Int. J. Cancer: 123, 8–13 (2008)
`' 2008 Wiley-Liss, Inc.
`
`Modes of action of the DNA methyltransferase inhibitors azacytidine
`and decitabine
`
`Carlo Stresemann and Frank Lyko*
`Division of Epigenetics, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany
`
`0
`-deoxy-
`The cytosine analogues 5-azacytosine (azacytidine) and 2
`5-azacytidine (decitabine) are the currently most advanced drugs
`for epigenetic cancer therapies. These compounds function as
`DNA methyltransferase inhibitors and have shown substantial
`potency in reactivating epigenetically silenced tumor suppressor
`genes in vitro. However, it has been difficult to define the mode of
`action of these drugs in patients and it appears that clinical
`responses are influenced both by epigenetic alterations and by ap-
`optosis induction. To maximize the clinical efficacy of azacytidine
`and decitabine it will be important to understand the molecular
`changes induced by these drugs. In this review, we examine the
`pharmacological properties of azanucleosides and their interac-
`tions with various cellular pathways. Because azacytidine and dec-
`itabine are prodrugs, an understanding of the cellular mechanisms
`mediating transmembrane transport and metabolic activation will
`be critically important for optimizing patient responses. We also
`discuss the mechanism of DNA methyltransferase inhibition and
`emphasize the need for the identification of predictive biomarkers
`for the further advancement of epigenetic therapies.
`' 2008 Wiley-Liss, Inc.
`
`Key words: azacytidine; decitabine; DNA methyltransferase; DNA
`methylation; cancer
`
`More than 40 years ago, the azanucleosides 5-azacytidine (aza-
`0
`-deoxy-5-azacytidine (decitabine) were developed
`cytidine) and 2
`as classical cytostatic agents.1 Several years later, it was shown
`that these compounds inhibit DNA methylation in human cell
`lines, which provided a mechanistic explanation for their differen-
`tiation-modulating activity.2 In addition, this observation also ini-
`tiated the development of azanucleosides as epigenetic drugs. Af-
`ter substantial refinements in their clinical dosing schedules, both
`azacytidine and decitabine have now shown significant clinical
`benefits in the treatment of myelodysplastic syndrome (MDS), a
`preleukemic bone marrow disorder.3,4 As a consequence, these
`drugs have now received FDA approval for the treatment of MDS.
`There are substantial ongoing efforts to identify and develop novel
`DNA methyltransferase inhibitors.5,6 However,
`the currently
`available compounds appear to have weaker gene reactivation
`potencies than azanucleosides7,8 and none of the candidate drugs
`has reached an advanced clinical testing stage for epigenetic indi-
`cations yet. This has established azacytidine and decitabine as
`archetypal drugs for epigenetic cancer therapies.9
`Despite the renewed interest in azacytidine and decitabine, sur-
`prisingly little is known about the molecular mode of action of
`these drugs. It is clear that azanucleosides have cytotoxic effects
`and that they can cause DNA demethylation, but the relationships
`between these characteristics and their respective significance for
`clinical responses has not been established yet. A comprehensive
`understanding of drug characteristics will be critically important
`for defining their modes of action and to further advance their clin-
`ical development.
`
`Demethylation therapies: Proof of mechanism
`and proof of concept
`
`Azanucleosides are established molecular tools for the induc-
`tion of DNA demethylation in cellular model systems. However, it
`is also known that high doses of these drugs can induce pro-
`nounced toxicities in patients. When dosing schedules were
`adapted to optimize epigenetic effects it became increasingly
`
`~ UiCC
`
`Publication of the International Union Against Cancer
`
`important to provide proof of mechanism data, i.e., to demonstrate
`DNA demethylation in patients. Several studies have now shown
`that decitabine can induce significant demethylation in the
`approved indication10,11 and additional results suggest that azacy-
`tidine might have comparable epigenetic effects in patients.12,13
`The rationale behind demethylation therapies is the ability of
`DNA methyltransferase inhibitors to revert hypermethylation-
`induced gene silencing.14 Hypermethylation-induced gene silenc-
`ing of tumor suppressor and other cancer-related genes plays a
`fundamental role in human tumorigenesis.15 The reversion of
`these epigenetic mutations can therefore restore proliferation con-
`trol and apoptosis sensitivity. The identification of such events in
`patients undergoing demethylation therapy has been notably diffi-
`cult. Most studies in this context have focused on the p15 tumor
`suppressor gene, which can be hypermethylated in MDS and
`AML patients and can be demethylated and reactivated in patients
`undergoing decitabine therapy.16 Similar observations were also
`made in other clinical studies with azacytidine, but a close connec-
`tion between demethylation and reactivation of p15 and clinical
`responses could not be confirmed.12,13 The identification of hyper-
`methylated genes that become demethylated and reactivated by
`drug treatment and the establishment of statistically robust associ-
`ations between epigenetic
`reactivation events
`and patient
`responses will be an important area for future research.
`
`Chemical stability of azacytidine and decitabine
`
`Azanucleoside drugs are widely considered to be unstable, and
`have therefore been handled with considerable care, both in the
`laboratory and in the clinic. In alkaline solutions azanucleosides
`undergo a rapid and reversible opening of the 5-azacytosine ring,
`followed by irreversible decomposition.17,18 In acidic solutions
`the glycosidic bond of azanucleosides is cleaved, which also inter-
`feres with a potential oral administration of these drugs.19
`To determine the half-life times of azacytidine and decitabine
`in neutral aqueous solutions, we used a capillary electrophoresis-
`based analytical assay. Both azacytidine and decitabine, respec-
`tively, were dissolved in neutral buffer, together with a chemically
`stable internal standard (adenine). Solutions were stored at 4, 20
`and 37°C, respectively, and samples were taken at various time
`points. The results revealed that both drugs were stable at 4°C
`with half-life times of 21 days (azacytidine) and 7 days (decita-
`bine; Fig. 1a). At 20°C, compound degradation became more
`rapid and half-life times were calculated to be 37 hr for azacyti-
`dine and 96 hr for decitabine (Fig. 1a). At 37°C the half-life times
`were 7 hr for azacytidine and 21 hr for decitabine (Fig. 1a). A
`very similar half-life time (20 hr) for decitabine solutions stored at
`37°C was also found in an independent, recent study.21 Our results
`
`Grant sponsors: German National Genome Research Network (NGFN-
`2); Helmholtz Association of National Research Center (Ideenwettbewerb
`Gesundheit).
`*Correspondence to: Deutsches Krebsforschungszentrum, Im Neuen-
`heimer Feld 580, 69120 Heidelberg, Germany. Fax: 149-6221-423-802.
`E-mail: f.lyko@dkfz.de
`Received 21 December 2007; Accepted after revision 14 March 2008
`DOI 10.1002/ijc.23607
`Published online 18 April 2008 in Wiley InterScience (www.interscience.
`wiley.com).
`
`CELGENE 2029
`APOTEX v. CELGENE
`IPR2023-00512
`
`

`

`AZANUCLEOSIDE MODE OF ACTION
`
`9
`
`0
`
`10
`log llme {hi
`
`100
`
`1000 0
`
`10
`log time {hi
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`100
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`g 2.5
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`! 0.5
`
`0
`
`4.5
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`control
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`Smln
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`1 h
`
`6.5 h
`
`48 h
`
`FIGURE 2 – Azacytidine-induced DNA demethylation requires
`extended drug exposure. Global methylation analysis was performed
`by capillary electrophoresis,20 after treatment of HCT116 cells with 2
`lM azacytidine. Cells were incubated in drug-containing medium for
`the time indicated. The medium was then exchanged for drug-free
`medium and cells were grown for a total of 48 hr.
`
`these drugs. For example, it has been shown that subcutaneous
`
`administration of azacytidine results in an 2-fold higher beta
`
`(substance elimination) half-life time compared to intravenous
`administration.22
`Both azacytidine and decitabine show wide distribution in body
`fluids of rabbits and dogs, with a comparably low alpha (substance
`distribution) half-life time of about 5 min.23 Nevertheless, both
`compounds are rapidly cleared from systemic circulation. Sys-
`temic clearance exceeds the glomerular filtration rate and total re-
`nal blood flow, which suggests an important role of nonrenal elim-
`ination. In this context it has been proposed that deamination by
`cytidine deaminase in the human liver and spleen appears to be an
`important pathway.24 A detailed quantification of azacytidine
`plasma levels in 3 patients treated s.c. with 25 mg/m2 showed a
`maximal plasma concentration of 374 ng/mL (1.5 lM), which
`occurred in the first hour after administration and the observed
`beta half-life time in plasma was 1.8 hr.25 When patients were
`treated with the established standard treatment schedule for MDS
`patients (75 mg/m2), plasma levels of 5-azacytidine in the range of
`3–11 lM could be achieved.22 Similar analyses with samples from
`AML patients that had been treated intravenously with 15 mg/m2
`decitabine revealed a maximum concentration of 103 ng/mL (0.5
`lM) and a beta half-life time of less than 1.5 hr.26
`The peak plasma concentrations observed in patients are com-
`parable to the concentrations that are being used to achieve DNA
`demethylation in vitro.8 However, substance elimination half-life
`times in patients are substantially shorter than the drug incubation
`times used for in vitro experiments (usually 48 or 72 hr). We
`therefore analyzed the possibility that short incubation times in
`drug-containing medium would be sufficient for the induction of
`azacytidine-mediated DNA demethylation. To this end, HCT116
`cells were incubated with 2 lM azacytidine. After various time
`points ranging from 5 min to 48 hr, cells were washed and incu-
`bated in drug-free medium for the remaining time until the end of
`the experiment was reached (after 48 hr). Analysis of global cyto-
`sine methylation levels in genomic DNA from these cells failed to
`indicate any demethylation after 5 min of drug incubation, but
`showed progressive demethylation after 1, 6.5 and 48 hr (Fig. 2).
`The maximum demethylation was observed after 48 hr, which
`confirmed that prolonged drug exposures cause more pronounced
`demethylation responses.27
`Because of the association between length of drug exposure
`and DNA demethylation, various clinical studies have tried to
`maximize demethylation responses in patients by continuous
`infusion of azanucleosides over several days. Analysis of DNA
`
`-
`-
`- - -
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`- - - - -
`-
`-
`. -
`· - - - - -
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`- -
`-
`-
`-
`-
`-
`-
`-control ...... -2 ... o_• ___ •_• __ R..,T ..... _..,37 ...... •
`
`AZA
`
`DAC
`
`FIGURE 1 – Chemical stability of neutral azacytidine and decitabine
`solutions. (a) Temperature-dependent decomposition of azacytidine
`(AZA) and decitabine (DAC). Compounds were dissolved in neutral
`0.9% NaCl solutions, stored at 4, 20 and 37°C, respectively, and snap-
`fozen in liquid nitrogen at the time points indicated. Samples were
`then diluted to 0.45 mg/mL and mixed with adenine as an internal
`standard (400 lM final concentration). Analyses were performed on a
`Beckman Coulter capillary electrophoresis system (MDQ Molecular
`Characterization System) with UV detection at 254 nm. Separation
`occurred in an untreated fused-silica column of 60 cm (effective
`length 50 cm) in a 10 mM phosphate buffer system, pH 7.0, with 150
`mM SDS. Analyses were performed at 25 kV and a capillary tempera-
`ture of 25°C. (b) Pharmacological potency of stored azacytidine and
`decitabine solutions in inhibiting DNA methylation. Genomic cytosine
`methylation levels were analyzed by capillary electrophoresis.20 Drug
`solutions were dissolved in neutral aqueous buffer and stored under
`the conditions indicated for 24 hr. HCT116 cells were treated with 2.0
`lM azacytidine (AZA) or 0.5 lM decitabine (DAC). A significant
`reduction in pharmacological potency could only be observed after
`storage of decitabine at 37°C.
`
`indicate a considerable chemical stability of azanucleosides at
`temperatures that are relevant for their general handling and use.
`To confirm the stability of azanucleoside solutions we tested
`their ability to inhibit DNA methylation in the human HCT116 co-
`lon carcinoma cell line. Drug samples were stored for 24 hr at
`220, 4, 20 or 37°C and then added to the tissue culture medium.
`Cells were harvested after 72 hr and their global DNA methylation
`level was analyzed by capillary electrophoresis. This revealed that
`azanucloside solutions stored at room temperature were still effec-
`tive in inhibiting genomic cytosine methylation (Fig. 1b) and pro-
`vided an important confirmation for the relative stability of these
`drugs.
`
`Drug pharmacokinetics
`
`In vivo, several additional factors limit the stability and bioa-
`vailability of azanucleosides. Refinements in dosing and adminis-
`tration have continuously improved the clinical performance of
`
`

`

`10
`
`STRESEMANN AND LYKO
`
`methylation levels showed that continuous administration of deci-
`tabine caused pronounced demethylation, but the study design did
`not permit the analysis of correlations between demethylation and
`clinical responses.28 More comprehensive studies may become
`feasible through the development of oral decitabine.29 Alterna-
`tively, more prolonged drug exposure might also be achieved by
`chemical modifications that improve plasma stability of azanu-
`cleosides. One example for this approach is the development of
`the decitabine-containing dinucleotide S110. This compound
`showed comparable growth inhibiting and demethylating effects
`in tumor cell lines and improved resistance to enzymatic deami-
`nation.21
`A potential safety concern for continuous DNA demethylation
`is the increased induction of illegitimate transcription events.
`Mouse models with strongly and permanently reduced DNA meth-
`ylation levels have shown genetic amplification and insertional
`activation of oncogenic loci.30,31 These events are probably linked
`to the epigenetic reactivation of mobile DNA elements and need
`to be monitored and minimized during demethylation therapy. In
`this context, it is interesting to notice that clinical decitabine
`administration schedules with relatively high dose intensity have
`shown better response rates than schedules that continuously
`maintain low plasma drug levels.32 Thus, downstream effects of
`demethylation might also be important for clinical drug activity
`and optimized clinical schedules will probably cause balanced
`DNA demethylation and apoptosis induction.
`
`Cellular uptake of azanucleosides
`
`Conceivably, the effectiveness of azanucleoside therapy is also
`influenced by the relative transport capacities of the target tissue.
`Of particular interest are the membrane transporters that mediate
`cellular drug uptake. Early studies in a leukemic mouse model
`showed a connection between azacytidine resistance and impaired
`uptake of uridine and cytidine, which supported an important role
`of cellular transport mechanisms in mediating drug effects.33
`However, the role of transport processes in the treatment with aza-
`nucleosides has not been characterized on the molecular level yet.
`In human cells, four different classes of proteins mediate the
`transport of nucleosides across membranes (Fig. 3)34: (i) equlibra-
`tive uniporters (SLC29A family), (ii) substrate exchange trans-
`porters (SLC22 and SLC15 family), (iii) concentrative transporters
`(SLCA28 family), and (iv) ATP-dependent exporters (ABC fam-
`ily). The transporter family members responsible for equilibrative
`uniport (ENTs/SLC29A) or the concentrative uptake (CNTs/
`SLC28A) of nucleosides have been directly linked to the uptake
`of chemotherapeutic nucleoside analogues in the treatment of
`leukemias.35–38
`Experimental data from in vitro treated patient cells showed a
`significant correlation between the expression levels of nucleoside
`transporters and the sensitivity to nucleoside chemotherapeutics,
`like gemcitabine,36,39,40 fludarabine37 or cytarabine,35 which
`might indicate a role of these proteins in mediating azanucleoside
`uptake. A statistically significant correlation between the expres-
`sion level of the equilibrative transporter ENT-1 and the sensitiv-
`ity of ex vivo cultivated mononuclear cells from 50 AML patients
`could also be demonstrated for decitabine.35 Similarly, an array-
`based study of transport-associated genes in 60 human cancer
`cell lines also identified a positive correlation between ENT-1
`expression and azacytidine chemosensitivity.41 A role of ENT-1
`in azacytidine uptake was also suggested by the observation that
`azacytidine-induced cytotoxicity could be reduced by nitrobenzyl-
`mercaptopurine ribonucleoside, a specific inhibitor of ENT-1.41
`Nevertheless, functional data demonstrating a role of specific
`nucleoside transporters in mediating the cellular uptake of azanu-
`cleosides is still lacking. This is an important area for future
`research, because nucleoside transporters expression patterns
`could potentially be used as predictive biomarkers for therapy
`responses with nucleoside therapeutics.42
`
`intracellular metabolism
`
`5-aza-dCR
`
`5-aza-CMP
`
`5-aza-dCMP
`
`5-aza-CR ! I Urd-Cyd kinase I
`I dCyd kinase I !
`!t
`!t
`I ribonucleotlde reductase I
`5-aza-COP ----------+ 5-aza-dCDP
`!t
`!t
`11 RNA polymerase I
`I DNA polymerase 11
`
`-10-20o/,
`
`5-aza-CTP
`
`5-aza-dCTP
`
`RNA
`
`DNA
`
`FIGURE 3 – Membrane transport and intracellular metabolism of
`azanucleosides. Four candidate transporter protein families (black and
`gray arrows) are believed to mediate the transport of nucleosides and
`nucleoside metabolites across the cell membrane (double line). After
`cellular uptake, azacytidine (5-aza-CR) and decitabine (5-aza-dCR)
`are modified by different metabolic pathways. It is assumed that 80–
`90% of azacytidine is incorporated into RNA, because ribonucleotide
`reductase limits the conversion of 5-aza-ribonucleotides to 5-aza-
`deoxyribonucleotides.
`
`Intracellular metabolism of azanucleosides
`
`After their cellular uptake, azanucleosides need to be activated and
`metabolically converted into the active nucleotide for DNA methyla-
`0
`0
`tion inhibition, 5-aza-2
`-deoxycytidine-5
`-triphosphate (Fig. 3).
`A first limiting step in this cascade is the ATP-dependent posphor-
`ylation of the nucleoside to the monophosporylated nucleotide. It
`is generally assumed that this reaction is catalyzed by different
`enzymes for azacytidine (uridine-cytidine kinase) and decitabine
`(deoxycytidine kinase), and phosphorylation of decitabine by
`deoxycytidine kinase has been confirmed experimentally.43 How-
`ever, recombinant human uridine cytidine kinase 1 and 2 enzymes
`failed to phosphorylate azacytidine, while showing detectable ac-
`tivity for cytidine, uridine and some derivatives.44 These results
`suggested that azacytidine phosphorylation is mediated by differ-
`ent enzymes. Selection for decitabine resistance in rat leukemic
`cell lines has been shown to be associated with the occurrence of
`mutations in the deoxycytidine kinase gene.45 This suggested that
`deoxycytidine kinase plays an essential role in the metabolic acti-
`vation of decitabine. Similarly, enzymes that negatively regulate
`the conversion of azanucleosides to azanucleotides could also play
`an important role in modulating drug responses. It has been shown
`that cytidine deaminase can deaminate azacytidine and decitabine
`to inactive aza-uradine nucleosides,24 and retroviral overexpres-
`sion of human cytidine deaminase in murine cells caused a signifi-
`cant drug resistance against decitabine.46 It should be noted, how-
`ever, that there is presently no published data supporting a role of
`human cytidine deaminase or deoxycytidine kinase in modifying
`decitabine responses in patients.
`Because of its deoxyribonucleoside structure, decitabine is gen-
`erally believed to be a more potent DNA methylation inhibitor
`
`

`

`AZANUCLEOSIDE MODE OF ACTION
`
`11
`
`than the ribonucleoside analogue azacytidine. Early incorporation
`studies in L1210 leukemic cells have shown that 80–90% of aza-
`cytidine are incorporated directly into RNA.47 A rate-limiting step
`for the conversion of ribonucleotides to deoxyribonucleotides is
`the activity of the ribonucleotide reductase enzyme. Surprisingly,
`treatment of cancer cell lines with hydroxyurea, a ribonucleotide
`reductase inhibitor, blocked the ability of both azacytidine and
`decitabine to induce DNA demethylation.48 This block was linked
`to the overall depletion of the nucleotide pool and a concomitant
`cell cycle arrest. It will be important to use more specific inhibi-
`tors for similar experiments and to investigate the role of the
`nucleotide metabolism as a potential response modifier in deme-
`thylation therapies.
`
`Incorporation of azanucleosides into nucleic
`acids and DNA demethylation
`
`0
`
`-deoxy-
`After azanucleosides have been metabolized to 5-aza-2
`cytidine-triphosphate, they can become substrates for the DNA
`replication machinery and will be incorporated into DNA, where
`azacytosine can substitute for cytosine. Azacytosine-guanine dinu-
`cleotides are recognized by the DNA methyltransferases as natural
`substrate and the enzymes will initiate the methylation reaction by
`a nucleophilic attack. This results in the establishment of a cova-
`lent bond between the carbon-6 atom of the cytosine ring and the
`enzyme.49,50 The bond is normally resolved by beta-elimination
`through the carbon-5 atom, but the reaction is blocked with azacy-
`tosine, where carbon-5 is substituted by nitrogen (Fig. 4a). Thus,
`the enzyme remains covalently bound to DNA and its DNA meth-
`yltransferase function is blocked. In addition, the covalent protein
`adduction also compromises the functionality of DNA and triggers
`DNA damage signaling, resulting in the degradation of trapped
`DNA methyltransferases (Fig. 4b). As a consequence, methylation
`marks become lost during DNA replication.
`Covalent trapping of mouse Dnmt1 has been confirmed experi-
`mentally by photobleaching approaches and has been shown to be
`dependent on the presence of the catalytic cysteine residue that is
`required for covalent complex formation.51 A different study indi-
`cated that the azacytidine-dependent degradation of DNA methyl-
`transferases was not affected by mutations in the catalytic cysteine
`residue of human DNMT1 and that the drug induced specific pro-
`teasomal targeting of the enzyme.52 Whereas the latter results are
`difficult to reconcile with the covalent trapping paradigm, they
`raised the possibility that additional pathways could also contrib-
`ute to azanucleoside-induced DNA demethylation. In this respect
`it is interesting to notice that a recent study has provided detailed
`insight into the DNA damage response induced by decitabine.53 It
`was shown that decitabine caused the formation of double strand
`breaks in human cancer cell lines and that DNMT1 might play a
`role in mediating the cellular damage response to the drug. The
`induction of DNA damage by decitabine (and, presumably, also
`by azacytidine), combined with a role of DNMT1 in DNA repair54
`indicates that drug-induced demethylation patterns might be influ-
`enced by DNA repair mechanisms.
`To characterize the mechanisms of drug-induced demethylation
`in greater detail, it will be critically important to quantitatively
`determine azanucleoside incorporation rates into genomic DNA.
`An early study with 10T1/2 mouse cells and radioactively labeled
`decitabine suggested that the substitution rate of 5-azacytosine for
`cytosine in genomic DNA could be as high as 10%.55 However,
`the corresponding assays cannot be easily adapted to the require-
`ments of clinical sample analysis and most laboratories use the
`drug-dependent depletion of DNMT1 protein as a non-quantitative
`substitute assay for confirming azanucleoside incorporation.56,57 It
`should be feasible to establish analytical methods for the quantita-
`tive determination of azacytosine levels in genomic DNA and it
`will be interesting to evaluate this parameter as a potential bio-
`marker for response prediction.
`
`a
`cytosine
`
`b
`5-azacytosine
`
`' H o,~:J:. . { "
`o.l.... =1-. w
`
`H
`
`o.l....
`
`' . ,.,...
`'
`
`.• t:
`o.l...•
`
`N
`
`o.l....
`
`' . .,,...
`'
`'
`
`enzyme release
`
`enzyme degradation
`
`FIGURE 4 – Trapping mechanism of azacytosine. (a) A nucleophilic
`attack of the protein-thiol group (from a catalytic cysteine residue of
`the DNA methyltransferase enzyme, DNMT) at the C6 position of cy-
`tosine drives the subsequent transfer of the methyl group from the
`methyl donor S-adenosyl-L-methionine. The transfer proceeds through
`a covalent complex at position C6 between the DNA and the DNMT
`protein. The complex is resolved through a b-elimination reaction
`resulting in the release of the active DNA methyltransferase enzyme.
`(b) Mechanism-based inhibition of DNMTs by azacytosine-containing
`DNA. The covalent complex at C6 cannot be resolved through b-elim-
`ination, because of the presence of a nitrogen atom at position 5. Co-
`valently trapped DNMTs are degraded, resulting in the depletion of
`cellular DNMTs.
`
`

`

`12
`
`Outlook
`
`STRESEMANN AND LYKO
`
`Understanding the mode of action of azanucleosides will
`require continued translational approaches on the molecular and
`the clinical level. It will be important to identify and validate bio-
`markers that predict the response of patients. Depending on their
`association with epigenetic regulation (i.e., demethylation of spe-
`cific markers) or apoptosis induction (i.e., activation of damage
`signaling), these biomarkers might provide detailed insight into
`the cellular pathways that are influenced by azanucleosides. Simi-
`larly, determining the mechanisms of azanucleoside resistance and
`sensitivity will ultimately allow a better understanding of the
`drugs’ mode(s) of action and facilitate the development of molec-
`ular markers for response prediction. For example, it will be inter-
`
`esting to analyze potential associations between clinical responses
`and polymorphisms in the genes encoding the transporters for aza-
`nucleoside uptake. Lastly, it will be important to obtain a better
`understanding of the drug-induced epigenetic changes in patients.
`Genome-wide methylation profiling technologies should be used
`in order to maximize epigenetic reprogramming events at silenced
`tumor suppressor genes and to minimize epigenetic side effects,
`like the activation of silenced retroelements.
`
`Acknowledgement
`
`The author thank Mr. Dirk Stach for his support in determining
`the chemical stability of azycytidine and decitabine.
`
`References
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`
`2.
`
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`8.
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`Sorm F, Piskala A, Cihak A, Vesely J. 5-Azacytidine, a new, highly
`effective cancerostatic. Experientia 1964;20:202–3.
`Jones PA, Taylor SM. Cellular differentiation, cytidine analogs and
`DNA methylation. Cell 1980;20:85–93.
`Silverman LR, Demakos EP, Peterson BL, Kornblith AB, Holland JC,
`Odchimar-Reissig R, Stone RM, Nelson D, Powell BL, DeCastro CM,
`Ellerton J, Larson RA, et al. Randomized controlled trial of azaciti-
`dine in patients with the myelodysplastic syndrome: a study of the
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