REVIEWS

DNA Methylation of ADME

P Fisel1,2, E Schaeffeler1,2 and M Schwab1,3,4

The epigenetic regulation of expression of genes involved in the absorption, distribution, metabolism, and (ADME) of contributes to interindividual variability in response. Epigenetic mechanisms include DNA methylation, histone modifications, and miRNAs. This review systematically outlines the influence of DNA methylation on ADME expression and highlights the consequences for interindividual variability in drug response or drug-induced and the implications for personalized .

ADME GENES AND INTERINDIVIDUAL VARIABILITY fications are dynamic and may change over time or in response 3–5 OF DRUG RESPONSE to stimuli. They are influenced by patient characteristics Approximately 300 genes contribute to the ADME of xenobiotics, (e.g., gender, ethnicity), environment, lifestyle, and disease state including the majority of drugs (http://pharmaadme.org/). Interin- or concomitant medication and contribute to individual pheno- dividual variability in expression and function of ADME genes are types. , in particular, address the epigenetic known to affect the individual response of patients to drugs with basis of interindividual variability of ADME genes, thereby con- respect to therapeutic efficacy as well as adverse drug reactions tributing to the elucidation of unknown reasons for interindivid- (ADR). The heterogeneity in expression and function of ADME ual variability in drug response. Furthermore, epigenetic genes among individuals is determined by the interaction of genetic regulation of ADME genes can affect endogenous metabolism and nongenetic factors, as well as epigenetic modifications.1 The and deregulation can lead to the pathogenesis of various diseases. influence of genetic variation in ADME genes and of nongenetic Recently, over 60 ADME genes have been considered to be influ- enced by histone modifications, DNA methylation, or miR- host factors, e.g., sex, age, disease state, or drug exposure, on drug 6,7 response is the concept of personalized medicine. Meanwhile, NAs and evidence for an epigenetic component for the major efforts are spent on implementing the pharmacogenomic regulation of ADME gene expression has been rising ever since. Best evidence does exist for findings related to in vitro experi- (PGx) knowledge into clinical practice (http://www.fda.gov/drugs/ ments using cell lines treated with the DNA methyltransferase scienceresearch/researchareas/pharmacogenetics/ucm083378.htm; (DNMT) inhibitor 5-aza-20-deoxycytidine or the histone deace- https://www.pharmgkb.org/page/cpic). PGx information is also tylase (HDAC) inhibitor trichostatin A. Moreover, these findings part of labels of drugs used in various medical disciplines. Of are supported and/or supplemented by data from cancer tissues, note, about 70% of PGx information for drugs approved by the where epigenetic deregulation of gene expression is a common European Agency (EMA) between 1995 and 2014 is 8 2 phenomenon. However, the clinical relevance of epigenetic regu- related to ADME processes. In contrast, the significance of epige- lation by DNA methylation on ADME gene expression and netic regulation of gene expression in the context of drug response implications for drug safety and efficacy is so far limited. This and ADME is just evolving and currently not part of drug labels. review provides a summary and update of the currently available The term “epigenetics” in general refers to heritable changes of knowledge of the impact of DNA methylation on ADME genes, gene expression without underlying changes in the encoding with particular focus on consequences for drug response. DNA sequence.3 Epigenetic modifications regulating gene expres- sion include DNA methylation, histone modification, and non- DNA METHYLATION 4 coding RNAs (miRNAs). The interaction of these mechanisms One of the three major epigenetic mechanisms for the regulation forms a complex network and determines developmental or dif- of gene expression is DNA methylation. DNA methylation is a ferentiation stage-specific expression patterns. In addition, the stable, covalent addition of a methyl (CH3) group, occurring at interplay of epigenetic modifications contributes to the regulation 50Cs of cytosines primarily within CpG dinucleotides (5-mC). of tissue- and cell type-specific gene expression. Epigenetic modi- The modification is catalyzed either by de novo DNA

1Dr. Margarete Fischer-Bosch Institute of Clinical , Stuttgart, Germany; 2University of Tubingen,€ Tubingen,€ Germany; 3Department of , University Hospital Tubingen,€ Tubingen,€ Germany; 4Department of and , University of Tubingen,€ Tubingen,€ Germany. Correspondence: M Schwab ([email protected]) Received 4 December 2015; accepted 20 January 2016; advance online publication 27 January 2016. doi:10.1002/cpt.343

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Figure 1 Epigenetic regulation of ADME gene expression by DNA methylation and DNA hydroxymethylation. Unmethylated DNA in gene promoters allows binding of transcription factors (TF) and coactivators (CoA) leading to active gene transcription. DNA methylation, i.e., the covalent addition of aCH3-group to 50Cs of cytosines within CpG dinucleotides, of gene promoters is catalyzed by DNA methyltransferases (DNMT) and leads to suppression of gene expression. 5-methylcytosine (5-mC) underlies oxidative DNA demethylation catalyzed by members of the ten-eleven translocation (TET) family, resulting in hydroxymethylated DNA (5-hmC). Further intermediates of the DNA demethylation process include 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). DNA methylation as well as DNA hydroxymethylation are dynamic and can be influenced by environmental or host factors (e.g., age, sex, disease state). methyltransferases (DNMT3a and DNMT3b) during develop- of ADME genes. With respect to drug response, the dynamic ment or by maintenance DNA methyltransferases (DNMT1) nature of DNA methylation represents a major challenge. Thus, during DNA replication. In general, DNA methylation in pro- age dependency of DNA methylation and subsequent alteration moter regions of genes suppresses their expression by directly of ADME gene expression need to be taken into consideration, inhibiting the binding of transcription factors or indirectly by especially in subpopulations such as children and the elderly. At recruiting methyl-CpG-binding , which in turn modulate the beginning of life and even in preterm infants there is convinc- chromatin structure (Figure 1).4 DNA methylation marks are ing evidence that expression and function of drug metabolizing heritable and can be transmitted transgenerationally.9 Despite (DME) are significantly reduced compared to adults. heritability and stability, DNA methylation represents a dynamic For instance, CYP3A4 levels increase very gradually dur- process which changes throughout the course of a lifetime and in ing the first year of life, and even 5–15-year-old children show response to environmental stimuli and therefore contributes to significantly lower CYP3A4 levels compared with adults.12 phenotypic variation in humans. This phenomenon is best corro- Related to drug transporters, a similar picture can be borated by studies examining genetically identical monozygotic assumed.13,14 Independent from other factors, there is first evi- twins, which show similar epigenetic marks during younger ages dence that differences in DNA methylation patterns may con- compared to older age, when remarkable epigenetic differences tribute to the ontogeny of the expression of DME in can be observed.10,11 childhood.15 A higher risk for ADRs is well established in older Aberrant DNA methylation is associated with the pathogenesis patients, which may not only be related to, e.g., different body and treatment response of cancer and various other diseases, e.g., composition and impaired organ function due to underlying dis- psychiatric or autoimmune diseases,8 and also affects expression eases as well as polypharmacy, but also to the reduced capability

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to metabolize and/or transport drugs.16 Major research activities REGULATION OF ADME GENE EXPRESSION are warranted to fully comprehend the impact of DNA methyla- BY DNA METHYLATION tion on ADME gene expression and function related to these dif- As genetic variation in ADME genes cannot fully account for ferent subgroups. interindividual variability of drug response, current research aims In addition to interindividual variability of DNA methylation, to identify the epigenetic component of ADME gene regulation. marked intraindividual differences, particularly in DNA methy- A comprehensive summary of currently available knowledge lation patterns between various normal tissues and cell types, regarding regulation of ADME gene expression by DNA methyl- are observed. These differences may be even more pronounced ation is given in Tables 1–3, distinguishing between drug- between tumor and normal tissues.5 metabolizing enzymes, drug transporters, and modifiers like DNA hydroxymethylation is a further recently identified cova- nuclear receptors. The role of DNA methylation for selected lent modification of DNA with an impact on gene expression. genes is highlighted below. Instead of a methyl group, cytosines are substituted with a 0 Phase I and phase II enzymes hydroxymethyl (CH2-OH) group at the 5 C (5-hmC). DNA hydroxymethylation is suggested to be an intermediate in the oxi- The hepatic expression of one of the major drug-metabolizing dative DNA demethylation pathway mediated by TET enzymes,17 phase I enzymes, CYP3A4, is extremely variable among individu- 22 and is proposed to interact with transcriptional and chromatin als (>100-fold), but the contribution of genetic variants to this 23 modifiers as well. The effect of DNA hydroxymethylation on interindividual variability is rather low. Habano et al. showed 0 expression of the respective gene is not yet fully understood and is that DNA methylation of a CpG island in the 5 UTR about presumably context-dependent. DNA hydroxymethylation in gene 25 kb distal of the CYP3A4 transcription start site does not bodies, i.e., in the gene region downstream of the first exon and affect gene expression. However, as demonstrated by Kacevska 24 upstream of the 30UTR (untranslated region), has been shown to et al., DNA methylation at CpG sites near or within binding be associated with active transcription. However, 5-hmC in prox- sites for various transcription factors (e.g., C/EBP or HNF4a)in imity to transcription start sites can either have a repressive or an a 12 kb upstream promoter region has an influence on the expres- activating effect.17 The abundance of 5-hmC is tissue-specific, with sion of CYP3A4. DNA methylation at several CpG sites was high levels occurring, e.g., in brain, liver, and kidney.18 In addition, shown to be highly variable and correlated with CYP3A4 expres- DNA hydroxymethylation is dependent on developmental stage sion in adult liver. The lack of CYP3A4 expression in fetal liver (fetus vs. adults)19 and varies among individuals of different age,20 could be explained by DNA hypermethylation within the investi- thereby contributing significantly to interindividual variability of gated region. In contrast, regions with important transcription gene expression. An important aspect to be considered when look- factor binding sites were hypomethylated in adult liver where ing at previous DNA methylation studies is that the majority of CYP3A4 is expressed. The expression of CYP1A1 has also been these are based on bisulfite treatment of genomic DNA, which has described to be influenced by DNA methylation. Hypermethyl- been accepted as the gold standard to examine DNA methylation ation of a CYP1A1 enhancer region has been shown to be associ- profiles. However, methods based on bisulfite treatment cannot ated with dioxin responsiveness in prostate cancer cell lines.25 distinguish between methylated and hydroxymethylated DNA. Furthermore, CYP1A1 methylation levels are influenced by Meanwhile, methods have been developed, e.g., TET-assisted smoking. A dose-dependent reduction of the CYP1A1 promoter bisulfite sequencing (TAB-seq) or oxidative bisulfite sequencing DNA methylation from nonsmoker to heavy smokers was (OxBS-seq),21 which are able to differentiate between 5-mC and observed, suggesting a role of DNA methylation for the induc- 5-hmC, thereby allowing examination of the individual influence tion of CYP1A1 in human lung due to tobacco smoking.26 of DNA methylation or hydroxymethylation on gene expression. Tekpli et al.27 report that CYP1A1 enhancer methylation was The specific influence of DNA hydroxymethylation on the regula- associated both with mRNA expression and smoking-induced tion of ADME genes in adults has been addressed in a pilot study DNA adducts in normal human lung. Interestingly, only recently by Ivanov et al., investigating DNA methylation as well as hydroxy- He et al.28 reported an association of CYP1A1 hypermethylation methylation in a set of 191 ADME genes using targeted TAB-seq with a higher susceptibility to antituberculosis drug-induced liver in 20 human liver samples, demonstrating a correlation of the injury (ADLI) in a cohort of 127 tuberculosis patients developing abundance of 5-mC and 5-hmC with interindividual variability in ADLI and 127 tuberculosis patients without liver injury. The ADME gene expression (Abstract: 13th European ISSX Meeting influence of DNA methylation on the expression of CYP1B1 has 2015; Ivanov, M. et al). However, large-scale studies that evaluate been shown in various cancer tissues. Overexpression of CYP1B1 the role of hydroxymethylation for ADME genes have not yet has been related to decreased promoter and enhancer DNA been reported. Such studies will be essential to better delineate the methylation at ARNT and Sp1 transcription factor binding sites impact of hydroxymethylation in normal as well as tumor tissues in prostate cancer.29 Widschwendter et al.30 found that CYP1B1 in terms of drug safety and effectiveness. hypermethylation levels are associated with better survival in Finally, two further intermediates, 5-formylcytosine (5-fC) and tamoxifen-treated breast cancer patients. Although CYP1B1 5-carboxylcytosine (5-caC), are involved in the oxidative DNA expression levels were not determined in that study, assuming demethylation process. Both 5-fC and 5-caC are considered that patients with higher methylation levels expressed less unstable intermediates and their potential functional influence CYP1B1, these patients would be expected to retain higher levels remains to be elucidated.17 of the active drug, consistent with the observed improved survival

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Table 1 Epigenetic regulation of phase I and II enzymes by DNA methylation (for references see Supplementary Material) Gene name DNA methylation Reference ALDH1A2 promoter hypermethylated in human prostate cancer cell lines; expression induced after Kim et al., 2005 5-aza-20-deoxycytidine; reduced expression and hypermethylation in human primary prostate tissue; association with recurrence-free survival evidence for association of DNA methylation status at CpG sites near ALDH1A2 gene Harlaar et al., 2014 with loss of control over drinking ALDH1A3 promoter hypermethylation associated with favorable prognosis in G-CIMP-primary Zhang et al., 2013 glioblastoma methylation status is associated with reduced ALDH1A3 gene expression and disease Kim et al., 2013 recurrence and progression in nonmuscle invasive bladder cancer epigenetic silencing by DNA methylation suggested as prognostic biomarker in Ma et al., 2015 glioblastoma suppressed ALDH1A3 expression due to increased DNA methylation in cervical cancer Lee et al., 2015 CYP1A1 smoking induces hypomethylation of CYP1A1 promoter in lung tissue Anttila et al., 2003 hypermethylation of CYP1A1 in prostate cancer cell line associated with reduced Okino, 2006 induction of CYP1A1 mRNA expression by TCDD; hypermethylation also in human prostate tumors CYP1A1 promoter methylation is not associated with mRNA expression in colorectal Habano et al., 2009 cancer cell lines in utero exposure to tobacco is associated with promoter hypomethylation and Suter et al., 2010 increased mRNA levels in the placenta methylation is not associated with tamoxifen resistance in breast cancer Kim et al., 2010 hypermethylation of CYP1A1 associated with head and neck cancer Sharma et al., 2010 CYP1A1 enhancer methylation inversely linked to mRNA expression and the level of Tekpli et al., 2012 hydrophilic DNA adducts in normal lung tissue; smoking induces hypomethylation; enhancer hypermethylation and reduced mRNA levels in lung cancer higher CYP1A1 promoter methylation in tuberculosis patients with anti-tuberculosis He et al., 2015 drug-induced liver injury compared to tuberculosis patients without liver injury CYP1A2 variation in methylation status at specific CpG site in the 50flanking region of the Hammons et al., 2001 CYP1A2 gene associated with interindividual differences in CYP1A2 expression in human liver DNA methylation in the CYP1A2 promoter correlates inversely with hepatic CYP1A2 Ghotbi et al., 2009 mRNA levels; 5-aza-20-deoxycytidine treatment induces expression in hepatoma cell line methylation of GC box in the CYP1A2 promoter contributes to tissue specific regulation Miyajima et al., 2009 of mRNA expression inverse correlation of mRNA expression and DNA methylation level in normal adult liver Habano et al., 2015 CYP1B1 CYP1B1 promoter methylation is associated with better survival in patients receiving Widschwendter et al., 2004 tamoxifen therapy and with inferior survival in patients without tamoxifen therapy CYP1B1 overexpression in prostate cancer is regulated by hypomethylation in the Tokizane et al., 2005 promoter 5-aza-20-deoxycytidine treatment results in increased CYP1B1 mRNA expression in Habano et al., 2009 colorectal cancer cell lines, increased CYP1B1 methylation found in a minor fraction of colorectal cancer tissues CYP1B1 inducibility by dioxin is decreased in HepG2 cells due to promoter methylation Beedanagari et al., 2010 correlation of CYP1B1 methylation with expression, evidence for association of CYP1B1 DiNardo et al., 2013 methylation with outcome and disease-free survival in a cohort of adolescent and young adult patients with acute lymphoblastic leukemia CYP2A13 CYP2A13 methylation contributes to regulation of gene expression in human lung cancer Ling et al., 2007 cells hypermethylation of CYP2A13 associated with head and neck cancer Sharma et al., 2010 Table 1 Continued on next page

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Table 1 Continued

Gene name DNA methylation Reference CYP2C19 inverse correlation of mRNA expression and DNA methylation level and high variability in Habano et al., 2015 methylation status in normal adult liver CYP2D6 methylation is not associated with tamoxifen resistance in breast cancer Kim et al., 2010 inverse correlation of mRNA expression and DNA methylation level in normal adult liver Habano et al., 2015 methylation status of CYP2D6 gene is associated with age and sex and is dependent on Tiili et al., 2015 genotype, poor metabolizer phenotype frequently linked to higher methylation level, low methylation level was associated with heavy smoking CYP2E1 CYP2E1 is not expressed in fetal liver due to methylation in the 50region, but in adult Jones et al., 1992 liver without methylation in this region demethylation of 50region of CYP2E1 gene along with increased mRNA in fetal liver Vieira et al., 1996 during neonatal period no CYP2E1 expression in human lung and kidney due to methylation in the 50flanking Vieira et al., 1998 region of CYP2E1 gene CYP2E1 hypomethylation and mRNA overexpression in brains of patients Parkinson’s Kaut et al., 2012 disease prenatal exposure to serotonin reuptake inhibitor antidepressants and maternal Gurnot et al., 2015 depressed mood leads to aberrant CYP2E1 DNA methylation, which is suggested to be associated with birth weight CYP2R1 higher methylation levels in the gene promoter in vitamin D nonresponders than in Zhou et al., 2014 vitamin D responders CYP2W1 CpG island in the first exon/intron junction methylated in cell lines without CYP2W1 Kalgren et al., 2006 expression; CYP2W1 expression restored after 5-aza-20-deoxycytidine treatment CYP2W1 expression is regulated by DNA methylation of CpG island in the first exon/ Gomez et al., 2007 intron junction in cell lines as well as in colon tumors and normal colon tissue CYP2W1 expression in fetal but not in adult colon samples, associated with higher Choong et al., 2015 methylation of 6 CpG sites in the region of the first exon/intron junction in adult colon samples CYP3A4 DNA methylation levels among individuals are variable in 50region of the CYP3A4 region Kacevska et al., 2012 in key regulatory binding sites; hypermethylation contributes to lacking expression in fetal liver; methylation status at single CpG sites correlates with CYP3A4 expression no significant association between interindividual variability in CYP3A4 expression and Habano et al., 2015 DNA methylation observed CYP3A7 5-aza-20-deoxycytidine treatment contributes to upregulation of CYP3A7 mRNA expres- Dannenberg and Edenberg, sion in HepG2 cells 2006 CYP7B1 methylation of CYP7B1 promoter region has an influence on gene expression in Olsson et al., 2007 high-grade prostatic intraepithelial neoplasia and adenocarcinomas CYP24A1 CYP24A1 promoter methylated in human full term placenta, but not in other somatic Novakovic et al., 2009 tissues inverse correlation of CYP24A1 expression and promoter DNA methylation in prostate Luo et al., 2010 cancer cell lines; 5-aza-20-deoxycytidine treatment induces expression, in vitro methylation reduces promoter activity; DNA promoter methylation contributes to reduced CYP24A1 expression in human prostate cancer hypermethylation of 50region of CYP241 gene in calcitriol sensitive tumor-derived Johnson et al., 2010 endothelial cells, treatment with 5-aza-20-deoxycytidine induces resistance higher methylation levels in the CYP24A1 gene in vitamin D nonresponders than in Zhou et al., 2014 vitamin D responders CYP26C1 DNA methylation of CYP26C1 is associated with poor prognosis in neuroblastoma Abe et al., 2008 CYP27B1 DNA methylation of the CYP27B1 promoter contributes to suppression of gene Kim et al., 2007 expression by the vitamin D Table 1 Continued on next page

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Table 1 Continued

Gene name DNA methylation Reference CYP39A1 CYP39A1 is hypermethylated in ovarian cancer Huang et al., 2009 DHRS4L2 DHRS4L2 expression is affected by DNA methylation in human neuroblastoma cells Zhang et al., 2009 DPYD DPYD promoter methylation contributes to the regulation of DPYD expression and Noguchi et al., 2004 influences sensitivity to 5-fluorouracil in cancer cell lines association of aberrant DPYD promoter methylation and unexplained DPYD enzyme Ezzeldin et al., 2005 deficiency in patients with 5-fluorouracil toxicity no association of DPYD promoter methylation and mRNA expression in colorectal Yu et al., 2006 cancer, suggesting major importance of CpG site location and tissue specificity DPYD promoter methylation is not associated with 5-fluorouracil severe toxicity in Savva-Bordalo, 2010 patients with gastrointestinal cancer GPx3 increased DNA methylation in the first exon of GPx3 leads to reduced GPx3 expression Yu et al., 2007 in prostate cancer DNA hypermethylation close to the transcription start site correlates inversely with Peng et al., 2009 reduced mRNA expression levels in Barrett’s adenocarcinoma GPx3 expression downregulated due to increased promoter methylation in gastric Zhang et al., 2010 cancer CpG island in the GPx3 promoter hypermethylated accompanied by decreased GPx3 He et al., 2011 mRNA expression in esophageal squamous cell carcinoma GPx3 promoter methylation is associated with resistance to cisplatin treatment in Chen et al., 2011 patients with head and neck cancer GPx3 promoter hypermethylation correlates with reduced expression and is associated Peng et al., 2012 with lymph node metastasis in gastric cancer downregulation of GPx3 expression by DNA methylation involved in progression of Mohamed et al., 2014 inflammatory breast cancer promoter DNA methylation is associated with reduced GPx3 expression, GPx3 Zhang et al., 2014 expression is associated with lymph node metastasis and progression in cervical cancer GPx3 hypermethylation is associated with poor survival in non-M3 acute myeloid Zhou et al., 2015 leukemia inverse correlation of GPx3 expression and methylation in chronic myeloid leukemia; Yao et al., 2015 re-expression of GPx3 after 5-aza-20-deoxycytidine in CML cell line GPx3 expression silenced due to promoter hypermethylation in clear cell renal cell Liu et al., 2015 carcinoma GPx7 DNA hypermethylation close to the transcription start site correlates inversely with Peng et al., 2009 reduced mRNA expression levels in Barrett’s adenocarcinoma DNA methylation in a specific region of the GPx7 promoter CpG island correlates Peng et al., 2014 inversely with expression in oesophageal adenocarcinoma GSTA4 inverse correlation of mRNA expression and DNA methylation level and high variability in Habano et al., 2015 methylation status in normal adult liver GSTO2 DNA methylation contributes to a larger part to the variation of GSTO2 gene expression Bonder et al., 2014 than SNP genotypes GSTP1 hypermethylation of GSTP1 promoter suggested as biomarker for various cancer for review see Schnecken- entities, including prostate and breast cancer burger et al., 2014 haplotype structure has an influence on the degree of GSTP1 promoter methylation in Ronneberg et al., 2008 breast cancer hypermethylation of GSTP1 promoter negatively correlates with expression and is Dejeux et al., 2010 associated with improved survival in doxorubicin treated breast cancer patients methylation of GSTP1 proposed as predictive marker for therapy with DNMT inhibitors in Chiam et al., 2011 prostate cancer Table 1 Continued on next page

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Table 1 Continued

Gene name DNA methylation Reference higher GSTP1 promoter methylation in tuberculosis patients with anti-tuberculosis He et al., 2015 drug-induced liver injury compared to tuberculosis patients without liver injury GSTM1 hypermethylation of GSTM1 associated with head and neck cancer Sharma et al., 2010 epigenetic repression due to promoter methylation in retina pigment epithelium (RPE)/ Hunter et al., 2012 choroid of patients with age-related macular degeneration DNA methylation contributes to a larger part to the variation of GSTM1 gene expression Bonder et al., 2014 than SNP genotypes GSTM2 DNA hypermethylation close to the transcription start site correlates inversely with Peng et al., 2009 reduced GSTM2 mRNA expression levels in Barrett’s adenocarcinoma GSTM5 DNA hypermethylation close to the transcription start site correlates inversely with Peng et al., 2009 reduced GSTM5 mRNA expression levels in Barrett’s adenocarcinoma inverse correlation of mRNA expression and DNA methylation level and high variability in Habano et al., 2015 methylation status in normal adult liver GSTT1 DNA methylation at a specific CpG site in the GSTT1 promoter/enhancer region is Liu et al., 2013 associated with mRNA expression with high significance in human monocytes DNA methylation contributes to a larger part to the variation of GSTT1 gene expression Bonder et al., 2014 than SNP genotypes inverse correlation of mRNA expression and DNA methylation level in normal adult liver Habano et al., 2015 NAT1 NAT1 promoter shows reduced methylation levels in breast cancer tissue, associated Kim et al., 2008 with NAT1 mRNA expression higher DNA methylation level in the NAT1 gene is related to tamoxifen-resistant breast Kim et al., 2010 cancer PON1 DNA methylation contributes to a larger part to the variation of PON1 gene expression Bonder et al., 2014 than SNP genotypes SULT1A1 SULT1A1 is silenced and methylated in breast cancer tissue Kwon et al., 2006 methylation is not associated with tamoxifen resistance Kim et al., 2010 inverse correlation of mRNA expression and DNA methylation level in normal adult liver Habano et al., 2015 UGT1A1 expression variable in primary colon tumors, expression inversely correlated to DNA Gangon et al., 2006 methylation with potential influence on irinotecan sensitivity UGT1A1 expression is repressed due to promoter methylation in colon cancer cell lines Belanger et al., 2010 UGT1A1 promoter methylation contributes to tissue-specific expression in liver and Oda et al., 2013 kidney DNA methylation contributes to a larger part to the variation of UGT1A1 gene expression Bonder et al., 2014 than SNP genotypes UGT1A10 expressed in intestine, but not in liver; promoter hypomethylated in LS180 cell line and Oda et al., 2014 intestine, but hypermethylated in HuH7 and hepatocytes; 5-aza-20-deoxycitidine treat- ment increased UGT1A10 expression only in HuH7 cells UGT2B15 DNA methylation is associated with metabolic traits in human blood Petersen et al., 2014

rate. In a cohort of adolescent and young adult patients with mRNA expression. Interestingly, CYP2E1 overexpression due to acute lymphoblastic leukemia, hypermethylation and reduced DNA hypomethylation has been shown in patients with Parkin- CYP1B1 expression in peripheral blood cells was associated with son’s disease.34 CYP2W1 is inherently expressed only in fetal worse overall survival.31 In 1992, Jones et al.32 found DNA meth- colon, but not in normal adult tissue. Re-expression of CYP2W1 ylation in the 50 flanking region of the CYP2E1 gene to be has been observed in colorectal cancer and has therefore been responsible for the lack of transcription in fetal liver, in contrast suggested as a prognostic marker for malignancy in colon can- to adult liver, where CYP2E1 is expressed and not methylated. cer.35 In addition, the cancer-specific expression can be exploited Vieira et al.33 subsequently showed that demethylation of the 50 for the treatment of colon cancer with prodrugs bioactivated by region during the neonatal period resulted in increased CYP2E1 CYP2W1 (e.g., duocarmycin) to selectively kill cancer cells.36

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Table 2 Epigenetic regulation of drug transporters by DNA methylation (for references see Supplementary Material) Gene name DNA methylation Reference ABCA1 higher DNA methylation in ABCA1 gene in individuals exposed to prenatal famine Tobi et al., 2009 larger interindividual variability of DNA methylation in the ABCA1 gene in old compared to Talens et al., 2012 young monozygotic twin pairs ABCA1 promoter methylation negatively correlates with HDL cholesterol levels and is Guay et al., 2012 associated with coronary artery disease in patients with familial hypercholesterolemia ABCA1 hypermethylated in prostate cancer associate with reduced expression and prostate Lee et al., 2013 tumor aggressiveness with potential influence for the response to treatment with LXR ABCA1 methylation and expression at maternal side of placenta is associated with maternal Houde et al., 2013 metabolic profile and at the fetal side with cord blood lipid profile with consequences for the susceptibility of newborn to obesity, dyslipidemia and cardiovascular diseases higher ABCA1 promoter DNA methylation associated with age, occurrence of coronary artery Guay et al., 2014 disease and low-density cholesterol levels; therapy with acetylsalicylic acid reduces ABCA1 promoter methylation reduced ABCA1 expression and increased promoter methylation in ovarian cancer cell lines; Chou et al., 2015 higher methylation and lower expression in ovarian cancer samples associated with inferior survival ABCB1 hypomethylation of ABCB1 is associated with resistance to treatment in various cancer cell for review see Baker and lines El-Osta, 2004 hypomethylation of the ABCB1 promoter is associated with ABCB1 expression and is Kantharidis et al., 1997 associated with treatment resistance in a T-cell leukemia cell line; association of DNA methylation with expression also seen in chronic lymphocytic leukemia samples less ABCB1 promoter methylation and increased gene expression in multi-drug resistant David et al., 2004 breast cancer cells hypomethylation of CpG island in the promoter associated with response to doxorubicin Dejeux et al., 2010 acquisition of is accompanied by temporal loss of ABCB1 promoter Reed et al., 2010 methylation and increased gene expression in breast cancer cells ABCB4 DNA hypermethylation of the ABCB4 promoter silences gene expression in human cancer cell Kiehl et al., 2014 lines and in human lung and breast cancer ABCC6 ABCC6 gene methylation included in a set of DNA methylation markers to detect bladder Yu et al., 2007 cancer ABCG1 DNA methylation is associated with metabolic traits in human blood Petersen et al., 2014 DNA methylation at CpG site within the ABCG1 gene is associated with fasting insulin levels Hidalgo et al., 2014 and HOMA-IR ABCG2 DNA methylation of the ABCG2 gene contributes to reduced expression in renal cell carcinoma To et al., 2006 cell lines topotecan treated patients show higher ABCG2 expression, regulated in parts by promoter Turner et al., 2006 demethylation ABCG2 expression correlates inversely with DNA methylation of the ABCG2 gene in small cell Nakano et al., 2008 and non-small cell lung cancer cells, ABCG2 overexpression and hypomethylation in a SN-38 treatment resistant lung cancer cell line ABCG2 promoter is demethylated, accompanied by increased gene expression in T-ALL and Bram et al., 2009 ovarian carcinoma cell lines in response to treatment with antitumor drugs SLC5A8 SLC5A8 expression is silenced by DNA methylation in human colon aberrant crypt foci and Li et al., 2003 cancers DNA methylation in the SLC5A8 promoter silences gene expression in lung cancer Park et al., 2013 SLC6A4 promoter DNA hypermethylation is associated with depression and a better response rate of Domschke et al., 2014 depressed patients treated with escitalopram SLC16A3 DNA methylation of the SLC16A3 promoter region correlates with expression and has Fisel et al., 2013 prognostic potential in patients with clear cell renal cell carcinoma Table 2 Continued on next page

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Table 2 Continued

Gene name DNA methylation Reference SLC19A1 CpG island in the SLC19A1 promoter is hypermethylated and not expressed in a Worm et al., 2001 methotrexate resistant breast cancer cell line, promoter is unmethylated in a SLC19A1 expressing and methotrexate sensitive breast cancer cell line rate of complete remission in response to methotrexate therapy is associated with SLC19A1 Ferreri et al., 2004 promoter methylation reverse correlation of SLC19A1 promoter methylation with expression in a panel of malignant Yang et al., 2008 cell lines reduced expression and hypermethylation in a methotrexate resistant lymphoblast cell line Kastrup et al., 2008 and in treatment na€ıve diffuse large B-cell lymphomas SLC22A1 SLC22A1 expression is reduced due to hypermethylation of the promoter region in Schaeffeler et al., 2011 hepatocellular carcinoma with potential consequences for cisplatin-based chemotherapy long-term cisplatin exposure leads to increased SLC22A1 promoter methylation conferring Lin et al., 2013 cisplatin resistance in human esophageal cancer cell lines SLC22A2 SLC22A2 promoter DNA methylation regulates kidney-specific expression Aoki et al., 2008 SLC22A3 DNA methylation of the SLC22A3 promoter region regulates gene expression in prostate and Chen et al., 2013 colon cancer cell lines; low expression accompanied by high promoter methylation levels in prostate cancer SLC22A8 DNA methylation contributes to the regulation of SLC22A8 gene expression in Caco2 and Kikuchi et al., 2006 HEK293 cells SLC26A4 methylation of the SLC26A4 gene is suggested to be an early event in the tumorigenesis of Xing et al., 2003 thyroid cancer SLCO1B3 5-aza-20-deoxycytidine treatment increased SLCO1B3 mRNA expression in HepG2 and Caco-2 Ichihara et al., 2010 cells SLC22A18 increased promoter methylation contributes to downregulation of SLC22A18 expression in Chu et al., 2011 gliomas; expression is increased upon 5-aza-20-deoxycytidine treatment in glioma cell lines SLC22A18 protein expression is a better predictive marker than promoter DNA methylation in Chu et al., 2013 glioblastoma cells receiving temozolomide therapy

Interestingly, Gomez et al.37 showed that DNA methylation in rant UGT1A1 expression, influenced by altered DNA the CpG-rich first exon/intron junction was involved in the regu- methylation, contributes to differential sensitivity to irinotecan lation of gene expression in colon tumor tissue. Only recently, treatment. In a study by Oda et al.,41 methylation of the developmental stage-specific CYP2W1 expression in human fetal UGT1A1 promoter was identified to contribute to the lack of colon was also found to be governed by methylation in this gene UGT1A1 expression in human kidney. The N-acetyltransferase region.38 Glutathione peroxidase 3 (GPx3) functions to protect 1 (NAT1), a phase II enzyme, is involved in the detoxification cells from oxidative damage by catalyzing the reduction of reac- (N-acetylation) or bioactivation (O-acetylation) of arylamine car- tive oxygen species (ROS). GPx3 promoter hypermethylation is a cinogens. NAT1 has been shown to be overexpressed in ER1 major contributor to suppressed GPx3 expression in multiple breast cancer and to be associated with tamoxifen responsive- human cancer tissues, such as breast, prostate, renal, cervical, and ness.42 According to Kim et al.,43 reduced promoter DNA meth- gastric cancer or acute myeloid leukemia (see Table 1). Accord- ylation of NAT1 leads to the increased expression in breast ing to a study by Chen et al.,39 GPx3 hypermethylation is tightly cancer tissue.44 In a subsequent study, they compared tamoxifen- related with cisplatin resistance and poor survival in patients with resistant and nonresistant breast cancer patients and suggested head and neck cancer. that higher NAT1 methylation was associated with nonresponse. The enzyme UGT1A1, involved in phase II , In addition to candidate gene studies, recently a genome-wide catalyzes the inactivation of SN-38, the active metabolite of analysis has been performed on evaluating the biological signifi- irinotecan, a drug used to treat metastatic colon cancer. cance of DNA methylation of DME genes on their mRNA UGT1A1 is variably expressed in colon cancer. In a study by expression in adult human liver tissues by combining these results Gagnon et al.,40 most colon cancer tissues showed only low with corresponding data from the Gene Expression Omnibus UGT1A1 expression; however, some exhibited high expression (GEO) database of the National Center for Biotechnology Infor- levels. They could prove that in colon cancer cell lines UGT1A1 mation (NCBI). Of note, 37 DME genes revealed highly variable expression was tightly associated with promoter DNA methyla- DNA methylation, of which DNA methylation was inversely tion levels. From these results, the authors hypothesize that aber- correlated with mRNA expression for seven DME genes

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Table 3 Epigenetic regulation of nuclear receptors by DNA methylation (for references see Supplementary Material) Gene name DNA methylation Reference HNF1A 5-aza-20-deoxycytidine treatment restores HNF1A expression in colon cancer cell lines Belanger et al., 2010 HNF1A gene methylation silences its expression and is associated with the composition of Zoldos et al., 2012 glycans on plasma glycoproteins PPARG hypomethylation of PPARG promoter is associated with gastric cancer Hong et al., 2010 PPARG promoter hypermethylation leads to reduced expression and is associated with Pancione et al., 2010 advanced tumors and poor outcome in patients with colorectal cancer PPARG promoter DNA methylation leads to reduced mRNA expression in patients with chronic Zhao et al., 2013 hepatitis B and is associated with liver inflammation and fibrosis PXR hypermethylation of PXR and reduced gene expression in aggressive neuroblastomas Misawa 2005 low expression of PXR in colon cancer cells by promoter methylation in the PXR gene, linked to Habano et al., 2011 reduced transactivation of the CYP3A4 gene, hypomethylation of the PXR promoter accompanied by increased gene expression in colorectal cancer tissue RARB RARB expression silenced by DNA methylation in breast cancer Widschwendter et al., 2000 aberrant RARB methylation found in head and neck cancer Youssef et al., 2004 RXRA higher methylation level at specific CpG site in the RXRA gene in umbilical cord tissue is Godfrey et al., 2011 associated with maternal pregnancy diet and childhood adiposity RXRG downregulation of RXRG gene expression by DNA methylation may play different roles in Lee 2010 NSCLCs depending on smoking status of patients SOD2 SOD2 expression is repressed by DNA hypermethylation in the SOD2 gene promoter in breast Hitchler et al., 2006 cancer cell lines, expression is restored by 5-aza-20-deoxycytidine treatment SOD2 expression silenced by SOD2 gene DNA methylation in pancreatic cancer cell lines Hurt et al., 2007 downregulation of SOD2 gene expression in human cartilage with osteoarthritis accompanied Scott et al., 2010 by increased SOD2 promoter methylation SOD3 hypermethylation of the SOD3 promoter contributes to loss of SOD3 expression in lung cancer Teoh-Fitzgerald et al., 2012 decreased SOD3 expression due to promoter hypermethylation in mammary Teoh-Fitzgerald adenocarcinomas et al., 2014 SOD3 gene methylation associated with adiposity Agha et al., 2015 VDR VDR expression is silenced in due to VDR promoter methylation leading to calcitriol Marik et al., 2010 insensitivity in breast cancer DNA hypermethylation in the VDR promoter in reduced VDR expression in HIV infected T-cells Chandel et al., 2013

(CYP1A2, CYP2C19, CYP2D6, GSTA4, GSTM5, GSTT1, and Interestingly, Petersen et al.46 recently suggested that DNA SULT1A1). Moreover, the authors provide evidence that DNA methylation affects human metabolism. They performed an methylation status around the first exon is responsible for tissue- epigenome-wide association study (EWAS) between DNA meth- specific (e.g., hepatic vs. intestinal tissue) and age-dependent ylation and metabolic traits in human blood and found signifi- expression of UGT1A isoforms by guiding correct alternative cant associations also for CpG loci associated with the ADME splicing of the UGT1A gene.45 genes UGT2B15 and ABCG1. However, in general the effects of Very recently, the contribution of genetic variation and epige- DNA methylation on the metabolic traits were not as strong as netic factors to the regulation of gene expression including the effects observed for genotypes. Direct monitoring of DNA ADME genes in human liver was investigated by a genome-wide methylation-based biomarkers in body fluids, such as plasma or approach. Interestingly, a larger effect of methylation level at sin- serum, as currently suggested for cancer or disease biomarkers, gle CpG sites on hepatic expression was found for five ADME would presumably allow better prediction and could facilitate genes (GSTT1, GSTM1, UGT1A1, GST01, and PON1) whose clinical implementation.47 expression levels are altered additionally by gene-specific variants. This study provides first evidence that a combination of genetic Transporters and epigenetic regulation by DNA methylation may better pre- Epigenetic regulation of transporter gene expression by DNA dict interindividual variability of gene expression than either of methylation has been shown for members of the ABC as well as the two alone.15 the SLC transporter family (Table 2). It is well known that

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Figure 2 Expression and DNA methylation of OCT1/SLC22A1, OCT2/SLC22A2 and OCT3/SLC22A3 in liver and kidney. Immunofluorescent staining of OCT1, OCT2, and OCT3 and DNA methylation profiles of the regulatory 50UTR regions of SLC22A1, SLC22A2, and SLC22A3, investigated by MALDI-TOF mass spectrometry, in human liver (n 5 100) and kidney (n 5 18) tissue, respectively (see refs. 55, 57, 75, unpublished data). Median methylation lev- els at investigated CpG sites are represented by diamonds; 25%/75%-quantiles are depicted as shaded areas. OCT1 is expressed in human liver, but not in human kidney. Accordingly, DNA methylation in the 50UTR of the SLC22A1 gene is low in liver (blue) and high in kidney tissues (yellow). In contrast, OCT2 is a kidney-specific transporter, showing low methylation levels in the 50 UTR of the SLC22A2 gene in kidney (yellow), but high methylation levels in liver (blue), where OCT2 is not expressed. OCT3 is expressed in liver as well as in kidney. The 50UTR region of the SLC22A3 gene does not show differen- ces in methylation profiles between the tissues. increased expression of the ABCB1 gene, encoding for the multi- ABCG2 expression upon exposure to chemotherapeutic drugs drug resistance protein MDR1 (P-glycoprotein), may lead to accompanied by demethylation of the ABCG2 promoter, result- acquired cancer treatment resistance. Besides other mechanisms ing in a significant increase in drug resistance compared to (e.g., transcriptional induction by transcription factors such as untreated cells. HIF1a48), DNA methylation in the ABCB1 gene promoter Aberrant drug uptake mediated by members of the SLC trans- contributes to MDR1 overexpression in vitro (cancer cell lines). porter family can also mediate drug resistance or toxicity. The In vivo the expression of MDR1 in cancer tissues of patients diag- SLC19A1 gene encodes for a carrier mediating the uptake of nosed with acute myeloid leukemia, acute lymphocytic leukemia, reduced folate and antifolate drugs such as methotrexate. Hyper- chronic lymphocytic leukemia, colorectal cancer, and bladder methylation of the SLC19A1 promoter is associated with cancer was linked to promoter DNA methylation.49 In addition, reduced expression in breast cancer cell lines52 and has been asso- hypomethylation of the ABCB1 promoter was associated with ciated with a lower response rate to methotrexate treatment in poor drug response and, together with hypomethylation of the patients with CNS lymphomas.53 Furthermore, members of the GSTP1 promoter, related to inferior survival in vivo in breast SLC22A gene family have been shown to be regulated by DNA cancer patients treated with doxorubicin.50 For the breast cancer methylation. The organic cation transporters (OCT) 1, 2, and 3, resistance protein (BCRP), mediating efflux of chemotherapeutic encoded by the genes SLC22A1, SLC22A2, and SLC22A3 drugs, such as methotrexate, doxorubicin, or SN-38 (the active respectively, mediate the uptake of several clinically relevant drugs metabolite of irinotecan), drug resistance is linked to the DNA including cancer agents (e.g., cisplatin or oxaliplatin).54 OCT1 is methylation status in the ABCG2 promoter, which results in primarily expressed in the liver,55 but not in kidney, where the overexpression of this transporter protein. As shown by Bram 50UTR of the SLC22A1 gene is highly methylated compared to et al.,51 leukemic and ovarian cancer cell lines upregulated liver tissue (Figure 2).56,57 In hepatocellular carcinoma, OCT1

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expression is downregulated due to DNA methylation, indicating PPARg agonists are supposed to potentiate the efficacy for a impaired uptake and subsequent lower intracellular concentra- number of chemotherapeutics, these authors hypothesize that a tion of cancer agents, resulting in potential nonresponse.57 Lin rational combination of chemotherapeutics with a PPARg ago- et al.58 showed that long-term treatment with cisplatin leads to nist and an epigenetic drug reactivating PPARg expression in hypermethylation of the SLC22A1 gene and in consequence to patients with silenced PPARg would sensitize tumors to anti- cisplatin resistance in human esophageal cancer cells. The expres- cancer treatment. The vitamin D receptor (VDR/NR1I1) plays sion of OCT2 is kidney-specific. According to this tissue-specific an important role in mediating the response to calcitriol, which expression, DNA methylation levels in the SLC22A2 promoter is the active metabolite of vitamin D. Calcitriol has been ascribed region is low in kidney tissue as compared to human liver to mediate antiproliferative effects on tumor cells; however, can- missing OCT2 expression due to hypermethylation of SLC22A2 cer tissue and cell lines often show insensitivity to calcitriol treat- (Figure 2).56,57 OCT3 is expressed in liver as well as in kidney ment.64 The expression of the VDR receptor is influenced by and does not show different DNA methylation patterns in DNA methylation in colon and endometrial cancers. Interest- these organs (Figure 2). However, aberrant DNA methylation of ingly, Marik et al.64 show that combined treatment of breast can- the SLC22A3 promoter has been identified as a regulatory factor cer cell lines with the demethylating agent 50aza-20-deoxycytidine in prostate cancer, corroborating the strong tissue-specific nature and calcitriol enhanced growth inhibitory effects accompanied by of epigenetic regulation.59 The monocarboxylate transporter induction of VDR mRNA expression. Indeed, the NR1I1 pro- 4 (MCT4), encoded by the gene SLC16A3, is an important lac- moter was hypermethylated in breast cancer cell lines as well as tate transporter responsible for the extrusion of lactate in highly primary breast cancer tissue, resulting in reduced VDR expression glycolytic cells, such as tumor cells. Independent from unspecific and presumably to insensitivity to calcitriol treatment. MCT4 inhibitors (e.g., statins), specific MCT4 inhibitors as promising cancer agents are currently under investigation. Interest- CONSEQUENCES FOR DRUG THERAPY ingly, DNA methylation at a specific CpG site in the 50 regulatory Whereas the contribution of DNA methylation on interindivid- region of the SLC16A3 promoter has been shown to correlate ual variability of expression of a number of ADME genes related with expression of this lactate transporter and is strongly associated to drug metabolism and transport has repeatedly been shown, the with survival of patients with clear cell renal cell carcinoma.60,61 clinical relevance of DNA methylation to explain drug nonres- ponse and drug-induced toxicity needs further intensive evalua- Nuclear receptors tion. DNA methylation of specific ADME genes (e.g., GSTP1, Nuclear receptors are a family of binding transcription GPx3,orSLC16A3) is currently proposed as biomarker for the factors acting to induce expression of genes involved in various diagnosis and prognosis of various cancers and has been associ- physiological processes in response to endogenous or exogenous ated with acquired drug resistance (Tables 1–3). A major draw- stimuli, including the expression of ADME genes in response to back of findings from in vitro studies with cultivated cancer cell xenobiotics. Nuclear receptors not only contribute to epigenetic lines generally used to investigate associations between ADME regulation of gene expression by recruiting coactivators and core- gene expression and DNA methylation is that extrapolation to pressors mediating epigenetic modifications in target genes, but the in vivo situation is limited, since expression and DNA meth- are also regulated epigenetically themselves. It can be assumed ylation profiles do not necessarily resemble the profiles found in that aberrant expression of nuclear receptors due to changes in vivo. It is well known that gene expression and DNA methylation DNA methylation profiles results in aberrant activation of profiles of a number of genes involved in drug metabolism and responsive ADME genes, and hence drug response. A major transport are considerably different between cell lines and pri- xenobiotic sensor and regulator of several ADME genes, includ- mary tissues.65 Much less information is available with respect to ing CYP3A4, UGT1A1 or ABCB1, is the pregnane X receptor the impact of DNA methylation in ADME genes and conse- (PXR/NR1I2). A comparison of PXR low- and PXR high- quences for the treatment of diseases other than cancer. One expression colon cancer cell lines showed that decreased expres- explanation might be that investigations on DNA methylation sion of PXR is linked to promoter methylation in the NR1I2 are restricted methodologically by the availability of tissue- gene and reduced transactivation of the CYP3A4 gene. In colo- specific DNA, which is readily available from tumor biopsies or rectal cancer tissues DNA methylation alters PXR expression as resection material, but rarely from nontumor tissue. Nevertheless, well, in that PXR expression is upregulated due to NR1I2 pro- regarding psychiatric disorders, increased promoter DNA methyl- moter hypomethylation.23 Misawa et al.62 found a link between ation of the serotonin transporter SLC6A4 in peripheral blood NR1I2 DNA methylation in a CpG island in close proximity to leukocytes has recently been associated with an increased risk for exon3 and reduced NR1I2 gene expression in advanced neuro- depression.66 Interestingly, decreased promoter methylation levels blastoma tumors, suggesting PXR to target genes with growth in blood of depressed patients were associated with a lower inhibitory functions. The effect of lower PXR levels due to response rate to antidepressant treatment using escitalopram.67 hypermethylation on target gene expression like CYP3A4, how- In addition, DNA methylation of a CpG site in the ABCG1 ever, was not analyzed in that study. Regarding the peroxisome gene was associated with fasting insulin levels and homeostasis proliferator-activated receptor g (PPARg), hypermethylation in model assessment of insulin resistance (HOMA-IR) in an EWAS colorectal cancer tissue is accompanied by reduced gene expres- with metabolically healthy volunteers. This indicates that DNA sion, advanced tumor stage, and poor patient survival.63 Since methylation at this CpG site might represent a marker for risk of

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metabolic disease or insulin resistance, with potential consequen- ces for drug therapy.68 However, a major limitation of these stud- ies so far is the rather small sample size and limited phenotypic characterization of study populations. The continuous development of high-throughput technologies to systematically analyze DNA methylation profiles on a genome- wide level, i.e., EWAS, and their use in large-scale projects, such as the International Human Epigenome Consortium or the NIH Roadmap Epigenomics Mapping Consortium, will enable a more comprehensive understanding of the epigenetic component of ADME gene regulation, and will provide additional information for personalized drug treatment in the future. Moreover, the advancement of technologies to reliably and efficiently quantify 5-mC specifically in selected ADME genes, such as the method recently developed by Ivanov et al.,69 which uses next generation sequencing of bisulfite-converted DNA specifically enriched for coding and regulatory regions of 174 ADME genes, will also facil- itate high-throughput analysis of DNA methylation with respect to drug response. Most important, the function and relevance of DNA hydroxy- methylation needs to be further evaluated to elucidate its role within the epigenetic machinery. Thus, the consideration of novel high-throughput technologies for quantification of not only 5-mC but also 5-hmc and further intermediates (5-fC and 5-caC) opens a new avenue in the understanding of epigenetic control of ADME genes. The dynamic nature of DNA methylation, being influenced, e.g., by age, sex, environmental, and lifestyle factors, represents a major challenge for the integration of the knowledge into clinical practice. In contrast to genetic variations, DNA methylation, as epigenetic modification, is dynamic and reversible. Use of DNA Figure 3 Schematic overview of methods to interfere with aberrant DNA methylation. (a) Treatment with either 5-azacytidine or 5-aza-20- methylation profiles as contributors to interindividual variability deoxyazacytidine inhibits DNMT, eventually leading to global DNA hypome- of drug response would require repeated investigation of bio- thylation. (b) Targeted epigenetic editing at ADME gene promoters can be marker gene DNA methylation profiles depending on patient achieved by use of zinc finger proteins, TALEs, or the CRISPR/Cas9 sys- age, treated target organ, or during long-term treatment. More- tem, specifically targeting DNA sequences of genes of interest, coupled to over, not only interindividual variability of DNA methylation of effector proteins, i.e., DNMTs or TETs. ADME genes, but also of drug target genes may account for 0 altered response to drug treatment. For instance, hypermethyl- 5-azacytidine and 5-aza-2 -deoxycytidine rely on transporter- ation of the promoter of the epidermal growth factor receptor mediated uptake into the cells to be incorporated into the (EGFR) gene, for example, is associated with resistance to the DNA, via, e.g., the human concentrative nucleoside transporter 1 71 EGFR-tyrosine kinase inhibitor gefitinib in nonsmall-cell lung (hCNT1). Advancements in technologies to epigenetically edit cancer cells.70 The concept of systems pharmacology1 and the selected target DNA sequences holds much promise to overcome integration of epigenomic information not only on ADME genes the problem of global off-target effects. Different targeted but also other pharmacogenes will provide promising progress to approaches to epigenetically modify expression of specific genes better understand drug resistance and nonresponse. have been established as powerful tools in biological research. The On the other hand, the plasticity of DNA methylation pro- rationale of these approaches is to fuse a catalytically active effec- vides an attractive opportunity for therapeutic intervention with tor domain, e.g., DNA methylating (DNMTs) or demethylating so-called epidrugs. DNMT inhibitors, such as the nucleoside ana- (TETs) enzymes, to a DNA-binding domain selectively targeting logs 5-azacytidine and 5-aza-20-deoxycytidine, are approved by the the effector to the target gene sequence. The currently most com- US Food and Drug Administration (FDA) for the treatment of monly applied tools differ in the way of mediating target myelodysplastic syndrome and for elderly patients with acute sequence recognition, which can be achieved via protein/DNA- myeloid leukemia. Just as shown for tumor suppressor genes, based interaction (zinc finger proteins or transcription activator ADME-related gene expression could be increased by drugs inhib- like effectors [TALEs]) or RNA/DNA-based interaction (clus- iting DNA methylation. However, treatment with DNMT inhib- tered regularly interspaced short palindromic repeats/CRISPR- itors is unspecific and causes genome-wide DNA demethylation, associated 9 system [CRISPR/Cas9 system]) (Figure 3). Zinc with potentially serious side effects (Figure 3). Furthermore, both finger-DNA binding domains coupled to nucleases have already

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been shown to be useful for therapeutic approaches and are cur- ACKNOWLEDGMENTS rently in clinical trials for the treatment of HIV. TALE nucleases The first two authors contributed equally to this review. This work was supported by the Robert Bosch Foundation (Stuttgart, Germany), the and the CRISPR/Cas9 system have proven their usefulness as 72 Federal Ministry for Education and Research (BMBF, Berlin, Germany, well for clinical applications in vivo in animal models. Refine- FKZ 031L0037 and 01EK1509A), the Horizon 2020-PHC-2015 grant ment of these tools with respect to their use for targeted modifi- U-PGx 668353, the German Cancer Consortium (DKTK) and German cation of DNA methylation will not only push forward this area Cancer Research Center (DKFZ, Heidelberg, Germany), and ICEPHA of research, but will also further their application for therapeutic Graduate School Tuebingen-Stuttgart. The authors thank Bernd Borstel purposes including the alteration of responsiveness to drug for drafting the figures and Prof. Anne T. Nies for providing figure material. treatment. Although this review focuses specifically on the influence of DNA methylation on ADME genes, the contribution of his- CONFLICT OF INTEREST tone modifications and miRNAs to interindividual variability of The authors have no conflicts of interest to disclose. ADME gene expression needs to be considered carefully. Both histone modifications and miRNAs significantly affect ADME VC 2016 American Society for Clinical Pharmacology and Therapeutics gene expression, with a putative role for variability of drug response.73 There is evidence that all three epigenetic mecha- 1. Meyer, U.A., Zanger, U.M. & Schwab, M. Omics and drug response. Annu. Rev. Pharmacol. Toxicol. 53, 475–502 (2013). nisms interact with each other by a highly complex network of 2. Ehmann, F. et al. 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