Epigenomes as therapeutic targets.

JPT-06767; No of Pages 15 Pharmacology & Therapeutics xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Pharmacology & Therapeutics journal homepage: www.elsevier.com/locate/pharmthera

Associate editor: B. Teicher

Epigenomes as therapeutic targets Christopher A. Hamm a,⁎, Fabricio F. Costa a,b,c,d,⁎⁎ a Cancer Biology and Epigenomics Program, Ann & Robert H Lurie Children's Hospital of Chicago Research Center and Department of Pediatrics, Northwestern University's Feinberg School of Medicine, 225 E. Chicago Avenue, Box 220, Chicago, IL 60611-2605, USA b StartUp Health Academy, 2000 Broadway St, 18th Floor, New York, NY 10.023, USA c Genomic Enterprise, 2405 N. Sheffield Av., # 14088, Chicago, IL 60.614, USA d Genomic Sciences and Biotechnology Program, UCB - Brasilia, SGAN 916 Modulo B, Bloco C, 70.790-160 Brasilia, Brazil

a r t i c l e

i n f o

Keywords: Epigenomes Epigenetics DNA Methylation Histone Modifications Therapeutics Complex Diseases

a b s t r a c t Epigenetics is a molecular phenomenon that pertains to heritable changes in gene expression that do not involve changes in the DNA sequence. Epigenetic modifications in a whole genome, known as the epigenome, play an essential role in the regulation of gene expression in both normal development and disease. Traditional epigenetic changes include DNA methylation and histone modifications. Recent evidence reveals that other players, such as non-coding RNAs, may have an epigenetic regulatory role. Aberrant epigenetic signaling is becoming to be known as a central component of human disease, and the reversible nature of the epigenetic modifications provides an exciting opportunity for the development of clinically relevant therapeutics. Current epigenetic therapies provide a clinical benefit through disrupting DNA methyltransferases or histone deacetylases. However, the emergence of next-generation epigenetic therapies provides an opportunity to more effectively disrupt epigenetic disease states. Novel epigenetic therapies may improve drug targeting and drug delivery, optimize dosing schedules, and improve the efficacy of preexisting treatment modalities (chemotherapy, radiation, and immunotherapy). This review discusses the epigenetic mechanisms that contribute to the disease, available epigenetic therapies, epigenetic therapies currently in development, and the potential future use of epigenetic therapeutics in a clinical setting. © 2015 Elsevier Inc. All rights reserved.

Contents 1. Introduction to epigenetics . . . . . . . . . . . 2. DNA methylation: the fifth base . . . . . . . . . 3. The sixth base: 5-hydroxymethylcytosine (5-hmC) 4. DNA methylation as therapeutic targets . . . . . 5. Future directions of epigenomic therapy . . . . . 6. Conclusions . . . . . . . . . . . . . . . . . . 7. Conflicts of interest . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .

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1. Introduction to epigenetics ⁎ Correspondence to: Translational R&D – Oncology, Quintiles. 777 Oakmont Lane Suite 100, Westmont IL 60559. Tel.: +1 (857) 753 7136. ⁎⁎ Correspondence to: Cancer Biology and Epigenomics Program, Ann & Robert H Lurie Children's Hospital of Chicago Research Center and Department of Pediatrics, Northwestern University's Feinberg School of Medicine, 225 E. Chicago Avenue, Box 220, Chicago, IL 60611-2605, USA, Genomic Sciences and Biotechnology Program, UCB Brasilia, SGAN 916 Modulo B, Bloco C, 70.790-160 Brasilia, Brazil. E-mail addresses: [email protected] (C.A. Hamm), [email protected] (F.F. Costa).

The term epigenetics was described in the early 1940s and defines the interactions between the genome and the environment that leads to the formation of the phenotype (Waddington, 1939). Epigenetics is a phenomenon that involves heritable changes in gene expression that does not imply a change in the nucleotide sequence but impacts the conformation of the DNA (Bird, 2002). The way the chromatin structure is maintained and organized is key to a better understanding of

http://dx.doi.org/10.1016/j.pharmthera.2015.03.003 0163-7258/© 2015 Elsevier Inc. All rights reserved.

Please cite this article as: Hamm, C.A., & Costa, F.F., Epigenomes as therapeutic targets, Pharmacology & Therapeutics (2015), http://dx.doi.org/ 10.1016/j.pharmthera.2015.03.003

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C.A. Hamm, F.F. Costa / Pharmacology & Therapeutics xxx (2015) xxx–xxx

Table 1 Glossary of epigenetic terms. Term

Definition

Active DNA demethylation

Demethylation that results from the failure to methylate a newly synthesized DNA strand. As a result, the DNA methylation of the template strand is not passed along to the new DNA strand. Complex of DNA, proteins (histone proteins and non-histone proteins), and ncRNA (non-coding RNA) that form the structural matrix of a chromosome. A CG (cytosine–guanine) rich DNA region. Approximately 60% of genes contain a CpG island in their promoter region. An enzyme that transfers a “methyl” group to a cytosine molecule at CG sites throughout the genome. The study of heritable modifications throughout the genome that do not involve changes to the DNA sequence. The study of multiple epigenetic modifications throughout the entire genome. Histones are a protein family with a structural and functional role in the packaging of DNA within a nucleus. Histones contribute to the structure of the nucleosome. An enzyme that removes acetyl groups from core histones. A relative increase in methylated cytosine residues in a DNA sequence. Relative absence of methylated cytosines in a DNA sequence. A cytosine molecule that has a “methyl” group (−CH3) at the “carbon-5” position. Molecule composed of a base (cytosine, guanine, adenine, thymine or uracil) linked to a ribose (RNA) or deoxyribose (DNA) sugar. The basic structural unit of chromatin made up of DNA and histone proteins. Nucleoside with a phosphate group(s) attached to the sugar moiety. Nucleotides are the building blocks for DNA and RNA. Occurs when a methylated cytosine (5-mC) is physically replaced with an unmethylated cytosine.

Chromatin

CpG island

DNA methyltransferase (DNMT) Epigenetics

Epigenomics Histone

Histone deacetylase (HDAC) Hypermethylation Hypomethylation Methylated cytosine (5-mC) Nucleoside

Nucleosome Nucleotide

Passive DNA demethylation

the origins of epigenetic alterations in embryonic development, cell differentiation and disease. The cytosines in DNA methylation and post-translational modifications on histones are the most studied epigenetic modifications. In the last decade, however, new players such as non-coding RNAs were associated to different epigenetic mechanisms (Costa, 2008). Epigenetic changes are able to affect gene expression mostly by interfering with the accessibility of transcription factors to DNA

Table 2 Leading causes of death in the United States in 2010. Leading causes of death in the United States *': Disease with an epigenetic component Causes of death

Number of deaths

Heart disease: Cancer: Chronic lower respiratory diseases: Stroke (cerebrovascular diseases): Accidents (unintentional injuries): Alzheimer's disease: Diabetes: Nephritis, nephrotic syndrome, and nephrosis: Influenza and pneumonia: Intentional self-harm (suicide):

597,689* 574,743* 138,080* 129,476* 120,859 83,494* 69,071* 50,476* 50,097 38,364

Data are for the U.S. and represents final data from the Centers for Disease Control corresponding to the year 2010 http://www.cdc.gov/nchs/fastats/leading-causes-of-death.htm.

(Table 1). Thus, the same DNA sequence in the cells of a multicellular organism may show alternative phenotypes that are based on different epigenetic states (Jones, 2012). Epigenetics is one of the rapidly developing fields in biomedical research and clinical medicine. Given the importance of epigenetics in gene regulation, it is no surprise that epigenetics play a significant role in human disease. Epigenetic alterations are considered a hallmark of cancer (Sandoval & Esteller, 2012). In addition, seven of the top 10 leading causes of death in the US are diseases with an epigenetic component (Table 2) (Hamm & Costa, 2011). 1.1. Epigenetics — a burgeoning field Advances in technologies of molecular biology and next-generation DNA sequencing are providing insight into the breadth and functional significance of epigenetic alterations in disease. Epigenetics has seen substantial growth over the past 20 years, and the number of epigenetic research articles published in a given year reflects the growth of the field (Fig. 1). The records from the National Institutes of Health document the publication of 81 epigenetic articles in 1990, 321 epigenetic articles in 2000, and over 5300 epigenetic articles in 2013 (Fig. 1). The growth in epigenetics research has provided a greater understanding of the human epigenome and also created new avenues for therapeutic interventions in diseases. It is now well established that epigenetics plays an essential role in normal development, and not surprisingly, epigenetic alterations contribute to the development and progression of several types of disease. Several 1st generation epigenetic therapies have garnered FDA approval including drugs that treat some specific cancers such as leukemia, lymphomas and other tumor types that are in clinical trials (Table 3) (Costa, 2010; Hamm & Costa, 2011) and next-generation epigenetic therapies are currently in clinical development. Emerging therapies aim to improve on the 1st generation epigenetic treatments, and they provide an exciting opportunity to treat novel disease-related epigenetic pathways. Despite significant advances in the field, epigenetics is still in its infancy. We believe that advances in molecular biology and nextgeneration DNA sequencing will lead to a greater understanding of the functional role of epigenetics in disease. Defining and unraveling the myriad of epigenetic alterations in particular disease states will provide a significant opportunity for the development of therapeutics across a spectrum of human diseases. Epigenetics is the study of heritable modifications throughout the genome that do not involve changes to the DNA sequence (Table 1) (Wolffe & Matzke, 1999; Feinberg & Tycko, 2004; Pogribny & Beland, 2009). Epigenetic modifications occur at distinct regions throughout the genome, and epigenomics is the study of epigenetic modifications throughout the entire genome (Table 1). Epigenetics and epigenomics are the study of chromatin: the complex of DNA, proteins (histone proteins and non-histone proteins), and non-coding RNAs (ncRNAs) that form the structural matrix of a chromosome (Table 1). Chromatin is essential for nuclear DNA packaging and gene regulation. At least three types of epigenetic modifications regulate chromatin: DNA methylation, histone modifications, and ncRNAs. The most well documented epigenetic alterations are DNA methylation and histone modifications. Both DNA methylation and histone modifications influence gene expression through their ability to alter DNA/protein interactions. These epigenetic changes may inhibit gene expression by preventing a transcription factor from binding to a DNA sequence. Epigenetic alterations may also lead to changes in chromatin structure that promote the expression of a particular gene. While epigenetic alterations influence gene expression, it is important to emphasize that epigenetic changes are reversible (as opposed to genetic alterations which are not reversible). Epigenetic-based therapeutics offers an exciting opportunity to reverse disease-associated epigenetic abnormalities. For example, abnormal DNA methylation is well documented in cancer, and abnormal methylation may silence


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Fig. 1. Growing number of publications in the epigenetics field.



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Table 3 Approved clinical DNA methylation and HDAC inhibitors. FDA approved epigenetic therapies Compound

Mechanism of action

Indications

Vidaza (5-Azacytidine)

Nucleoside DNMT inhibitor

Dacogen (Decitabine) Zolinza (Vorinostat)

Nucleoside DNMT inhibitor HDAC inhibitor

Istodax (Romidepsin)

HDAC inhibitor

Beleodaq (Belinostat)

HDAC inhibitor

FDA approved for myelodysplastic syndromes and chronic myelomonocytic leukemia. FDA approved for myelodysplastic syndromes. FDA approved for the treatment of cutaneous T-cell lymphoma FDA approved for the treatment of cutaneous T-cell lymphoma and peripheral T-cell lymphoma FDA approved for the treatment of relapsed or refractory peripheral T-cell lymphoma

the expression of tumor suppressor genes. Drugs, such as Dacogen (Decitabine; Eisai Inc.) and Vidaza (5-azacytidine; Celgene), may reverse tumor-associated gene silencing through their ability to disrupt DNA methylation. Aberrant histone modifications occur in cancer, and the aberrant histone modifications may lead to inappropriate gene silencing. The histone deacetylase inhibitors Zolinza (Vorinostat; Merck) and Istodax (Romidepsin; Celgene) may reverse tumorassociated gene silencing through their ability to disrupt aberrant posttranslational histone alterations (Table 3). This article provides an introduction to the field of epigenetics, emphasizes the relevancy of epigenomics in disease, highlights FDA approved epigenetic therapies, and provides insight into novel epigenetic therapeutic pipelines. 1.2. Chromatin Consisting of 46 chromosomes, a typical human cell (diploid) has 6 billion base pairs of DNA. If the DNA from the 46 chromosomes is laid out end-to-end, the total length of DNA in a human cell is approximately 2.04 m (6.7 ft) (Annunziato, 2008). The two meters of DNA must fit into a small compartment: a nucleus with a diameter of 10−5 m. To organize an entire genome into a 10−5 m nucleus, the DNA must compact into a smaller volume relative to a linear strand of DNA. Linear DNA is organized into a more compact state through DNA modifications and interactions with proteins (histones) and also ncRNAs (Costa, 2008) that assist with DNA coiling/folding. Chromatin is the complex of DNA, proteins, and ncRNAs (Costa, 2008) that form the structural matrix of a chromosome. Prior to a more in-depth explanation of the function of specific epigenetic modifications, it is important to emphasize the vital role of chromatin in a human cell. Chromatin is essential for proper gene regulation throughout the genome and chromatin is also essential for the structural organization of the DNA within a human cell. Chromatin plays a structural and functional role in regulating the approximately 25,000 proteincoding genes in the human genome (Pertea & Salzberg, 2010). To prevent disease, each gene in the human genome must be tightly regulated in a time and tissue-specific manner. For example, specific cell division genes are essential for normal human development, and the same genes must be tightly regulated to prevent uncontrolled cell division. 1.2.1. Nucleosome: structural and functional unit of the chromatin Histones are proteins that condense DNA within the nucleus. Histones form a protein complex and the DNA coils around the protein complex to create a nucleosome. The nucleosome is the basic structural and functional unit of chromatin, and each nucleosome consists of approximately 147 base pairs of DNA coiled around a histone core (an octamer) (Table 1). The primary nucleosome structure repeats throughout the genome and ultimately leads to an array of nucleosomes

that further condenses the DNA. A diploid human cell contains an estimated 30 million nucleosomes, and these nucleosomes play an essential role in chromatin-mediated packaging of DNA within a cell (Alberts et al., 2002). In fact, the chromatin is capable of condensing DNA by 10,000- to 20,000-fold (Woodcock & Ghosh, 2010). Mammalian cells possess enzymes that modify DNA or histones and subsequently alter their biologic function. These enzymes will be discussed further in this article. 1.2.2. Chromatin regulation: histone modifications The ability of the nucleosome to condense and organize the genome is related to both the molecular characteristics of the DNA, as well as, the molecular characteristics of the proteins that form the nucleosome. The core of the nucleosome is made up of eight proteins, consisting of two of each of the following histone proteins: H2A, H2B, H3, and H4. Histones are proteins with an inherent positive charge that bind to the DNA (which has an inherent negative charge) and form the nucleosome. Post-translational histone modifications may affect the electrostatic charge of the histones, thereby altering the association between the histone proteins and the DNA. Depending on the electrostatic change, the posttranslational modification may promote a condensed chromatin state or a relaxed chromatin state. In addition, the posttranslational modifications may create binding sites for proteins that may further impact chromatin structure and/or gene expression. The histone acetylation was first described in the early 60s, but the chemical nature of the N-acetylation was completed only a few years later (Yang & Seto, 2007). The identification of enzymes related to the process of acetyltransferases and deacetylases was only performed in the mid-90s and was directly related to gene regulation (Brownell et al., 1996; Mizzen et al., 1996). The HAT catalyzes the transfer of an acetyl group from direct acetylCoA the ε-amino group of the lysine residue. With a water molecule HDAC promotes the removal of the acetyl group from acetyl–lysine (Ac–Lys) regenerating the ε-amino group. Histone acetylation is a widely studied post-translation modification with a function in transcriptional regulation (Struhl, 1998). Histone acetylation is a reversible process that involves the transfer of an acetyl group to a lysine amino acid in a histone protein. Lysine acetyltransferases catalyze the transfer of an acetyl group to a histone protein, and histone deacetylases (HDAC) remove the acetyl group. Histone acetylation is traditionally a sign of open chromatin structure and transcriptional activation (Hebbes et al., 1988). Whereas histone deacetylation, and HDAC activity, is characteristic of transcriptional repression and structurally repressive chromatin (Alland et al., 1997). In mammals, eighteen HDACs are classified into four distinct classes (Table 5). HDACs possess both distinct and overlapping cellular functions (Jurkin et al., 2011). While HDACs act on histone proteins, HDACs also play a role in the deacetylation of non-histone proteins (Glozak et al., 2005; Yao & Yang, 2011). The ability of HDACs to interact with non-histone proteins suggests that HDACs may have diverse cellular functions, which lie outside of their function in chromatin. For example, HDAC3 targets at least five non-histone protein substrates (Yao & Yang, 2011). Further insight into the diverse nature of the acetylome is provided by the observation that, in addition to nuclear protein substrates, HDACs are located in the cytoplasm and also target proteins in the cytoplasm. The clinical relevancy of HDACs in the cytoplasm is highlighted by the observation that cytoplasmic HDAC4 plays a pathogenic role in Huntington's disease (Robinson, 2013). HDACs are also found in the cytoplasm. The range of HDAC localization and function indicates that HDACs may have context-specific functions (Kelly & Cowley, 2013). Histone proteins are targets for a myriad of other post-translational modifications including: ADP-ribosylation, citrullination, clipping, methylation, phosphorylation, sumoylation, phosphorylation, ubiquitination, and others (Hassan et al., 2002; Lister et al., 2009; Sanchez & Zhou, 2009; Ray-Gallet & Almouzni, 2010) [9–12]. Post-translational modification can alter the electrostatic interaction between the histones and



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Table 4 Clinical and pre-clinical DNA methylation inhibitors. Drug

Mechanism

Developer/source

Stage of development

Findings

(−)-epigallocatechin-3-gallate (EGCG)

Nucleoside DNMT inhibitor. Polyphenol isolated from green tea.

PMID: 16037419

Preclinical.

5-fluoro-2′-deoxycytidine (FdCyD) with tetrahydrouridine (THU)

Nucleoside analog. FdCyD metabolite inhibits DNMT's. THU inhibits cytidine deaminase.

NCI

Currently in Phase I and Phase II trials of advanced solid tumors. NCT01534598 and NCT00978250.

ASTX727

Nucleoside DNMT inhibitor. Oral combination product; consisting of decitabine and E7727 (a novel cytidine deaminase inhibitor)

Astex Pharmaceuticals/Otsuka Pharmaceutical, Eisai Inc

Phase I trial with MDS Patients (NCT02103478).

cc-486

Nucleoside DNMT inhibitor. Oral formulation of 5-azacytidine.

Celgene

Phase I–III clinical trials. Single-agent trials and drug combination trials.

CP-4200

Aqualis (AQUA; Formerly Clavis Pharma)

Preclinical.

Decitabine (Dacogen)

Nucleoside DNMT inhibitor. 5-Azacytidine derivative: 5-azacytidine-5′-elaidate. 5-Azacytidine linked to a lipid/fatty acid (nucleoside transporter-independent). Nucleoside DNMT inhibitor.

Eisai Inc [Tokyo: 4523] (Licensed from Astex Pharmaceuticals)

FDA and EMA approval.

Hydralazine

Non-nucleoside DNMT inhibitor.

PMID: 12632429

MG98

Non-nucleoside DNMT1 inhibitor. Antisense oligonucleotide molecule designed against DNMT1.

Mirati Therapeutics Inc (formerly Methylgene Inc; TSX:MYG) PMID: 18413836 and 22571342

Arterial vasodilator approved by FDA for treatment of severe hypertension, heart failure and hypertension in pregnancy. Phase I–III development as a chemotherapeutic agent. Phase I studies in adult solid tumors and AML.

EGCG may or indirectly inhibit DNMT activity. EGCG also induces cytotoxic oxidative stress. Bioavailability issues and discrepancy with in vitro/in vivo data have hindered development. FdCyd is processed into cytotoxic metabolites that incorporate into DNA and RNA. Co-administration with THU allows for more efficient DNA hypomethylation by slowing the metabolism of FdCyD and preventing the formation of particular cytotoxic metabolites. Decitabine can be delivered orally with E7727 due to the inhibition of CDA by E7727. ASTX727 was designed for efficient oral delivery of decitabine at low doses. Once-daily oral azacitidine is active and well-tolerated in extended dosing schedules. One-half of high-risk MDS patients achieved a hematologic response to an extended dosing schedule of CC-486. Fatty acid moiety allows for the nucleoside transporter-independent uptake of 5-azacytidine. Potential to overcome nucleoside transporter–related drug resistance to 5-azacytidine. FDA approved for the treatment of patients with myelodysplastic syndromes. EMA approved for treatment of acute myeloid leukemia. Hydralazine/HDACi treatment demethylates promoters and leads to expression of tumor suppressor genes in patients. DNMT inhibitory activity not observed in certain cell types.

Procainamide and procaine (ester analog of procainamide)

Non-nucleoside DNMT inhibitors.

PMID: 16230360 and 12941824

Psammaplin A

Non-nucleoside DNMT inhibitor. HDAC and DNMT and SIRT deacetylase inhibitor

PMID: 23030799

Procainamide approved for the treatment of cardiac arrhythmias and approved as a local anesthetic. Procainamide and procaine are in the preclinical stages of chemotherapeutic development. Preclinical.

RG108 (N‐Phthaloyl‐L‐ tryptophan)/RG108 analogs

Non-nucleoside DNMT inhibitor. Blocks the active site of the DNMT enzyme. Non-nucleoside DNMT inhibitor. Pan-epigenetic anticancer agent. RRx-001 indirectly inhibits HDACs and Dnmt1 and 3a expression.

PMID: 24328113

Preclinical.

RADIORx (private company)

Phase I–Phase II.

PMID: 19417133 and 23637988

Preclinical.

RRx-001

SGI-1027

Non-nucleoside DNMT inhibitor. Inhibits DNMT by competing with methyl donor (S-adenosylmethionine).

MG98 binds to DNMT1 mRNA and prevents subsequent processing of the DNMT1 mRNA. MG98 treatment decreases the cellular level of the DNMT1 enzyme. In 2006, methylgene chose not to pursue further development of MG98. Procainamide and procaine have affinity for CpG-rich DNA. Procainamide and procaine reduce the affinity of DNMT1 for DNA, which ultimately results in loss of DNA methylation. Inhibits multiple enzymes (DNMTs, HDACs, DNA gyrase and Topoisomerase). Data indicates that psammaplin A has a stronger HDAC inhibitory activity relative to the inhibitory DNMT activity. RG108 analogs are more cytotoxic and more efficient at DNMT inhibition. Inhibition of HDAC, DNMT1, DNMT3a via reactive oxygen and nitrogen species (oxidation and inactivation of DNMT1). RRx-001 oxidative and nitrosative stress promotes apoptosis. Phase I data indicate that RRx-001 was well tolerated. DNMT1, DNMT3A and DNMT3B inhibitor.

(continued on next page)


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Table 4 (continued) Drug

Mechanism

Developer/source


Findings

SGI-110

Nucleoside DNMT inhibitor: dinucleotide of decitabine and deoxyguanosine.

Astex Pharmaceuticals/Otsuka Pharmaceutical

In aqueous solution, SGI-110 is as stable as decitabine, but SGI110 is more resistant to cytidine deaminase.

Vidaza (5-azacytidine)

Nucleoside DNMT inhibitor.

Celgene

Phase I-II clinical trial in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). FDA and EMA approval.

Zebularine

Nucleoside DNMT inhibitor. Cytidine deaminase inhibitor. Cytidine analog. Incorporated into DNA and RNA. DNMT1 inhibitor (also incorporated into RNA and is a cytidine deaminase inhibitor).

PMID: 24225777

Preclinical.

nearby DNA. These changes can alter accessibility to the transcriptional machinery (Ray-Gallet & Almouzni, 2010). Histone modifications can also alter the association of histones with protein complexes. For example, bromodomain proteins can bind to acetylated histones, and these bromodomain proteins may represent a subunit of a protein complex that further modulates chromatin structure and gene transcription (Hassan et al., 2002; Sanchez & Zhou, 2009). 1.2.3. Chromatin regulation: DNA methylation Epigenetic modifications (posttranslational histone modifications) impact the structure and function of proteins within the chromatin, and epigenetic modifications can also impact the structure and function of the DNA within chromatin. The most widely studied epigenetic DNA modification is methylation. DNA methylation involves the transfer of a methyl group (−CH3) to a cytosine nucleotide within in a given DNA sequence (Fig. 2). DNA methylation correlates with transcriptional silencing and typically occurs in DNA sequences that contain cytosines adjacent to a guanine nucleotide (known as a CpG site) (Table 1). Approximately 30 million CpG sites exist in the human genome (Cocozza et al., 2011). Although CpG sites are located throughout the genome, certain regions of DNA contain a high proportion of CpG sites, known as CpG islands. The methylation of CpG islands is associated with the silencing of the genes that are in proximity to the CpG islands. Aberrant CpG island methylation often leads to inappropriate gene silencing and disease (Jones & Baylin, 2002). 1.3. Epigenetic therapy in the clinic The FDA approval of 1st generation epigenetic therapies highlights the validity of the epigenome as a therapeutic target. These 1st Table 5 Histone deacetylase classification and localization in the cells. HDAC classification and subcellular localization Histone deacetylase (HDAC)

Class

Localization

HDAC1 HDAC2 HDAC3 HDAC4 HDAC5 HDAC6 HDAC7 HDAC8 HDAC9 HDAC10 HDAC11 Sirtuins (SIRT1–SIRT7)

I I I IIa IIa IIb IIa I IIa IIb IV III

Nucleus, Cytoplasmic Nucleus, Cytoplasmic Nucleus, Cytoplasmic Nucleus, Cytoplasmic Nucleus, Cytoplasmic Nucleus, Cytoplasmic Nucleus, Cytoplasmic Nucleus, Cytoplasmic Nucleus, Cytoplasmic Nucleus, Cytoplasmic Nucleus, Cytoplasmic Nucleus (SIRT1, 6, and 7), Mitochondria (SIRT3, 4, and 5), and Cytoplasm (SIRT2)

FDA and EMA approved for the treatment of patients with myelodysplastic syndromes and chronic myelomonocytic leukemia EMA approved for AML. Inefficient metabolic activation and preclinical toxicity has limited the use of zebularine as a monotherapy. PMID: 19509174 and 20846732.

generation epigenetic therapies provide a clinical benefit through their ability to disrupt epigenetic modifications throughout the entire genome. Despite the benefit of 1st generation epigenetic therapies, these therapies nonspecifically alter the epigenomic landscape and may have unwanted side effects. Ongoing epigenetic research will improve existing therapies and identify new epigenetic drug targets. The identification of novel epigenetic targets will lead to the next generation therapies with greater therapeutic efficacy and fewer side effects than the current global-epigenetic therapies. Several ongoing clinical trials are examining novel epigenetic therapies that may provide greater specificity and fewer side effects than the current FDA approved epigenetic therapies. While current clinical trials focus on epigenetic therapies in cancer, it is important to recognize that epigenetic alterations also occur in diseases ranging from asthma/immune disorders, to diabetes and psychiatric disorders (Hamm & Costa, 2011). The prevalence of epigenetic alterations in multiple diseases indicates that epigenetic drugs may have significant promise outside of the cancer therapeutics sector (Hamm & Costa, 2011). 2. DNA methylation: the fifth base DNA methylation involves the transfer of a methyl group to a cytosine molecule to form a methylated cytosine (5-mC) (Fig. 2). In the context of the mammalian genome, 5-mC is assigned the moniker “the fifth base”. Although 5-mC is technically not a distinct nucleotide, 5-mC is biologically unique from an unmodified cytosine nucleotide. During mammalian development, the nuclear DNA is subject to genome-wide changes in DNA methylation (Laurent et al., 2010). Genome-wide DNA methylation changes are necessary for proper development, and they contribute to the cellular diversity during embryonic development. At an early stage in embryonic development (post-fertilization), the majority of the genomic DNA becomes demethylated (Cantone & Fisher, 2013). Subsequently, DNA methylation is reestablished in a tissue-specific manner (Reik et al., 2001; Santos et al., 2002). Normal differentiation requires proper DNA methylation patterns and abnormal DNA methylation limits the capacity of a cell to differentiate into tissue/cell-specific lineages (Fisher & Fisher, 2011). DNA methylation may play a role in the silencing of genes that are necessary for early stages of development, but no longer needed for subsequent stages of development. Localized DNA methylation promotes a repressive chromatin structure and the silencing of gene expression. Failure to properly methylate a specific region of DNA may lead to abnormal gene expression. Alternatively, aberrant DNA methylation may lead to inappropriate gene silencing, which may promote disease. For example, DNA methylation is associated with the silencing of tumor suppressor genes in several types of cancer (Jones & Baylin, 2002). Although DNA methylation correlates with gene silencing, it is important to note that DNA methylation also plays an opposite role in the activation of specific genes (Rishi et al., 2010).



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Fig. 2. Multiple cytosine variants are present in the human genome. DNA methyltransferases (DNMTs) add a methyl group (−CH3) to the carbon-5 position in a cytosine molecule, which results in the formation of 5-methylcytosine (5-mC). Subsequently, 5-mC can be converted to 5-hydroxymethylcytosine (5-hmC) by Tet proteins (a family of oxygenases). Tet proteins can further oxidize 5-hmC to 5-formylcytosine (5-fC). Finally, the Tet proteins can oxidize 5-fC to 5-carboxylcytosine (5-caC). The complete function of the Tet proteins are not known, however, Tet proteins produce cytosine variants that may ultimately lead to DNA demethylation. Alternatively, the Tet-induced cytosine variants may have a functional role in cellular signaling through their ability to recruit or displace effector proteins. As a point of reference, the atoms of the cytosine molecule are numerically labeled. In the cytosine variants, the carbon-5 of the cytosine ring is labeled with “5” and the carbon-5 modification is noted in red. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

2.1. DNA methylation is a reversible signal Since DNA methylation directly influences gene expression, normal mammalian development requires spatial and temporal control of DNA methylation. To facilitate normal development, mammalian cells possess multiple pathways to establish, maintain, and modify DNA methylation throughout the genome. Enzymes in mammalian cells, known as DNA methyltransferases, are responsible for establishing and maintaining DNA methylation. Since DNA methylation is dynamic, mammalian cells also possess the ability to remove DNA methylation (DNA demethylation). DNA demethylation occurs through passive and active mechanisms. Passive DNA methylation requires DNA replication and the subsequent blocking of DNA methylation maintenance pathways (Piccolo & Fisher, 2014). Active DNA methylation is replicationindependent and occurs through the enzymatic removal of the methylcytosine (Piccolo & Fisher, 2014). Taken together, the DNA methylation/demethylation machinery allows mammalian cells to regulate gene expression throughout development. While proper DNA methylation is essential for normal development, the abnormal regulation of the DNA methylation machinery may contribute to disease development and progression (Gaudet et al., 2003). Although abnormal DNA methylation signals may contribute to disease, the reversible nature of epigenetic alterations makes the DNA methylation machinery an exciting therapeutic target (Wright, 2013). Therapies targeting the DNA methylation/demethylation machinery may disrupt abnormal epigenetic signals and subsequently restore normal DNA methylation profiles.

2.2. CpG methylation and CpG islands During DNA methylation, an enzyme – DNA methyltransferase – transfers a methyl group from a donor molecule to a cytosine located in a cytosine–guanine (CpG) dinucleotide. Specifically, a methyl group from S-adenosylmethionine is transferred to the Carbon-5 position of cytosine, in the context a CpG dinucleotide, to form a methylated cytosine (5-mC; Fig. 2). CpG sites exist throughout the human genome, however, particular regions of the genome have clusters of CpGs. These clusters are named CpG islands (Table 1). The human genome contains approximately 25,000 CpG islands (haploid) (Illingworth et al., 2010). Interestingly, CpG islands are located near genes and approximately 60% of genes contain a CpG island in their promoter region (Bird, 2002). While CpG is the most common form of DNA methylation, it is important to note that DNA methylation also occurs at other nucleotides (Ramsahoye et al., 2000; Yan et al., 2011).

Of the 60% of genes containing promoter region CpG islands, only 10% of these CpG islands are methylated (Bergman & Cedar, 2013). CpG island DNA methylation correlates with long-term gene silencing that plays a role in tissue-specific control of gene regulation (Jones, 2012). However, aberrant CpG methylation patterns are associated with human disease. For example, abnormal DNA methylation in cancer may lead to the silencing of tumor suppressor genes and subsequent uncontrolled cell growth and cancer progression. CpG island methylation can alter gene expression by disrupting the binding of transcription factors, by recruiting chromatin-modifying complexes, and by altering the conformation of the DNA (Temiz et al., 2012). CpG island methylation prevents transcription factors from binding to the promoter regions of genes (Miranda & Jones, 2007). The transcription factors cannot bind to the methylated promoter region of the DNA, which ultimately prevents gene transcription/expression (Fig. 3). In addition to disrupting the transcription factor binding, DNA methylation can also recruit proteins with methyl-CpG-binding domains (Chatagnon et al., 2011). Proteins with methyl-CpG-binding domains bind to methylated DNA and recruit additional protein complexes, which provide further control of gene expression through their ability to condense or relax the local chromatin structure (Chatagnon et al., 2011). 2.3. DNA methyltransferases (DNMTs) DNA methylation is established and maintained through a family of enzymes known as DNA methyltransferases (DNMTs). Mammals possess three DNMTs with catalytic activity towards DNA: DNMT1, DNMT3A, DNMT3B (Fig. 4). Other mammalian DNMTs have sequence similarity, however, these proteins have negligible DNA methyltransferase activity (DNMT2) or they lack catalytic activity (DNMT3L) (Schaefer & Lyko, 2010). DNMTs cooperate to establish and maintain methylation patterns during normal development. DNA methyltransferases share common features, mainly an N-terminal domain and a C-terminal catalytic domain (Fig. 4) (Jurkowska et al., 2011). The N-terminal domain contains DNA binding motifs and/or protein recognition sites (For example: chromatin recognition sites) (Cheng & Blumenthal, 2010). The C-terminal domain is responsible for the catalytic activity of the DNMT and contains several motifs, including a binding site for the methyldonor (S-adenosylmethionine) and a motif that recognizes the cytosine substrate. 2.3.1. DNMT1 DNMT1 is the most abundant DNMT, and DNMT1 has a greater affinity for hemimethylated DNA (hemimethylated DNA is DNA that


8


Fig. 3. CpG methylation influences the expression of nearby genes. Regions of the genome with a high density of cytosine–guanine (CpG) dinucleotide sites are known as CpG islands. Approximately 60% of genes contain a CpG island in their promoter region. Unmethylated CpG islands are associated with an open chromatin structure and active gene promoters. Whereas, methylated CpG islands are associated with gene silencing and a repressive chromatin structure. (A) In an unmethylated state, a transcription factor may bind to a CpG site and promote gene expression. (B) Alternatively, methylation of the CpG site may alter the affinity between the transcription factor and the CpG site, ultimately disrupting gene expression. In human disease, aberrant CpG methylation may be a mechanism that contributes to disease progression (ex. CpG methylation in cancer correlates with the silencing of tumor suppressor genes).

is methylated on only one of the complementary strands) than unmethylated DNA (Robertson et al., 1999). DNMT1 primarily acts on newly synthesized DNA and functions as a maintenance methyltransferase (Pradhan et al., 1999). DNMT is expressed at high levels in proliferating cells, but expressed at low levels in non-dividing cells (Robertson et al., 1999). A functional DNMT1 gene is necessary for proper embryonic development, and mutations in the DNMT1 gene result in embryonic lethality (Li et al., 1992). In normal cells, the disruption of DNMT1 leads to the formation of tumors (Eden et al., 2003; Gaudet et al., 2003). Although DNMT1 is traditionally thought of as a maintenance methyltransferase, it is important to note that DNMT1 has de novo methyltransferase activity in vitro, and research indicates that DNMT1 may also have de novo methyltransferase activity in vivo (Peters et al., 2013).

is required for imprinting during gamete development. Conversely, DNMT3B, but not DNMT3A, is required to prevent loss of DNA methylation in embryonic cells (Dodge et al., 2005). Given that each DNMT has distinct cellular functions, it is likely that individual DNMTs may have a unique role in epigenetic disease etiology. In primary T-cell lymphoma cells, DNMT1 activity is required for tumor cell proliferation/maintenance, and the function of DNMT1 is independent of DNMT3A and DNMT3B (Peters et al., 2013). While the complete function of DNMT1 in T-cell lymphoma is unknown, aberrant promoter methylation may induce tumor growth through the silencing of tumor suppressor genes (Portela & Esteller, 2010; Kaiser et al., 2013).

2.3.2. DNMT3A and DNMT3B DNMT3A and DNMT3B primarily act on unmethylated DNA. DNMT3A and DNMT3B play a role in establishing DNA methylation (Fernandez et al., 2012), and they are essential for development (Okano et al., 1999). DNMT3A and DNMT3B are commonly referred to as de novo methyltransferases based on the observation that these enzymes do not have a preference for hemimethylated DNA or unmethylated DNA (Okano et al., 1998). In addition to their roles as de novo methyltransferases, DNMT3A and DNMT3B also have a role in maintenance methylation. While DNMT3A and DNMT3B have similar biochemical functions in de novo DNA methylation, these enzymes also have distinct functions. For example, DNMT3A, but not DNMT3B,

The enzyme, activation-induced cytidine deaminase (AID), contributes to active DNA demethylation. AID was initially characterized as an enzyme that plays a role in antibody maturation (Muramatsu et al., 2000). More recently, AID has also been implicated in DNA demethylation. AID-mediated deamination of 5-mC produces a thymidine residue, which becomes a target for the BER repair pathway. The base-excisionrepair machinery will replace the thymidine residue with an unmethylated cytosine. Therefore, the AID enzyme initiates a process that ultimately leads to DNA demethylation through the replacement of a 5-mC with an unmethylated cytosine. AID also plays a role in tumorigenesis (Okazaki et al., 2007), and AID may promote aberrant gene expression by decreasing the promoter DNA methylation of specific genes

2.4. Activation-induced cytidine deaminase (AID) — active DNA demethylation

Fig. 4. Overview of mammalian DNA methyltransferases. Depictions of three enzymatically active mammalian DNA methyltransferases are DNMT1, DNMT3a, and DNMT3b. The active DNMTs consist of an N-terminal region and a C-terminal region. The N-terminal portion of the DNMTs is responsible for cellular localization and nucleic acid/protein (chromatin) interactions. The C-terminal region of DNMTs contains the catalytic motifs responsible for methyltransferase activity. DNMT1 is responsible for the “maintenance” of DNA methylation patterns throughout cell division, whereas, DNMT3a and DNMT3b are de novo DNMTs. Although the DNMTs have overlapping structural domains, the functional significance of each DNMT is highlighted by knockout studies. Knockout of any one of the three aforementioned DNMT genes is lethal and leads to genome-wide loss of methylation.



(Isobe et al., 2013; Munoz et al., 2013). An increase in AID activity may lead to aberrant gene expression that may ultimately influence tumorigenesis. 3. The sixth base: 5-hydroxymethylcytosine (5-hmC) Recent epigenetic studies revealed the existence of 5hydroxymethylcytosine (5-hmC) in mammalian genomes (Tahiliani et al., 2009). The recent discovery of 5-hmC, the “6th base” of the genome (Munzel et al., 2011), adds another layer of complexity to the epigenetic code. As with 5-mC, 5-hmC is not truly a new nucleotide, but a nucleotide that arises from the conversion (hydroxylation) of 5-mC to 5-hmC. Members of the ten–eleven translocation (Tet) protein family catalyze the conversion of 5-mC to 5-hmC. The Tet protein family consists of three members (Tet1, Tet2, and Tet3), and all three Tet proteins are capable of converting 5-mC to 5-hmC (Fig. 2) (Ito et al., 2010). 5-hmC is present in embryonic and adult genomes and the level of 5hmC varies between tissues (Ye & Li, 2013). Loss of 5-hmC correlates with poorer prognosis in particular types of cancer (Orr et al., 2012; Jawert et al., 2013; Liu et al., 2013; Yang et al., 2013), and loss of 5hmC is now considered a hallmark of melanoma (Lian et al., 2012). On the other hand, elevated levels of 5-hmC exist in the neurons of Alzheimer's disease brain (Coppieters et al., 2013). The function of 5hmC is likely dependent on the genomic context. Global changes in 5hmC levels may contribute to disease, however, more research is required to elucidate the biologic significance of 5-hmC. A greater understanding of the biologic significance of 5-hmC will provide further insight into the epigenetics in normal and disease states. 5-hmC may play a role in maintaining a border between genomic regions of high methylation and regions with low levels of DNA methylation (canyons) (Jeong et al., 2014). In addition, 5-hmC may also play a role in active DNA methylation. It is noteworthy that the Tet proteins can catalyze the conversion of 5-mC to 5-hmC. Interestingly, the Tetmediated conversion of 5-mC to 5-hmC is also the first step in a particular DNA repair pathway that ultimately leads to DNA demethylation. The DNA repair pathway, known as the base excision repair (BER) pathway, leads to the replacement of the 5-hmC with an unmethylated cytosine (Hackett et al., 2013). Therefore, the Tet proteins provide a mechanism for mammalian cells to elicit DNA demethylation, a process that is essential for normal development and may contribute to human disease (Delatte & Fuks, 2013). In addition to a function in active DNA demethylation, 5-hmC may also play a functional role in epigenetic signaling pathways. Recently, several proteins have been shown to bind to 5-hmC, revealing the possibility that specific proteins may be able to interpret the 5-hmC epigenetic mark and subsequently influence chromatin structure and gene expression (Mellen et al., 2012; Iurlaro et al., 2013;). 3.1. Epigenetic complexity: additional cytosine variants The Tet proteins, which oxidize 5-mC to 5-hmC, can further oxidize 5-hmC into additional cytosine variants. 5-hmC can be oxidized to 5formylcytosine (5-fC), and 5-fC can be further oxidized to 5carboxylcytosine (5-caC; Fig. 2) (He et al., 2011). As with 5-hmC, both 5-fC and 5-caC are substrates for a DNA repair pathway, which replaces a 5-fC/5-caC base with an unmethylated cytosine (active DNA demethylation) (He et al., 2011). In terms of the entire genome, 5-fC/5-caC appear to be less abundant than 5-hmC. However, 5-fC might be biologically relevant based on the observation that specific proteins have a higher affinity for 5-fC than 5-hmC. Interestingly, proteins with binding affinity towards 5-fC are also involved in transcription, DNA repair, and chromatin regulation (Iurlaro et al., 2013). Taken together, the Tet-mediated cytosine variants (5-hmC, 5-fC and 5-caC) are epigenetic modifications that have a unique role in demethylation and epigenetic signaling pathways.

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3.2. Interplay between Ezh2 and Tet In addition to the role of the Tet proteins in 5-mC modification, the Tet proteins may have another function in transcriptional regulation. For example, Tet1 can bind to a promoter that contains methylated histone proteins. Interestingly, knockdown of the Tet1 protein impaired the binding of Ezh2 to promoters with a specific histone methylation mark (histone H3 lysine 4 trimethylation) (Wu et al., 2011). Although Tet1 did not bind directly to Ezh2, Tet1 contributes to the recruitment of Ezh2 to distinct promoter regions (Wu et al., 2011). The relationship between Tet1 and Ezh2 is noteworthy because Ezh2 is a histone methyltransferase that methylates histone H3 at lysine 27, which is an epigenetic mark associated with gene silencing. Ezh2 is overexpressed in several cancers and plays a role in tumor progression and metastasis (Chang & Hung, 2012; Crea et al., 2012). Interestingly an Ezh2 inhibitor is currently in clinical trials (Epizyme; EPZM [NASDAQ]). Since Tet may play a role in the Ezh2 signaling, Tet may also make an interesting target for therapeutic intervention (Chia, 2012). 3.3. Activation-induced cytidine deaminase (AID) — active DNA demethylation The enzyme, activation-induced cytidine deaminase (AID), contributes to active DNA demethylation. AID was initially characterized as an enzyme that plays a role in antibody maturation (Muramatsu et al., 2000). More recently, AID has also been implicated in DNA demethylation. AID-mediated deamination of 5-mC produces a thymidine residue, which becomes a target for the BER repair pathway. The base-excisionrepair machinery will replace the thymidine residue with an unmethylated cytosine. Therefore, the AID enzyme initiates a process that ultimately leads to DNA demethylation through the replacement of a 5-mC with an unmethylated cytosine. AID also plays a role in tumorigenesis (Okazaki et al., 2007), and AID may promote aberrant gene expression by decreasing the promoter DNA methylation of specific genes (Isobe et al., 2013; Munoz et al., 2013). An increase in AID activity may lead to aberrant gene expression that may ultimately influence tumorigenesis. 4. DNA methylation as therapeutic targets Unlike genetic abnormalities, epigenetic changes are reversible and represent a promising avenue for therapeutic intervention. As discussed before, epigenetic alterations are considered a hallmark of cancer (Sandoval & Esteller, 2012), and global DNA hypomethylation and gene-specific hypermethylation are features of cancer cells (Feinberg, 2007). DNA hypermethylation occurs in the promoter regions of tumor suppressor genes and correlates with gene silencing (Greger et al., 1989). The silencing of tumor suppressor genes may also contribute to tumor development. Current epigenetic therapies aim to reverse disease-associated epigenetic changes and restore normal gene expression. Some of these therapies will be discussed below and on Table 4. 4.1. 1st generation DNMT inhibitors DNA methylation inhibitors are at the forefront of DNA-targeted epigenetic therapy. DNMT inhibitors disrupt abnormal DNA methylation and restore the expression of aberrantly silenced genes (Table 4). In addition, DNMT inhibition prevents the transmission of DNA methylation marks during DNA replication, ultimately leading to a loss of DNA methylation (hypomethylation) (Stresemann & Lyko, 2008). DNMT inhibitors are classified in two main groups: nucleoside inhibitors and nonnucleoside inhibitors. Nucleoside inhibitors are analogs of nucleosides that disrupt DNMT activity and prevent subsequent DNA methylation. 5-Azacytidine (Vidaza; Celgene) and decitabine (Dacogen; Eisai) are nucleoside analogs that are FDA approved for the treatment of Myelodysplastic Syndrome or Myelodysplasia (Table 4) (5-azacytidine


10


is also approved for the treatment of Chronic Myelomonocytic Leukemia). Decitabine and 5-azacytidine are cytidine analogs (Fig. 5). Upon uptake, cellular enzymes convert decitabine and 5-azacytidine nucleosides to nucleotides that can integrate into the genome during DNA synthesis (Fig. 6). Decitabine contains deoxyribose (the sugar found in DNA), therefore, the decitabine nucleoside can directly undergo a series of phosphorylation reactions that ultimately produce the nucleotide 5-aza-2′-deoxycytidine-5′-triphosphate (5-aza-dCTP) (Fig. 6). Since 5-azacytidine contains the sugar ribose, phosphorylation of the 5-azacytidine nucleoside results in the formation of a ribonucleotide. To be incorporated into DNA, the 5-azacytidine nucleotide must be enzymatically converted from a ribonucleotide to a deoxyribonucleotide. In both cases, the decitabine and 5-azacytidine can be metabolically processed to 5-aza-2′-deoxycytidine-5′-triphosphate (Fig. 6). 4.1.1. Decitabine Decitabine is a cytidine analog with a carbon atom (carbon number 5) replaced by a nitrogen atom (Fig. 5). Upon entering a cell, decitabine is metabolized into a nucleotide, known as an azanucleotide (Fig. 6). The azanucleotides compete with cytidine nucleotides during DNA synthesis. As a result, azanucleotides incorporate into the DNA regions that are usually occupied by cytidine nucleotides. The azanucleotides disrupt normal cellular functions, most notably, the methylation of DNA. Under normal conditions, DNA methylation occurs at cytidine–guanosine dinucleotides (CpG sites), but in the presence of decitabine, an azanucleotide can replace the cytidine in a cytidine–guanosine dinucleotide. The DNA methylation machinery recognizes the azanucleotide– guanosine dinucleotide, however, the DNA azanucleotide irreversibly binds to the DNMT. The irreversible DNMT-azanucleotide adduct triggers a DNA damage response that ultimately leads to DNMT degradation and the subsequent loss of DNA methylation (Stresemann & Lyko, 2008). During DNA synthesis, decitabine substitutes for approximately 10% of cytidine nucleotides (Flatau et al., 1984). Greater decitabine doses promote higher substitution rates, but cytotoxicity limits the therapeutic decitabine dosage (Qin et al., 2009). Decitabine is approved by the FDA for the treatment of myelodysplastic syndromes (http://www. cancer.gov/cancertopics/druginfo/fda-decitabine). In 2012, the FDA's

Oncologic Drug Advisory Committee concluded that decitabine did not improve mortality rates in patients (≥65 years) with acute myelogenous leukemia (AML). In the primary analysis of overall survival (the primary endpoint; 81.6% patients deceased) decitabine did not statistically improve survival time; decitabine (7.7 months; 95% CI, 6.2 to 9.2) versus TC (TC: supportive care or cytarabine; 5.0 months; 95% CI, 4.3 to 6.3; P = .108) (Kantarjian et al., 2012). A subsequent survival analysis (95% of patients deceased) continued to show an advantage in the decitabine arm (median survivals of 7.7 vs. 5.0 months; HR = 0.82, 95% CI = 0.68–0.99, p = 0.037). Based on the AML data, the European Medicines Agency (EMA) granted approval for decitabine in the treatment of AML. Although there was statistically significant survival benefit in the secondary analysis, the FDA did not approve decitabine for use in the treatment of AML. Despite the FDA's decision, decitabine appears to offer a benefit for the treatment of AML (Kantarjian, 2013), further highlighting the importance of epigenetic therapeutics.

4.1.2. 5-Azacytidine 5-azacytidine is a cytidine analog that is structurally similar to decitabine, however, the azanucleotide is linked to the sugar ribose (decitabine contains a deoxyribose; Fig. 5). Due to a slight difference in structure, 5-azacytidine has two possible cellular fates; 5azacytidine can be incorporated into both DNA and RNA (Fig. 6). Although 5-azacytidine affects RNA metabolism and DNA metabolism, approximately 80–90% 5-azacytidine incorporates into RNA (Li et al., 1970). Consequently, 5-azacytidine is regarded as a less potent DNA methylation inhibitor than decitabine because a greater amount of 5azacytidine is necessary to achieve the level of DNA hypomethylation that is obtained with a lower dosage of decitabine (Stresemann & Lyko, 2008; Hollenbach et al., 2010). Although 5-azacytidine is a less potent DNA methylation inhibitor than decitabine, 5-azacytidine can interfere with other cellular processes relevant to cancer. For example, 5-azacytidine incorporates into RNA disrupts the translation of RNA into proteins and ultimately disrupt the viability of the cancer cell (Hollenbach et al., 2010). Cell division is not a requirement for the incorporation of 5-azacytidine into RNA (cell division/DNA synthesis is necessary for the incorporation of 5-azacytidine into DNA). Since 5-azacytidine can influence both dividing cells and

Table 6 Selective overview of HDAC inhibitors. Drug

Mechanism

Developer/source


Abexinostat (PCI-24781)

Pan-histone Deacetylase Inhibitor (HDACi)

Pharmacyclics

Belinostat (PXD 101; TopoTarget A/S; Spectrum SPPI) CHR-5154

HDAC class I and class II inhibitor

PMID: 22046971

Macrophage-targeted HDAC inhibitor

Chroma therapeutics

CUDC-907

HDAC inhibitor and PI3K inhibitor

Curis, Inc.

Entinostat, SNDX-275, MS-275

class I HDACs 1 and 3; benzamide histone deacetylase inhibitor HDAC class 1 and class 2 inhibitor

Syndax (compound licensed from Bayer AG) Italfarmaco S.P.A. (Cinisello Balsamo, Italy) Celgene

Phase I trials in metastatic solid tumors and sarcomas. FDA approved for the treatment of relapsed or refractory peripheral T-cell lymphoma. Phase I trial, rheumatoid arthritis. Trial terminated (NCT01934101). Phase I trial in Multiple myeloma and lymphoma. Phase II trials for breast cancer, lung cancer, kidney cancer, and lymphoma. Phase i trial in muscular dystrophy and polycythemia vera. FDA approved for the treatment of cutaneous T-cell lymphoma and peripheral T-cell lymphoma. Phase I trial of mocetinostat in combination with azacitidine in MDS Phase I and II trials for leukemia, lymphoma, myeloma, and melanoma Phase I trial in colorectal carcinoma.

Givinostat (ITF2357) Istodax (romidepsin) (formerly FK228, acyclic peptide principally active against class 1 HDACs); nanomolar potent Mocetinostat (MGCD01030)

Iso-selective inhibitor HDAC inhibitor (Class I HDAC inhibitor) HDAC inhibitor: class I HDACs 1, 2, and 3

Mirati Therapeutics, Inc. (MRTX)

Panobinostat

Pan HDAC inhibitor

PMID: 22943463

Resminostat (RAS2410, 4SC-201, BYK408740)

Resminostat is an inhibitor of HDACs 1, 3 and 6; pan-inhibitor of class I and II HDAC enzymes Trichostatin A inhibits HDACs 1–9

4SC AG′ (subsidiary of 4SC discovery GmbH), PMID: 24065624 PMID: 21386836

Pan-histone deacetylase inhibitor (HDACi)

Merck

Trichostatin A (TSA) Zolinza (vorinostat) (suberoylanilide hydroxamic acid, SAHA)

Side-effects have limited the clinical development of TSA. FDA approved for the treatment of cutaneous T-cell lymphoma.


C.A. Hamm, F.F. Costa / Pharmacology & Therapeutics xxx (2015) xxx–xxx NH2

NH2

N O

O

N

HO O

OH

Phosphorylation

O

OH

Phosphorylation

OH

Nucleoside: Cytidine

Nucleoside: Deoxcytidine

NH2 N O HO

N

N

O HO

OH

Analog: 5-Azacytidine

Additional Phosphorylation

Approximately 10-20% of azacytidine incorportaes into DNA

5-aza-2’-deoxycytidine5’-diphosphate (5-aza-dCDP) Phosphorylation

N N

O

Cytidine

Ribonucleotide Reductase

NH 2

N

O

OH

Decitabine

N N

HO

5-Azacytidine

11

5-azacytidine5-triphosphate (5-aza-CTP)

5-aza-2’-deoxycytidine5’-triphosphate (5-aza-dCTP)

OH

Deoxcytidine Analog: Decitabine NH2 F

N O HO

N

O

OH

Deoxcytidine Analog: 5−Fluoro−2− Deoxycytidine

RNA

DNA

Fig. 6. Incorporation of 5-azacytidine and decitabine to nucleic acids. Cellular processing of 5-azacytidine and decitabine leads to incorporation into nucleic acids (RNA and/or DNA). DNMT inhibition requires the conversion of the azanucleosides (5-azacytidine and decitabine) into nucleotides. 5-Azacytidine can be incorporated into RNA and DNA. For RNA metabolism, 5-azacytidine undergoes three rounds of phosphorylation to form 5azacytidine-5-triphosphate. For DNA metabolism, 5-azacytidine undergoes two rounds of phosphorylation, followed by the conversion of ribose to deoxyribose (ribonucleotide reductase) to form 5-aza-2′-deoxycytidine-5′-diphosphate. 5-aza-2′-deoxycytidine-5′-diphosphate is further phosphorylated to form 5-aza-2′-deoxycytidine-5′-triphosphate. Approximately 10–20% of 5-azacytidine is incorporated into DNA. Decitabine incorporates into DNA but not RNA. For DNA metabolism, decitabine undergoes three rounds of phosphorylation to form 5-aza-2′-deoxycytidine-5′-triphosphate. 5-aza-2′-deoxycytidine-5′triphosphate that can be directly incorporated into DNA (Issa et al., 2013b).

non-dividing cells, it offers a different treatment option than decitabine, which requires cell division for a therapeutic benefit.

4.1.4. Additional DNMT inhibitors 5-azacytidine treatment can alter DNA methylation in dividing cells, and it also offers the potential to alter the viability of cancer cells that are not actively dividing during the treatment window. Decitabine and 5azacytidine are powerful DNMT inhibitors, but the inhibitory effect of these compounds directly correlates to the drug exposure time. In vitro, decitabine and 5-azacytidine have a long half-life, however, in vivo, the half-life of decitabine is reduced 10-fold (Tellez et al., 2014). The decrease in half-life is due to the enzyme “cytidine deaminase” (CDA), which converts decitabine and 5-azacytidine to inactive 5-azauridine compounds (Yoo et al., 2007). Subsequent drug screens have searched for compounds that are less susceptible to the CDA. For example, zebularine is a DNMT inhibitor that also inhibits the activity of CDA. Zebularine held promise in a preclinical setting, but inefficient metabolic activation and preclinical toxicity has limited the use of zebularine in a clinical setting (Issa & Kantarjian, 2009; Yang et al., 2010).

4.1.3. Oral formulation of 5-azacytidine: CC-486 5-azacytidine is currently an intravenous therapy. Intravenous 5azacytidine allows for immediate bioavailability in the blood stream. However, intravenous 5-azacytidine has a mean elimination half-life of approximately 4 h (Kaminskas et al., 2005). As an alternative to intravenous 5-azacytidine, Celgene is currently investigating an oral formulation of 5-azacytidine known as CC-486. CC-486 allows for alternative dosing schedules that may improve on the safety and efficacy of intravenous 5-azacytidine (Laille et al., 2014). Celgene is currently conducting several trials on CC-486, including a Phase III trial in AML (NCT01757535). It is also noteworthy that CC-486 is being studied as an epigenetic priming agent prior to the treatment of non-small-cell lung carcinoma (NSCLC) with an immunotherapy (Nivolumab [BMY]; NCT01928576).

4.1.4.1. 5-fluoro-2′-deoxycytidine (FdCyd) and the CDA inhibitor tetrahydrouridine (THU). 5-fluoro-2′-deoxycytidine (FdCyd) is a pyrimidine analog that inhibits DNA methylation (Fig. 5) (Zhao et al., 2012). FdCyd competes with cytidine nucleotides during DNA synthesis. FdCyD is structurally similar to a cytidine nucleotide, except that a fluorine atom is linked to the “carbon-5” position in FdCyd (Fig. 5). The fluorine atom prevents methylation of the FdCyD molecule, and as a result, incorporation of FdCyd leads to subsequent loss of DNA methylation (Ren et al., 2011). FdCyD also forms a covalent bond with, and this covalent bond traps the DNMT and prevents the enzyme from methylating additional DNA (Reither et al., 2003). In tumor cells, lower doses of FdCyD are more cytotoxic than larger doses (10-fold) than 5-azacytidine (Zhao et al., 2012). The cytotoxic effects of FdCyD are related to the inhibition of DNA methylation,

Fig. 5. Nucleoside analogs and DNMT inhibitors. A nucleoside is a molecule composed of a base (ex. cytosine) linked to a sugar. The sugar in DNA is deoxyribose and the sugar in RNA is ribose. Cytidine is a building block for RNA, whereas deoxycytidine is a building block for DNA. The DNMT inhibitor 5-azacytidine is a cytidine analog (attached to ribose) in which a carbon atom (carbon number 5) is replaced by a nitrogen atom (denoted with a red font). The DNMT inhibitor decitabine is a cytidine analog (attached to deoxyribose) in which the carbon-5 atom is replaced by a nitrogen atom (noted with red font). The DNMT inhibitor 5-fluoro-2′-deoxycytidine (FdCyd) is a deoxycytidine analog with a fluorine atom is linked to the carbon-5″ position of the cytosine (denoted with a red font). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


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however, FdCyD also has several pharmacologically active metabolites. Most notably, the metabolites 5-fluoro-2′-dUMP (FdUMP; a thymidylate synthase inhibitor) and 5-fluoro-2′-deoxyuridine (FdUrd) promote cell toxicity through the disruption of DNA and/or RNA synthesis (Beumer et al., 2006). Although FdCyD is cytotoxic, the half-life of the drug is limited due to its metabolism by the CDA enzyme (Beumer et al., 2006). To prolong the in vivo half-life, FdCyD is administered with the CDA inhibitor tetrahydrouridine (THU). THU inhibits certain FdCyD metabolic pathways, prolongs FdCyD exposure time, and ultimately promotes more efficient hypomethylation (Beumer et al., 2008). FdCyD and THU are available in intravenous and oral formulations (Beumer et al., 2006). The National Institutes of Health and the National Cancer Institute are currently sponsoring Phase I and Phase II trials of FdCyD and THU in solid tumors (NCT01534598 and NCT00978250). 4.1.4.2. 2nd generation DNMT inhibitor: SGI-110. A recent screen for more stable DNMT inhibitors, led to the identification of SGI-110 (Otsuka Pharmaceutical [TYO: 4768]; formerly Astex Pharmaceuticals) that is a deoxyguanosine-decitabine dinucleotide. The SGI-110 dinucleotide interferes with the ability of the CDA to recognize decitabine, and as a result, SGI-110 is more resistant to the CDA. SGI-110 delivers decitabine with a longer half-life and a longer exposure (Issa et al., 2013a). SGI110's extended exposure time may increase the likelihood of restoring the expression of aberrantly silenced/methylated genes. SGI-110 is currently in a Phase I–II clinical trial of MDS and AML patients. The first clinical data from a Phase II trial revealed an overall remission rate of 43% in treatment-naïve AML patients (≥65 years old) and an overall remission rate of 16% in patients with relapsed/refractory AML (Issa et al., 2013b). In addition, SGI-110 is tolerable in the patients as myelosuppression and injection site pain are the most common adverse events (Issa et al., 2013b). Final data collection for the Phase II trial was expected in 2014 (NCT01261312). 4.2. Priming cancer: adding epigenetic therapeutics to traditional treatment regimens

with immunotherapeutic agents. Treatment with SGI-110 increases the expression of cancer testis antigens (CTAs) and co-stimulatory molecules that are necessary for an efficient immune response to cancer cells (Costa et al., 2007; Coral et al., 2013). Preliminary data indicates a synergistic anti-tumor effect when SGI-110 is combined with immunostimulatory antibodies (ex. anti-CTLA-4; Ipilimumab, or antiPD-1; Pembrolizumab or Nivolumab) (Coral et al., 2013). Clinical data indicates that immune-checkpoint blockades (anti-PD1) are useful for the treatment of Non-Small Cell Lung Cancer (NSCLC) (Brahmer et al., 2012). Additional research indicates that 5azacytidine activates immune stimulating pathways in NSCLC cells, and may produce a synergistic effect when combined with an inhibitor of the immune checkpoint blockade (Wrangle et al., 2013). While 5azacytidine may promote immune stimulating pathways, it is also important to note that 5-azacytidine upregulates the expression of an immune inhibitory ligand, PD-L1 (Wrangle et al., 2013), which may alter the anti-tumor immune response. 5-Azacytidine-induced PD-L1 expression may disrupt an anti-tumor immune response, indicating that epigenetic therapies must be carefully matched with appropriate immunotherapies for maximum clinical benefit (ex. in certain settings an anti-PD-L1 antibody may be more appropriate than an anti-PD1 antibody). In a Phase I ovarian cancer trial, treatment with decitabine and a cancer vaccine (NY-ESO-1 vaccine) produced a clinical benefit of 60% (Odunsi et al., 2014). Interestingly, the ovarian cancer patients were immunized against only one antigen, but immune responses were also detected against three other antigens (Odunsi et al., 2014). This phenomenon, known as “antigen spreading”, may result from the ability of decitabine to influence the expression of additional tumor antigens or MHC Class I molecules (Karpf, 2006; Odunsi et al., 2014). A followup Phase II trial will examine the impact of decitabine and the NYESO-1 cancer vaccine therapy on progression-free survival in ovarian cancer (Odunsi et al., 2014). 4.3. Resistance to DNA hypomethylating agents

4.2.1. Conventional chemotherapy While SGI-110 and CC-486 (oral 5-azacytidine) are being studied as single-agent therapies for the treatment of MDS and AML, new clinical data indicates that these epigenetic therapies are useful when combined with other treatments. SGI-110, CC-486, decitabine, and 5-azacytidine are currently under investigation in combination based therapeutic regimens. Adding an epigenetic therapy may increase the efficacy of other cancer therapies by “priming” the cancer cells for subsequent treatments (Jones, 2013). For example, epigenetic priming may allow tumor cells to become resensitized to conventional chemotherapies (Balch & Nephew, 2013). Pre-treatment with a DNA hypomethylating agent may restore the expression of transcriptionally silenced genes, such as tumor suppressors, and the reactivation of these genes may increase the efficacy of cytotoxic chemotherapies. In heavily pretreated ovarian cancer patients, decitabine restored the sensitivity to carboplatin (Matei et al., 2012). In a Phase I trial of solid tumors, the addition of 5-azacytidine to an EGFR inhibitor (erlotinib), showed promising clinical responses (Bauman et al., 2012). In solid tumors, response to DNMT inhibitors is noteworthy as current epigenetic therapy is FDA approved for hematological malignancies. More efficient epigenetic dosing regimens (dosage and timing) are providing clinical benefits to solid tumors. Current solid tumor dosing schedules aim to provide exposure necessary for epigenetic reprogramming, reduce toxicity, and provide an opportunity for sequential treatments (Azad et al., 2013; Balch & Nephew, 2013).

It is already known that mutations in DNMT3A impact the effectiveness of hypomethylating agents (Metzeler et al., 2012), and evidence suggests that a resistance to decitabine may be related to the level of the enzyme CDA (Qin et al., 2009). In vitro resistance to decitabine is related to the levels of the enzymes that are involved in nucleoside transport and metabolism. For example, resistance to decitabine correlates with a decrease in the expression of deoxycytidine kinase, an enzyme that phosphorylates decitabine. Sequencing analysis indicates that preexisting and spontaneous deoxycytidine kinase mutations contribute to decitabine resistance (Qin et al., 2009). The cellular uptake of decitabine relies on the nucleoside transporters hENT1 and hENT2, and decitabine resistant cell lines have low levels of hENT1 mRNA (Qin et al., 2009). As discussed previously, the CDA enzyme regulates the half-life of decitabine, and not surprisingly, decitabineresistant cell lines also have high levels of CDA. Interestingly, certain cell lines with resistance to decitabine are susceptible to 5-azacytidine. Resistance to decitabine but not 5-azacytidine, highlights the diversity in the metabolic processing of these compounds, which may be clinically relevant in situations where a patient develops resistance to one particular type of hypomethylating treatment (Borthakur et al., 2008).

4.2.2. Immunotherapy Given the recent clinical success of immunotherapeutic agents (Ipilimumab and Nivolumab; Bristol–Myers), it is important to note that DNA methylation inhibitors may also be useful in combination

Due to their reversible nature, epigenetic modifications are an attractive therapeutic target. The FDA approved drugs, decitabine and 5-azacytidine, offer a potential benefit in the treatment of human malignancies. Increasing knowledge of the mechanism of action of DNMT

5. Future directions of epigenomic therapy 5.1. DNMT inhibitors



inhibitors has already translated into a clinical benefit; low doses of DNMT inhibitors allow for disruption of DNA methylation while reducing the toxicity observed in earlier (higher dosage) treatment regimens (Yoo & Jones, 2006). New dosing schedules and the addition of DNMT inhibitors to chemotherapy and immunotherapy regimens may further prove the utility of current epigenetic therapies. Traditional DNMT inhibitors will continue to have a role in the clinical setting, and the clinical development of second-generation DNMT inhibitors may lead to epigenetic therapies with greater efficacy and fewer side effects. Drugs such as SGI-110 (decitabine prodrug) aim to improve the pharmacokinetic profile of the 1st generation DNMT inhibitors. The development of oral DNMT inhibitors, such as CC-486, may provide opportunities for alternative/extended dosing regimens. 5.2. HDAC inhibitors Aberrant histone modifications are documented in cancer, immune disorders, diabetes, and neurodegenerative diseases (Khan & Khan, 2010). As with other reversible epigenetic modifications, acetylation is an attractive therapeutic target. Therapies targeting aberrant histone modifications are of great clinical interest. Three HDAC inhibitors (HDACi) are currently approved by the FDA (Table 3) and several HDACi are in development (Table 6). Currently approved HDACi target multiple HDAC family members (HDAC pan-inhibitors or HDAC isoselective inhibitors), however, a greater understanding of the acetylome may lead to the identification of HDACi that selectively target one specific HDAC family member. For example, Rocilinost (Acetylon Pharmaceuticals), is a selective inhibitor of HDAC6 that is currently in clinical trials for the treatment of multiple myeloma (Table 6). The selective targeting of specific HDACs may prove to be an effective therapy without the side-effects that accompany the use of pan-HDAC inhibitors. While HDACi are currently approved for cancer treatment, HDACi may also provide a benefit in the treatment diseases including neurodegenerative disease, HIV, heart disease, diabetes, and respiratory disease (Coppede, 2014; Jayaraman, 2014; Royce & Karagiannis, 2014; Tao et al., 2014; Wei et al., 2014). Given the limited treatment options for neurodegenerative diseases, HDACi may prove to be an attractive therapeutic option. HDACi can cross the blood brain barrier, and disrupt the progression of neurodegenerative disease in animal models (Coppede, 2014). While single agent HDACi therapy may provide therapeutic benefit, HDACi may also be used in combination with other therapies to provide a synergistic therapeutic response (Dokmanovic et al., 2007). In cancer treatment, HDACi may sensitize tumor cells to radiotherapy (Groselj et al., 2013). Additionally, the HDACi romidepsin is currently in a clinical trial as part of a treatment regimen with a candidate HIV vaccine (NCT02092116). Romidepsin can activate latent HIV reservoirs in infected patients, which may make infected cells more susceptible to the immune response that is induced by the HIV vaccine (Bionor_Pharma, 2014). 6. Conclusions While this report provides an overview of clinically advanced nucleoside DNMT inhibitors, it is important to note that several nonnucleoside DNMT inhibitors are under development (Table 4). Nonnucleoside DNMT inhibitors offer a unique clinical benefit since these compounds do not need to incorporate into the DNA. For example, the preclinical compound RG108 blocks the active site of DNMT1 and leads to DNA demethylation (Asgatay et al., 2014). Alternatively, the compound SGI-1027 inhibits DNMT activity by competing with the methyl donor molecule (S-adenosylmethionine) (Yoo et al., 2013). As epigenetic therapies advance in the clinic, it will be important to use genomic/epigenomic tools to identify patients that will most likely benefit from hypomethylating therapeutics. For example, mutations in DNMT3A impact the effectiveness of hypomethylating agents (Metzeler et al., 2012), and evidence suggests that a resistance to

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decitabine might be related to the level of the enzyme CDA (Qin et al., 2009). Finally, it is important to recognize that DNA methylation is only one of the players in the epigenomic landscape. Post-translational histone modifications play an essential role in the structure and function of chromatin. As discussed in this article, therapies targeting aberrant histone modifications are of great clinical interest. Three HDAC inhibitors (HDACi) are currently approved by the FDA (Table 3) and several HDACi are in development. Other players include non-coding RNAs such as microRNAs and long RNA molecules that are emerging as important epigenomic modifiers. We believe that in the near future, a synergistic combination of histone modification inhibitors, DNMT inhibitors and also molecules targeting non-coding RNAs may provide a clinically relevant reversal of epigenomic disease states. In addition, we believe that epigenetic modifying agents may provide a means to improve the effectiveness of existing immunotherapies through alterations in immune related genes. Although the future of epigenomic therapies has several challenges ahead, it is a promising field for clinical interventions not just for cancer, but for several complex diseases. That is just the beginning of the epigenomics era. 7. Conflicts of interest The authors declare that they have no conflicts of interest and no relationship with any of the companies discussed in this article. Acknowledgments FFC was supported by the generosity of the Maeve McNicholas Memorial Foundation. References Alberts, B., J. A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2002). Molecular biology of the cell. (4th ed.). Garland Sci. Alland, L., Muhle, R., Hou, H., Jr., Potes, J., Chin, L., Schreiber-Agus, N., & DePinho, R.A. (1997). Role for N-CoR and histone deacetylase in Sin3-mediated transcriptional repression. Nature 387, 49–55. Annunziato, A. (2008). DNA packaging: Nucleosomes and chromatin. Nat Educ (nature. co006D). Asgatay, S., Champion, C., Marloie, G., Drujon, T., Senamaud-Beaufort, C., Ceccaldi, A., Erdmann, A., Rajavelu, A., Schambel, P., Jeltsch, A., Lequin, O., Karoyan, P., Arimondo, P.B., & Guianvarc'h, D. (2014). Synthesis and evaluation of analogues of N-phthaloyl-l-tryptophan (RG108) as inhibitors of DNA methyltransferase 1. J Med Chem 57, 421–434. Azad, N., Zahnow, C.A., Rudin, C.M., & Baylin, S.B. (2013). The future of epigenetic therapy in solid tumours—Lessons from the past. Nat Rev Clin Oncol 10, 256–266. Balch, C., & Nephew, K.P. (2013). Epigenetic targeting therapies to overcome chemotherapy resistance. Adv Exp Med Biol 754, 285–311. Bauman, J., Verschraegen, C., Belinsky, S., Muller, C., Rutledge, T., Fekrazad, M., Ravindranathan, M., Lee, S.J., & Jones, D. (2012). A phase I study of 5-azacytidine and erlotinib in advanced solid tumor malignancies. Cancer Chemother Pharmacol 69, 547–554. Bergman, Y., & Cedar, H. (2013). DNA methylation dynamics in health and disease. Nat Struct Mol Biol 20, 274–281. Beumer, J.H., Eiseman, J.L., Parise, R.A., Joseph, E., Holleran, J.L., Covey, J.M., & Egorin, M.J. (2006). Pharmacokinetics, metabolism, and oral bioavailability of the DNA methyltransferase inhibitor 5-fluoro-2′-deoxycytidine in mice. Clin Cancer Res 12, 7483–7491. Beumer, J.H., Parise, R.A., Newman, E.M., Doroshow, J.H., Synold, T.W., Lenz, H.J., & Egorin, M.J. (2008). Concentrations of the DNA methyltransferase inhibitor 5-fluoro-2′deoxycytidine (FdCyd) and its cytotoxic metabolites in plasma of patients treated with FdCyd and tetrahydrouridine (THU). Cancer Chemother Pharmacol 62, 363–368. Bionor_Pharma (2014). First evidence that romidepsin “kicks” HIV out of reservoirs. See more at: http://www.bionorpharma.com/First+Evidence+that+Romidepsin+%E2% 80%9CKicks%E2%80%9D+HIV+out+of+Reservoirs.b7C_wlvYWU.ips#sthash.niStZLPd. dpuf (Bionor Pharma) Bird, A. (2002). DNA methylation patterns and epigenetic memory. Genes Dev 16, 6–21. Borthakur, G., Ahdab, S.E., Ravandi, F., Faderl, S., Ferrajoli, A., Newman, B., Issa, J.P., & Kantarjian, H. (2008). Activity of decitabine in patients with myelodysplastic syndrome previously treated with azacitidine. Leuk Lymphoma 49, 690–695. Brahmer, J.R., Tykodi, S.S., Chow, L.Q., Hwu, W.J., Topalian, S.L., Hwu, P., Drake, C.G., Camacho, L.H., Kauh, J., Odunsi, K., Pitot, H.C., Hamid, O., Bhatia, S., Martins, R., Eaton, K., Chen, S., Salay, T.M., Alaparthy, S., Grosso, J.F., Korman, A.J., Parker, S.M., Agrawal, S., Goldberg, S.M., Pardoll, D.M., Gupta, A., & Wigginton, J.M. (2012). Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med 366, 2455–2465.


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