pharmaceuticals
`
`
`
`Review
`
`Hypomethylating Chemotherapeutic Agents as Therapy
`for Myelodysplastic Syndromes and Prevention of Acute
`Myeloid Leukemia
`
`Vincent G. Sorrentino +, Srijan Thota !+®, Edward A. Gonzalez !, Pranela Rameshwar 2©,Victor T. Chang 23
`and Jean-Pierre Etchegaray '*
`
`1 Departmentof Biological Sciences, Rutgers University—Newark, Newark, NJ 07102, USA;
`vincent.sorrentino@rutgers.edu (V.G.S.); st981@scarletmail.rutgers.edu (S.T.);
`egonzalez1796@gmail.com (E.A.G.)
`Departmentof Medicine, Division of Hematology/Oncology, Rutgers New Jersey Medical School,
`Newark, NJ 07103, USA; rameshwa@njms.rutgers.edu
`3 Veteran Affairs New Jersey Health Care System, East Orange, NJ 07018, USA; Victor.Chang@va.gov
`Correspondence: jeanpierre.etchegaray@rutgers.edu
`+ These authors contributed equally.
`
`Abstract: Myelodysplastic Syndromes (MDSs) affect the elderly and can progress to Acute Myeloid
`Leukemia (AML). Epigenetic alterations including DNA methylation and chromatin modification
`may contribute to the initiation and progression of these malignancies. DNA hypomethylating agents
`such as decitabine and azacitidine are used as therapeutic treatments and have shown to promote
`expression of genes involved in tumor suppression, apoptosis, and immune response. Another
`anti-cancer drug, the proteasome inhibitor bortezomib, is used as a chemotherapeutic treatment
`for multiple myeloma (MM). PhaseIII clinical trials of decitabine and azacitidine used alone and
`in combination with other chemotherapeutics demonstrated their capacity to treat hematological
`malignancies and prolong the survival of MDS and AMLpatients. Although phaseII] clinical trials
`examining bortezomib’s role in MDS and AMLpatients are limited, its underlying mechanisms in
`MM highlightits potential as a chemotherapeutic for such malignancies. Further research is needed
`to better understand how the epigenetic mechanisms mediated by these chemotherapeutic agents
`andtheir targeted gene networksare associated with the development and progression of MDSinto
`AML.This review discusses the mechanisms by which decitabine, azacitidine, and bortezomib alter
`epigenetic programsandtheir results from phaseIII clinicaltrials.
`
`Keywords: epigenetic; myelodysplastic syndrome; acute myeloid leukemia; DNA methylation;
`decitabine; azacytidine; bortezomib; cancer; hypomethylating agent; myeloma
`
`
`1. Introduction
`
`Myelodysplastic syndrome (MDS) is a hematological malignancy characterized by
`aberrant hematopoiesis and most commonly affects the elderly [1]. From 2007 to 2011,
`the reported incidence of MDS per 100,000 individuals in the United States was 4.9 per
`year; today, the numberis likely higher due to increased awareness of the condition [2].
`MDSpatients display cytopenia, leading to a susceptibility for infection and bleeding[3].
`Principal diagnostic criteria for MDS include the presence of dysplasias in the peripheral
`blood and bone marrow;in bothtissues, more than onecell lineage can be affected [3]. MDS
`subtypesare defined by cytopenia type andcell lineage-specific dysplasias. For example,
`RARSor MDS-RSis defined by refractory anemia, dysplasia associated with erythropoiesis,
`the presence of ringed sideroblasts (RSBs), and erythroid precursorcells with abnormal
`iron accumulation in the mitochondria aroundthe nucleus [4]. Differential blood tests are
`helpful in prognostic classification of dysplasias and hematopoietic insufficiencies; higher
`blast counts in the bone marrow (5-10%) and low peripheral blood cell counts are often
`
`check for
`updates
`
`Citation: Sorrentino, V.G.; Thota,S.;
`Gonzalez, E.A.; Rameshwar,P.;
`Chang,V.T.; Etchegaray,J.-P.
`Hypomethylating Chemotherapeutic
`Agents as Therapy for
`Myelodysplastic Syndromes and
`Prevention of Acute Myeloid
`Leukemia. Pharmaceuticals 2021, 14,
`641. https://doi.org/10.3390/
`ph14070641
`
`Academic Editor: Marcin Ratajewski
`
`Received: 9 June 2021
`Accepted: 30 June 2021
`Published: 4 July 2021
`
`Publisher’s Note: MDPIstays neutral
`with regard to jurisdictional claims in
`published mapsandinstitutional affil-
`iations.
`
`Copyright: © 2021 by the authors.
`Licensee MDPI, Basel, Switzerland.
`This article is an open accessarticle
`distributed under
`the terms and
`conditions of the Creative Commons
`
`Attribution (CC BY)license (https://
`creativecommons.org/licenses/by/
`4.0/).
`
`CELGENE 2118
`APOTEX v. CELGENE
`IPR2023-00512
`
`Pharmaceuticals 2021, 14, 641. https: //doi.org/10.3390/ph14070641
`
`https: / /www.mdpi.com/journal/pharmaceuticals
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`associated with more severe form of the disease [3]. The 2016 World Health Organization
`classification of myelodysplastic syndromes and neoplasmsrecognizes categories of MDS
`with single lineage dysplasia, MDS with multilineage dysplasia, MDS with ringed siderob-
`lasts, MDS with excess blasts, and MDS Unclassifiable [5]. The IPSS-Rstratifies patients at
`diagnosis into risk groups based upon cytogenetics, marrow blast proportion, hemoglobin,
`absolute neutrophil count, and platelet count [6]. High risk MDSpatients can progress
`to acute myeloid leukemia (AML)[1]. AML is a hematological cancer characterized by
`immature myeloid cell proliferation with blasts >20% and often accompanied by bone mar-
`row failure [7]. Cytological features of the bone marrow such as Auerbodies,crystalline
`rod-like structures found in the cytoplasm of leukemic myeloid cells, are common in AML
`and can be helpful in diagnosis of the disease [8].
`The last decade of research has characterized various epigenetic and genetic abnor-
`malities linked to both MDS and AML,resulting in impaired genetic machinery and
`hematopoietic stem cell function, all of which are more commonin the aged popula-
`tion [1,7]. More than 30 driver mutations have been identified in MDSpatients; these
`mutations affect DNA methylation pathways, RNA splicing, chromatin modifications,
`transcription, and signal transduction [9]. Non-random chromosomal mutations have been
`identified as genetic events that promote disease initiation and progression and are com-
`monin approximately 52% of AML patients [10]. Somatic mutations of genes that encode
`epigenetic, transcription factors and signaling proteins in AMLpatients [11], include DNA
`methyltransferase 3A (DNMT3<A), Ten-Eleven Translocation 2 (TET2), CCAAT Enhancer
`Binding Protein « (CEPBA), p53 and tyrosine kinase receptors [7]. This review selects three
`specific epigenetic drugs capable of inhibiting DNA methylation: decitabine, azacitidine,
`and bortezomib.
`Decitabine wasfirst synthesized in 1964 [12] as a cytostatic compound and then
`identified as a suitable treatment for leukemia in murinestrains with distinct MHC-II class
`in 1968 [13]. Decitabine (5-aza-2'-deoxycytidine) has been the focus of numerousstudies
`to better understand the epigenetic basis for cancer treatments. Early experimentation
`pointed to decitabine’s anti-leukemic properties in mouse models. A 1978, leukemic mice
`injected with decitabine with an 8-h infusion showedanincreasein lifespan with little to
`no toxicity [14]. Additionally, in 1983, treatment of leukemic mice with longer infusion of
`decitabine resulted in long-term survivors [15]. These early studies formed the basis for
`the use of decitabine as a chemotherapy for leukemia.
`Studies on decitabine at lower doses uncoveredits specific inhibitory effects on DNA
`methylation, and thereby identified as a hypomethylating agent (HMA)[16]. One of the
`initial studies found decitabine and azacytidine, another cytidine-analog, to induced gene
`expression via DNA demethylation (Figure 1A-C) [16]. Subsequent research confirmed
`decitabine as a DNA methyltransferase inhibitor (DNMTIi) that targets 14,000 regulatory
`regionsof genesin different cancercell types derived from lung[17], prostate [18], colon [19]
`andblood cancers [20]. Animportant study reported the use of decitabine at three low doses
`in patients diagnosed with high-risk MDS and chronic myelomonocytic leukemia (CML).
`The study foundthe best results with a dose of 20 mg/m?intravenouslyfor 5 days [21]. In
`2006, the U.S. Food and Drug Administration (FDA) approveddecitabine as a treatmentfor
`MDSand CML [22].
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`Pharmaceuticals 2021, 14, 641
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`A Azacitidine
`
`NH
`
`2
`
`B Decitabine
`
`NH
`
`2
`
`C
`
`Cytidine
`
`NH
`
`2
`
`opt ek
`
`Ck
`
`¢
`OH
`
`HO
`
`Hot
`
`HO
`
`OH
`
`D
`Azacitidine
`
`Decitabine
`
`Vv
` yoe
` NMPKul
`
`
`
`
`“|
`yarn”
`
`
`
`
`5-aza-dCMP
`J
`{NMPK
`RNR
`5-aza-CDP >~C5-aza-dCDP
`{NDPK
`grerk
`
`
`
`RNA
`DNA
`
`
`Figure 1. Incorporating mechanismsof azacytidine and decitabine into RNA and DNA.Chemical
`structures of (A) azacytidine, and (B) decitabine, which are analogs of (C) cytidine.
`(D) Both
`azacitidine and Decitabine have similar mechanismsof integration into RNA and DNA,respectively.
`These nucleoside analogs are transportedinto the cell via human nucleoside transport (hNT) channel.
`Azacitidine (5-azacytidine) becomes phosphorylated via the uridine cytidine kinase (UCK) to form
`5-aza-CMP analog, which becomessuccessively phosphorylated via nucleoside monophosphate
`kinases (NMPK) and nucleoside diphosphatase kinase (NDPK) to create 5-aza-CDP and 5-aza-
`CTP, respectively. Decitabine (5-aza-2'-deoxycytodine) becomes phosphorylated by deoxycytidine
`kinase (DCK) to make 5-aza-dCMP analog, which becomessuccessively phosphorylated via NMUPK
`and NDPKto form 5-aza-dCDP and 5-aza-dCTP, respectively. Once these ribonucleoside and
`deoxyribonucleoside analogs are in their active states (5-aza-CTP, 5-aza-dCTP), they can replace
`cytosines in RNA and DNA,respectively. Note that the monophosphate analogs, 5-aza-CMP and
`5-aza-dCMP, can be deaminated into 5-aza-U and 5-aza-dU uridine analogs respectively, by cytidine
`deaminase (CDA), which helps maintain the surplus of pyrimidines. Additionally, the diphosphate
`analog 5-aza-CDP, can be converted into 5-aza-dCDPvia ribonucleotide reductase (RNR) by reducing
`the ribonucleoside into a deoxyribonucleoside.
`
`Azacitidine (5-azacytidine), an analog of cytidine with similar structure to decitabine,
`acts as a DNMTinhibitor by modifying the 5th carbon of the pyrimidine ring (Figure 1A).
`Instead of a carbon atom bondedto a hydrogen atom,azacitidine consists of a nitrogen atom
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`Pharmaceuticals 2021, 14, 641
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`in the sameposition [23]. The analog wasfirst synthesized in 1963 by Piskala and Sorm
`at the Institute of Organic Chemistry and Biochemistry in Prague [24,25]. Soon after, the
`azanucleoside wasisolated from the gram-positive bacteria Strepotoverticillium ladakanum,
`andits functionality with respect to cytotoxicity and anti-proliferation, were shown[26].
`After initial discovery and synthesis, azacitidine was used for its anti-metabolite properties
`in disturbing normal metabolic processes, leading to its use as a chemotherapeutic for
`leukemia [23]. In 1971, the nucleoside analog was used for its cytostatic properties in
`chemotherapy, as it provedefficacious in the treatment of childhood leukemia. In the 1980s,
`the demethylating properties of the drug were identified. This led to further studies and
`clinical trials to investigate the drug as an epigenetic modulator [25]. With the linkage
`between DNA hypermethylation and the development of cancer, azacitidine, as a hy-
`pomethylating agent, was quickly soughtafter for its antineoplastic properties, specifically
`throughreactivation of previously silenced genes, including tumor-suppressors[23,27].
`In 2004, nearly 40 yearsafter its discovery, Azacitidine (Vidaza; Celgene), an injectable
`suspension, was approved by the FDA, followed by approvalin 2008 by the European
`Medicines Agency (EMA)[23,27]. Vidaza, a bioavailable formulation of 100 mgof azaciti-
`dine and 100 mg mannitol, has been used for subcutaneous administration in MDS, AML
`and CMML patients [23]. Currently, the hypomethylating agents azacitidine and decitabine,
`are the recommendedtherapeutic treatment for MDSpatients classified by the International
`Prognostic Scoring System (IPSS) at intermediate-2/high risk [28]. Subcutaneousinjections
`of azacitidine, however, require the need for in-person treatments and potentially cause
`injection site infections; this can be detrimental for AML patients with severe alterations
`in bone marrowblasts. Recently, azacitidine has becomeavailable as an oral formulation
`CC-486 (ONUREG;Celgene), whichis being clinically tested in patients with MDS, AML,
`and CMML[29,30]. This version was approved by the FDA in 2020 as a maintenance
`treatment for AML patients whofailed intensive induction chemotherapy and achieved
`a first complete remission due to the successful trials in the QUAZAR AML-001 study
`(ClinicalTrials.gov Identifier: NCT01757535) on 1 September 2020 [31].
`In addition to hypermethylation, aberrant proteolysis has been associated with the
`developmentof particular malignancies; as such, targeting protein degradation processes
`has been the focus of many researchers interested in developing anticancer drugs [32].
`Oneof the crucial pathwaysinvolved in protein degradationis the ubiquitin-proteasome
`pathway (UPP), which is responsible for targeting degradation of approximately 80% of
`cellular proteins [33]. This pathway is executed by the proteasome: a multiprotein complex
`that is responsible for the recognition and degradation of ubiquitin-markedproteins[33].
`Malignantcells tend to accumulate defective proteins due to their increased synthesis
`capability; this increases their reliance on the proteasome’s disposal mechanism [34,35].
`Therefore, any compound that interferes with proteasomal disposal of proteins could
`result in the accumulation of defective proteins, increased cellular stress, and apoptosis
`of malignantcells [35]. Since the proteasome degrades and processes mediators of the
`cell-cycle and apoptosis such as cyclins, caspases and nuclear factor of kB (NF-kB) [35],
`this molecule also drives the cell cycle via regulation of protein levels that activate and/or
`inhibit phase transitions in cell growth and replication [36]. Moreover, several studies have
`demonstrated proteasomeinhibition results in cell death [33] due to impaired degradation
`of p53 [37] and p21 [38].
`The chemotherapeutic effect of bortezomibrelies on its ability to regulate protein
`turnover. Bortezomib is a proteasome inhibitor knownto induce apoptosis in malignant
`hematopoietic cells [35,39]. First synthesized by Myogenics in 1995, bortezomib showed
`early in vitro and in vivo results in clinical studies [32] and wasthe first proteasome in-
`hibitor to be usedclinically in the treatment of malignancies, namely refractory/relapsed
`myelomas[40]. After extensiveclinicaltrials, it was approved by the FDA in 2003 for the
`treatmentof relapsed multiple myeloma[32,41]. Current research investigates bortezomib-
`mediated mechanismsassociated with epigenetic pathways implicated in cancer develop-
`ment and progression [42].
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`Pharmaceuticals 2021, 14, 641 5 of 36
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`Determining the mechanisms and targeted genes by decitabine, azacytidine and
`bortezomib remain anactive area of research in the context of hematological malignancies
`and other cancers. The purposeof this review is to underline relevantfindings with respect
`to each of these drugs in MDS and AML.Theidentification of new epigenetic biomarkers
`linked to these drugs, in the context of MDS and AML,can be used for developing new
`targeted therapies to increase efficacy of existing treatments.
`
`2. Main Body of Review
`2.1. Decitabine (5-Aza-2'-Deoxycytidine)
`2.1.1. Mechanism of Action
`
`Decitabineis a cytidine analog where nitrogen replaces the carbonfive in the pyrimi-
`dine ring (Figure 1B,D). In general, decitabine represents a DNA hypomethylating agent
`with two main mechanismsofaction (Figure 2). At low doses, decitabine reactivatessi-
`lenced genes and promotescellular differentiation. At high doses,it elicits cytotoxic effects,
`leading to cell death [16,22]. In general, azanucleosides like decitabine are chemically
`unstable and considerations must be taken whenidentifying safe and effective methods to
`administer such drugs[43]. Decitabine and its metabolites bind moretightly to enzymes
`that mediate its incorporation into DNA when comparedto the natural substrate; for exam-
`ple, the drug binds 10 times moretightly to cytidine deaminase than deoxycytidine [44].
`Like many drugs, the effectiveness of decitabine treatment is dependent upon the
`targeted cells to transport the drug [43]. Mechanistically, decitabine is incorporated
`intracellularly by nucleoside transport proteins, including the equilibrative uniporters
`(ENTs; SLC29A family) and concentrative uptake transporters (CNTs; SLC28A family),
`which have both been directly linked to the uptake of chemotherapeutic analogues in
`the treatment of leukemias [43]. The drug is then targeted by deoxycytidine kinase,
`which converts decitabine (5-aza-dCR) to 5-aza-2'-deoxycytidine monophosphate (5-aza-
`dCMP)(Figure 1D) [43]. Through a series of phosphorylation reactions by nucleoside
`di-phosphokinase[44,45], 5-aza-dCMPis further converted into the active nucleotide form
`5-aza-2'-deoxycytidine-5’-triphosphate (5-aza-dCTP), which can substitute for cytosine
`during DNAreplication and thereby incorporated into DNA (Figure 1D)[43,46]. DNA
`methyltransferase enzymeswill then recognize 5-aza-dCTP-guanine dinucleotides for DNA
`methylation (Figure 2) [43]. DNMT1 becomesinactivated by forming an irreversible cova-
`lent bond with the 5-aza-dCTP-guanine dinucleotide (Figure 2) [47]. This covalent bond
`results in a rapid loss of DNMTactivity as bound enzymesare unableto carry out further
`downstream methylation activity [48], leading to global hypomethylation (Figure 2) [45].
`This process is known as enzyme DNA adduct formation. Specifically targeting the S-phase
`of the cell cycle [44], decitabine leads to antitumoraction via inhibition of proper DNA
`replication in cancerouscells; however, the drug also interferes with transcription and
`DNArepair processes (Figure 2) [49].
`Experimental evidence suggests that the effectiveness of low-dose decitabine in ma-
`lignancies stems from its ability to demethylate silenced tumor-suppressor genesrelated
`to leukemic malignancies and other myeloid disorders [49] such as p15'NS4>, E-cadherin
`and MYOD1 [50,51]. Moreover, various distinct methylation patterns have been iden-
`tified in AMLpatients, some predictive of clinical outcomes [20]. As mentioned above,
`nucleoside uptake is an important process in the effectiveness of decitabine treatment
`regimens. Reduced expression of uptake transporters like SLC22A4 have been reported
`as strong predictors of poor event-free and overall survival in AMLpatients [52]. DNA
`methylation-based epigenetic repression could be a contributing factor to such poor out-
`comes, as pre-treatment with decitabine restored SLC22A mRNA expression, increased
`cellular uptake of anthracyclines, and wasassociated with increased sensitivity to cytara-
`bine, a chemotherapeutic, in human AML cell lines [52].
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`SESSASes Genomic DNA @ Methylated Cytosine
`
`© Unmodified Cytosine
`
`— —> —> 6—__-9+—__A+—_ ©_Decitabine
`DCK
`NMPK NDPK
`NDPK
`RNR
`NMPK UCK
`© 5-aza-dCTP
`|
`W@W Azacitidine
`
`DNAReplication
`
`Maintenance
`
`DNA methylation <p «me DNA methylation
`
`De novo
`
`@ 5-azadCDP
`
`X_
`
`Irreversible bond with DNMTs
`
`"WV Newly synthesized DNA strand
`
`AAA. Parent DNA strand
`
`Inactivated DNMTs
`
` O
`SUSASASASASATAURNRTAN
`
`OPO
`OOO
`Anti-tumoreffect:
`{|
`.
`Inhibition of cell cycle S-phase
`DNA Hypomethylation Cc Activation of tumor suppressors
`Loss of epigenetic memory
`Activation of apoptotic genes
`¢
`Impairs DNA repair
`
`Figure 2. Inhibition of DNMTs uponincorporation of decitabine and azacytidine into DNA. The genomic DNA methylation
`landscape consists of methylated and unmethylated cytosines. Upon multiple phosphorylation steps by DCK (deoxycytidine
`kinase), NMPK (nucleoside monophosphate kinases) and NDPK(nucleoside diphosphatase kinase), decitabine becomes
`5-aza-dCTP, which gets incorporated into DNA. Upon phosphorylation steps by UCK(uridine cytidine kinase) and NMPK
`(nucleoside monophosphate kinases), azacytidine is converted into 5-aza-CDP, which is recognized by ribonucleotide
`reductase (RNR) leading to the formation of 5-aza-dCDP. This reactive analog is further phosphorylated to 5-aza-dCTP by
`NDPK(nucleoside diphosphatase kinase), which is then integrated into genomic DNA. Upon DNAreplication, DNMT1
`maintains the methylation status of the genome. However, DNMT1 becomescovalently boundto the cytosine analog, 5-aza-
`dCTP, which prevents DNMT1activity leading to genomic hypomethylation andloss of epigenetic memory. Additionally,
`other DNMT enzymes, DNMT3a and DNMT3b,can also form anirreversible interaction with azanucleosides leading to
`hypomethylation of the genome. Collectively, this drug-mediated DNA hypomethylation causes anti-tumorigenic effects
`includinginhibition of the cell cycle, DNA repair impairments, activation of pro-apoptotic and tumor suppressor genes.
`
`Decitabine has also been shownto induce the expression of tumor-associated anti-
`gens, resulting in induced immunecytotoxiceffect, indicating its indirect role in immune
`therapy [49]. MDSpatients treated with 1.3 uM decitabine at a dose of 15 mg/m?/day
`showed improvedexpression of cancer-testis antigens (CTAs; ie. MAGE-A1, MAGE-A3,
`and SP17) against solid tumors [53]. Decitabine treatments were accompanied by en-
`hanced T-lymphocyte recognition of MDScells, specifically in response to increased CTA
`expression [53]. This study also found decitabine treatments in MDSpatients to increase
`T-lymphocyte function, expression of HLAclass antigen and ICAM-1, a cell adhesion and
`co-stimulatory molecule in adaptive immunity responses [53,54]. This underlines oppor-
`tunities for decitabine treatment regimensto be used in-tandem with immunotherapies
`alreadyin use and highlights the wide variety of genetic targets affected by decitabine’s
`mechanism ofaction.
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`2.1.2. Decitabine Hematological Malignancies
`Hypomethylating agents are attractive because of their reduced toxicity in elderly
`patients when compared to standard induction chemotherapy [55]. As mentioned above,
`anti-tumoractivity of decitabine in hematological malignancies results from multiple mech-
`anismsincluding, induction tumor-suppressor genes upon hypomethylation, enzyme DNA
`adduct formation, activation of apoptotic pathways and induction of tumor-associated
`antigens [49,53]. In AML cell lines such as OCI-AML2, decitabine was found to induce
`the expression of 81 genes while inhibiting the expression of 96 genes; whereas, normal
`peripheral blood cells displayed significantly fewer changes in gene expression [56]. In-
`terestingly, this study showedthat nearly 50% of decitabine-induced genes are deprived
`of CpG methylation sites in their promoter regions, which suggest a decitabine-mediated
`effect that is independent of DNA methylation status.
`In patients with hematological malignancies like MDS and AML, decitabineis at its
`most effective when used over a prolonged period with fractionated exposures [57,58].
`Moreover, responserates to decitabine tend to be slow, with mostpatients requiring at least
`twoto five monthly cycles to achieve maximum clinical results [59]. DNA demethylation
`in responseto decitabine is a function of the dose, in which lowerdoses (0.1 uM) induced
`higher levels of DNA hypomethylation, whereas very low (0.01 uM) and high (10 uM)
`decitabine doses induced significantly lower DNA hypomethylation with concomitant
`higherrate of cytotoxicity. A 2017 study examinedtheeffect of low dose (20 mg/m?/day x
`5 days) decitabine treatments in lower risk MDSpatients. The results show 70% of patients
`displayed an overall response rate (ORR) to decitabine treatments and 32% of these pa-
`tients became blood transfusion-independent[60], demonstrating an overall hematological
`improvement. Onetrial interested in dose/schedule dependent responses to the drug
`enrolled MDSpatients in one of twodistinct treatment regiments: 3-day treatments (3 h
`IV infusion of 15 mg/m? given every 8 h for 3 consecutive days every 6 weeks) or 5-day
`treatments (1 h IV infusion of 20 mg/m? given once daily on days 1-5, every 4 weeks;[61]).
`Overall Response Rate for the 3-day group was 29.4% and 25.5% for the 5-day group
`with the median AML-free survival time was 23.8 months, and 24-month overall survival
`was 48.9%[61].
`Recent phaseIII clinical trials have demonstrated varied success with decitabinetreat-
`ments. Thesetrials typically use criteria like progression-free survival (PFS) defined as
`duration from the date of treatment to progressive disease or death, event-free survival
`(EFS) defined as the time from study entry until relapse/malignancy/death, and overall
`response rate (ORR) defined as the percentage of participants who achieved stringent
`complete response, very good partial response, or partial response, to quantify the effec-
`tiveness of the drug therapy in question. In a study comparing decitabine (15 mg/m? every
`8 h for 3 days) with best supportive care (BSC)in elderly patients (>60 years) with MDS,
`15%of those receiving decitabine showed complete/partial remission and hematological
`improvement; patients receiving decitabine also had longer PFS than those receiving BSC,
`but similar overall survival (OS) in both groups [62]. Another phase III randomized study
`looking at decitabine in individuals with MDS found an overall improvementin patient
`outcomes whentreated with the drug. Patients received either 15 mg/m? over 3 h every
`8 h decitabine IV repeated every 6 weeks, or BSC; decitabine patients showed an ORR of
`17%, with 9% complete responses, compared to 0% response for BSC group [63]. Decitabine
`responseswereclassified as durable (median, 10.3 months) and prolonged patients’ median
`time to AML progression when comparedto BSC patients [63].
`Additionally, researchers have been searching for improved biomarkers of predictive
`of clinical outcomes in MDS/AML patients undergoing decitabine treatment. A trial in
`patients with MDS whohave unfavorable-risk cytogenetic profiles and TP53 mutations,
`have shown increased response rates to decitabine in MDS treatmentprotocols [19,59];
`therefore, higher-risk individuals may be moresensitive to the drug. Anothertrial looking
`at high-risk MDSpatients with varying cytogenetic profiles found decitabine responses
`specific to patients’ cytogenetic profile, namely regarding autosomal monosomies (MK-—,
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`Pharmaceuticals 2021, 14, 641 8 of 36
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`MK+, MK1, MK2+) [64]. ORR in cytogenetically normal (CN)patients was 36.1%, 16.7% in
`MK-patients, and 43.6% in MK+ patients; PFS was prolonged in CN and MK2+ groups
`but not MK—/MK+/MkK1 subgroups when compared to BSC patients [64]. One in vivo
`study found elevated fetal hemoglobin (HbF)to beareliable predictor of overall survival
`in MDS/AMLpatients, with decitabine elevating HbF levels in 81% of MDSpatients and
`54% of AML patients [65]. These studies underline the importance of patient karyotype
`whendeciding which treatment regimens may be mosteffective in MDS/AMLtreatment
`and whatphysiological signals clinicians should assess for reliable predictors of patient
`outcome.
`
`2.1.3. Side Effects and Complications
`During decitabine’s initial development phase in the mid-1980s, toxicities associated
`with the drug’s use included myelosuppression, nausea, and mild to moderate fatigue [16].
`Contemporary studies report neutropenia, febrile neutropenia, thrombocytopenia, leukope-
`nia, anemia, fatigue, nausea, diarrhea, and constipation [66]. In general, one of the main
`concerns with the use of decitabine is its cytotoxic effects at high doses [45], especially
`at non-target sites. Despite this concern, decitabine and azacitidine tend to haveless cy-
`totoxic effects compared to other more conventional chemotherapeutics; this is because
`the dosage neededto elicit desirable epigenetic effects is relatively low comparedto the
`dosage that elicits cytotoxic effects [16]. Possible myelosuppressive complications like
`infection/bleeding, or prolonged myelosuppression [defined as hypocellular marrow]
`havealso been reported but tend to be less common than the milder adverse side effects
`mentioned above [21].
`Despite success with decitabine in the treatment of hematological malignancies, some
`MDS/AML patients do not respondeffectively to the drug [67]. Many patients fail to respond
`to initial decitabine treatments (primary resistance); those that do respondinitially often
`relapse and become unresponsive to subsequent exposures (secondary resistance) [68,69].
`Moreover, treatment with decitabineis not curative; different forms of resistance to the drug
`have been identified over the years [70], most notably dueto alterations in the pathwaysthat
`activate and metabolize decitabine, as well as downstream mutations of genes involved in
`DNAmethylation/ demethylation dynamics such as TET2 [71], DNMT3A, and ASXL1[72].
`Thus, a better understanding of these contributing pathways can help researchers and
`clinicians identify which mechanisms should be targeted at different stages of MDS and
`AMLprogression to develop salvage therapies.
`Researchers have begun looking for waysto bolster the effects of decitabine using
`combination therapies with other drugs. Polo-like kinases regulate thecell cycle [73], vari-
`ousportions of mitosis, and contributes to DNA damagerepair and replication stress [74];
`therefore, these proteins make a reasonable target for anticancer therapeutics. Volasertib
`andrigosertib, two polo-like kinase inhibitors currently under phaseIII trial review, are
`currently being used in combination with decitabine in the treatment of MDS/AML pa-
`tients whoare ineligible for intensive remission therapy [75]. 3-Dezazneplanocin (DZNep),
`a histone methyltransferase inhibitor, has also been identified as a drug that could bolster
`decitabine’s efficacy in MDS/AMLpatients [67]. Together, decitabine and DZNep have
`demonstrated synergistic activation of several tumor suppressor genes and synergistic
`activation of apoptosis in human AML cell lines HL-60 and AML-30 [67]. These types of
`studies widen options for developing new combination treatment regimens.
`
`2.2. Azacitidine
`2.2.1. Mechanism of Action
`
`Similar to decitabine, azacitidine elicits two distinct properties, cytotoxicity and DNA
`hypomethylation, depending on dosage schedules. At high dosage, azacitidine promotes
`cytotoxicity due to its incorporation into both RNA and DNA,while low dosage prompts
`DNAhypomethylation effects (Figures 1D and 2D)[76]. The anti-proliferation effect on
`abnormally dividing hematopoietic cells in the S-phase of the cell cycle results from
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`interference with nucleic acid metabolism [77]. Thereby, further nucleic acid synthesis and
`cellular proliferation are impaired, and apoptotic pathwaysareactivated.
`Azacitidine’s effect is initiated after intake of the oral CC-486 or injection by Vidaza
`via cellular uptake carried out by specific transmembraneproteins; the nucleoside analog
`is transported into a cell via a humanconcentrative nucleoside transport 1 (hCNT1) which
`is part of the SLC28 gene family of three subtypes that transport naturally occurring
`nucleosides and synthetically engineered nucleoside analogs [23]. Upon transport into
`the cell, azacytidine is phosphorylated by uridine cytidine kinase to 5-azacitidine 5’-
`triphosphate, its active conformation [23]. The compoundis then degradedor integrated
`into the nucleic acids. Triphosphate nucleosides are degraded in the cells by cytidine
`deaminase (CDA) and converted to 5-azauridine compounds, rendering them inactive [23].
`Concentration and synthesis of CDA in humanliver and spleen influences the half-life
`of the drug, making it approximately 41 min in vivo. Moreover, reducedlevels of active
`drug by degradation decreases the drug’s efficacy and potency. The various inactive
`metabolites of azacitidine are primarily discarded via urine secretion with minimal (<1%)
`fecal excretion [78].
`Azacitidine is incorporated into the genomeof rapidly proliferating cells during the S-
`phase[23,79] and does not show sufficient affinity for non-proliferating cells [80]. Upon in-
`tegration into RNA,azacitidine interrupts RNA metabolism andtranslation processes. Fur-
`thermore, azacitidine can also incorporate into DNAbyribonucleotide reductase-mediated
`conversion of azacitidine diphosphate into 5-aza-2'-deoxycytidine diphosphate. Phospho-
`rylation of the diphosphate into triphosphate enables azacitidine to be integrated into DNA
`during replication [27]. Azacitidine has an increased affinity to RNA over DNA with a
`ratio of incorporation of 65:35, respectively, in AMLcell lines [76].