemia requires dose modification. Severe thrombocytopenia and gastrointestinal bleeding have been reported with concomitant use of vorinostat with other HDAC inhibitors. It is a pregnancy category D drug. Safety and effectiveness in pediatric patient has not been established.


Romidepsin is a potent HDAC inhibitor which was isolated from Chromobacterium violaceum [106] .  Chemical structure of romidepsin is shown in figure 3C. Romidepsin is a prodrug which inside a cell undergoes reduction of the disulfide bond by glutathione to release a monocyclic dithiol. One of the thiol groups of reduced romidepsin can bind with Zn ions of enzyme that prevents the binding of substrate. The reduced form inhibits HDAC1 and 2 enzymes of class I and thus inhibits deacetylation of lysine residues of N-terminal histone tails making chromatin open and transcriptionally active. Romidepsin also causes G1 phase cell cycle arrest via acetylation of non-histone proteins. In vitro, romidepsin causes decrease in cyclin D1 and c-myc with increase in p53-independent p21 WAF1/Cip 1 induction. Increase p21 inhibits cyclin dependent kinase (decrease Cdc2/Cdk-1 and cyclin B1) and dephosphorylation of Rb protein leading to G1 phase cell cycle arrest. Romidepsin leads to cell apoptosis via the death receptor (hyper acetylation of TNF, death receptor-5, Fas ligand and Fas) and intrinsic pathways (accumulation of ROS and disruption of mitochondrial membrane). Romidepsin inhibits angiogenesis by downregulating VEGF, VEGF receptor, FLT1 and FLK1 and upregulates angiogenic inhibitory factors VHL and neurofibrin 2. However, the exact mechanisms leading to cell cycle arrest and apoptosis is still unclear [107,108] .

Romidepsin was approved by FDA on November 5, 2009 for the treatment of CTCL in patients who have received at least one prior systemic therapy and for treatment of peripheral T-cell Lymphoma (PTCL) in patients who have received at least one prior therapy.  It is marketed as trade name Istodax by company Celgene [109] . This indication is based on RR in two large phase II studies [110,111] . Clinical benefit such as improvement in OS has not been demonstrated. A multicenter phase II trial done in 71 patients with CTCL shows an ORR of 34%(CI 95%, 23%-46%) with a median duration of response being 13.7 months. In an initial cohort of 27 patients who received no more than 2 prior cytotoxic regimens, 3 patient receive a complete response and 8 achieved PR for an ORR of 41 %( 95% CI, 22%-61%). The results supported the approval of romidepsin for CTCL [110] . Other multicenter study done in 96 patients with stage IB to IVA CTCL who had received one or more systemic therapies demonstrated an overall response rate of 34% including 6 patients with complete response with median duration of reduction of pruritus being 6 months [111] .  In Phase II study, 47 patients with PTCL of various subtypes who had received prior therapy with a median of 3 previous treatment was enrolled and the ORR was 38 %( 95% CI 24%-53%) with a median duration of response of 8.9 months (range 2-4) [112] . All these studies concluded that romidepsin is a significant and sustainable single agent for CTCL and PTCL with an acceptable safety profile. An open label phase II study done in 131 patients among whom 130 had histologically confirmed PTCL, refractory to prior systemic therapy demonstrated objective response rate of 25% including 15% with complete response or unconfirmed complete response (CR/CRu) . The median duration of response was 17 months and the drug is well tolerated with manageable toxicity. The results lead to FDA approval for PTCL [113] .

The FDA approved dose is similar to treatment schedules of those two phase II studies [112] , which is 14 mg/m2 IV over a 4 hour period on days 1, 8, and 15 of a 28 day cycle. The cycle is repeated every 28 days provided the patient has benefitted and tolerates the drug. Interruption or discontinuation of treatment can be done to manage toxicity. 4 hours IV infusion of romidepsin exhibits linear pharmacokinetics from doses 1.0 to 24.9 mg/m2. In vitro, it is primarily metabolized by CYP3A4 with minor contribution from CYP3A5, CYP1A1, CYP2B6, and CYP2C19. After 4 hours of infusion (usual dose), romidepsin is rapidly cleared from circulation, half-life being around 3.5 hours [110,111,114] .

The most common drug related side effects of romidepsin are gastrointestinal disturbances and asthenic conditions. The most common adverse effect are neutropenia, lymphopenia, thrombocytopenia, infections, nausea, fatigue, vomiting, anorexia, anemia and T wave changes in EKG [110,111,113] . Therefore, it should be used cautiously in patient with thrombocytopenia, leukopenia and anemia. Careful monitoring of blood count is necessary and may require dose modification and therapy modification. Fatal and serious infections and reactivation of EBV and Hepatitis B virus (HBV) can occur [112] . Because some asymptomatic T wave flattening and ST-segment depression was observed in phase I trials, detailed cardiac evaluation was done in phase II trial that revealed no cardiac damage based on serial troponin I values, multiple gated acquisition scans and echocardiograms   [111] . There is an increase in QT interval median being 14 millisecond and 0.2% of the evaluated EKG had QTcB (Bazett’s correction) interval of >500 millisecond which proves that romidepsin is safe enough [110] . QTc changes were observed in other study as well, but EKG changes returned to baseline within 24 hours with no associated functional cardiovascular symptoms [111] . Careful monitoring of cardiovascular status and magnesium and potassium electrolytes is important in patients with prior heart disease and congenital QT prolongation.  Patients with advance disease should be monitored for tumor lysis syndrome (though very low incidence). The drug is a potential hazard for the fetus and pregnancy should be avoided when on romidepsin. Careful monitoring of PT/INR is necessary when there is concurrent administration of warfarin. Its use with rifampin and strong CYP3A4 inducers should be avoided.  Six deaths were observed in phase II trial with 96 patients, however, none were drug related. Three of them was due to disease progression alone, one was due to disease progression and dyspnea, one due to disease progression and acute renal failure, and one initially thought to be related to the treatment, but later was concluded to be most likely due to disease progression [111] . Romidepsin is relatively safe and has no obvious contraindications.

Combined use of DNMT and HDAC inhibitors

Combination therapy with DNMT inhibitor and HDAC inhibitor theoretically has great biologic rationale. Physical and functional interactions of DNA with histones make it a sensible combination treatment strategy.  Results of in vitro experiments have shown the possibility of maximizing epigenetic response by combining DNMT inhibitor and HDAC inhibitor [115] . This combination is proven to have synergistic effect in inducing apoptosis and cell growth arrest in various cancers, such as lung, breast, colon, leukemia, MDS and thoracic cancers [116] . Combination epigenetic therapy with the use of DNMT inhibitor first followed by HDAC inhibitor has shown a significant re-expression of DNA methylation silenced genes [115,117] . One challenge with combination treatment is to adjust dosage and treatment schedules during treatment. Multiple trials were done in the past to evaluate its efficacy and response rates. A phase I trial with DAC 20mg/m2/day and valproic acid (VA) done in AML failed to show a synergistic response [118] . Another phase I/II trial of DAC+VA in leukemia patient demonstrated decreased global and promoter methylation in both responders and non-responders. Histone acetylation was observed in small number of patients. The combination showed significant clinical activity and was safe but further exploration with new HDAC inhibitors was recommended due to limitations in the study like small number of patients and lack of correlation between VA induced histone acetylation and response rate [119] .  Other study with AZA or DAC with VA also did not demonstrate clinical benefit which can be attributed to dosing schedule of VA(VA given as a course instead of continuously) and/or lack of VA potency in inhibiting HDAC [120] . Whereas the other study done in MDS and AML patients with AZA(dose ranging from 25-75/m2 for 5 to 14 days) followed by sodium phenyl butyrate(375mg/kg/day for 7 day continuous infusion) demonstrated a promising results. Six of six responding patients with pre-treatment methylation of p15 or CDH-1 have methylation reversal after the first cycle of treatment.  Combination showed increased acetylation of histone H3 and H4, promoter methylation reversal and clinical responses [121] . Superiority of combination therapy over single drug therapy is yet to be proven but is an area of interest based on in vitro and preclinical trials. In the future, combination therapy could be a good alternative to chemotherapies, especially in patients who cannot tolerate chemotherapies and who are not a suitable candidate for bone marrow transplants.

Epigenetic drugs in clinical trial

In the recent years, with approval of some epigenetic drugs by the FDA and EMA, these agents have been extensively studied in cancer treatment. Promising results of these drugs in pre-clinical and in vitro trials provides a foundation for their use in cancer treatment. These drugs either alone or in combination have been tested for almost all types of cancer including solid tumors [122] . Early phase I and phase II trials in breast cancer with AZA shows response rate as high as 18%. The dose used was higher (up to 188mg/m2) and has higher risk of cytotoxicity. AZA as a single agent is under trial using 75mg/m2/day to induce expression of ER and PR genes in patient with triple negative breast cancer who are awaiting surgery [123] . Multicenter phase II clinical trials with combination of AZA and entinostat in advanced triple negative or hormone resistant breast cancer are being performed with the dose of AZA 40mg/m2 subcutaneously on days 1-5 and 8-10, and entinostat 7mg orally on day 3 and 10 of a 28 day cycle [124] . Various trials have been done combining epigenetic drugs with chemotherapies as well. In a phase I trial, done in 35 patients (colon 7, breast  5, ovary 5, melanoma 4, sarcoma 4, gall bladder 2, pleural mesothelioma 2 and other cancers 6 patients), 33 received DAC and carboplatin, while 2 patient received only DAC. Although less demethylation was observed in tumor biopsies than peripheral blood cell (PBC), up to 60% demethylation was observed in patient tumors in xenograft at chemosensitising doses [125] . In a phase II trial, 360mg/m2 of MG 98 (a second generation DNMT1 inhibitor) was given to 17 untreated adult patient with metastatic renal cell carcinoma as 2 hour IV infusion twice weekly for three consecutive weeks. This study did not demonstrated antitumor activity of MG 98 which can be explained by lack of target effect or the choice of tumor type [126] . Another multicenter study demonstrated that MG 98 combined with interferon resulted in clinical activity and is safe in metastatic renal cell carcinoma [127] . Describing every trial of epigenetic drugs in detail is beyond the reach of this review, therefore, we tried to summarize the trials in cancer, sponsored by NCI (National Cancer Institute at National Institute of Health) and have NCT identifiers in table 3 to 15 [128,129] . Among DNMT inhibitors only AZA, DAC and SGI-110 are in clinical trial. Because AZA is widely studied and there are more than 300 trials for AZA alone, we have included only those AZA trials in the table that have been performed in the USA. There are no clinical trials for zebularine and fazarabine in clinicaltrial.gov web site. RG 108 is in trial for Rheumatoid arthritis, cardiovascular disease, Diabetes mellitus, Chronic Obstructive Pulmonary Disease (COPD), hyperlipidemia and post-menopausal osteoporosis but not for cancer. Active status of trial means they are recruiting patients. Closed status suggests that the trial is not recruiting patients and does not necessarily mean study is completed.

Future directions of use of epigenetic drugs

Reversing the epigenetic changes in solid tumors seems to be the challenge both at this time and in future. Optimal dose and schedule for solid tumors need to be determined [123] . That dose needs to kill cancer cells but not the normal cells. These epigenetic drugs are cytotoxic at higher dose and with the lower dose, maintaining the therapeutic window is necessary [130] .Convenient route of administration such as oral instead of SC or IV should be goal in treatment with epigenetic drugs. A number of trials are going on with oral form of AZA (CC486), and it has been found to be effective and well tolerated [131,132] . Development of advanced epigenetic analogues to prevent drug resistance and improve pharmacokinetics is also a matter of interest in future. An example being SGI-110, a second generation hypomethylating agent in phase I development, that is a dinucleotide of decitabine and is resistant to deamination by cytosine deaminase(CDA). Thus SGI-110 has longer half-life and resistance to therapy can be avoided with this agent [54] . DNMT and HDAC inhibitors selective for enzyme subtype, targeting only the relevant enzyme involved in that particular cancer needs to be developed in the future. Rational combination of epigenetic drugs with other anticancer agent such as, chemotherapy or radiation therapy may have a greater impact in treatment and survival which needs to be further explored [59] . In the future, for further development of epigenetic drugs, it will be important to know drug dependent methylation level in cancer tissue, and to do evaluation of epigenetic markers to know the treatment effect. It seems technologies for assessing methylation markers are needed to evaluate drug induced methylation changes for guiding clinical decision. Identifying predictive biomarkers of response to DNMT inhibitors and HDAC inhibitors can help identify which patient will be benefitted from therapy. Presence ofTET2 and/or DNMT3A mutations are associated with higher ORR in treatment with AZA. The nucleoside transporter expression pattern could also potentially be used as predictive biomarker for therapy with nucleoside analogues. Another area to think about is reducing and managing toxicity with these drugs.  Such as ENT-1 has a role in AZA uptake and AZA induced cytotoxicity can be reduced by nitrobenzyl mercaptopurine ribonucleoside (an inhibitor of ENT-1) [57,133,134] . Combination therapy with epigenetic drugs seems useful but validation is required with further successful clinical trials [121,135] . With FDA approval, these drugs are being used in treatment of MDS and CTCL successfully, but what the next step should be if the patient does not respond or in case of treatment failure is also a matter of concern. The outcome of patients who fail to respond with DAC is poor, and future research is needed to prevent relapse [90] . Studies show that only 50% of patients respond to therapy with AZA and DAC and a majority these relapse within two years. This further mandated the use of biomarkers for response prediction [58] . Improved outcome of the patient relies on improved understanding of the molecular mechanisms of epigenetic drugs in detail, and in identifying the biomarkers to evaluate response [136,137] . Furthermore, promising clinical trials of AZA and DAC for treatment of AML proves their potential utility and could be the therapy of choice in elderly patients and patients not suitable for bone marrow transplantation.


Understanding epigenetics and the discovery of agents to reverse the epigenetic changes that lead to cancer are the phenomenal achievements in cancer research and are an evolving area of interest in cancer therapy. Combination treatment of DNMT inhibitors with HDAC inhibitors and other anticancer agents seems to be a promising strategy of cancer therapy in the future. FDA approval of these agents has raised hope for further development of these drugs and further understanding of epigenetic modifications in pathogenesis of cancer. The ongoing clinical trials in this field provide us further insights about new treatment strategies for cancer. With all this, perhaps, we are getting closer to using the reversal of unwanted epigenetic modification in cancer as a pertinent therapy, for which all types of cancer patients might benefit.


This work was supported by The Pennsylvania Cancer Cure Grant 6914101 to Dr Jose Russo, and by NIH (National Institute of Health) Core grants CA06927 to Fox Chase Cancer Center. 


  1. Rodríguez-Paredes M, Esteller M (2011). Cancer epigenetics reaches mainstream oncology. Nat Med , 17(3): 330-339.
  2. Feinberg AP (2004). The epigenetics of cancer etiology. Semin Cancer Biol , 14(6): 427-432.
  3. Laird, P.W., (2005). Cancer epigenetics. Human Molecular Genetics, 14(suppl 1): p. R65-R76.
  4. Gal-Yam EN, Saito Y, Egger G, Jones PA (2008). Cancer epigenetics: modifications, screening, and therapy. Annu Rev Med , 59: 267-280.
  5. Hatzimichael E, Crook T (2013). Cancer epigenetics: new therapies and new challenges. J Drug Deliv , 2013: 529312.
  6. Jones PA, Baylin SB (2002). The fundamental role of epigenetic events in cancer. Nat Rev Genet , 3(6): 415-428.
  7. Gebhard C, Benner C, Ehrich M, Schwarzfischer L, Schilling E, et al. (2010). General transcription factor binding at CpG islands in normal cells correlates with resistance to de novo DNA methylation in cancer cells. Cancer Res , 70(4): 1398-1407.
  8. Herman JG, Baylin SB (2003). Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med , 349(21): 2042-2054.
  9. Das PM, Singal R (2004). DNA Methylation and Cancer Journal of Clinical Oncology, 22(22): p. 4632-4642.
  10. Bestor TH (2000). The DNA methyltransferases of mammals. Hum Mol Genet , 9(16): 2395-2402.
  11. Okano M, Bell DW, Haber DA, Li E (1999). DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell , 99(3): 247-257.
  12. Goll MG, Kirpekar F, Maggert KA, Yoder JA, Hsieh CL, et al. (2006). Methylation of tRNAAsp by the DNA methyltransferase homolog Dnmt2. Science , 311(5759): 395-398.
  13. Li KK, Luo LF, Shen Y, Xu J, Chen Z, et al. (2013). DNA methyltransferases in hematologic malignancies. Semin Hematol , 50(1): 48-60.
  14. Svedruzic ZM (2008). Mammalian cytosine DNA methyltransferase Dnmt1: enzymatic mechanism, novel mechanism-based inhibitors, and RNA-directed DNA methylation. Curr Med Chem , 15(1): 92-106.
  15. Goffin J, Eisenhauer E (2002). DNA methyltransferase inhibitors-state of the art. Ann Oncol , 13(11): 1699-1716.
  16. Robertson AK, et al. (2004). Effects of chromatin structure on the enzymatic and DNA binding functions of DNA methyltransferases DNMT1 and Dnmt3a in vitro. Biochemical and Biophysical Research Communications, 322(1): p. 110-118.
  17. Robertson KD (2001). DNA methylation, methyltransferases, and cancer. Oncogene , 20(24): 3139-3155.
  18. Lustberg MB, Ramaswamy B (2009). Epigenetic targeting in breast cancer: therapeutic impact and future direction. Drug News Perspect , 22(7): 369-381.
  19. Akhavan-Niaki H, Samadani A (2013). DNA Methylation and Cancer Development: Molecular Mechanism. Cell Biochemistry and Biophysics, 67(2): p. 501-513.
  20. Jjingo D, Conley AB, Yi SV, Lunyak VV, Jordan IK (2012). On the presence and role of human gene-body DNA methylation. Oncotarget , 3(4): 462-474.
  21. Luczak MW, Jagodziński PP (2006). The role of DNA methylation in cancer development. Folia Histochem Cytobiol , 44(3): 143-154.
  22. Pan LN, Lu J, Huang B (2007). HDAC inhibitors: a potential new category of anti-tumor agents. Cell Mol Immunol , 4(5): 337-343.
  23. Jenuwein T, Allis CD (2001). Translating the histone code. Science , 293(5532): 1074-1080.
  24. Turner BM (1998). Histone acetylation as an epigenetic determinant of long-term transcriptional competence. Cell Mol Life Sci , 54(1): 21-31.
  25. Ho AS, Turcan S, Chan TA (2013). Epigenetic therapy: use of agents targeting deacetylation and methylation in cancer management. Onco Targets Ther, 6: p. 223-32.
  26. Barneda-Zahonero B, Parra M (2012). Histone deacetylases and cancer. Mol Oncol , 6(6): 579-589.
  27. Lane AA, Chabner BA (2009). Histone deacetylase inhibitors in cancer therapy. J Clin Oncol , 27(32): 5459-5468.
  28. Dokmanovic M, Clarke C, Marks PA (2007). Histone deacetylase inhibitors: overview and perspectives. Mol Cancer Res , 5(10): 981-989.
  29. Glozak MA, Seto E (2007). Histone deacetylases and cancer. Oncogene , 26(37): 5420-5432.
  30. Glaser KB (2007). HDAC inhibitors: Clinical update and mechanism-based potential. Biochemical Pharmacology, 74(5): p. 659-671.
  31. Adams H, Fritzsche FR, Dirnhofer S, Kristiansen G, Tzankov A (2010). Class I histone deacetylases 1, 2 and 3 are highly expressed in classical Hodgkin's lymphoma. Expert Opin Ther Targets , 14(6): 577-584.
  32. Fritzsche FR, Weichert W, Röske A, Gekeler V, Beckers T, et al. (2008). Class I histone deacetylases 1, 2 and 3 are highly expressed in renal cell cancer. BMC Cancer , 8: 381.
  33. Weichert, W., et al. (2008). Class I Histone Deacetylase Expression Has Independent Prognostic Impact in Human Colorectal Cancer: Specific Role of Class I Histone Deacetylases In vitro and In vivo. Clinical Cancer Research, 14(6): p. 1669-1677.
  34. Choi JH, et al. (2001). Expression Profile of Histone Deacetylase 1 in Gastric Cancer Tissues. Cancer Science, 92(12): p. 1300-1304.
  35. Sun JY, et al. (2011). Histone Deacetylase Inhibitors Demonstrate Significant Preclinical Activity as Single Agents, and in Combination with Bortezomib in Waldenström's Macroglobulinemia. Clinical Lymphoma Myeloma and Leukemia, 11(1): p. 152-156.
  36. OzdaÄŸ H, Teschendorff AE, Ahmed AA, Hyland SJ, Blenkiron C, et al. (2006). Differential expression of selected histone modifier genes in human solid cancers. BMC Genomics , 7: 90.
  37. Stark M, Hayward N (2007). Genome-wide loss of heterozygosity and copy number analysis in melanoma using high-density single-nucleotide polymorphism arrays. Cancer Res , 67(6): 2632-2642.
  38. Sjöblom T, Jones S, Wood LD, Parsons DW, Lin J, et al. (2006). The consensus coding sequences of human breast and colorectal cancers. Science , 314(5797): 268-274.
  39. Lachenmayer A, Toffanin S, Cabellos L, Alsinet C, Hoshida Y, et al. (2012). Combination therapy for hepatocellular carcinoma: additive preclinical efficacy of the HDAC inhibitor panobinostat with sorafenib. J Hepatol , 56(6): 1343-1350.
  40. Krusche CA, Wülfing P, Kersting C, Vloet A, Böcker W, et al. (2005). Histone deacetylase-1 and -3 protein expression in human breast cancer: a tissue microarray analysis. Breast Cancer Res Treat , 90(1): 15-23.
  41. Zhang Z, Yamashita H, Toyama T, Sugiura H, Ando Y, et al. (2005). Quantitation of HDAC1 mRNA expression in invasive carcinoma of the breast*. Breast Cancer Res Treat , 94(1): 11-16.
  42. Minamiya Y, Ono T, Saito H, Takahashi N, Ito M, et al. (2011). Expression of histone deacetylase 1 correlates with a poor prognosis in patients with adenocarcinoma of the lung. Lung Cancer , 74(2): 300-304.
  43. Oehme I, Deubzer HE, Wegener D, Pickert D, Linke JP, et al. (2009). Histone deacetylase 8 in neuroblastoma tumorigenesis. Clin Cancer Res , 15(1): 91-99.
  44. Osada H, et al. (2004). Reduced expression of class II histone deacetylase genes is associated with poor prognosis in lung cancer patients. International Journal of Cancer, 112(1): p. 26-32..
  45. Copeland RA, Olhava EJ, Scott MP (2010). Targeting epigenetic enzymes for drug discovery. Curr Opin Chem Biol , 14(4): 505-510.
  46. Lubbert M (2000). DNA methylation inhibitors in the treatment of leukemias, myelodysplastic syndromes and hemoglobinopathies: Clinical results and possible mechanisms of action. DNA Methylation and Cancer, 249: p. 135-164.
  47. Juttermann R, Li E, Jaenisch R (1994). Toxicity of 5-aza-2'-deoxycytidine to mammalian cells is mediated primarily by covalent trapping of DNA methyltransferase rather than DNA demethylation. Proceedings of the National Academy of Sciences, 91(25): p. 11797-11801.
  48. D'Incalci M, Covey JM, Zaharko DS, Kohn KW (1985). DNA alkali-labile sites induced by incorporation of 5-aza-2'-deoxycytidine into DNA of mouse leukemia L1210 cells. Cancer Res , 45(7): 3197-3202.
  49. Stresemann C, Brueckner B, Musch T, Stopper H, Lyko F (2006). Functional diversity of DNA methyltransferase inhibitors in human cancer cell lines. Cancer Res , 66(5): 2794-2800.
  50. Lyko F, Brown R (2005). DNA methyltransferase inhibitors and the development of epigenetic cancer therapies. J Natl Cancer Inst , 97(20): 1498-1506.
  51. Singh BN, Shankar S, Srivastava RK (2011). Green tea catechin, epigallocatechin-3-gallate (EGCG): mechanisms, perspectives and clinical applications. Biochem Pharmacol , 82(12): 1807-1821.
  52. Brueckner B, Kuck D, Lyko F (2007). DNA methyltransferase inhibitors for cancer therapy. Cancer J , 13(1): 17-22.
  53. Fleming G, et al. (2014). Abstract 2320: Clinical epigenetic resensitization of platinum-resistant, recurrent ovarian cancer patients with SGI-110, a novel, second-generation, subcutaneously administered hypomethylating agent (HMA). Cancer Research, 74(19 Supplement): p. 2320.
  54. Jabbour E, et al. (2013). First Clinical Results Of a Randomized Phase 2 Study Of SGI-110, a Novel Subcutaneous (SQ) Hypomethylating Agent (HMA), In Adult Patients With Acute Myeloid Leukemia (AML). , Vol. 122, 497-497.
  55. Scholl J, et al. (2010). SGI-110, a Novel Second Generation Potent DNA Methylation Inhibitor, In Development for the Treatment of MDS and AML. Preclinical Safety, Pharmacokinetics, and DNA Methylation Results of a Low Volume Subcutaneous (SC) Formulation. Blood, AMER SOC HEMATOLOGY 1900 M STREET. NW SUITE 200, WASHINGTON, DC 20036 USA, .
  56. Billam M, Sobolewski MD, Davidson NE (2010). Effects of a novel DNA methyltransferase inhibitor zebularine on human breast cancer cells. Breast Cancer Res Treat , 120(3): 581-592.
  57. Stresemann C, Lyko F (2008). Modes of action of the DNA methyltransferase inhibitors azacytidine and decitabine. Int J Cancer , 123(1): 8-13.
  58. Derissen EJ, Beijnen JH, Schellens JH (2013). Concise drug review: azacitidine and decitabine. Oncologist , 18(5): 619-624.
  59. Gravina GL, Festuccia C, Marampon F, Popov VM, Pestell RG, et al. (2010). Biological rationale for the use of DNA methyltransferase inhibitors as new strategy for modulation of tumor response to chemotherapy and radiation. Mol Cancer , 9: 305.
  60. Kaminskas E, Farrell AT, Wang YC, Sridhara R, Pazdur R (2005). FDA drug approval summary: azacitidine (5-azacytidine, Vidaza) for injectable suspension. Oncologist , 10(3): 176-182.
  61. Robert MF, Morin S, Beaulieu N, Gauthier F, Chute IC, et al. (2003). DNMT1 is required to maintain CpG methylation and aberrant gene silencing in human cancer cells. Nat Genet , 33(1): 61-65.
  62. Szyf M (2001). Towards a pharmacology of DNA methylation. Trends Pharmacol Sci , 22(7): 350-354.
  63. McCabe MT, Brandes JC, Vertino PM (2009). Cancer DNA methylation: molecular mechanisms and clinical implications. Clin Cancer Res , 15(12): 3927-3937.
  64. Ambinder RF, Robertson KD, Tao Q (1999). DNA methylation and the Epstein-Barr virus. Semin Cancer Biol , 9(5): 369-375.
  65. Greenberg PL, Attar E, Bennett JM, Bloomfield CD, De Castro CM, et al. (2011). NCCN Clinical Practice Guidelines in Oncology: myelodysplastic syndromes. J Natl Compr Canc Netw , 9(1): 30-56.
  66. Estey EH (2013). Epigenetics in clinical practice: the examples of azacitidine and decitabine in myelodysplasia and acute myeloid leukemia. Leukemia , 27(9): 1803-1812.
  67. FDA (2014). Azacitidine. Available from: http://www.accessdata.fda.gov/drugsatfda_docs/label/2014/050794s026lbledt.pdf, .
  68. Silverman LR, Demakos EP, Peterson BL, Kornblith AB, Holland JC, et al. (2002). Randomized controlled trial of azacitidine in patients with the myelodysplastic syndrome: a study of the cancer and leukemia group B. J Clin Oncol , 20(10): 2429-2440.
  69. Malcovati L, Hellström-Lindberg E, Bowen D, Adès L, Cermak J, et al. (2013). Diagnosis and treatment of primary myelodysplastic syndromes in adults: recommendations from the European LeukemiaNet. Blood , 122(17): 2943-2964.
  70. Silverman LR, et al. (2006). Further Analysis of Trials With Azacitidine in Patients With Myelodysplastic Syndrome: Studies 8421, 8921, and 9221 by the Cancer and Leukemia Group B. Journal of Clinical Oncology, 24(24): p. 3895-3903.
  71. Cheson BD, Bennett JM, Kantarjian H, Pinto A, Schiffer CA, et al. (2000). Report of an international working group to standardize response criteria for myelodysplastic syndromes. Blood , 96(12): 3671-3674.
  72. Itzykson R, Thépot S, Quesnel B, Dreyfus F, Beyne-Rauzy O, et al. (2011). Prognostic factors for response and overall survival in 282 patients with higher-risk myelodysplastic syndromes treated with azacitidine. Blood , 117(2): 403-411.
  73. Voso MT, et al. (2013). Revised International Prognostic Scoring System (IPSS) Predicts Survival and Leukemic Evolution of Myelodysplastic Syndromes Significantly Better Than IPSS and WHO Prognostic Scoring System: Validation by the Gruppo Romano Mielodisplasie Italian Regional Database. Journal of Clinical Oncology, 31(21): p. 2671-2677.
  74. Duong VH, Lancet JE, Alrawi E, Al-Ali NH, Perkins J, et al. (2013). Outcome of azacitidine treatment in patients with therapy-related myeloid neoplasms with assessment of prognostic risk stratification models. Leuk Res , 37(5): 510-515.
  75. Santini V, Fenaux P, Mufti GJ, Hellström-Lindberg E, Silverman LR, et al. (2010). Management and supportive care measures for adverse events in patients with myelodysplastic syndromes treated with azacitidine*. Eur J Haematol , 85(2): 130-138.
  76. Batty GN, Kantarjian H, Issa JP, Jabbour E, Santos FP, et al. (2010). Feasibility of therapy with hypomethylating agents in patients with renal insufficiency. Clin Lymphoma Myeloma Leuk , 10(3): 205-210.
  77. Kantarjian HM, Thomas XG, Dmoszynska A, Wierzbowska A, Mazur G, et al. (2012). Multicenter, randomized, open-label, phase III trial of decitabine versus patient choice, with physician advice, of either supportive care or low-dose cytarabine for the treatment of older patients with newly diagnosed acute myeloid leukemia. J Clin Oncol , 30(21): 2670-2677.
  78. Quintás-Cardama A, Ravandi F, Liu-Dumlao T, Brandt M, Faderl S, et al. (2012). Epigenetic therapy is associated with similar survival compared with intensive chemotherapy in older patients with newly diagnosed acute myeloid leukemia. Blood , 120(24): 4840-4845.
  79. Fenaux P, Mufti GJ, Hellström-Lindberg E, Santini V, Gattermann N, et al. (2010). Azacitidine prolongs overall survival compared with conventional care regimens in elderly patients with low bone marrow blast count acute myeloid leukemia. J Clin Oncol , 28(4): 562-569.
  80. Sudan N, Rossetti JM, Shadduck RK, Latsko J, Lech JA, et al. (2006). Treatment of acute myelogenous leukemia with outpatient azacitidine. Cancer , 107(8): 1839-1843.
  81. Bally C, et al. (2013). Azacitidine in the treatment of therapy related myelodysplastic syndrome and acute myeloid leukemia (tMDS/AML): A report on 54 patients by the Groupe Francophone Des Myelodysplasies (GFM). Leukemia Research, 37(6): p. 637-640.
  82. Momparler RL (2005). Pharmacology of 5-Aza-2'-deoxycytidine (decitabine). Semin Hematol , 42(3 Suppl 2): S9-16.
  83. FDA (2015). Decitabine. Available from: http://www.accessdata.fda.gov/drugsatfda_docs/label/2010/021790s006lbl.pdf, .
  84. Kantarjian H, Issa JP, Rosenfeld CS, Bennett JM, Albitar M, et al. (2006). Decitabine improves patient outcomes in myelodysplastic syndromes: results of a phase III randomized study. Cancer , 106(8): 1794-1803.
  85. Wijermans P, Lübbert M, Verhoef G, Bosly A, Ravoet C, et al. (2000). Low-dose 5-aza-2'-deoxycytidine, a DNA hypomethylating agent, for the treatment of high-risk myelodysplastic syndrome: a multicenter phase II study in elderly patients. J Clin Oncol , 18(5): 956-962.
  86. Kantarjian H, Oki Y, Garcia-Manero G, Huang X, O'Brien S, et al. (2007). Results of a randomized study of 3 schedules of low-dose decitabine in higher-risk myelodysplastic syndrome and chronic myelomonocytic leukemia. Blood , 109(1): 52-57.
  87. Lee YG, Kim I, Yoon SS, Park S, Cheong JW, et al. (2013). Comparative analysis between azacitidine and decitabine for the treatment of myelodysplastic syndromes. Br J Haematol , 161(3): 339-347.
  88. Karahoca M, Momparler RL (2013). Pharmacokinetic and pharmacodynamic analysis of 5-aza-2'-deoxycytidine (decitabine) in the design of its dose-schedule for cancer therapy. Clin Epigenetics , 5(1): 3.
  89. Cashen AF, Shah AK, Todt L, Fisher N, DiPersio J (2008). Pharmacokinetics of decitabine administered as a 3-h infusion to patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Cancer Chemother Pharmacol , 61(5): 759-766.
  90. Santos FP, Kantarjian H, Garcia-Manero G, Issa JP, Ravandi F (2010). Decitabine in the treatment of myelodysplastic syndromes. Expert Rev Anticancer Ther , 10(1): 9-22.
  91. Cashen AF, Schiller GJ, O'Donnell MR, DiPersio JF (2010). Multicenter, phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia. J Clin Oncol , 28(4): 556-561.
  92. Drummond DC, Noble CO, Kirpotin DB, Guo Z, Scott GK, et al. (2005). Clinical development of histone deacetylase inhibitors as anticancer agents. Annu Rev Pharmacol Toxicol , 45: 495-528.
  93. Lin HY, Chen CS, Lin SP, Weng JR, Chen CS (2006). Targeting histone deacetylase in cancer therapy. Med Res Rev , 26(4): 397-413.
  94. Prince HM, Dickinson M (2012). Romidepsin for cutaneous T-cell lymphoma. Clin Cancer Res , 18(13): 3509-3515.
  95. Ververis K, Hiong A, Karagiannis TC, Licciardi PV (2013). Histone deacetylase inhibitors (HDACIs): multitargeted anticancer agents. Biologics , 7: 47-60.
  96. Grant S, Easley C, Kirkpatrick P (2007). Vorinostat. Nat Rev Drug Discov , 6(1): 21-22.
  97. Richon VM (). Cancer biology: mechanism of antitumour action of vorinostat (suberoylanilide hydroxamic acid), a novel histone deacetylase inhibitor. Br J Cancer, 95(S1): p. S2-S6.
  98. Xu WS, Parmigiani RB, Marks PA (2007). Histone deacetylase inhibitors: molecular mechanisms of action. Oncogene , 26(37): 5541-5552.
  99. Zhang C, Richon V, Ni X, Talpur R, Duvic M (2005). Selective induction of apoptosis by histone deacetylase inhibitor SAHA in cutaneous T-cell lymphoma cells: relevance to mechanism of therapeutic action. J Invest Dermatol , 125(5): 1045-1052.
  100. Duvic M, Talpur R, Ni X, Zhang C, Hazarika P, et al. (2007). Phase 2 trial of oral vorinostat (suberoylanilide hydroxamic acid, SAHA) for refractory cutaneous T-cell lymphoma (CTCL). Blood , 109(1): 31-39.
  101. FDA (2014). vorinostat. Available from: http://www.accessdata.fda.gov/drugsatfda_docs/label/2011/021991s002lbl.pdf, .
  102. Mann BS, Johnson JR, Cohen MH, Justice R, Pazdur R (2007). FDA approval summary: vorinostat for treatment of advanced primary cutaneous T-cell lymphoma. Oncologist , 12(10): 1247-1252.
  103. Olsen EA, Kim YH, Kuzel TM, Pacheco TR, Foss FM, et al. (2007). Phase IIb multicenter trial of vorinostat in patients with persistent, progressive, or treatment refractory cutaneous T-cell lymphoma. J Clin Oncol , 25(21): 3109-3115.
  104. Iwamoto M, Friedman EJ, Sandhu P, Agrawal NG, Rubin EH, et al. (2013). Clinical pharmacology profile of vorinostat, a histone deacetylase inhibitor. Cancer Chemother Pharmacol , 72(3): 493-508.
  105. Mann BS, Johnson JR, He K, Sridhara R, Abraham S, et al. (2007). Vorinostat for treatment of cutaneous manifestations of advanced primary cutaneous T-cell lymphoma. Clin Cancer Res , 13(8): 2318-2322.
  106. Nakajima H, Kim YB, Terano H, Yoshida M, Horinouchi S (1998). FR901228, a potent antitumor antibiotic, is a novel histone deacetylase inhibitor. Exp Cell Res , 241(1): 126-133.
  107. VanderMolen KM, McCulloch W, Pearce CJ, Oberlies NH (2011). Romidepsin (Istodax, NSC 630176, FR901228, FK228, depsipeptide): a natural product recently approved for cutaneous T-cell lymphoma. J Antibiot (Tokyo) , 64(8): 525-531.
  108. Radhakrishnan V, Song YS, Thiruvengadam D (2008). Romidepsin (depsipeptide) induced cell cycle arrest, apoptosis and histone hyperacetylation in lung carcinoma cells (A549) are associated with increase in p21 and hypophosphorylated retinoblastoma proteins expression. Biomedicine and Pharmacotherapy, 62(2): p. 85-93.
  109. FDA (2014). Romidepsin. , .
  110. Piekarz RL, et al. (2009). Phase II Multi-Institutional Trial of the Histone Deacetylase Inhibitor Romidepsin As Monotherapy for Patients With Cutaneous T-Cell Lymphoma. Journal of Clinical Oncology, 27(32): p. 5410-5417.
  111. Whittaker SJ, Demierre MF, Kim EJ, Rook AH, Lerner A, et al. (2010). Final results from a multicenter, international, pivotal study of romidepsin in refractory cutaneous T-cell lymphoma. J Clin Oncol , 28(29): 4485-4491.
  112. Piekarz RL, Frye R, Prince HM, Kirschbaum MH, Zain J, et al. (2011). Phase 2 trial of romidepsin in patients with peripheral T-cell lymphoma. Blood , 117(22): 5827-5834.
  113. Coiffier B, Pro B, Prince HM, Foss F, Sokol L, et al. (2012). Results from a pivotal, open-label, phase II study of romidepsin in relapsed or refractory peripheral T-cell lymphoma after prior systemic therapy. J Clin Oncol , 30(6): 631-636.
  114. Woo S, Gardner ER, Chen X, Ockers SB, Baum CE, et al. (2009). Population pharmacokinetics of romidepsin in patients with cutaneous T-cell lymphoma and relapsed peripheral T-cell lymphoma. Clin Cancer Res , 15(4): 1496-1503.
  115. Cameron EE, Bachman KE, Myöhänen S, Herman JG, Baylin SB (1999). Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat Genet , 21(1): 103-107.
  116. Zhu W G, Otterson G A (2003). The Interaction of Histone Deacetylase Inhibitors and DNA Methyltransferase Inhibitors in the Treatment of Human Cancer Cells. Current Medicinal Chemistry -Anti-Cancer Agents, 3(3): p. 187-199.
  117. Griffiths EA, Gore SD (2008). DNA methyltransferase and histone deacetylase inhibitors in the treatment of myelodysplastic syndromes. Semin Hematol , 45(1): 23-30.
  118. Blum W, Klisovic RB, Hackanson B, Liu Z, Liu S, et al. (2007). Phase I study of decitabine alone or in combination with valproic acid in acute myeloid leukemia. J Clin Oncol , 25(25): 3884-3891.
  119. Garcia-Manero G, Kantarjian HM, Sanchez-Gonzalez B, Yang H, Rosner G, et al. (2006). Phase 1/2 study of the combination of 5-aza-2'-deoxycytidine with valproic acid in patients with leukemia. Blood , 108(10): 3271-3279.
  120. LUbbert M, Kuendgen A (2014). Combining DNA methyltransferase and histone deacetylase inhibition to treat acute myeloid leukemia/myelodysplastic syndrome: Achievements and challenges. Cancer, .
  121. Gore SD, Baylin S, Sugar E, Carraway H, Miller CB, et al. (2006). Combined DNA methyltransferase and histone deacetylase inhibition in the treatment of myeloid neoplasms. Cancer Res , 66(12): 6361-6369.
  122. Nebbioso A, Carafa V, Benedetti R, Altucci L (2012). Trials with 'epigenetic' drugs: an update. Mol Oncol , 6(6): 657-682.
  123. Connolly R, Stearns V (2012). Epigenetics as a therapeutic target in breast cancer. J Mammary Gland Biol Neoplasia , 17(3-4): 191-204.
  124. Connolly R, et al. (2011). OT3-01-06: A Phase 2 Study Investigating the Safety, Efficacy and Surrogate Biomarkers of Response of 5-Azacitidine (5-AZA) and Entinostat (MS-275) in Patients with Advanced Breast Cancer. Cancer Research, 71(24 Supplement): p. OT3-01-06.
  125. Appleton K, Mackay HJ, Judson I, Plumb JA, McCormick C, et al. (2007). Phase I and pharmacodynamic trial of the DNA methyltransferase inhibitor decitabine and carboplatin in solid tumors. J Clin Oncol , 25(29): 4603-4609.
  126. Winquist E, et al. (2006). Phase II trial of DNA methyltransferase 1 inhibition with the antisense oligonucleotide MG98 in patients with metastatic renal carcinoma: a National Cancer Institute of Canada Clinical Trials Group investigational new drug study. Invest New Drugs, 24(2): p. 159-67.
  127. Amato RJ, Stephenson J, Hotte S, Nemunaitis J, Bélanger K, et al. (2012). MG98, a second-generation DNMT1 inhibitor, in the treatment of advanced renal cell carcinoma. Cancer Invest , 30(5): 415-421.
  128. NCI (2015). None Available from: http://www.cancer.gov/clinicaltrials/search, .
  129. NIH (). None Available from: https://www.clinicaltrials.gov/, .
  130. Azad N, Zahnow CA, Rudin CM, Baylin SB (2013). The future of epigenetic therapy in solid tumours--lessons from the past. Nat Rev Clin Oncol , 10(5): 256-266.
  131. LoRusso P, et al. (2013). Abstract A120: A Phase Ib study of CC-486 (Oral Azacitidine) as a priming agent for carboplatin or NAB-paclitaxel in subjects with relapsed and refractory solid tumors. Molecular Cancer Therapeutics, 12(11 Supplement): p. A120.
  132. Garcia-Manero G, et al. (2012). Safety and Efficacy of Oral Azacitidine (CC-486) Administered in Extended Treatment Schedules to Patients with Lower-Risk Myelodysplastic Syndromes. ASH Annual Meeting Abstracts, 120(21): p. 424.
  133. Hubeek I, et al. (). The human equilibrative nucleoside transporter 1 mediates in vitro cytarabine sensitivity in childhood acute myeloid leukaemia. Br J Cancer, 93(12): p. 1388-1394.
  134. Marce S, et al. (2006). Expression of human equilibrative nucleoside transporter 1 (hENT1) and its correlation with gemcitabine uptake and cytotoxicity in mantle cell lymphoma. , Vol. 91, 895-902.
  135. Juergens RA, Wrangle J, Vendetti FP, Murphy SC, Zhao M, et al. (2011). Combination epigenetic therapy has efficacy in patients with refractory advanced non-small cell lung cancer. Cancer Discov , 1(7): 598-607.
  136. Traina F, Visconte V2, Elson P3, Tabarroki A2, Jankowska AM2, et al. (2014). Impact of molecular mutations on treatment response to DNMT inhibitors in myelodysplasia and related neoplasms. Leukemia , 28(1): 78-87.
  137. Itzykson R, Kosmider O, Cluzeau T, Mansat-De Mas V, Dreyfus F, et al. (2011). Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias. Leukemia , 25(7): 1147-1152.