Diesch et al. Clinical Epigenetics (2016) 8:71 DOI 10.1186/s13148-016-0237-y REVIEW Open Access A clinical-molecular update on azanucleoside-based therapy for the treatment of hematologic cancers Jeannine Diesch1, Anabel Zwick2, Anne-Kathrin Garz2,3, Anna Palau1, Marcus Buschbeck1* and Katharina S. Götze2,3* Abstract The azanucleosides azacitidine and decitabine are currently used for the treatment of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) in patients not only eligible for intensive chemotherapy but are also being explored in other hematologic and solid cancers. Based on their capacity to interfere with the DNA methylation machinery, these drugs are also referred to as hypomethylating agents (HMAs). As DNA methylation contributes to epigenetic regulation, azanucleosides are further considered to be among the first true “epigenetic drugs” that have reached clinical application. However, intriguing new evidence suggests that DNA hypomethylation is not the only mechanism of action for these drugs. This review summarizes the experience from more than 10 years of clinical practice with azanucleosides and discusses their molecular actions, including several not related to DNA methylation. A particular focus is placed on possible causes of primary and acquired resistances to azanucleoside treatment. We highlight current limitations for the success and durability of azanucleoside-based therapy and illustrate that a better understanding of the molecular determinants of drug response holds great potential to overcome resistance. Keywords: Azanucleoside, Azacitidine, Decitabine, MDS, AML, HMA, Methylation, Chromatin Background able to induce terminal differentiation of mouse embryo Historical view of azanucleosides cells, which was later associated with changes in DNA Azanucleosides (AZN) are pyrimidine analogues of the nu- methylation [2, 4]. This was the first link of DNA methy- cleoside cytidine that were originally developed as classical lation to cellular differentiation. As patterns of methyla- cytostatic agents to be used at higher doses [1]. Nowadays, tion and chromatin structure are known to be severely these compounds are known to be potent inhibitors of altered in many forms of cancer, often with gene silen- DNA methylation when used at lower doses and are often cing of key tumor suppressor genes as a result of pro- referred to as hypomethylating agents (HMAs) [2]. As such, moter hypermethylation, this important observation AZN are considered the first epigenetic drugs. Azanucleo- opened an avenue for developing DNA methylation in- sides currently used in clinic are 5-azacytidine (azacitidine) hibitors for cancer treatment. In particular, acute leuke- and 5-aza-2′-deoxycytidine (decitabine). mias as well as myelodysplastic syndromes had been AZN were first synthesized in 1964 [1] and clinical tri- shown to exhibit multiple silenced tumor suppressor als examining their anticancer activity commenced as genes, one reason why AZN were tested in these dis- early as 1967 [3]. In 1978, it was shown that AZN are eases. Importantly, in the initial experiments of Jones and Taylor [2, 4], optimal inhibition of DNA methylation and subsequent differentiation of cells was observed at * Correspondence: mbuschbeck@carrerasresearch.org; katharina.goetze@tum.de lower drug concentrations with prolonged exposure, Jeannine Diesch, Anabel Zwick, and Anne-Kathrin Garz shared first whereas higher concentrations led to a decrease in de- authorship. methylation and differentiation. The original studies of 1 Josep Carreras Leukaemia Research Institute (IJC), Campus ICO—Germans Trias i Pujol, Campus Can Ruti, Badalona, Spain decitabine and azacitidine in the 1980s for the treatment 2 Department of Medicine III, Klinikum rechts der Isar, Technische Universität of leukemia employed these drugs at the maximum tol- München, Ismaninger Strasse 22, Munich, Germany erated dose (e.g., 2500 mg/m2 decitabine per course) but Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Diesch et al. Clinical Epigenetics (2016) 8:71 Page 2 of 11 had to be discontinued due to prolonged myelosuppres- have also been shown to exhibit distinctly different effects sion. Subsequently, both drugs were explored at much and have shown varied clinical efficacy in clinical trials. lower doses allowing optimal hypomethylation as sug- Some of the differences in efficacy may stem from dosing gested by Jones and Taylor. This led to the design of the issues as well as differences in incorporation into RNA first successful clinical trial in myelodysplastic syndromes and DNA as delineated below, while others may have to using low doses of azacitidine (75 mg/m2) administered do with the specific disease characteristics of the treated over a prolonged period of 7 days repeated every 28 days, patients within each trial. demonstrating superiority over best supportive care [5]. After cellular uptake of AZN by human equilibrative On the basis of these data, azacitidine was approved by and concentrative nucleoside transporters 1 and 2 [9], aza- the US Food and Drug Administration (FDA) in 2004 for citidine and decitabine are modified by different metabolic the treatment of myelodysplastic syndromes (MDS) and pathways to achieve their active forms (Fig. 2). The first thus is the first HMA agent approved for treatment of this limiting step in this cascade is the ATP-dependent phos- disease [6]. Similarly, decitabine was shown to have ac- phorylation of the nucleoside to the monophosphorylated tivity in higher-risk MDS at a low-dose schedule of nucleotide catalyzed by uridine-cytidine kinase for azaciti- 15 mg/m2 every 8 h for 3 days repeated every 6 weeks dine and deoxycytidine kinase for decitabine [10]. Subse- [7]. Decitabine was approved by the FDA for the treat- quent phosphorylation by two different kinases yields the ment of MDS using this schedule in 2006. A lower- active metabolites 5-aza-CTP for azacitidine or 5-aza- dose regimen with higher-dose intensity of 20 mg/m2 over dCTP for decitabine. During replication, decitabine- 5 days repeated every 28 days was later suggested as a derived 5-aza-dCTP is incorporated in newly synthesized superior regimen within a randomized phase III trial [8]. DNA. In contrast, 80–90 % of azacitidine is incorporated in RNA as 5-aza-CTP, while 10–20 % are incorporated Molecular drug action into DNA after multistep conversion to 5-aza-dCTP by AZN are analogues of the naturally occurring pyrimidine the enzyme ribonucleotide reductase (Fig. 2). nucleoside cytidine (Fig. 1) and have so far been shown to have two main mechanisms of antitumor activity: (i) cyto- DNA demethylation toxicity due to incorporation into DNA (and RNA for aza- At lower doses, DNA incorporation of 5-aza-dCTP im- citidine) leading to induction of DNA damage response pairs DNA methylation by irreversible inhibition of DNA and (ii) DNA hypomethylation through inhibition of DNA methyltransferases (DNMT), particularly DNMT1, which methyltransferase, enabling restoration of normal growth is responsible for the maintenance of methylation fol- and differentiation. Although azacitidine and decitabine lowing DNA replication [11]. Effects on other DNMTs, are considered to be mechanistically similar drugs, they such as DNMT3A and DNMT3B, have only been observed a b c Cytidine 5-azacytidine (azacitidine) 5-aza-2’-deoxycytidine Vidaza® (decitabine) Dacogen® NH2 NH2 NH2 N N 5 N N 5 N HO N O HO N O HO N O O O O 2’ OH OH OH OH OH Fig. 1 Cytidine nucleoside (a) and azanucleoside (b, c) chemical structures. Sugar moieties are indicated in grey and chemical changes between cytidine nucleoside and azanucleosides are highlighted in red Diesch et al. Clinical Epigenetics (2016) 8:71 Page 3 of 11 5-aza 5-aza-dC 5-aza/ 5-aza-dU 5-aza 5-aza-dC CDA/AID UCK DCK 5-aza 5-aza -CMP -dCMP hENT/hCNT/ SLC15/SLC22 NMPK NMPK 5-aza RNR 5-aza -CDP ABC 10-20% -dCDP NDPK NDPK phosphate group 5-aza 5-aza -CTP -dCTP 80-90% RNA TET2 mut DNA DNMT3A mut IDH1/IDH2 mut ASXL1 mut SF3B1 mut Fig. 2 Azanucleoside uptake and intracellular metabolism. Human equilibrative and concentrative nucleoside transporters (hENT/SLC29A and hCNT/SLC28A, respectively) and the SLC15 and SLC22 transporter families mediate azanucleosides (5-aza and 5-aza-dC) uptake. Once inside the cell, the drugs are activated through consecutive ATP-dependent phosphorylation steps: the first one is mediated by uridine-cytidine kinase (UCK) for 5-aza and by deoxycytidine kinase (DCK) in the case of 5-aza-dC; the enzyme nucleoside monophosphate kinase (NMPK) incorporates the second phosphate group in both drugs; then, ribonucleotide reductase (RNR) partly converts (10–20 %) 5-aza-CDP into its deoxy form 5-aza-dCDP. Finally, nucleoside diphosphate kinase (NDPK) adds the third phosphate group and 5-aza-CTP is incorporated into RNA while 5-aza-dCTP is incorporated into DNA. Enzymes involved in resistance are highlighted in red, while mutated genes, which have been described to increase the sensitivity to AZN treatment or improve overall survival in patients, are highlighted in green at higher AZN doses [12]. DNMT1 recognizes incorporated decoupling DNA damage and DNA demethylation in 5-aza-dCTP as natural substrate and becomes irreversibly response to AZN [19]. This further highlights that bound to the cytidine analogue, inhibiting DNA methyl- other mechanisms, besides DNMT inhibition, are in- transferase function and leading to its degradation [13]. As a volved in AZN action. consequence, methylation marks become lost during DNA replication. This hypomethylation of DNA can then lead to re-activation of silenced tumor suppressor genes [14]. RNA-dependent effects Covalent DNMT-5-aza-dCTP-DNA adducts have also Since most of the drug azacitidine is incorporated into been shown to induce DNA damage ATM/ATR response RNA, it is assumed that its efficacy, at least in part, is pathways resulting in growth inhibition, G2 cell cycle arrest, due to RNA-dependent effects, which are cell-cycle in- and apoptosis [15]. This activation is represented by strong dependent. Incorporation of 5-aza-CTP into RNA in- induction of γ-H2AX and activation of DNA repair pro- hibits tRNA methylation and processing [20] by teins including CHK1, CHK2, and RAD51 [16]. The DNA reducing tRNA methyltransferase levels [21], giving rise damage caused by AZN is repaired by the base excision re- to defective messenger and transfer RNAs. Furthermore, pair (BER) machinery and is susceptible to inhibition of it disrupts rRNA processing ultimately leading to inhib- poly-ADP ribose polymerase (PARP) [17]. As DNMT1 has ition of mRNA and protein synthesis and thus inducing also been demonstrated to have a role in DNA repair, in- apoptosis [22]. Recently, it has been shown that RNA in- hibition of DNMT1 by AZN may also indirectly influence corporation of 5-aza-CTP is able to repress the expres- DNA repair mechanisms [18]. Recent findings support sion of the M2 subunit of ribonucleotide reductase this by showing that depletion of DNMT1 by RNA (RRM2) and therefore interferes with the conversion of interference in combination with AZN treatment can ribonucleotides to deoxyribonucleotides, leading to in- reduce AZN-induced DNA damage formation, thereby hibition of DNA synthesis and repair [23]. As decitabine Diesch et al. Clinical Epigenetics (2016) 8:71 Page 4 of 11 is not incorporated into RNA, it does not have any dir- response [34]. The maximum plasma concentration of ect effects on RNA processing. active AZN-derived metabolites achieved by standard treat- ment is 3–11 μM for azacitidine or 0.5 μM for decitabine Immune effects [35, 36, 37]. These concentrations have been shown to in- In addition to their demethylating activity, AZN have re- duce transient demethylation in various hypermethylated cently been shown to induce specific immune responses loci in patients and thus provide valuable proof of in cancer cells [24, 25, 26]. Analysis of expression changes mechanism [37, 38]. However, a clear correlation between in response to azacitidine treatment in different cancer the extent of genome-wide DNA hypomethylation, re- cell lines (breast, colorectal, and ovarian) led to the identi- expression of tumor suppressor genes, and clinical response fication of a subset of azacitidine-induced immune genes to AZN has not been demonstrated thus far. (AIM), which can be used to classify primary tumors into Due to its primary incorporation into newly synthe- low and high expression groups. For the latter, treatment sized DNA, decitabine has been shown to be more po- with azacitidine combined with immunotherapy has been tent than azacitidine in vitro, leading to inhibition of suggested to be beneficial [25]. In addition, it has been DNMT1, DNA hypomethylation, and DNA damage in- demonstrated that treatment with DNA methylation in- duction at concentrations two- to tenfold lower than hibitors sensitizes murine melanoma cells to anti-CTLA4 azacitidine [10, 39, 40, 41]. Unlike azacitidine, decitabine immunotherapy [24], suggesting that a treatment combin- is an S-phase specific agent that only targets proliferating ation of AZN and PD1/PDL-1 antibodies may show syner- cells in S-phase while other stages of the cell cycle remain gistic effects [25]. unaffected [42]. Taking into account the short half-life, to A recent study on ovarian cancer has provided an intri- improve treatment success, shorter intervals of decitabine guing molecular mechanism linking AZN treatment to treatment or continuous infusion are needed to increase the immune response. DNA methylation inhibitors were the probability that all cancer cells enter S-phase and thus shown to upregulate endogenous retroviruses in tumor the therapeutic time window [43]. Hypomethylation after cells, leading to a tumor-inhibiting immune response. This decitabine treatment is dose dependent, peaking at 10– type I interferon immune response is thought to be a 15 days after administration and recovering to baseline result of induction of the viral defense pathway, which at 4–6 weeks [44]. Although preclinical data suggested detects cytosolic double-stranded RNA originating from that continuous infusion of decitabine may be advanta- endogenous retrovirus genes (ERVs) re-expressed upon geous, dose-finding trials indicated that short bolus infu- AZN treatment [24]. However, if a similar mechanism also sions in patients with a poor hematological status may be occurs in hematologic cancers in response to AZN treat- better than continuous infusion, lower doses are better ment will need to be determined. than higher doses, and dose intensity is important for effi- In addition to inhibition of DNA methylation and RNA cient DNA demethylation [8, 45]. These observations led metabolism, AZN also inhibits the nuclear factor kappa B to the current standard dosing schedule of 20 mg/m2 per (NFkB) pathway through inhibition of phosphorylation of day over 5 days. However, because this trial had only a the NFkB-activating kinase IKKalpha/beta [27]. As a con- small number of patients in the dosing regimens deemed sequence, AZN can specifically inhibit regulatory T cells as inferior, novel continuous dosing schedules merit in MDS patients [28]. clinical investigation. In contrast to decitabine, azaci- Furthermore, azacitidine has been reported to impair tidine is administered subcutaneously at a dose of de novo synthesis of pyrimidine through inhibition of 75 mg/m2/day over 7 days. Other schedules of azaciti- uridine monophosphate synthase, leading to a significant dine (e.g., over 5 or 10 days) have also been examined decrease in UTP and CTP levels [29], which results in but not proven superior. impairment of cholesterol and lipid homeostasis and Importantly, studies examining gene expression in hu- contributes to the cytostatic effect of azacitidine [30]. man acute myeloid leukemia (AML) cell lines showed that patterns of drug-modulated gene expression were non- Differences between azacitidine and decitabine overlapping between azacitidine and decitabine [46], indi- AZN are most toxic during S-phase of the cell cycle [31], cating that both drugs indeed may have different target and while cytotoxicity requires relatively high doses, the genes. This observation in addition to the biochemical dif- effect on DNA methylation preferentially occurs at lower ferences may explain why one drug may work in a patient doses [32]. AZN are intrinsically instable and after admin- while the other may not. istration undergo spontaneous hydrolysis as well as de- amination by cytidine deaminase leading to degradation Current status of clinical application of [33]. At low doses, the effects of AZN are rapidly lost hypomethylating agents in hematologic cancers upon drug withdrawal and therefore continuous adminis- Azacitidine (Vidaza®) and decitabine (Dacogen®) were tration and several treatment cycles are required to sustain first tested for the treatment of MDS decades ago with Diesch et al. Clinical Epigenetics (2016) 8:71 Page 5 of 11 the first large randomized trial published in 2002 [5] and compared to patients receiving best supportive care, but have emerged as very promising drugs in this field. MDS overall survival was not improved. The drug has there- are a heterogeneous group of clonal hematopoietic stem fore not been licensed for treatment of MDS in Europe cell disorders characterized by dysplastic and ineffective so far, although it is approved in the USA in this indica- hematopoiesis with clinically apparent cytopenias in the tion. Results of a schedule which can be administered in peripheral blood and a predisposition to AML. Primarily the outpatient setting using 20 mg/m2 decitabine as a disease of the elderly, treatment options are limited intravenous infusion for five consecutive days yielded due to comorbidities and a high progression rate into comparable clinical efficacy [56]. secondary AML in one third of the patients [47]. The Based on the encouraging results of the MDS trials for only proven curative approach for MDS is allogenic hypomethylating agents, these drugs are also being stem cell transplantation (allo-SCT), which is not a evaluated for the treatment of AML, in particular or feasible option in the majority of MDS patients. patients not eligible for intensive chemotherapy. As in- Since the FDA approval of azacitidine for all MDS sub- cidence of AML increases with age and our population types in 2004 and the positive results of several clinical tri- grows increasingly older, the median age at diagnosis is als, the drug has been widely used in the clinic [5, 47, 48] now approximately 70 years. Treating elderly AML pa- and has become the standard of care for higher-risk MDS tients presents a significant therapeutic challenge as inten- patients (IPSS intermediate-2 and high or IPSS-R very high sive treatment is often not possible due to comorbidities and intermediate) not eligible for allo-SCT [48, 49]. The and poor performance status. In addition, AML of the international randomized phase III multicenter “AZA-001” elderly more often exhibit adverse cytogenetic and mo- trial revealed that patients benefit from hematologic im- lecular genetic features leading to poor outcome after in- provement (47 %) and a significant delay in progression to tensive chemotherapy. Thus, there is an urgent need for AML as well as a prolonged survival under azacitidine effective but less toxic therapies for elderly AML patients. treatment vs. conventional care regimens with a median First data on the efficacy of azacitidine in AML emerged overall survival 24.5 vs. 15 months [48]. These results make from the AZA-001 trial as a significant percentage of pa- azacitidine the first drug to confer a survival benefit to tients in the AZA-001-MDS trial were originally classified MDS patients. Most responders achieve a first response as the advanced MDS subtype RAEB-t with 20–30 % bone within 6 cycles of subcutaneous azacitidine injection marrow blasts. This subgroup no longer exists in the (75 mg/m2 per day for 7 days every 28 days), and continu- WHO classification of MDS and is now considered as ation of treatment after first response improves response AML which is defined by 20 % or more blasts in the bone quality [50]. Treatment continuation is recommended until marrow. A separate analysis of this AML subgroup within progression of the disease or unacceptable toxicity [51]. the AZA-001 trial showed that these patients benefitted Ongoing studies are currently examining the efficacy from a significantly improved overall survival compared to of oral azacitidine which shows a higher bioavailability conventional care [57]. Similarly, azacitidine treatment has [52, 53]. The oral formulation (maximal tolerated dose been associated with a median overall survival of ap- 480 mg) is under investigation to serve as a long-term low- proximately 9 to 10 months in patients with AML who intensity treatment option (extended to 14 or 21 days cycles) participated in the Austrian Azacitidine Registry or in a for lower-risk MDS with additional poor prognostic factors French compassionate use program [58, 59]. These en- such as severe thrombocytopenia (AZA-MDS-003 trial) or couraging results paved the way for a large randomized as a post-transplant maintenance therapy for MDS as well phase III trial in AML patients older than 65 years and as AML patients (CC-486-AML-002 trial) [53, 54, 55]. with bone marrow blast counts >30 % (AZA-AML- In contrast to the results of the AZA-001 study for 001). Patients with AML were randomized to receive azacitidine, clinical trials for decitabine have thus far not either azacitidine (75 mg/m2 days 1–7 q28) or investi- been able to show an overall survival benefit in patients gator’s choice of conventional care (CCR, consisting of with MDS. Initial phase II trials in Europe using decita- BSC, low-dose ARA-C or intensive chemotherapy) [60]. bine at a total dose of 225 mg/m2 as a continuous infu- Although this trial did not reach its primary endpoint, the sion over 72 h or at a total dose of 135 mg/m2 using a results showed an improved overall survival for patients 3-day intravenous administration over 4 h three times a treated with azacitidine compared to conventional care day showed a favorable overall response rate between 49 (10.4 vs. 6.5 months, p = 0.1). Remission rates were not in- and 54 %, comparable to that of azacitidine. In a ran- creased in the azacitidine arm compared to CCR but the domized phase III trial testing decitabine in a schedule subgroup of patients with adverse cytogenetics or AML of 15 mg/m2 for a total of nine doses over 72 h com- with myelodysplasia-related changes showed a significantly pared to best supportive care, the overall response rate improved overall survival. This trial demonstrates that aza- was 30 % [7]. Patients receiving decitabine had a pro- citidine is superior to CCR in biologically poor-risk sub- longed median time to progression to AML or death groups and makes azacitidine the only drug so far to show Diesch et al. Clinical Epigenetics (2016) 8:71 Page 6 of 11 a survival benefit in elderly AML patients with adverse Table 1 Comparison of patient characteristics between the cytogenetics [60]. Based on the positive results of the AZA- AZA-001 trial and the EORTC 0611 trial for high-risk MDS AML-001 trial, azacitidine was licensed for the treatment AZA-001 trial: EORTC 0611 trial: of AML in patients older than 65 years in 2015. Azacitidine azacitidine decitabine was also evaluated as an addition to standard intensive Eligibility criteria IPSS INT2/high IPSS INT1/INT2/high chemotherapy with the cytotoxic agents daunorubicin and MDS with 5–30 % blasts MDS with 11–30 % cytosine arabinoside (ARA-C) for the treatment of elderly blasts or <10 % blasts and poor cytogenetics AML patients older than 60 years but showed increased toxicity and failed to improve overall survival for unse- CMML with >10 % CMML independent of blasts and WBC blast counts or WBC lected patients [61], although patients with mutations <13 G/L counts in DNMT3A seemed to derive a survival benefit from No t-MDS allowed t-MDS allowed the addition of azacitidine (Müller-Tidow, personal 2 Treatment schedule 75 mg/m 15 mg/m2 3× day q42 communication). days 1–7, q28 Similarly, decitabine was also evaluated for the treat- Treatment until Maximum number ment of elderly AML in a large phase III randomized progression of 8 cycles trial (DACO-16), where the drug was tested against BSC Patient cohort or low-dose ARA-C [62]. Decitabine significantly im- IPSS high 46 % 38.70 % proved response rates compared to BSC/low-dose ARA- C (17.8 vs. 7.8 months, p = 0.001) but overall survival Poor cytogenetics 28 % 48 % was not significantly increased 7.7 vs. 5 months, p = 0.1). t-MDS 0 12.60 % Nevertheless, decitabine was licensed for the treatment Median cycle number 9 4 of elderly AML in Europe in 2013. It is currently not ap- proved in this indication in the USA. A remarkable clinical hallmark of both hypomethylating and decitabine in MDS and AML in Table 2. The more agents is that they achieve significant clinical efficacy and direct metabolic activation pathway to DNA incorpor- can confer a survival benefit in MDS and AML despite in- ation of decitabine compared to the primarily RNA in- ducing only low complete remission rates of around 20 %. corporation of azacitidine has been suggested to make This challenges the long-standing dogma that achieve- decitabine a more potent compound than azacitidine. A ment of complete remission is necessary for improvement large retrospective matched-pair analysis of patient out- of overall survival. It also further demonstrates that we comes in 300 patients treated with either azacitidine or lack a sufficient understanding of the systemic and cel- decitabine suggested that while remission rates were lular actions of these drugs, which might involve still similar between the two drugs, overall survival was de- unrecognized molecular mechanisms. creased in patients >65 years treated with decitabine compared to azacitidine [63]. This seemed primarily due Differences in outcome between azacitidine and to an increased infection rate in patients receiving deci- decitabine trials tabine, perhaps due to higher cytotoxicity and possibly A direct comparison between azacitidine and decitabine an additional contributing factor to the inferior outcome in terms of efficacy within a controlled clinical trial has in terms of overall survival. not been performed so far. It is therefore difficult to ac- curately assess why the survival outcomes in the ran- Limitations of the treatment and possible causes domized MDS trials were so different between both of acquired and primary resistance drugs. Although remission rates were similar for azaciti- The clinical outcome of patients with MDS is quite vari- dine and decitabine, the overall survival in the experi- able even within the same WHO subtype and when classi- mental arm was significantly shorter in the decitabine fied according the revised IPSS score, which has become trial compared to the azacitidine trial [7, 48]. Besides the the standard prognostic model in MDS [47]. Since every possibility that this may be due to differing mechanisms second patient has no clinical benefit from azacitidine of action, it is also clear that the patient characteristics treatment and at least 4 to 6 cycles are required before in the two large randomized MDS trials were divergent. treatment failure becomes apparent [50], the development In addition, patients enrolled in the decitabine trial re- of response predicting biomarkers is an unmet need for ceived a maximum of 8 cycles of therapy (median 4 cy- improving patient selection and designing better thera- cles) while patients in the azacitidine trial were treated peutic options. until progression (median 9 cycles). A detailed compari- In the past few years, a large number of recurring son of patients’ characteristics in both trials is shown in somatic gene mutations have been discovered in patients Table 1 and the current approval status for azacitidine with MDS [64, 65, 66]. First studies analyzing the Diesch et al. Clinical Epigenetics (2016) 8:71 Page 7 of 11 Table 2 Approval status for azacitidine and decitabine in MDS and AML Azacitidine (Vidaza) Decitabine (Dacogen) MDS AML MDS AML USA (FDA) All subtypes AML 20–30 % blasts All subtypes AML <30 % blasts (formerly RAEB-t) (formerly RAEB-t) Dose 75 mg/m2 s.c. days 75 mg/m2 s.c. days 15 mg/m2 i.v. 3× daily days 15 mg/m2 i.v. 3× daily 1–7 q28 1–7 q28 1–3 q42 or 20 mg/m2 i.v. days 1–3 q42 or 20 mg/m2 i.v. days 1–5 q28 days 1–5 q28 Europe (EMA) INT2/high-risk MDS according AML ≥65 years regardless of Not approved AML ≥65 years not candidates for to IPSS, CMML 10–29 % blasts, blast counts, not eligible for standard induction chemotherapy not eligible for allogeneic SCT allogeneic SCT Dose 75 mg/m2 s.c. days 1–7 q28 75 mg/m2 s.c. days 1–7 q28 n/a 20 mg/m2 i.v. days 1–5 q28 mutation status of a panel of genes failed to provide pre- to AZN treatment [70, 71, 72]. This correlation was more dictive markers that would allow identification of re- significant in the absence of ASXL1 mutations [70]. Despite sponders prior to treatment initiation with correlation with response, mutated TET2 was not associ- hypomethylating agents [67]. Moreover, higher-risk MDS ated with improved overall survival [70, 72]. Furthermore, patients frequently progress to AML even when therapy is these studies could not identify a mutational pattern asso- continued [48]. Eventually, all patients including those ciated with the absence of response and thus the pres- initially achieving remissions or hematologic improvement ence of specific mutations cannot be used to identify develop resistances to AZN leading to treatment failure non-responders. Mutations in other genes involved in [48]. These failures can be divided into two broad cate- epigenetic regulation, such as DNMT3A, ASXL1, and gories: refractory or primary AZN failure, which is seen IDH1/IDH2, have also been suggested to affect AZN re- in patients that show no response to at least 6 cycles of sponse [72]. Moreover, the expression of BCL2L10, a therapy, and secondary failure in patients that either member of the Bcl2 family preventing cell apoptosis, progress under treatment or relapse after termination positively correlates with AZN resistance [73]. of treatment. Although the processes responsible for Despite these reports, efforts to correlate response to both primary and secondary AZN failures have been a AZN with hypermethylation patterns have not yielded topic of intense research, the molecular mechanisms of clear-cut results. None of the studies were able to reveal resistance are not well understood. clinical or molecular patterns to identify non-responders, First causes of AZN resistance have been found both and no diagnostic tool can yet be used as a basis for forgo- in the metabolic pathways that activate AZN and path- ing HMA treatment. Neither the measurement of pre- ways linked to DNA methylation. A recent study analyzing treatment DNA methylation [66] nor the presence of TP53 decitabine response in MDS patients described alterations mutations is predictive for response to azacitidine in MDS, in nucleoside metabolism leading to difference in response although as expected TP53 mutated patients have a [68]. In this study, 32 responders and non-responders as poor overall survival despite response [74]. ASXL1-mu- well as 14 patients showing complete remission and subse- tated clones seem to mediate a partial resistance to aza- quent relapse were examined. In responders, the ratio of citidine, as they showed a tendency to lower likelihood cytidine deaminase (CDA) to deoxycytidine kinase (DCK) of response [70]. But even in responding patients, the drug was higher compared to non-responders [68]. This sug- is not curative and response is lost over time. Recently, gests that increased deamination and decreased phosphor- Meldi et al. identified 167 differentially methylated DNA ylation of decitabine is able to confer primary resistance to regions at baseline in patients with the subtype of chronic the drug, although the exact mechanisms leading to a myelomonocytic leukemia (CMML) that could predict changed CDA/DCK ratio are not known. Similar results response to decitabine at the time of diagnosis [75]. This were obtained in patients treated with azacitidine, in study also showed that upregulation of the cytokines which the expression of uridine cytidine kinase was lower CXCL4 and CXCL7 may contribute to primary decita- in patients without a response than in patients responding bine resistance, as these molecules were overexpressed to azacitidine treatment [69]. in non-responding patients. Ongoing studies are currently Downstream of the AZN action, several gene muta- investigating similar correlations between methylation sta- tions have been proposed to affect treatment outcome. tus and response in MDS. For example, several groups have found that mutations in It has been shown that azacitidine does not eradicate TET2, an enzyme that converts 5-methylcytosine to 5- the leukemia stem cell (LSC)-containing population hydroxymethylcytosine correlated with increased sensitivity even in patients achieving a complete remission. A later Diesch et al. Clinical Epigenetics (2016) 8:71 Page 8 of 11 expansion of this LSC-containing population leads to re- toxicity [82]. Decitabine in combination with the monoclo- lapse [76] and thus provides a possible explanation for nal antibody gemtuzumab has shown improved response AZN treatment failure over time. Recently, it was also rates in MDS and AML patients compared to historical shown for CMML that the mutation allele burden in controls [83]. Furthermore, next-generation epigenetic responding patients remains stable during treatment with agents, such as other DNMT inhibitors, compounds dir- AZN despite changes in DNA methylation and gene ex- ectly targeting mutated or dysregulated proteins, including pression [77]. These data indicate that hematologic re- Idh1, Idh2, Ezh2, and Brd2/4, as well as kinase inhibitors sponse is primarily due to epigenetic modulation rather (rigosertib, volasertib) [84] and immune checkpoint inhib- than eradication or suppression of the leukemic clone. itors (PD-1/PD-L1) are currently being tested [85]. Why this epigenetic modulation fails to be effective over Ongoing phase II/III trials are also examining the ad- time is not understood at present and is the focus of on- ministration of azacitidine for the prevention or treat- going research efforts. Acquired resistance to AZN may ment of relapse in patients after hematopoietic stem cell conceivably also be due to acquisition of new gene muta- transplantation (RELAZA trial, NCT01462578). Also, the tions over time or a growth advantage of primary resistant oral formulation of azacitidine is currently being tested in clones such as those with mutant ASXL1. These hypoth- a phase III trial for continuous administration and ex- eses are currently being examined in several studies analyz- tended low dose schedules as a maintenance therapy in ing serial bone marrow samples of patients over time as AML (Quazar AML-001 trial, NCT01757535) as well as well as other efforts developing and analyzing novel for lower-risk MDS patients with low platelet counts murine models of MDS. (AZA-MDS-003 trial, NCT01566695). In addition to myeloid malignancies, azacitidine is also Unmet needs and ongoing clinical trials being investigated in lymphoid malignancies such as re- MDS patients failing treatment with AZN have a very lapsed aggressive B-cell lymphomas (DLBCL-001 trial, poor outcome with a median survival of 5.6 months NCT02343536) or T-cell lymphomas in combination with [78]. Switching the AZN agent at progression (i.e., from chemotherapy and other agents such as proteosome inhibi- azacitidine to decitabine or vice versa) is an option that tors like bortezomib or HDAC inhibitors like romidepsin. has been described as successful in some cases although extensive data is lacking [79]. Unmet medical needs are Hypomethylating agents for treatment of solid therefore improvement of response rates and response tumors duration for patients receiving AZN as well as develop- Due to its promising results in hematologic malignan- ment of alternative therapies after failure of AZN. cies, AZN are further being tested in phase I/II clinical To this end, the effectiveness of several combination ther- trials for advanced solid tumors—mainly colorectal cancer, apies is currently being investigated. Since DNA methyla- small-cell lung carcinomas, ovarian cancer, and breast tion and hypoacetylation often occur at the same time and cancer. Low-dose decitabine in combination with cyto- ensure robust inhibition of gene expression, the combin- toxic drugs has shown encouraging results with a response ation of AZN with histone deacetylase (HDAC) inhibitors rate up to 60 % [86]. Furthermore, combination of low- has been proposed to improve treatment outcome. Indeed, dose azacytidine with the HDAC inhibitor entinostat in in vitro experiments combining AZN with HDAC inhibi- refractory advanced non-small cell lung cancer led to im- tors have shown synergistic re-expression of a subset of pressive responses in a subset of patients [87]. genes [80]. Based on this observation, several clinical trials A detailed description of epigenetic therapy (including have evaluated the combination of AZN with HDAC in- AZN) in solid tumors has recently been reviewed [88]. hibitors, and while some had promising results, others demonstrated significant toxicity (reviewed in [81]). Thus, Conclusions combination therapy with HDAC inhibitors requires fur- AZN have provided a significant improvement in the ther investigation. Currently, combination therapies of treatment of higher-risk MDS and elderly AML. However, mocetinostat (NCT02018926) or pracinostat (MEI-005, while they show significant efficacy, these patients con- NCT01993641) with azacitidine are being tested. tinue to have an overall poor prognosis. Thus, it will be Azacitidine and decitabine have also been tested in important to obtain a better understanding of the AZN combination with (i) immunomodulatory drugs (lenalido- action and to identify and validate biomarkers that predict mide, monoclonal antibodies) or (ii) chemotherapy. Sim- treatment response as well as understand the mechanisms ultaneous administration of azacitidine and lenalidomide leading to AZN failure. was highly effective in higher-risk MDS patients with an Although preclinical studies indicate that decitabine is a overall response rate of 72 % [82]. The best response was more potent antileukemic agent than azacitidine [40, 41], observed in patients with at least one mutation in TET2, the clinical data suggest that azacitidine is more effective IDH1/2, or DNMT3A but also induced significantly higher than decitabine. In order to elucidate this apparent Diesch et al. Clinical Epigenetics (2016) 8:71 Page 9 of 11 contradiction, future investigations of decitabine should 9. Rius M, Stresemann C, Keller D, et al. Human concentrative nucleoside be performed to optimize the current dose schedule. transporter 1-mediated uptake of 5-azacytidine enhances DNA demethylation. Mol Cancer Ther. 2009;8:225–31. 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J Mol Biol. 1982; Acknowledgements 162(3):679–92. Collaborative clinical, basic research in the groups of Katharina Götze and 14. Bender CM, Zingg JM, Jones PA. DNA methylation as a target for drug Marcus Buschbeck is supported by a joint grant from the Deutsche Jose design. Pharm Res. 1998;15(2):175–87. Carreras Leukämie Stiftung (DJCLS R 14/16). Research in the Buschbeck lab is 15. Juttermann R, Li E, Jaenisch R. Toxicity of 5-aza-2_-deoxycytidine to further supported by the MINECO (BFU2015-66559-P), AGAUR (2014-SGR-35), mammalian cells is mediated primarily by covalent trapping of DNA AFM Téléthon (AFM 18738), Fundació Internacional Josep Carreras, Foundation methyltransferase rather than DNA demethylation. Proc Natl Acad Sci U S A. “Obra Social la Caixa,” the European Commission (H2020-MSCA-ITN-2015-675610), 1994;91:11797–801. and the Asociación Española Contra el Cáncer (AECC—Junta de Barcelona). 16. Palii SS, Van Emburgh BO, Sankpal UT, et al. DNA methylation inhibitor 5-Aza-2'- JD holds a Juan de la Cierva from MINECO. Research in the Götze lab is further deoxycytidine induces reversible genome-wide DNA damage that is distinctly supported by grants from the German Research Foundation (FOR2033 GO 713/2- influenced by DNA methyltransferases 1 and 3B. Mol Cell Biol. 2008;28(2):752–71. 1 and SFB 1243). A-KG is supported by the German Cancer Consortium (DKTK) 17. Orta ML, Höglund A, Calderón-Montaño JM, et al. The PARP inhibitor and German Cancer Research Center (DKFZ), Heidelberg, Germany. Olaparib disrupts base excision repair of 5-aza-2'-deoxycytidine lesions. Nucleic Acids Res. 2014;42(14):9108–20. 18. Mortusewicz O, Schermelleh L, Walter J, et al. Recruitment of DNA Authors’ contributions methyltransferase I to DNA repair sites. Proc Natl Acad Sci U S A. 2005; JD, AZ, and A-KG carried out the search of literature. AP designed the figures. 102(25):8905–9. JD, AZ, A-KG, KSG, and MB wrote the manuscript. 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