Epigenetic mechanisms of importance for drug treatment

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Epigenetic mechanisms of importance for drug treatment Maxim Ivanov*, Isabel Barragan*, and Magnus Ingelman-Sundberg Pharmacogenetics Section, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden

There are pronounced interindividual variations in drug metabolism, drug responses, and the incidence of adverse drug reactions. To a certain extent such variability can be explained by genetic factors, but epigenetic modifications, which are relatively scarcely described so far, also contribute. It is known that a novel class of drugs termed epidrugs intervene in the epigenetic control of gene expression, and many of these are now in clinical trials for disease treatment. In addition, disease prognosis and drug treatment success can be monitored using epigenetic biomarkers. Here we review these novel aspects in pharmacology and address intriguing future opportunities for gene-specific epigenetic editing. Pharmacoepigenetics During the last decade much information has accumulated regarding the genetic causes of differences in drug responses. This has led to the identification of many pharmacogenomic biomarkers important for individualized drug therapy, particularly in cancer treatment, that are currently in clinical use. Such biomarkers help to avoid severe complications of drug treatment, which are estimated to cost billions of dollars and cause 100,000 deaths in the USA annually. However, only 20–30% of the interindividual variations important for adverse drug reactions (ADRs) or drug efficacy can be explained by genetic factors [1–3]. Additional factors for such variability include drug–drug interactions and endocrine, environmental, and pathophysiological causes. Epigenetic control of the expression of genes encoding drug absorption, distribution, metabolism, and excretion (ADME) proteins, as well as drug target proteins, has been emphasized in recent years. Much has still to be learned about the extent and mechanisms by which epigenetic modification of gene expression contributes to short- or long-term variability in drug action. In certain cases, epigenetic modifications resulting from disease progression or drug treatment can be monitored not only in the affected tissue, but also in body fluids [4]. Such circulating DNA elements constitute a novel class of pharmacoepigenetic biomarkers amenable for use to improve and individualize drug therapy.

Corresponding author: Ingelman-Sundberg, M. ([email protected]). Keywords: pharmacoepigenetics; DNA methylation; 5-hydroxymethylcytosine; histone modifications; epidrugs; epigenetic editing. * These authors contributed equally to this work. 0165-6147/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tips.2014.05.004

Drugs that interfere with the epigenetic control of gene expression are now being developed, and the potential of these so-called epidrugs (see Glossary) in the chemotherapy of cancer has already been demonstrated [5]. Apart from epidrugs, other xenobiotics may also possess epigenetic activity. For example, it was demonstrated that exposure of mice to certain drugs or drug receptor ligands during fetal life can trigger epigenetic modifications of specific genes, causing altered ADME gene expression in adult mice [6,7]. In addition, a new therapeutic concept is being developed based on the ability of transcription factors and long noncoding RNAs (lncRNAs) to target epigenetic proteins including enzymes to specific loci in the genome. The aim of this review is to introduce readers to the novel and exciting field of pharmacoepigenetics. After a brief description of the major epigenetic mechanisms, the most recent data relevant to epigenetic regulation of ADME genes, epigenetic biomarkers of drug response, and the biological effects of epidrugs are summarized. The concept of epigenetic editing, which opens an intriguing possibility to interrogate disease-specific epigenetic signatures, is also briefly discussed.

Glossary Bromodomains: protein domains responsible for recognition of acetylated lysine residues in histones, thus mediating the effect of histone acetylation on gene transcription. DNA hydroxymethylation: modification of 5-methylcytosine by oxidation of the methyl group (-CH3) to a hydroxymethyl group (-CH2-OH), yielding 5hydroxymethylcytosine (5hmC). DNA methylation (DNAme): modification of cytosine residues in DNA by addition of a methyl group (-CH3) to the fifth position of a cytosine base, yielding 5-methylcytosine (5mC). Epidrugs: drugs that inhibit or activate disease-associated epigenetic proteins with the aim of ameliorating, curing, or preventing the disease. Epigenetic editing: intentional overwriting of epigenetic signatures by artificial targeting of epigenetic enzymes to specific loci. Epigenetic enzymes: epigenetic proteins belonging to the writer and eraser groups. Epigenetic proteins: proteins that can either covalently modify DNA or histones, thus yielding epigenetic signatures (writers); or remove such epigenetic modifications (erasers); or recognize and bind to modified chromatin, thus mediating the effect of epigenetic signatures on gene transcription (readers). Epigenetics: changes in gene function that can be inherited via cellular divisions and cannot be explained by any change in the primary sequence of nucleic acids. Histone modification: post-translational modification of N terminus of histones (histone tail) that protrudes from the core nucleosome. Histone-modifying enzymes: epigenetic enzymes responsible either for posttranslational modification of histones or for removal of such modifications. Trends in Pharmacological Sciences xx (2014) 1–13

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Basics of epigenetics The current definition of epigenetics is changes in gene function that can be inherited via cellular divisions and cannot be explained by any change in the primary sequence of nucleic acids. In contrast to germline genetic variants, which are inherited from progenitors and are stable across lifespan, epigenetic signatures can be generated in response to various external or internal stimuli. Moreover, epigenetic regulation constitutes a means whereby an organism can ‘remember’ the altered gene expression pattern resulting from a stimulus (to be prepared for any eventual reoccurrence of a similar condition). To date, the conventional epigenetic mechanisms identified in mammals include DNA methylation (DNAme) and hydroxymethylation, as well as various histone modifications. DNA methylation The majority of CpG sites throughout the genome are fully methylated, whereas not more than 15% of them are

(A)

MLL1

Me1/2

K4

5mC

H3 MB D4 5mC D1 MeCP2 MB D2

HDAC1

HD

Methylated DNA (repressed chroman)

AC

MT

K4

MT

HD

C

M

5hmC

H3

H3

1

P2

TET1-3

Me3

C

Ac

eC

DN

K27

M

SET

DN

Me2/3

1

Unmethylated DNA (acve chroman) (D)

EZH2

C

Ac

MB

AC

C

H3

Ac

HD

MT

Me3

K9

(C)

DN

HP1

(B)

DNMT

G9a

hypomethylated and thus may be subject to variable DNAme [8]. Hypomethylated CpG sites are usually associated with CpG islands (CGIs) and their flanking regions (CGI shores). It has become evident that variable DNAme is more frequently observed at CGI shores and single CpG sites than in CGIs [9]. DNAme is known to be involved into the regulation of gene expression. Hypomethylated genomic elements are often associated with bound transcription factors (TFs), and this cytosine modification either inhibits (e.g., CREB, E2F, CTCF, Sp1, AhR) or promotes (e.g., C/EBPa) TF binding [10–12]. In addition, DNAme also regulates transcription via recruitment of methyl-CpG-binding proteins such as MBD1, MBD2, MBD4, and MeCP2 [13]. These proteins can in turn recruit additional factors such as the nucleosome remodeling deacetylase (NuRD) complex, which possesses ATP-dependent chromatin remodeling activity and contains histone deacetylases HDAC1 and HDAC2, thereby causing transcriptionally silenced chromatin (Figure 1A).

P eC

5hmC AC

Ac 1

MeCP2

2

PcG-silenced chroman

MB

D2

MB

D1

MBD4

Hydroxymethylated DNA (acve chroman) TRENDS in Pharmacological Sciences

Figure 1. Crosswise interactions between epigenetic proteins and epigenetic signatures. Four major epigenetic states of chromatin are represented. (A) Methylated DNA (repressed chromatin). Methylated CpG sites (5-methylcytosine, 5mC) are usually present together with histone H3, which is mono- or dimethylated at Lys9 (H3K9me1/2). This co-occurrence is caused by physical interactions between DNA methyltransferases DNMT3A/B and the histone methyltransferase G9a. In addition, DNA methylation is accompanied by a hypoacetylated state of histone H3 because methyl-CpG-binding proteins (MeCP2, MBD1, MBD2, MBD4) are able to recruit histone deacetylases HDAC1 and HDAC2. Thus, 5mC, methylated H3K9, and deacetylated H3 together represent a repressive epigenetic signature. (B) Unmethylated DNA (active chromatin). Trimethylated histone H3 (H3K4me3) is found together with unmodified cytosine because of the binding of H3K4 methyltransferase MLL1 to unmethylated DNA and the preference of DNMT3A for unmodified H3K4. Methyl-CpG-binding proteins do not recognize unmethylated DNA and thus do not recruit histone deacetylases to H3K4me3containing loci. Histone acetylation promotes active gene transcription. Taken together, unmethylated DNA, methylated H3K4, and acetylated H3 represent an activating epigenetic signature. (C) PcG-silenced chromatin. Di- and trimethylation of histone H3 at Lys27 (H3K27me2/3) is catalyzed by histone methyltransferase EZH2, which is a part of polycomb repressive complex 2 (PRC2; other components not shown). Inheritance of PcG silencing does not seem to depend on DNA methylation, but is rather achieved through self-recruitment of PRC2 to H3K27me2/3-containing loci. (D) Hydroxymethylated DNA (active chromatin). TET enzymes (TET1, TET2, TET3) oxidize 5mC to 5-hydroxymethylcytosine (5hmC). 5hmC can interact with MeCP2, but not with other methyl-CpG-binding proteins (MBD1, MBD2, MBD4), which thus determines the lack of HDAC1 recruitment and the acetylated state of histone H3. In addition, TET2 and TET3 proteins can activate histone methyltransferase SET1 and thus promote trimethylation of histone H3 at Lys4 (H3K4me3). These histone modifications determine transcriptionally active chromatin.

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It is believed that DNAme is the most stable epigenetic modification, and in some cases altered DNAme can persist through the whole lifespan of an individual. Thus, altered methylation of multiple genes, including ABCA1, has been revealed in individuals exposed to prenatal famine either periconceptionally (n = 60) or in late pregnancy (n = 62) during the Dutch hunger winter of 1944 [14]. DNA hydroxymethylation 5-Hydroxymethylcytosine (5hmC) was discovered in 2009 as a novel epigenetic determinant [15]. TET1, TET2, and TET3 enzymes, belonging to the ten–eleven translocation family of dioxygenases, are responsible for oxidation of 5mC to 5hmC (Figure 2). Under moderate local TET enzyme activity, 5hmC can persist as a stable epigenetic signature. In contrast to the general repressive effect of cytosine methylation, 5hmC correlates with active transcription, probably because this modified base presents a different pattern of DNA binding proteins than 5mC does (Figure 1A,D) [16]. It was shown that 5hmC serves as an intermediate of active DNA demethylation during primordial germ cell (PGC) reprogramming for totipotency, as well as in the paternal pronucleus during preimplantation development [17]. Moreover, 5hmC might play a role in cancer progression and metastasis because the level of 5hmC is strongly reduced in different tumor types [18]. In addition, 5hmC may be involved in neurodevelopment, because siRNAdirected knockdown of TET2 and TET3 causes accumulation of incompletely differentiated neurons in ventricular and intermediate zones of the mouse cortex [19].

NH2 N O

~

N NH2

TDG → BER

C

NH2

DNMTs

HO-C=O

H3C

N

N

5caC O

O

~

N

~

N

5mC

Excess TET1-3

TET1-3

5hmC

5fC NH2

NH2 H2C-OH

HC=O

Excess TET1-3

~

N

O

N N

O

~

N

TRENDS in Pharmacological Sciences

Figure 2. Cytosine modification cycle. Cytosine (C) is methylated by transfer of the methyl group from S-adenosylmethionine to the C5 position of cytosine, yielding 5-methylcytosine (5mC). This process is catalyzed by DNA methyltransferases (DNMTs), which in mammals include de novo (DNMT3A and DNMT3B) and maintenance (DNMT1) methyltransferases. Thereafter, 5mC can be oxidized to 5hydroxymethylcytosine (5hmC) by the TET1, TET2, and TET3 enzymes. Excessive TET activity can promote further oxidation of 5hmC to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). The carboxyl group is recognized and removed by thymine-DNA glycosylase (TDG), and the resultant abasic site is regenerated by the base excision repair (BER) pathway, yielding unmodified cytosine. Thus, the abundance of 5hmC is dictated by the balance between DNMT and TET enzyme activities at a given locus.

Until recently it was believed that significant amounts of 5hmC can be found exclusively in mammalian embryonic stem cells and in nervous tissue (0.2% and 1% of total cytosine, respectively). However, we found that 5hmC can account for up to 1% of total cytosine in adult human liver, and genome-wide mapping of 5hmC in multiple liver samples demonstrated that 5hmC is over-represented in the coding regions of actively transcribed genes and in hepatic enhancers, but is under-represented in genes with low expression levels. Pathway analysis revealed that 5hmC signatures in adult liver are especially enriched in genes involved in catabolic and metabolic processes [20]. These findings underline the importance of using methods that allow discrimination between 5mC and 5hmC in the liver, such as Tet1-assisted bisulfite sequencing (TAB-Seq) and oxidative bisulfite sequencing (oxBS-Seq), because conventional methods based on bisulfite conversion do not distinguish between these two cytosine modifications. This issue has to be considered in all previous epigenetic studies in human liver for which bisulfite-based methods were used for determination of cytosine modifications. TAB-Seq uses recombinant Tet1 enzyme for in vitro oxidation of 5mC to 5caC (which is converted to uracil by bisulfite treatment and thus appear as thymine when sequenced), whereas 5hmC remains protected from oxidation because of specific labeling with a glucose residue (and thus appears as cytosine in subsequent bisulfite sequencing) [21]. By contrast, oxBS-Seq relies on specific oxidation of 5hmC to 5fC by potassium perruthenate (KRuO4); when treated with bisulfite, 5fC is converted to uracil, whereas 5mC is resistant to bisulfite [22]. Both methods have been designed to allow profiling of absolute levels of 5mC and 5hmC with singlebase resolution, which is crucial for unbiased analysis of cytosine modifications in 5hmC-rich samples. Histone modifications Post-transcriptional modifications of N-terminal tails of histone proteins, which protrude outwards from the core nucleosome, are numerous and not yet fully characterized [23]. However, methylation and acetylation of histones H3 and H4 seem to be the most widespread and functionally important modifications, playing a major role in the regulation of gene expression. DNAme, together with H3K9me, constitutes a stable repressive epigenetic signature, whereas histone acetylation in general and H3K4me are modifications found in association with unmethylated or hydroxymethylated DNA and active gene transcription (Figure 1A,B,D). It is important to note that not all histone modifications are functionally linked to DNAme. For example, the mechanism of epigenetic silencing of polycomb target genes, which is critically important for mammalian development, does not require the action of DNA methyltransferases, and the trimethylated state of histone H3 (H3K27me3) is achieved by self-recruitment of histone methyltransferase EZH2, which is part of polycomb repressive complex 2 (PRC2), to H3K27me3-containing loci (Figure 1C) [24]. Proteins participating in epigenetic regulation of gene transcription can be collectively called epigenetic proteins. They are more than 200 in number, and can be divided into writers, readers, and erasers of epigenetic signatures. 3

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Writers and erasers are subsequently grouped into epigenetic enzymes and include enzymes that are able to covalently modify DNA or histone proteins. By contrast, readers are able to recognize and bind to modified chromatin, and thus mediate the effect of epigenetic signatures on gene transcription [25]. It is widely accepted that the vast majority of epigenetic enzymes lack intrinsic DNA binding activity and need to be recruited to genomic DNA by other factors, such as TFs or lncRNAs. In particular, histonemodifying enzymes account for a significant proportion of transcriptional coactivators and co-repressors; they interact in vivo with TFs and are targeted to particular genomic loci owing to the site-specific DNA binding activity of TFs (Figure 3A) [26]. In addition, certain histone-modifying enzymes physically interact with lncRNAs and are recruited to specific genomic loci by complementary base (A)

pairing between lncRNA and genomic DNA (Figure 3B) [27]. Targeting of epigenetic enzymes is considered to be one of the primary functions of lncRNAs and provides a good explanation for their important roles in cells. For example, HOTAIR (HOX antisense intergenic RNA) lncRNA might be an important force driving metastasis in breast cancer because overexpression of HOTAIR not only alters the genomic distribution of H3K27me2/3 signatures, thus causing profound gene expression changes, but also increases lung metastasis tenfold in a mouse xenograft model of breast cancer [28]. miRNAs miRNAs, which are conceptually not epigenetic marks, are central to post-transcriptional regulation of mRNA expression. We briefly consider them here as an example of an

Transcripon factor:

Coacvators:

Corepressors:

CBP/p300

HDAC2

PRMT1

H3

H4

Ac

Ac

H3

HDAC3

YY1

YY1

CGCCATNTT

CGCCATNTT

R3

H2A H2B H3

Ac

Ac

Ac

H4

Ac

Me Repressed transcripon

Acve transcripon

(B)

Histone-modifying complexes:

PRC1 monoubiquinase (H2AK119ub1 ↑) BMI1

mPh1/2

CBX RING1A/B

LncRNAs:

PRC2 methyltransferase (H3K27me2/3 ↑)

SUZ12

EZH1/2

ANRIL

p15INK4b

CoREST

EED

Xist

Genomic loci:

LSD1 demethylase (H3K4me2/3 ↓)

chrX inacvaon

REST

LSD1

HOTAIR

HOXD (+ over 800 other loci) TRENDS in Pharmacological Sciences

Figure 3. Sequence-specific recruitment of epigenetic enzymes. (A) Recruitment by transcription factors (TFs). This mechanism is exemplified here by Yin Yang 1 (YY1), a ubiquitously expressed TF, that plays pivotal roles in development, apoptosis, and cancer progression. This TF recognize its consensus sequence CGCCATNTT and can either upregulate or downregulate gene transcription, depending on the context. The underlying transcriptional effects are mediated by YY1-dependent recruitment of either coactivators (e.g., histone acetyltransferases p300 and CBP, histone arginine methyltransferase PRMT1) or co-repressors (e.g., histone deacetylases HDAC2 and HDAC3) to the cognate loci. (B) Recruitment by long noncoding RNAs (lncRNAs). Another mechanism is recruitment of epigenetic enzymes by lncRNAs, which has been demonstrated for certain protein complexes containing histone-modifying enzymes. For example, polycomb repressive complex 1 (PRC1) contains ubiquitin E3 ligase RING1A/B (which is responsible for monoubiquitination of histone H2A at Lys119) as a catalytic subunit. PRC2 is responsible for di- and trimethylation of histone H3 at Lys27 exerted by its catalytic subunit EZH1 or EZH2. Histone demethylase LSD1 is found within the LSD1–CoREST–REST complex, which also contains histone deacetylase HDAC1 or HDAC2 (not shown). lncRNA ANRIL (antisense noncoding RNA in the INK4 locus), which is encoded by the p15INK4b–p14ARF–p16INK4a gene cluster, interacts with both PRC1 and PRC2 and targets them to the p15INK4b gene, and thus plays an important role in establishment of PcG silencing in this locus. lncRNA Xist (X-inactive specific transcript) interacts with the PCR2 complex and plays a key role in epigenetic inactivation of the extra copy of chromosome X in females. Another lncRNA, HOTAIR (HOX antisense intergenic RNA), which is transcribed in the antisense orientation from the HOXC locus, recruits both PRC2 and LSD1 to the nearby HOXD gene, and to multiple loci on other chromosomes. In all the examples shown, lncRNA-mediated recruitment of histone-modifying complexes results in the establishment of repressive epigenetic signatures.

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Review orchestrated scenario of transcriptional regulation whereby epigenetic signatures in genes encoding relevant miRNAs and TFs can participate in the final transcriptional outcome of an ADME gene (see Epigenetic regulation of human ADME genes). In addition, we would like to emphasize that miRNAs are responsible for mediation of epigenetic effects in trans because they are susceptible to epigenetic regulation of their expression, like proteincoding genes [29]. Therefore, DNAme and histone modifications in the vicinity of miRNA genes can influence the post-transcriptional regulation of protein-coding genes located in distant genomic regions. Epigenetic regulation of ADME genes Epigenetic modifications and other factors within the complex regulatory network are able to influence ADME gene expression in cis and in trans [30–32]. In addition, the epigenetic regulation of gene expression can be modulated by environmental factors such as drugs, diet, and environmental pollutants [33]. Therefore, knowledge of the intrinsic and extrinsic factors that influence epigenetic signatures in ADME genes is of importance for understanding interindividual variations in drug kinetics and response.

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Phase I enzymes

Phase II enzymes

Transporters

Modifiers

ADH1B ALDH1A2 ALDH1A3 CYP17A1 CYP26C1 CYP39A1 CYP3A5

CYP3A7 CYP7B1 DPYD FMO3 GPX1 GPX3 GPX7

ADH1C CYP19A1 CYP1A1 CYP1A2 CYP1B1 CYP24A1 CYP26A1

GSTM1 GSTM5 SULT1A1 SULT1C2 UGT1A6 UGT2B15 UGT2B28

GSTA1 GSTA2 GSTT2 NNMT SULT2B1 UGT2B11 UGT2B7

GSTM2 GSTP1 NAT1 UGT1A1

ABCB4 ABCC6 SLC22A18AS SLC22A1 SLC22A3 SLC22A6

SLC22A8 SLC26A4 SLC29A1 SLCO1B3 SLCO1C1

ABCA1 ABCB1 ABCG2 SLC19A1 SLC22A2 SLC2A5

CAT HNF1A PXR RARB

CFTR MPO PPARA PPARG SOD2 SOD3

SLC5A5

CYP27B1 CYP2A13 CYP2E1 CYP3A4 DHRS4L2 SULF1

SLC5A8 SLCO2A1

TRENDS in Pharmacological Sciences

Acquisition of epigenetic memory Epigenetic signatures can be acquired owing to various external stimuli and are able to govern the expression of genes even after cessation of the inducing conditions, and thus provide the basis for the molecular memory of previous exposure. For example, transient pharmacological activation of the constitutive androstane receptor (CAR) by its ligand TCPOBOP in neonatal mice induced lifelong enhanced expression of the hepatic Cyp2b10 and Cyp2c37 genes and thus decreased sensitivity to zoxazolamine treatment in adult mice. It was demonstrated that H3K4 methylation and H3K9 demethylation are involved in the long-term activation of these genes [6]. Acquisition of drug-induced epigenetic changes can either occur very quickly or appear after repeated exposure to the stimulus. For example, short- and long-term exposure to phenobarbital (PB) induced changes in gene expression in murine livers within hours for some genes, whereas others were only modified after several weeks of exposure. Changes in gene expression were associated with a decrease in 5mC and an increase in 5hmC signatures in the corresponding gene promoters, thus suggesting DNA (hydroxy)methylation as the most probable mechanism of drug-induced transcriptional memory. These PB-induced epigenetic alterations were highly conserved between mice and provoked changes in the expression of multiple drug-metabolizing genes, including cytochromes P450 and GST enzymes [7]. Thus, altered epigenetic signatures can influence drug responses some months or even years after the initial induction. To predict the possible spectrum of such memorized metabolic states, it is important to know about the epigenetic regulation of ADME genes.

epigenetic factors at both transcriptional and post-transcriptional levels, as reviewed recently [30–32]. In Figure 4 we update and summarize current knowledge on epigenetic regulation of ADME genes. The majority of the epigenetic regulation hitherto described (89%) concerns DNAme, but an increasing number of studies have reported variations in ADME gene expression that involve alterations in histone modifications, which in most cases (36 out of 44) are accompanied by a change in DNAme. The most paradigmatic example of epigenetic influence at the different levels of regulation is probably the CYP3A4 gene. The critical transcriptional control of CYP3A4 expression by several nuclear receptors (PXR, CAR, HNF4a, HNF3b, VDR, PPARa) is well established. Almost all of these nuclear receptors are regulated by epigenetic mechanisms, either by DNAme in cis or by miRNAs in trans (Figure 5). A recent study has indicated that carbamazepine-induced inhibition of HDAC1 binding to the promoter of CYP3A4 causes increased CYP3A4 gene transcription [34]. In addition, the extent of DNAme of the CYP3A4 promoter may also contribute to the expression of this gene, as evident from the decrease in DNAme in regions corresponding to important TF binding sites, including the proximal promoter, XREM, and CLEM4, as well as C/EBP and HNF4a binding regions in adult compared to fetal livers, where the gene is not expressed [35].

Epigenetic regulation of human ADME genes It is now evident that the activities of enzymes and transporters involved in ADME processes are regulated by

Epigenetic biomarkers of drug response An understanding of the epigenetic mechanisms controlling the expression of ADME genes may provide a basis for

Figure 4. Current knowledge on the epigenetic regulation of genes encoding drug absorption, distribution, metabolism, and excretion (ADME) proteins. Genes relevant to drug metabolism for which epigenetic regulation has been demonstrated are grouped by functional class (Phase I enzymes, Phase II enzymes, transporters, and modifiers). Green denotes genes regulated by DNA methylation; blue denotes genes modulated by histones modifications; orange denotes genes influenced by both mechanisms. See the supplementary material online for relevant references.

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5mC

VDR

5mC

VDR mRNA

miR-125b

PPARA

miR-27b

miR-27b

miR-27b

PPARA mRNA

miR-21

HDAC1

Ac VDR

HNF3β

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PPARα H3 PXR

HNF4α

miR-24a

miR-449

HNF4A mRNA

CAR

PXR mRNA

miR-148a

5mC

miRNA genes

miR-34

5mC

PXR TRENDS in Pharmacological Sciences

Figure 5. Epigenetic regulation of the CYP3A4 gene. The pregnane xenobiotic receptor (PXR), which is one of the most important transcription factors controlling xenobiotic-inducible expression of CYP3A4, is regulated by DNA methylation (in neuroblastoma and colon cancer cells) and probably by miR-148a. Another liver-enriched transcription factor, hepatocyte nuclear factor 4a (HNF4a), which plays an important role in PXR- and constitutive androstane receptor (CAR)-mediated induction of CYP3A4, is post-transcriptionally regulated by miR-24a, miR-34, and miR-449. DNA demethylation and histone acetylation are involved in inducible expression of peroxisome proliferator-activated receptor a (PPARa); in addition, miR-21 and miR-27b have been described as regulators of its expression. The vitamin D receptor (VDR) is regulated by DNA methylation (in breast cancer cell lines and primary tumors) and by miR-125b and miR-27b. Interindividual differences in the levels of these miRNAs may in turn be determined by DNA methylation and/or histone modifications, and thus mediate epigenetic regulation of CYP3A4 in trans. Interestingly, it was demonstrated that miR-27b interacts with at least three different mRNAs belonging to the same cellular pathway (CYP3A4, PPARA, and VDR), suggesting that this miRNA may act as a master regulator of xenobiotic metabolism. In addition, carbamazepine induces CYP3A4 by inhibiting binding of HDAC1 to the CYP3A4 promoter, independently of the presence of PXR. In contrast to these examples, there are no reports so far that two important transcriptional regulators of CYP3A4, CAR and hepatocyte nuclear factor 3b (HNF3b), are regulated by DNA methylation, histone modifications, or miRNAs.

the development of pharmacoepigenetic biomarkers predicting drug responses. However, there are only a few examples of genes encoding drug-metabolizing enzymes proposed as pharmacoepigenetic biomarkers. One of these is GSTP1, which may predict the response of cancer cells to treatment with doxorubicin and DNA methyltransferase (DNMT) inhibitors [31,36]. Some of the most important epigenetic biomarkers of drug responses are summarized in Table 1 and in recent reviews [4,31,32,36]. Promoter methylation in the MGMT gene, which encodes O-6-methylguanine-DNA methyltransferase, is associated with increased progression-free survival (PFS) in glioma patients treated with alkylating agents such as temozolomide [37], and a clinical test for this biomarker is now commercially available. The DNAme status of the Wnt antagonist SFRP5 predicts treatment outcome in cases of non-small-cell lung cancer (NSCLC) treated with EGFR-tyrosine kinase inhibitors [38]. Similarly, DNAme in the promoter of the progesterone receptor (PR) gene might constitute a biomarker of progesterone sensitivity in endometrial tumors [39]. Circulating epigenetic biomarkers of drug response A key prerequisite for the clinical use of pharmacoepigenetic biomarkers is the possibility to monitor them in 6

accessible biological fluids. Recent reports have demonstrated a high level of concordance of altered DNAme in tumor biopsies and matched DNA samples extracted from body fluids such as serum, plasma, urine, and sputum [4]. One of the most relevant examples is hypermethylation of the MGMT gene, which has been detected in cell-free DNA fragments in serum and saliva. Moreover, the methylation levels corresponded to those in the original primary tumor. It has been found that the methylation level of MGMT is informative for the progression of glioma, head and neck cancer, and NSCLC (Table 1) [36]. Epidrugs Epidrugs can be defined as drugs that inhibit or activate disease-associated epigenetic proteins for ameliorating, curing, or preventing the disease. In many human diseases, particularly cancer, the expression or activity of epigenetic proteins is altered. Epigenetic alterations are observed very early during cell transformation, and thus are driver rather than passenger events in cancer [40,41]. The use of epidrugs may thus represent a step forward in the therapy of cancer and other diseases in which epigenetic regulation plays a role. The effects of epidrugs have been validated in relevant biological models [5,42–45].

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Table 1. Relevant examples of epigenetic biomarkers of drug responses Drug Anthracyclines

Alkylating agents

Gene ABCB1 GSTP1

Function Transport Detoxification

PITX2

Cell proliferation

BRCA1

DNA damage response

GPX3

Detoxification of hydrogen peroxide

MGMT

a

DNA repair

PLK2

Tumor suppression

Fluoropyrimidines

TFAP2E

Transcriptional regulation

DNMTi

GSTP1

Detoxification

Tyrosine kinase inhibitors

OSCP1

Transport

SFRP5

WNT signaling

RASSF1A

Cell cycle, DNA repair

Docetaxel

Evidence for epigenetic regulation Promoter methylation correlated with survival in breast cancer patients and may be a marker for the efficacy of doxorubicin treatment Promoter methylation correlated with clinical outcome for anthracycline-based chemotherapy Promoter hypermethylation of BRCA1 predicted enhanced sensitivity to platinum-derived drugs in cancer cell lines and xenografted tumors; it also predicted increased time to relapse (P = 0.0087) and survival (P = 6.4  107) in ovarian cancer patients under cisplatin treatment Loss of GPX3 expression due to promoter hypermethylation correlated with resistance to cisplatin (P = 0.014) and with reduced disease-free survival (P = 0.02) in head and neck cancer patients Promoter methylation of MGMT associated with improved overall survival (21.2 vs 14 months; HR 1.74, P < 0.001), progression-free survival (8.7 vs 5.7 months; HR 1.63, P < 0.001), and response (P = 0.012) in glioma patients treated with temozolomide Promoter methylation of PLK2 associated with a higher risk of relapse in ovarian cancer patients Hypermethylation of TFAP2E associated with clinical nonresponsiveness in colorectal cancer patients DNA methylation of GSTP1 correlated with efficiency of DNMTi therapy in prostate cancer cells Patients with higher methylation of OSCP1 were resistant to imatinib treatment DNA methylation of SFRP5 correlates with lower progression-free survival rate in non-small-cell lung cancer patients in response to EGFR tyrosine-kinase inhibitors Promoter methylation of RASSF1A is associated with nonresponsiveness to docetaxel in breast cancer patients

Power n = 75, P = 0.004 (validation cohort n = 163)

Refs [75]

n = 241, P = 0.002

[76]

n = 30

[77]

n = 46

[78]

n = 411

[79]

n = 54, P = 0.003

[80]

n = 220, P < 0.001

[81]

[82]

n = 90, P = 0.0003

[83]

n = 155, P = 0.011

[38]

n = 45, P = 0.042

[84]

Bold font denotes biomarkers also reported as circulating biomarkers. HR, hazard ratios. a

Tested using a commercially available pharmacoepigenetic test for the efficiency of glioma chemotherapy.

Table 2 lists the most representative small-molecule epidrugs, with a focus on those in clinical trials. Table S1 in the supplementary material online presents a more comprehensive overview of the biological effects observed in in vitro systems or in experimental animals on epidrug treatment. Multiple inhibitors of DNMTs are now considered as promising anticancer agents owing to their ability to reverse the epigenetic silencing of tumor suppressor genes [46]. Two such inhibitors, Vidaza and Dacogen, have already been approved by the FDA for the treatment of myelodysplastic syndrome. Interestingly, inhibition of DNMT activity was also demonstrated for certain drugs currently on the market, such as the cardiovascular

drugs hydralazine, procainamide, and procaine, as well as the antibiotics mitoxantrone, mitramycin A, and nanaomycin A [47]. It has also been reported that the global level of DNAme in macrophages was altered in a cohort of healthy women (n = 77) after taking oral contraceptives compared to women who did not take these drugs (n = 85) [48]. Diethylstilbestrol (DES), the first orally active synthetic estrogen, was withdrawn from the market because of the increased incidence of vaginal tumors and breast cancer in women exposed in utero. Studies in mice demonstrated that the side effects of DES are mediated by epigenetic mechanisms, including genespecific changes in DNAme, as well as altered expression of epigenetic enzymes such as DNMT3A, MBD2, HDAC2, 7

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Table 2. Epidrugs currently in clinical use, under investigation in clinical studies, or being evaluated in clinical trials according to the type of epigenetic enzyme target Type of enzyme DNMT inhibitors

HDAC inhibitors

Drug 5-Azacytidine (Vidaza)

Target enzyme DNMT1

5-Aza-20 -deoxycytidine (aza-dC, decitabine, Dacogen)

DNMT1

Hydralazine

DNMT1

Valproic acid

HDAC

Suberoylanilide hydroxamic acid (vorinostat, Zolinza)

HDAC

Romidepsin (depsipeptide, Istodax) Sodium butyrate

HDAC

HDAC

Panobinostat

HDAC

Entinostat

HDAC1, HDAC2

Mocetinostat

HDAC1, HDAC2

Selisistat

SIRT1

and EZH2, and HOTAIR lncRNA [49]. These examples raise the possibility that the mechanisms of action of certain approved drugs may involve previously uncharacterized epigenetic processes. Inhibitors of histone deacetylases (HDACs) constitute another important group of epidrugs that are highly relevant to the treatment of cancer. Two HDAC inhibitors, vorinostat and romidepsine, were approved by the FDA for treatment of advanced cutaneous T-cell lymphoma. In addition to their potential in cancer, HDAC inhibitors are also considered as promising neuroprotective agents, because sodium butyrate, a known inhibitor of HDACs, stimulates ischemia-induced neurogenesis in rat brain, whereas valproic acid (VPA), which also inhibits HDACs, 8

Clinical application Approved by the FDA in 2004 for treatment of myelodysplastic syndromes (MDS) Approved by the FDA in 2006 for treatment of MDS Reversed platinum resistance in ovarian cancer when coadministered with carboplatin (Phase II trial) Clinical benefit in cancer patients with refractory solid tumors when added to valproic acid (Phase II trial) Reversed imatinib resistance in patients with chronic myeloid leukemia when coadministered with magnesium valproate Promising nontoxic and effective therapy for MDS in combination with hydralazine (Phase II trial ongoing) Under evaluation in metastatic cervical cancer in combination with hydralazine (Phase III trial ongoing) Combined with aza-dC for non-small-cell lung cancer (Phase I trial) Approved by the FDA in 2006 for treatment of advanced cutaneous T-cell lymphoma Reversed hormone resistance in patients with ER+ metastatic breast cancer, when coadministered with tamoxifen (Phase II trial) Approved by the FDA in 2009 for treatment of advanced cutaneous T-cell lymphoma Induces antimicrobial peptide LL-37 in the rectum of shigellosis patients (Phase II trial) Promising results in monotherapy of heavily pretreated Hodgkin’s lymphoma patients (Phase II trial) Recaptures responses in bortezomibresistant multiple myeloma patients (Phase II trial) Improved survival in women with ER+ advanced breast cancer when added to exemestane (Phase II trial) Promising effect in monotherapy of relapsed Hodgkin’s lymphoma (Phase II trial) Under evaluation for treatment of Huntington’s disease (Phase II trial ongoing)

Refs

[65]

[85]

[86]

[87]

NCT00532818

[88]

[89]

[90] [91]

[66]

[92]

[93]

NCT01521585

has a neuroprotective effect in rats after experimental stroke (Table S1 in the supplementary material online). HDACs deacetylate not only histones, but also multiple non-histone proteins, thus contributing to pleiotropic effects of HDAC inhibition on gene transcription and cellular physiology. Furthermore, certain HDACs can demonstrate opposite effects on the same biological process, as exemplified by cardiac hypertrophy, which is promoted by class I and repressed by class IIa HDACs [50]. Therefore, isoform-specific HDAC inhibitors (e.g., entinostat, mocetinostat) may be advantageous over the first-generation socalled pan-HDAC inhibitors [vorinostat, romidepsine, trichostatin A (TSA), VPA, sodium butyrate] because less profound side effects are anticipated.

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Review Methylation of histones at lysine residues is catalyzed by histone lysine methyltransferases (KMTs) and their demethylation by histone lysine demethylases (KDMs) [51]. Besides the anticancer properties of KMT inhibitors (such as DZNep, GSK126, EPZ005687), which are well studied in vitro and in mouse xenograft models (Table S1 in the supplementary material online), the KMT inhibitor BIX01294 displayed unexpected antiparasitic activity, as evidenced by efficient killing of Plasmodium falciparum both in vitro and in infected mice owing to its action on histone methyltransferases of the parasite [52]. Bromodomains (called after the first reported brahma, or brm, gene in Drosophila) are responsible for recognition of acetylated lysine residues in histones and thus serve as readers of acetylation modifications. Moreover, they are often tightly linked to the transcription machinery, and thus potentially mediate the effects of histone acetylation on gene transcription. In particular, a subset of bromodomain-containing proteins is represented by the BET (bromodomain and extra C-terminal domain) family, which is involved in cancer and inflammation. Small-molecule inhibitors targeting BET bromodomain proteins such as BRD4 have demonstrated anti-tumor and anti-inflammatory effects in rodents (Table S1 in the supplementary material online). Another interesting effect of BET inhibitors was observed during treatment of experimental heart failure. Administration of the small-molecule BET inhibitor JQ1 prevented cardiac hypertrophy in mice via suppression of the BRD4-dependent pathologic gene expression program in murine cardiomyocytes in vivo [53]. A striking example of the effect of JQ1 is its ability to control male fertility via reversible inhibition of the testis-specific protein BRDT in mice [54]. Notably, some drugs currently on the market can also bind bromodomains, as recently demonstrated for alprazolam and midazolam [55], but the importance of these interactions in their therapeutic effects is unknown. Epidrugs for treatment of drug abuse The addiction phenotype represents a long-term molecular memory of drug consumption, suggesting the existence of strong epigenetic components. Drugs of abuse might thus influence the activity of certain epigenetic proteins, and epidrugs might possibly be developed as a novel pharmacological intervention for drug addiction. Indeed, morphine-treated mice demonstrated decreased HDAC activity and increased histone H3 acetylation in the spinal cord, and administration of the histone acetyltransferase inhibitor curcumin significantly reduced the development of opioid-induced changes, such as hyperalgesia, tolerance, and physical dependence [56]. In rats injected with ethanol, the anxiolytic response was caused by inhibition of HDAC activity in the amygdala, resulting in upregulation of neuropeptide Y (NPY). Intracellular compensatory mechanisms contributed to the development of rapid ethanol tolerance (RET), which is considered an important factor in human alcoholism. Therefore, a second exposure within 24 h to the same dose of ethanol did not cause any anxiolytic effect, and an increased amount of ethanol was required to overcome RET. The HDAC inhibitor trichostatin A was able to reverse RET, and thus counteracted the epigenetic effects of acute ethanol exposure [57].

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In addition to histone acetylation, other epigenetic mechanisms are involved in drug addiction. For example, it was shown that ethanol, cocaine, and opiates downregulate the histone methyltransferases G9a and GLP in mice, and consequently the global level of histone methylation in the nucleus accumbens (NAc, the brain reward center). It was suggested that this drives gene expression changes resulting in altered synaptic plasticity of neurons [58]. In line with this, it has been demonstrated that inhibition of G9a in NAc increases behavioral responses to drugs, whereas its activation blunts them [59]. Finally, DNAme changes on exposure to drugs of abuse have been observed. For example, opiate-dependent hypermethylation of CpG sites in the promoter of the OPRM1 gene (encoding the m-opioid receptor) results in decreased binding of the Sp1 TF and in subsequent downregulation of the m-opioid receptor, and thus plays a role in the development of opioid addiction [60]. Epidrugs and acquired drug resistance Increasing evidence suggests that acquired resistance to chemotherapy is a multifactorial phenomenon in which epigenetic changes could play a major role. Induced epigenetic changes in gene expression causing resistance in cancer therapy can involve (i) impaired proliferation inhibition, (ii) inhibited induction of cell differentiation, (iii) inhibited cell apoptosis, (iv) altered DNA damage response, or (v) altered drug transport [61]. The beststudied example of epigenetically induced tumor resistance is promoter modification of multidrug resistance genes belonging to the ATP-binding cassette (ABC) transporter family. Resistance may arise from upregulated expression of the ABCB1 (MDR1) gene, which can be caused by DNA demethylation or histone H3 hyperacetylation in the promoter region, thus contributing to increased drug efflux [62]. Tumors may contain, albeit at low numbers, cells with a mesenchymal or stem cell-like phenotype that are intrinsically resistant to anticancer agents [63]. Interestingly, an altered epigenetic state of such drug-tolerant subpopulations of cancer cells requires the activity of H3K4 demethylase KDM5A, and these cells can be eradicated by HDAC inhibition, which was suggested as a phenocopy of KDM5A knockdown [64]. This and other examples from in vitro and animal models studies (Table S1 in the supplementary material online) suggest that epigenetic therapy could erase the epigenetic signatures associated with the drugresistant phenotype and therefore sensitize drug-resistant tumors to chemotherapy. In a few clinical trials, epigenetic drugs have been tested for cancer treatment either alone or in combination with conventional cytotoxic therapy (Table 2). The DNMT inhibitor decitabine is able to reverse platinum resistance in ovarian cancer when coadministered with carboplatin, resulting in a high response rate and prolonged progression-free survival in a Phase II clinical trial [65]. The HDAC inhibitor panobinostat was able to revert bortezomib resistance and induce better responses in refractory myeloma patients in a Phase II trial [66]. Interestingly, acquired drug resistance involving epigenetic mechanisms can be observed not only in human 9

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cancers but also in infectious or parasitic diseases. It has been demonstrated that fluconazole-induced resistance in Candida albicans strains [67] and blasticidin S resistance in Plasmodium falciparum involve epigenetic mechanisms and can potentially be reversed by epidrugs. Blasticidin Sresistant parasites silence their clag2 and clag3 genes to reduce the encoded protein levels at the host membrane and curtail channel-mediated uptake of this antibiotic. It is hypothesized that this silencing is mediated by a decrease in histone marks H3K9ac and H3K4me3 [68]. Epigenetic editing As mentioned, a common feature of epigenetic proteins is that they lack an intrinsic sequence recognition capability, and modulation of their activity by epidrugs can theoretically cause genome-wide gene expression changes. Hence, the general disadvantage of epidrugs is their potential lack of specificity, which can result in serious side effects. An alternative to epidrugs that may counteract this limitation is the novel approach of epigenetic editing, which can be defined as intentional overwriting of epigenetic signatures by artificial targeting of epigenetic enzymes to specific loci [69]. The methodologies for epigenetic editing can be divided into nucleic acid-based and protein-based, depending on whether the target genomic sequence is recognized by a DNA or RNA probe, or by a sequence-specific DNA-binding protein, respectively. Further development of such approaches could give rise to a completely new pharmacological treatment class, which might contribute to erasing of disease-associated epigenetic signatures. A relevant example of epigenetic editing using nucleic acid-based methodology is the delivery of siRNA molecules into the nuclei of mammalian cells. It has been demonstrated that siRNA acts in a completely different manner in the nucleus than in cytoplasm; cytoplasmic siRNAs contribute to post-transcriptional degradation of mRNAs,

whereas nuclear siRNAs can induce heritable DNA hypermethylation of the cognate gene (Figure 6) [70]. Protein-based epigenetic editing involves fusion of a sequence-recognizing artificial protein (which represents the DNA-binding domain) to a certain effector domain that can modify epigenetic patterns of the chromatin (e.g., DNMT3A). Currently, two options for engineering of DNA-binding domains are available: ZF (zinc finger) and TALE (transcription activator-like effector) domains. The effector domain can be a DNA methyltransferase (e.g., the DNMT3A catalytic domain), a transcriptional activator (e.g., p65 transactivation subunit of the NF-kB complex), or a repressor domain (e.g., Kruppel-associated box or KRAB). In the latter cases, the resultant fusion proteins are called artificial TFs. Transcriptional activation/repressor domains are able to recruit multiple chromatin-modifying enzymes, which can work synergistically to establish activating or repressing epigenetic signatures. For example, in a proof-of-principle study, a ZF domain recognizing the promoter of the SOX2 oncogene was fused to the catalytic domain of DNMT3A, and the resulting protein was expressed from a retroviral vector in breast cancer cells. This caused stable hypermethylation of the target gene, resulting in its transcriptional repression by 60% for at least 50 cellular generations, even after the artificial protein was no longer expressed in the cells [71]. In another study, the same DNA-binding ZF domain was fused to the transcriptional repressor KRAB domain, resulting in 95% transcriptional downregulation of the SOX2 oncogene [72]. In a Phase II clinical trial, a ZF domain against the vascular endothelial growth factor A (VEGFA) gene was fused to the transcriptional activator p65, and the resulting SB-509 plasmid was intramuscularly injected into patients suffering from diabetic neuropathy [73]. Unfortunately, this trial was stopped because of an insufficient

G9a

Transcriponal silencing complex (TSC)

HDAC1

Ago-1/2 DNMT3

EZH2

siRNA

Me1/2

Duplex

Ac

pRNA

K9

5mC

RNAPII Promoter

H3 5mC

H3

H3

Gene

K27 Me2/3

Repressive epigenec signature TRENDS in Pharmacological Sciences

Figure 6. Epigenetic editing by siRNA. siRNA, when delivered to the nucleus, can hybridize with homologous low-copy promoter-associated RNA (pRNA), which is usually associated with transcribed promoters. Then the nuclear Ago-1 and Ago-2 proteins (which are enriched at the gene promoters, probably because of their interaction with RNA polymerase II) recognize the pRNA–siRNA duplexes, which in turn are responsible for recruitment of epigenetic enzymes such as DNMT3A, histone deacetylase HDAC1, and histone methyltransferases EZH2 and G9a to the targeted promoter, and thus establish repressive epigenetic marks on both histones and DNA.

10

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Pharmacoepigenecs Epigenec regulaon of genes

ADME genes

• • •

Drug response Adverse drug reacons Acquired drug resistance

Disease-related genes

• • •

Disease diagnosis Disease prognosis Straficaon of paents

Pharmacoepigenec biomarkers (invasive or circulang)

Increased efficacy of drug treatment

Epigenec effects of drugs

Drugs on market Drugs of abuse

Epigenecs of drug acon

Epidrugs

Epigenec eding

Correcon of epigenec signatures

New paradigms for the drug treatment TRENDS in Pharmacological Sciences

Figure 7. Overview of the mechanisms and consequences of epigenetic regulation of drug actions and responses. Genes encoding drug absorption, distribution, metabolism, and excretion (ADME) proteins can be epigenetically regulated and thereby influence the risk of adverse drug reactions, drug resistance, and drug responses in general. Epigenetic modification of disease-related genes occurs, and analysis of invasive and circulating epigenetic biomarkers can contribute to disease prognosis and thus establish a basis for personalized drug therapy. Such pharmacoepigenetic biomarkers can also indicate the risk of acquired drug resistance. Drugs can also directly act to modify the activity of epigenetic proteins and thus influence the genomic distribution of epigenetic signatures. For future applications, epigenetic editing might be used to modify specific epigenetic signatures causing a disease.

response, probably because the naked plasmid DNA injected is not stable enough; in this case, a more robust method for delivery of the DNA drug would be advantageous. Apart from the provision of appropriate delivery systems, the protein-based methodology for epigenetic editing needs to be further developed to address the challenge of reactivating a silenced gene, which by definition is more demanding than silencing of an actively transcribed gene. Epigenetically silenced chromatin is usually highly compacted and thus can be inaccessible to artificial reactivating proteins. It is likely that this hurdle can be overcome by coadministration of transcription-activating epidrugs and an epigenetic editing construct. For example, TALE-based activators against a silent OCT4 gene were efficient in neural stem cells only in the presence of either 5-aza-20 deoxycytidine or VPA, but not alone [74]. Concluding remarks The field of pharmacoepigenetics is rapidly growing and our understanding of the roles of epigenetic mechanisms in drug action is increasing, as summarized in Figure 7. It is apparent that epigenetic modifications induced by drugs and other xenobiotics can act as short- and long-term regulatory mechanisms for drug dependence, drug resistance, and altered drug metabolism and action. There is a need to investigate to what extent xenobiotic exposure can influence epigenetic signatures in the human body, and how epigenetic variability in ADME genes and disease-related genes can be translated into interindividual differences in drug

pharmacokinetics, toxicity, and responses. We can already use pharmacoepigenetic biomarkers to monitor disease prognosis and to predict individual responses to anticancer drugs, and use of such biomarkers will probably increase in importance in the years to come. In addition, novel therapeutic approaches based on the epigenetic action of drugs opens up new horizons in the field of cancer, inflammation, and other diseases. Moreover, the developing technology of epigenetic editing, which allows us to direct the activity of epigenetic enzymes towards specific genes, has the potential to bring epigenetic-based interventions to a completely new level. Acknowledgments Research at our laboratory is funded by grants from The Swedish Research Council, The Swedish Cancer Fund, FP-7/Seurat 1 project NOTOX, IMI-JU project MIP-DILI, and Marie Curie grant CIG 322283.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.tips.2014.05.004.

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