Review Targeting DNA methylation for epigenetic therapy

Xiaojing Yang1, Fides Lay1,2, Han Han1,3 and Peter A. Jones1

1 Department of Urology, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA 2 Program in Genetics, Molecular and Cellular Biology, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA 3 Department of Pharmacology and Pharmaceutical Sciences, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

Patterns of DNA methylation are established during and also CpHpG nucleotide sequences in embryonic stem embryonic development and faithfully copied through cells (ES) [2]. In somatic cells, most CpG dinucleotides are somatic cell divisions. Based on current understanding methylated except those located in CpG islands [3],which of DNA methylation and other interrelated epigenetic are defined as regions of DNA >500 bp with a GC content modifications, a comprehensive view of the ‘epigenetic 55%, and a ratio of observed and expected CpG >0.65 [4]. landscape’ and cancer epigenome is evolving. The can- Nearly 60% of mammalian gene promoters are located in cer methylome is highly disrupted, making DNA meth- CpG islands [5].DNAmethylationoftheseCpG-rich ylation an excellent target for anticancer therapies. promoters silences gene expression by changing the ac- During the last few decades, an increasing number of cessibility of DNA to transcription factors (TFs) or by drugs targeting DNA methylation have been developed recruiting additional silencing-associated proteins [6]. to increase efficacy and stability and to decrease toxicity. Nevertheless, there is also growing evidence suggesting The earliest and the most successful epigenetic drug to that even in CpG-poor regions such as the Oct4 promoter date, 5-Azacytidine, is currently recommended as the DNA methylation still has a role in gene regulation [7,8]. first-line treatment of high-risk myelodysplastic syn- Patterns of DNA methylation are established and main- dromes (MDS). Encouraging results from clinical trials tained by DNA methyltransferases (DNMTs). During early have prompted further efforts to elucidate epigenetic embryogenesis, de novo DNA methylation is mediated by alterations in cancer, and to subsequently develop new DNMT3A and DNMT3B associated with DNMT3L, which epigenetic therapies. This review delineates the latest lacks a methyltransferase domain [9–11]. To maintain pat- cancer epigenetic models, the recent discovery of hypo- terns of DNA methylation in daughter cells, ubiquitin-like, methylation agents as well as their application in the containing PHD and RING finger domains 1 (UHRF1) clinic. recognizes hemi-methylated DNA and directs DNMT1 to methylate the appropriate cytosine in newly synthesized The epigenetic landscape DNA strands during successive replications [12,13]. Recent Primary heritable genetic information is dictated by the studies have proposed an updated model, suggesting that DNA sequence packed into chromatin. The basic repeating DNMT3A/B are also required for the maintenance of pat- unit of chromatin is a nuclesome consisting of 146–147 terns of DNA methylation in somatic cells, particularly of base pairs (bp) of DNA wrapped around a histone octamer. repeat regions and imprinted genes (Figure 1a) [6,14]. DNA and histone proteins can undergo various post- In contrast to DNA methylation, the patterns of histone synthetic modifications. These modifications, along with modifications are more labile. The N-termini of histones histone variants and nucleosome remodelers, can strongly can undergo various post-synthetic modifications, includ- influence chromatin structure and transcriptional regula- ing a diverse combination of methylation, acetylation, tion without changing the underlying DNA sequence, a ubiquitylation, phosphorylation and sumoylation. In re- process called ‘epigenetic regulation’. Epigenetic regula- cent years, the development of modification-specific anti- tion can be separated into three inter-related layers: bodies in chromatin immunoprecipitation (ChIP), coupled nucleosome positioning, histone modifications, and DNA with microarray (ChIP-chip) or direct sequencing technol- methylation [1]. ogy (ChIP-seq), has revolutionized assessment of the global DNA methylation is probably the most extensively distribution of histone modifications and their roles in studied epigenetic mark. It plays an important part in gene regulation [15–17]. For instance, H3K4me3 has been genomic imprinting, in X-chromosome inactivation, and in found to occur near the transcription start sites (TSS) of the silencing of retrotransposon, repetitive elements and genes or miRNA, and is positively correlated with tran- tissue-specific genes. In mammalian cells, a methyl group scription level [18–21]. Similarly, H3K9/14 acetylation is is covalently added to cytosine in the context of cytosine- highly associated with gene transcriptional activation phosphate-guanine (CpG) dinucleotides in somatic cells [22]. By contrast, H3K9me and H3K27me are enriched in the vicinity of suppressed or silenced gene promoters Corresponding author: Jones, P.A. ([email protected]). (Figure 1a) [15]. The enzymes that catalyze these

536 0165-6147/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tips.2010.08.001 Trends in Pharmacological Sciences, November 2010, Vol. 31, No. 11 [(Figure_1)TD$IG]Review Trends in Pharmacological Sciences Vol.31 No.11

Figure 1. Epigenetic regulation in normal and cancer cells. Shown are schematic promoters. Arrows represent transcription start sites (TSS); filled circles represent methylated CpG dinucleotide, and empty circles represent unmethylated CpG dinucleotide. In normal cells (a), genes such as MLH1, CDKN2A are, in general, unmethylated and packaged with active modified histone proteins containing active marks (e.g. H3K4me3) as well as histone variants (e.g. H2A.Z). These epigenetic modifications constitute an ‘open’ chromatin structure which, with a nucleosome-depleted region (NDR), favor transcription. In other genomic regions, such as in the repetitive elements, the CpG sites are methylated and thereby maintain a closed chromatin structure. In cancer cells (b), epigenetic modifications are disrupted. Besides cancer-specific hypomethylation (e.g. in repetitive sequences), there are two inter-related epigenetic mechanisms to repress gene expression. Some genes (e.g. FBXO32) could be recognized by polycomb proteins such as EZH2 which catalyses H3K27 methylation, and are consequently repressed. By contrast, CpG sites within gene promoters could undergo de novo methylation by DNMT3A/B, which are compartmentalized to these regions to complete the methylation process. The methylated CpG sites attract methyl-binding proteins such as MBD, which is coupled with HDAC proteins to remove histone acetylation as well as histone methyltransferase to methylate H3K9. Associated with all the repressive factors shown, nucleosomes cover the promoter region, generating a tightly closed chromatin status to shut down gene expression. modifications and the crosstalk between these complexes cing the suppressive nature of promoters (Figure 1) [36– have been summarized in several important review arti- 37]. The interplay between DNA methylation and histone cles [23–26]. modifications can be further observed in ES cells. Some Other crucial components of the epigenetic landscape studies have proposed that histone modifications and as- are histone variants which partially determine nucleosome sociated proteins (e.g., EZH2 or G9a) take part in establish- stability and ultimately affect gene expression. H2A.Z is ing de novo patterns of DNA methylation during early commonly localized in promoter regions. The differences in development [38–41]. the amino-acid sequence of H2A.Z compared with H2A might affect the interactions of the protein with the H3/ Cancer epigenetics H4 tetramer, thereby altering nucleosome stability [27– It has been documented that epigenetic alterations are 28]. In mammalian cells, the role of H2A.Z in gene regula- involved in the initiation and progression of cancer in tion is incompletely understood, but, there is increasing addition to abnormal genetic events. Early studies that evidence suggesting that H2A.Z assists in the transcrip- measured the global content of 5-methylcytosine of tumors tion initiation of a special group of genes such as endoplas- showed that hypomethylation was a common feature of mic reticulum (ER) target genes [29–32]. carcinogenesis, leading to abnormal chromosomal instabil- The interplay between histone modifications, histone ity and transcriptional regulation [42–44]. However, most variants, and DNA methylation constitutes a global regu- cancer epigenetic studies subsequently concentrated on lation network. Extensive studies have elucidated the focal CpG island hypermethylation in cancer, and revealed biological and functional interactions among these three many tumor suppressor genes, cellular functional genes, epigenetic layers. For example, DNA methylation anti- and miRNAs silenced by promoter DNA methylation [45– correlates with the active mark H3K4me3, H3ac or 48]. Recent genome-wide studies have demonstrated dis- H2A.Z [33–35]. Meanwhile, highly methylated promoter tinct patterns of DNA methylation in cancerous tissues regions are bound by methylcytosine binding proteins such compared with their normal counterparts [49–52]. as MeCP2, which subsequently recruits histone deacety- The detailed mechanisms by which these discrete lase (HDAC) to deacetylate histones, thus further reinfor- regions undergo hyper- or hypomethylation are unclear.

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Early evidence suggested that elevated DNA methyltrans- interaction between DNA and DNMTs through the nitro- ferase levels might trigger hypermethylation of tumor sup- gen in the 5 position of the modified pyrimidine, and traps pressor gene promoters, which would consequentially result DNMTs for proteosomal degradation [74–75] (Figure 3b). in proliferation of cancer cells [53]. In addition to this The depletion of DNMTs results in the passive loss of ‘selection’ model, an alternative mechanism has been pro- cytosine methylation in the daughter cells after replica- posed that takes advantage of the current genome-wide tion. This is associated with reduced H3K9me3, increased epigenetic studies in stem cells. Investigators have sug- H3ac and H3K4me3 modifications around gene promoter gested that the establishment of aberrant epigenetic profiles regions, as well as the formation of a nucleosome-deficient in cancer undergoes a process similar to epigenetic repro- region [66,76]. A recent genome-wide study of alterations gramming during development [54]. During cancer initia- in the epigenetic landscape after 5-Aza-CR treatment fur- tion, the promoters of genes (which are repressed by histone ther validated this concept [77]. The demethylation func- H3K27me3 in normal differentiated cells) might become tion of 5-Aza-CR and 5-Aza-CdR is most evident at low methylated and thereby set up for long-term silencing. This drug concentration because the drugs exhibit greater cy- ‘epigenetic switch’ could be regulated by the cooperation of totoxicity, interfere with DNA synthesis, and cause DNA polycomb proteins and DNMTs [55–57] (Figure 1b). damage at higher concentrations [78]. The S-phase is In cooperation with DNA methylation, other epigenetic required for the selective and effective incorporation of mechanisms also exhibit abnormal regulation in cancer. these two drugs into the DNA of rapidly proliferating cells, For example, histone deacetylases (HDACs) are often thereby limiting unwanted hypomethylation in cells un- found to be overexpressed in various types of cancer, dergoing the normal cell cycle. resulting in histone deacetylation around the TSS region Besides inhibiting DNMTs, 5-Aza-CR incorporates into and the formation of a more compact structure to silence RNA and interrupts normal cellular processes by inducing genes [58–59]. In addition, H3K4me is also selectively ribosomal disassembly and preventing the translation of demethylated by the histone lysine demethylase (LSD1), oncogenic proteins [73,79]. The ability of 5-Aza-CR to be which is upregulated in cancer, making it a potential drug incorporated into DNA and RNA increases its side effects target [60–61]. In some loci, polycomb-group (PcG) proteins in vitro and in vivo because it can function in resting and associated with H3K27me work independently of DNA dividing cells [80]. However, 5-Aza-CR and 5-Aza-CdR are methylation to aberrantly repress genes in cancer cells readily hydrolyzed in aqueous solution and subject to [62–65]. Nucleosome occupancy is also switched from an deamination by cytidine deaminase. The instabilities of ‘open’ to a ‘covered’ status in gene regulation elements in these compounds inevitably present a challenge to their neoplastic cells [66–67] (Figure 1b). clinical applications. To improve the stability and efficacy of 5-Azanucleo- In vitro study of DNMT inhibitors (DNMTi) sides, several other cytidine analogs have been developed Epigenetic modifications play a crucial part in regulating (Figure 2a). For example, zebularine (a cytidine analog normal cells, but these processes are disrupted during that lacks an amino group in the 4 position of the pyrimi- tumorigenesis. The relatively reversible character of epi- dine ring) can inhibit DNMTs and cytidine deaminase after genetic alterations (in contrast to genetic changes) has oral administration [81–83]. Studies have shown that inspired the development of therapeutic strategies target- zebularine induces hypomethylation in breast cancer cell ing various epigenetic components. Among them, DNA lines and reactivates silenced tumor suppressor genes [84– methylation and its associated enzymes have been well- 85]. The inefficient metabolic activation of this compound studied. The understanding of their fundamental mecha- has, however, delayed its clinical use as a single agent. As nism of action and correlation with other epigenetic mod- an inhibitor of cytidine deaminase, its co-administration ifications makes them attractive drug targets. has increased the efficacy of 5-Aza-CdR [82]. The cytidine analog 5-Fluoro-20-deoxycytidine (5-F-CdR) had also been Nucleoside analogs demonstrated to have hypomethylating activity in mouse 5-Azacytidine (5-Aza-CR) and 5-aza-20-deoxycitidine (5- cells as well as in human breast and lung carcinoma cells Aza-CdR) are the two most potent DNMTi. They have [68,86]. Clinical studies further showed that co-adminis- been approved by the Food and Drug Administration tration of 5-F-CdR with the cytidine deaminase inhibitor (FDA) in the USA for the treatment of myeloid malignan- tetrahydrouridine (THU) improved the stability of 5-F- cies (Figure 2a). They were first synthesized as cytotoxic CdR [87]. The therapeutic potential of another stable agents. In the 1980s, these compounds were found to have analog, dihydro-5-azacytidine (DHAC), was also assessed hypomethylating activity after incorporation into the DNA for the treatment of malignant mesothelioma, but results of actively replicating tumor cells [68–70]. on this compound’s clinical efficacy have been inconsistent Upon transport into cells by the human concentrative [88]. nucleoside transporter 1 (hCNT1), 5-Aza-CR and 5-Aza- The effort to improve the stability of DNMTi includes CdR are phosphorylated by different kinases, converting the development of prodrugs of the nucleoside analogs. A them to their active triphosphate forms, 5-Aza-CTP and 5- preclinical study showed that NPEOC-DAC (a prodrug of Aza-dCTP, respectively [71–72] (Figure 3a). 5-Aza-CR can 5-Aza-CdR containing a 2-( p-nitrophenyl)ethoxycarbonyl be incorporated into RNA and DNA after the reduction of 5- (NPEOC) group at the 4 position of the pyrimidine ring) Aza-CDP by ribonucleotide reductase, whereas 5-Aza-CdR can be incorporated into DNA and inhibit DNMTs after its incorporates into DNA after its phosphorylation to 5-Aza- activation by human carboxylesterase 1 in a liver cancer dCTP [73]. The incorporated 5-azanucleoside disrupts the cell line. The NPEOC moiety protects 5-Aza-CdR from

538 [(Figure_2)TD$IG]Review Trends in Pharmacological Sciences Vol.31 No.11

Figure 2. Chemical structure of DNMTi:

A. Nucleoside analogs B. Non-nucleoside analogs. deamination, but the compound itself is less potent if Non-nucleoside analogs administered at the same concentration as 5-Aza-CdR. Unlike cytidine analogs, non-nucleoside DNMTis (Figure Moreover, the activity of NPEOC-DAC is dependent upon 2b) do not require incorporation into DNA, and thus might carboxylesterases, which are not expressed in all tissues. exhibit less cytotoxicity. Some of the compounds assessed Further studies are required to explore the use of this for their potential to induce hypomethylation in solid compound in combination therapy [89]. Alternatively, tumors are hydralazine and procainamide, the widely used S110 (a dinucleotide containing the 5-azacytosine ring) vasodilator and antiarrhythmic agents, respectively [91]. has also been shown to improve the efficacy of 5-Aza- Hydralazine has been reported to block the activity of CdR by protecting it from deamination. The compound DNMTs by the interaction of its nitrogen atoms with the is well tolerated and can reduce the level of DNA methyla- Lys-162 and Arg-240 residues of the enzyme, whereas tion in the CDKN2A promoter region in xenografts [90]. procainamide acts similarly as a competitive inhibitor by

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Figure 3. Metabolic pathway and mechanism of action of 5-Azanucleosides. (a) Human concentrated nucleoside transporter 1 (hCNT1) facilitates the entry of 5-Aza-CR and 5-Aza-CdR into cells, where they are phosphorylated by uridine-cytidine kinase and deoxycytidine kinase, respectively. 5-Aza-CMP and 5-Aza-dCMP are subsequently phosphorylated into their active triphosphate forms. 5 Aza-dCR can be incorporated solely into DNA, whereas 5-Aza-CR can be incorporated into RNA as well as DNA after the reduction of its 5-Aza-CDP form to the 5-Aza-dCDP form. Incorporation of 5-Azanucleosides into DNA induces hypomethylation of the daughter DNA strands, whereas incorporation of 5-Aza-CR into RNA disrupts crucial cellular process such as protein translation and causes ribosomal disassembly. 5-Azanucleosides, however, are also very unstable and can be deaminated to inactive uridine. (b). Schematic working model of 5-Azanucleosides. During DNA replication, 5-Azanucleosides are incorporated into DNA and trap DNMTs, which are subsequently targeted for proteosomal degradation. DNA-containing azanucleosides are hemi-methylated after the first round of DNA replication and become fully demethylated after several rounds of replication. Using hypomethylation agents, the silenced epigenetic modifications could be switched to an active status (e.g. H3K4me3 and AcH3). preferentially binding to DNMT1 [92–93]. These com- successful results encouraged the use of DNMTi to develop pounds, however, have limited DNA hypomethylation ac- novel clinical regimens. We have summarized several tivity in living cells [94]. The small molecule RG108 also recent important clinical trials in Table 1. shows the potential to reactivate tumor suppressor genes in human colon cancer cells [95–96]. Recently, the lipophil- Single-agent therapy ic, quinoline-based compound SGI-1027 was demonstrated 5-Azacytidine (Vidaza, Azacitidine) In early clinical trials, to be a novel DNMTi in vitro. In RKO cells, SGI-1027 5-Aza-CR was administered at maximum tolerated doses causes the degradation of DNMT1 and the demethylation (MTD) to patients with osteogenic sarcoma or other cancer- of the CDKN2A gene promoter as well as reactivating related diseases, but showed unfavorable toxicity [100– silenced genes [97]. 101]. Increasing knowledge of the mechanism of action An alternative strategy to inhibit DNMT1 includes the of 5-Aza-CR indicated that lower dosages of 5-Aza-CR use of short-chain oligodeoxynucleotides and microRNAs. could act as DNMTi with minimal effects on DNA MG98 is a 20-bp antisense oligonucleotide that specifically synthesis [102]. 5-Aza-CR was approved by the FDA for binds to the 30 UTR of human DNMT1 mRNA to prevent its the treatment of MDS based on the positive results from translation. Despite promising results in preclinical stud- the GALGB9221 clinical trial [103]. A reported 60% of 5- ies, the clinical use of MG98 has not been validated [98]. Aza-CR-treated patients exhibited various levels of MicroRNA miR29a, which targets DNMT3A/B directly and response, whereas only 5% of patients in the supportive DNMT1 indirectly in a similar way to MG98, can reduce group showed hematological improvement (HI). 5-Aza-CR global DNA methylation and reactivate CDKN2B [99]. also benefited patients by delaying progression time to acute myeloid leukemia (AML), improving quality of life, Clinical data on DNMTi and prolonging overall survival in Refractory Anemia with Accumulated in vitro evidence has demonstrated that Excess Blasts (RAEB) or Refractory Anemia with Excess of DNMTi can reverse abnormal DNA methylation, indirectly Blasts in Transformation (RAEB-T) subgroups [104–105]. affect histone modifications, and eventually restore normal To further validate the efficiencies of 5-Aza-CR in MDS gene expression profiles in various cancer cell lines. These patients, the European AZA-001 trial was conducted for

540 Table 1. Selected Recent Clinical Trials with DNMT Inhibitors Review Drug(s) Indication(s) Author (Year) Total Results Comments Ref. Patients Azacitidine MDS Silverman 191 7% CR, 16% PR, Low dose treatment improved quality of life and survival rate; delayed [102, (2002/2006) 37% HI progression time to AML(CALGB9221). 104] MDS Fenaux (2009) 358 17% CR, 12% PR, 42% SD Improved OS compared to CCR in high risk MDS patients (AZA-001). [105] MDS Lyons (2009) 151 44%, 45%, 56% HI RBC transfusion independence achieved in 3 alternative dosing regimens. [109] MDS Martin (2009) 25 27% ORR Response rates are comparable to CALGB 9221 and AZA-001 trials but shorter OS in this 5 [110] days treatment regimen. MDS/AML Muller-Thomas 32 15.6% CR, 25% HI, Survival advantage in responder group of high-risk MDS [111] (2009) 34.4% SD MDS Musto (2010) 74 10.8% CR, 9.5% PR, Survival benefits in responder group. [108] 20.3% HI Decitabine MDS Kantarjian (2006) 170 9% CR, 13% HI, RBC transfusion independence and delayed progression time toward AML (D-0007). [112] 17% ORR MDS Kantarjian (2007) 95 34% CR Better response rate at low dose. [114] MDS Steensma (2009) 99 17% CR, 18% HI, Alternative dosing and significant difference in ORR compared to single-center trial by [115] 32% ORR Kantarjian et al, (ADOPT). Refractory Stewart (2009) 31 Relative reduction of tumor size but no correlation between PBMC DNA methylation and [117] Solid Tumor tumor DNA methylation. MG98 MDS/AML Klisovic (2009) 23 26% SD No clinical benefits observed in administered doses and no correlation [119] between DNMT1 downregulation with SD. Advanced Solid Plummer (2009) 33 Suppression of DNMT1 expression in PBMC in all doses with mild toxicity observed. [121] Malignancies Azacitidine in Combination Azacitidine + Valproic Acid + AML/MDS Soriano (2007) 53 22% CR, 42% ORR The increase of histone acetylation level is not correlated with VPA dose level. [122] all-trans-Retinoic Acid Azacitidine + Thalidomide MDS/AML Raza (2008) 40 42% HI, 14% SD High expression of cellular proliferation genes was observed in non-responders. [135] Azacitidine + Valproic Acid Advanced cancer Braiteh (2008) 55 25%SD Safe dosage of azacitidine up to 75mg/m2 in advanced malignancies. [124] Azacitidine + Refractory Lin (2009) 27 No clear benefits seen in the three dosing regimens tested. [126] Sodium Phenylbutyrate Solid Tumor Azacitidine + Valproic Acid MDS Voso (2009) 62 30.7% CR/PR, 15.4% Increasing therapeutic level of VPA promoted efficacy of azacitidine [123] HI, 38.5% SD Azacitidine + Entinostat MDS/AML Fandy (2009) 30 42%ORR Lack of association between reversal of DNA methylation and clinical response. [125] Azacitidine + Allogeneic SCT MDS/AML Jabbour 17 High response rate in patients with recurrent disease after HSCT . [136] (2009) Azacitidine + Lenalidomide MDS Sekeres (2010) 18 44% CR, 17% HI, Higher CR is associated with normal cytogenetics and lower methylation levels [129] Sciences Pharmacological in Trends 67% ORR Azacitidine + Cytarabine MDS/AML Borthakur (2010) 34 Limited clinical activity in patients with relapsed, refractory AML. [137] Azacitidine + Etanercept MDS/CMML Scott (2010) 32 72% ORR Increased response rate and duration of response observed. [138] Decitabine in Combination Decitabine + Valproic Acid Advanced Garcia-Manero 54 19% CR Lower pretreatment CDKN2B methylation level correlates with [127] leukemia (2006) higher response rate. Decitabine + Carboplatin Solid Tumor Appleton (2007) 33 A correlation between different doses of decitabine with PBC DNA demethylation. [132] Decitabine + Valproic Acid AML Blum (2007) 25 44% ORR VPA increased treatment-related toxicity; Re-expression of ER associated with clinical [128] response. Decitabine + AML Chowdhury 12 42% CR Treatment facilitated subsequent HSCT. [131] Gemtuzumab ozogamicin (2009) Decitabine + Allogeneic SCT MDS De Padua Silva (2009) 17 No increased toxicity due to decitabine. [139] Decitabine + Imatinib CML Oki (2007) 28 32% CR, 4% PR, Higher activity in patients without BCR-ABL mutations. [130] Mesylate 7% HI o.1No.11 Vol.31 Decitabine + IL-2 Metastatic Gollob (2006) 21 Reexpreesion of immunomodulatory genes. [133] Melanoma; Renal Carcinoma

541 Abbreviations: CR, complete remission; PR, partial response; HI, hematologic improvement; ORR, overall response rate; SD, stable disease. CCR, conventional care regimens; RBC, red blood cells; HSCT, hematopoietic stem cells transplantation; PBMC, peripheral blood mononuclear cell; PBC, peripheral-blood cells. Review Trends in Pharmacological Sciences Vol.31 No.11 intermediate- and high-risk MDS patients. In that study, trial reported an ORR of 32%, suggesting that this 5-day patients treated with 5-Aza-CR showed a significant im- schedule was as effective as the approved inpatient provement in median overall survival (OS) than patients regimen [116]. 5-Aza-CdR is currently being investigated who received conventional care regimens (CCR): 24.5 in other cancer types to assess the best conditions for months vs 15.0 months. It was the first time that 5-Aza- administration [117–119]. CR treatment was demonstrated to prolong OS in high-risk MDS patients [106]. Detailed analyses of AZA-001 indicat- MG98 Several phase I/II clinical trials have been ed that low-dose cytarabine (one of CCR for patients with conducted to find out the tumor types sensitive to MG98 higher-risk MDS) less efficient and more toxic as compared and an appropriate working dosage. Some of the patients with 5-Aza-CR [107]. Fenaux et al. also demonstrated that already showed decreases in DNMT1 levels, but a 5-Aza-CR benefited older AML patients by prolonging the consistent correlation of DNMT1 level and dosage has OS from 16.0 months to 24.5 months while reducing the not been observed [120–121]. New evidence indicated prevalence of side effects [107]. Based on the outcome of that 7-day continuous dosing of MG98 was well the AZA-001 trial, the National Comprehensive Cancer tolerated for patients with advanced solid tumors, but Network (NCCN) recommended 5-Aza-CR as the preferred an objective clinical response was not reported [122]. therapy for patients with high-risk MDS. In addition to high-risk MDS, 5-Aza-CR can also be used as a potentially Combination therapy effective treatment for patients with low-risk MDS [108– HDAC inhibitors (HDACi) are a family of epigenetic drugs 109]. that increase acetylation of histone proteins and cyto- However, the current FDA-approved, 7-day course of 5- plasmic proteins such as p53. As discussed above, revers- Aza-CR treatment requires weekend treatment and is ing abnormal silencing of epigenetic genes is coordinated inconvenient for patients and care providers. To overcome by increasing promoter histone acetylation levels and DNA this problem, Lyons et al. [110] designed three alternative demethylation. Based on these observations, numerous regimens which avoided weekend treatment. Patients who clinical trials have been conducted to study the synergistic received any one of the three regimens showed similar effects of DNMTi and HDACi (Table 1). hematological improvement as the previously approved 7- Several research teams have combined 5-Aza-CR with day 5-Aza-CR regimen, as well as a higher transfusion- the HDACi valproic acid (VPA) to treat MDS and AML. independent rate. More patients who needed transfusion of Soriano et al. [123] conducted a phase-I/II clinical trial in red blood cells (RBCs) at baseline became independent of which patients were treated with 5-Aza-CR and VPA every RBC transfusion. Another phase-II trial administered an day for 7 days. An ORR of 42% indicated that this combi- alternative 5-day 5-Aza-CR intravenous schedule and nation strategy was clinically effective. Another phase-II reported a 27% partial response (PR) + complete remission study sequentially administered 5-Aza-CR after VPA and (CR) rate that was comparable with the 7-day subcutane- observed a CR+PR of 30.7%. As the plasma concentration ous regimen [111]. However, further studies are required of VPA increased, (>50 mg/mL vs <50 mg/mL), a better to illuminate the survival benefit of these modified regi- median survival rate was observed (18.7 vs 10 months), mens. Although one group reported that a limited number indicating HDACi have the potential to increase 5-Aza-CR of treatment cycles could achieve a overall response rate efficacy [124]. Combination therapy of 5-Aza-CR + VPA has (ORR) of 50% according to new criteria set by the Interna- also been investigated in breast cancer, colon cancer and tional Working Group, most clinical trials indicate that other advanced cancers [125]. Other HDACi have also been prolonged exposure to 5-Aza-CR will benefit patients [112]. combined with 5-Aza-CR. For example, 46% of patients with MDS or AML who received 5-Aza-CR supplemented 5-aza-2’-deoxycitidine (Decitabine) 5-Aza-CdR was also with entinostat (another potential HDACi) showed prom- approved by the FDA for MDS therapy, but there is no clear ising outcomes. Among them, 3 patients had CR, 4 patients evidence indicating 5-Aza-CdR improves OS. In the USA- had PR and 7 patients had hematologic improvement registered trial (D-0007), patients treated with 5-Aza-CdR [126]. In a phase-I study, researchers co-administered 5- had a 17% ORR, which was significantly higher than that Aza-CR and the first-generation HDACi sodium phenylbu- in the best supportive care (BSC) group (0%). When tyrate to patients with refractory solid tumors. The three- comparing the OS between the 5-Aza-CdR and control dose schedule administered in that study showed mild arms, a statistical improvement (14.0 vs 14.9 months) toxicity, albeit with few benefits for the patients [127]. was not observed, even though clinical benefits (e.g. Similar to 5-Aza-CR, 5-Aza-CdR has also been tested for independence from RBC transfusion or elongation of its therapeutic efficacy in combination with HDACi. 5-Aza- median time to AML progression) were seen after 5-Aza- CdR + VPA achieved a 22% objective response in patients CdR treatment [113]. Wijermans et al. presented a similar with leukemia although, in this case, the higher level of negative result of the OS advantage of 5-Aza-CdR for older VPA did not correlate with clinical activity [128]. A later patients with MDS or Chronic Myelomonocytic Leukemia study confirmed this conclusion, demonstrating that 5- (CMML) [114]. To increase the CR rate of patients with Aza-CdR itself had promising clinical activity in elderly these diseases taking 5-Aza-CdR in the outpatient setting, patients with AML. Adding 25 mg/kg/d of VPA neither several clinical trials explored alternative schedules. enhanced the efficacy of 5-Aza-CdR nor did it increase Kantarjian et al [115] showed that a 5-day intravenous the re-expression of CDKN2B [129]. schedule with the highest dose-intensity yielded the 5-Aza-CR and 5-Aza-CdR have also been combined with highest CR rate (39%). In a follow-up study, the ADOPT other conventional therapies. For example, a recent study

542 Review Trends in Pharmacological Sciences Vol.31 No.11 combining 5-Aza-CR with the FDA-approved MDS chemo- respond to hypomethylation therapy best are those who therapy drug lenalidomide revealed an impressive ORR of carry markers such as lower methylation levels in the 67% [130]. 5-Aza-CdR has been combined with carboplatin, CDKN2B promoter or higher levels of miR29 [128,143]. imatinib, or with gemtuzumab ozogamicin (monoclonal Ultimately, efforts to create a comprehensive picture of the antibody) for the treatment of various cancer types [131– epigenetic landscape should help in the understanding of 133]. The encouraging results from these clinical trials cancer pathology and the molecular basis of tumorigenesis. could broaden the application of DNMTi to various tumor This could lay the foundation for future drug development types, but further randomized clinical trials with control to improve the survival and quality of life of cancer arms are needed to demonstrate the feasibility of these patients. approaches. Acknowledgments Validating epigenetic targets in clinical trials This work was supported by a R01 grant from the National Institutes of The ability of 5-Aza-CR and 5-Aza-CdR to reduce global Health (‘Mechanisms of de novo methylation in cancer’ (CA082422-12)) DNA methylation in vivo has been validated in multiple and a grant from the organization ‘Stand Up To Cancer’. clinical trials in which they have been used as single agents or combined with other anticancer therapies [118,123,125– References 1 Jones, P.A. and Baylin, S.B. (2007) The epigenomics of cancer. 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