The Emerging Role of Cytidine Deaminase in Human Diseases: A New Opportunity for Therapy?

Abstract

The recycling activity of cytidine deaminase (CDA) within the pyrimidine salvage pathway is essential to DNA and RNA synthesis. As such, CDA deficiency can lead to replicative stress, notably in Bloom syndrome. Alternatively, CDA also can deaminate cytidine and deoxycytidine analog-based therapies, such as gemcitabine. Thus, CDA overexpression is often associated with lower systemic, chemotherapy-related, adverse effects but also with resistance to treatment. Considering the increasing interest of CDA in cancer chemoresistance, the aims of this review are to describe CDA structure, regulation of expression, and activity, and to report the therapeutic strategies based on CDA expression that recently emerged for tumor treatment.

Cytidine deaminase (CDA), previously considered as a “spare wheel” for recycling nucleotides, shows undeniable and specific properties, notably for genome stability and cancer chemoresistance. This review gathers current knowledge concerning CDA structure and function to better understand why this enzyme may soon become a key player in disease management.

Main Text

Cytidine deaminase (CDA) is a ubiquitous enzyme whose major role is to participate in the recycling of free pyrimidines. The pyrimidine salvage pathway appears to have two different aims: one is the recycling of pyrimidines for the synthesis of other nucleotides that will be integrated into DNA and RNA, the other is the degradation of pyrimidines to ensure a constant source of carbon and nitrogen to the cell. Interestingly, CDA has a pivotal role in both pathways. During the past decades, cancer researchers have also taken interest in this enzyme for its additional role on chemoresistance. Accordingly, this review aims to gather known data on CDA, from structure to activity regulation, and the first evidence of CDA relevance to cancer treatment.

CDA Gene Organization

CDA (EC 3.5.4.5) can also be referred to as cytidine aminohydrolase, CD, or CDD, and it is responsible for cytidine (and deoxycytidine) transformation to uridine (and deoxyuridine), respectively. This enzyme belongs to the cytidine and deoxycytidylate deaminase family that relies on zinc binding.¹ The CDA gene is located on chromosome 1 (1p36.12)^2,3 and spans a 29,961-bp region, but the open reading frame (divided in 4 exons) is encoded only by 441 bp.^4,5 CDA gene and protein sequences are well conserved among species. Orthologous CDA genes are found in many species with a high percentage of sequence homology with human CDA (hCDA). For instance, the hCDA gene is 83.79% identical to the mouse CDA gene, and 99.77%, 68.99%, and 51.88% identical to chimpanzee, zebrafish, and Saccharomyces cerevisiae CDA genes, respectively.³ The protein sequence of hCDA is also 39% identical to the Escherichia coli CDA protein sequence.¹

CDA Protein Structure

CDA is composed of a CMP (cytidine monophosphate)/dCMP (deoxycytidine monophosphate)-type deaminase domain that consists of a central β sheet with one or more α helices on each side.⁶ This feature is shared with all APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) proteins. However, unlike its counterpart AI-CDA (activation-induced CDA; extensively described by Kumar et al.⁷), CDA does not belong to the APOBEC family and thus processes only free pyrimidines. The CDA gene codes for a 146-aa protein (15 kDa) that associates with a zinc ion (Zn²⁺)⁸ and is organized into a 52-kDa homotetramer to ensure its enzymatic function.^9,10 The enzyme isoelectric point is 4.5, and the pH range in which it is active is between 3.5 and 10.5.¹⁰ As the crystallization of CDA revealed a second-order axis of symmetry, it is assumed that the enzyme is actually a dimer of dimers.¹¹ Vincenzetti and colleagues¹² elucidated the roles of different amino acids in CDA using targeted mutagenesis. They found that each monomer binds to a zinc atom in coordination with the cysteines C99 and C102 while C65 is essential to the correct orientation of the zinc ion. E67 has a central role in the initiation of catalysis since it is the glutamate carboxylic moiety that authorizes the deprotonation and protonation of the water molecule and amine group of the substrate, respectively, which initiates the nucleophilic attack of the hydroxide on the C4 of the pyrimidine ring.¹³ The reaction intermediate is a tetrahedral interaction between the pyrimidine ring of the substrate, the hydroxyl group formed by the water molecule, the glutamate carboxylate moiety, and the zinc ion (Figure 1).¹⁴ Each catalytic site is formed by the interaction of three monomers, which explains why it needs to be tetrameric to be active.¹¹ Among others, F137, Y33, and R68 are important for interaction with the substrate since they participate in the formation of the catalytic site and Y60 in the recognition of the substrate.^15,16 Interestingly, all of the amino acids of interest mentioned in this section are conserved in the mouse CDA protein sequence.¹⁷

Figure 1.

Tetrahedral Reaction Intermediate of Cytidine following Deamination to Uridine by Cytidine Deaminase

CDA Function

The molecular landmark of CDA is its deaminase function, which catalyzes the transformation of cytidines and deoxycytidines into uridines and deoxyuridines, respectively,¹⁸ by hydrolyzing the amine moiety into ketone with ammonia release (Figure 2).¹¹ The deamination of cytidine to uridine contributes, on the one hand, to maintaining nucleotide pool balance for DNA and RNA synthesis. On the other hand, such conversion is also necessary for the catabolism of the pyrimidine cycle, a source of carbon and nitrogen, which results in the formation of β-alanine (Figure 2).^19,20 Therefore, CDA has a complementary role within the pyrimidine salvage pathway to the de novo pyrimidine synthesis pathway (Figure 3). Large-scale interactome studies revealed many CDA potential partners (Figure 4A; Table 1), for which gene ontology analysis is provided (Figure 4B).²¹ This could lead to the discovery of new functions and new roles for this enzyme. However, none of these potential molecular interactions has been studied further.

Figure 2.

Cytidine Deaminase Reaction Equation from Cytidine to Uridine and Catabolism of Uridine to β-Alanine

Figure 3.

De Novo Pyrimidine Synthesis, Pyrimidine Salvage Pathways, and Metabolism of Pyrimidine Nucleosides to Nucleotides

Figure 4.

Putative Interactome of CDA

(A) Theoretical interactome network of CDA using BioGRID (https://www.thebiogrid.org). Physical edges are shown in yellow. (B) Gene ontology analysis of CDA potential partners. Pie charts were generated with PANTHER14.1 from the 2018_04 release.

Table 1.

Abbreviated and Full Name of CDA Potential Interactants

Acronym	Full Name
AAR2	AAR2 splicing factor homolog (S. cerevisiae)
APBA3	amyloid beta (A4) precursor protein-binding, family A, member 3
C20ORF195	chromosome 20 open reading frame 195
CDKN2C	cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4)
CLIC4	chloride intracellular channel 4
CPNE1	copine I
CPNE3	copine III
DPH2	DPH2 homolog (S. cerevisiae)
DPYSL2	dihydropyrimidinase-like 2
EVPL	envoplakin
G6PD	glucose-6-phosphate dehydrogenase
GBE1	glucan (1,4-alpha-), branching enzyme 1
LIN7A	lin-7 homolog A (C. elegans)
LNX1	ligand of numb-protein X 1, E3 ubiquitin protein ligase
LYPLA1	lysophospholipase I
MCM2	minichromosome maintenance complex component 2
MPP2	membrane protein, palmitoylated 2 (MAGUK p55 subfamily member 2)
MPP6	membrane protein, palmitoylated 6 (MAGUK p55 subfamily member 6)
MTMR6	myotubularin related protein 6
MTMR9	myotubularin related protein 9
PFKP	phosphofructokinase, platelet
PLEKHB2	pleckstrin homology domain containing, family B (evectins) member 2
PSMA1	proteasome (prosome, macropain) subunit, alpha type, 1
PTGER3	prostaglandin E receptor 3 (subtype EP3)
RC3H1	ring finger and CCCH-type domains 1
RNF181	ring finger protein 181
SDCBP	syndecan binding protein (syntenin)
SH3KBP1	SH3-domain kinase binding protein 1
TIMM8A	translocase of inner mitochondrial membrane 8 homolog A (yeast)
TRIM25	tripartite motif containing 25
TTC38	tetratricopeptide repeat domain 38
WARS	tryptophanyl-tRNA synthetase
WDYHV1	WDYHV motif containing 1
WIBG	within bgcn homolog (Drosophila)
XPO1	exportin 1
ZBTB24	zinc finger and BTB domain containing 24
ZMYM6	zinc finger, MYM-type 6

Subcellular Localization of CDA

Databases describe CDA as a cytoplasmic protein,²² which is in agreement with recycling free pyrimidines from the cytoplasm. Pyrimidines are then passively transported into the nucleus or actively transported into the mitochondria by the family of adenine nucleotide carriers ANT or SLC25A19 where they are integrated into DNA or RNA.^23,24 It is therefore commonly accepted that CDA is present mainly in the cytoplasmic fraction of cells. However, Somasekaram et al.²⁵ reported that CDA might also be nuclear, due to a bipartite NLS sequence (a specific nuclear localization signal composed of two clusters of basic amino acids, separated by 10 aa) recognized by the importin receptor. CDA can also be found in the serum, which demonstrates release from the cell.²⁶ CDA serum activity has been monitored in several cancers and it is correlated with chemotherapy response or chemotherapy-related toxicities.27, 28, 29, 30 Moreover, CDA serum activity is measured to predict abnormal pregnancy with complications,³¹ or during acute inflammation in rheumatoid arthritis.³² Indeed, as CDA leaks out into serum when the membrane of PMNs (polymorphonuclear neutrophils) is damaged,³³ one can speculate that the CDA serum activity increase during this time may reflect higher numbers of damaged cells in tissues expressing high levels of this enzyme. However, addressing CDA activity in blood is not (yet) part of routine clinical practice.

Regulation of CDA Expression

CDA expression is heterogeneous between tissues. CDA is most commonly expressed in bone marrow and liver, and more moderately or even undetectably in other tissues.³⁴ In white blood cells, CDA activity is 279 nmol/h/mg protein in peripheral lymphocytes, and 2,443 nmol/h/mg protein in granulocytes.³⁵ More generally, the activity of CDA is higher in mature hematopoietic cells than in immature cells.³⁶ In cancer cells, CDA gene is not lost, nor amplified, meaning that regulation of CDA expression is mainly at the transcriptional level.³⁷ Several binding sites to transcription factors have been described in the literature in CDA promoter or enhancer regions (cEBPs, SP1, AP1, Myc/Max, HFH2, Lyf1, ARP1 and GATA1).^5,38

As several natural mutations of the CDA gene are found in the population, and in particular within the promoter region,³⁹ this is likely to modify transcription factors binding sites and to alter the level of transcription of CDA, as exemplified by Ferrell and colleagues.³⁹ More recently, Amor-Guéret and colleagues⁴⁰ showed that the level of expression of CDA in tumors is negatively regulated by DNA methylation of the CDA promoter, as epigenetic drugs increase CDA expression. Watanabe and Uchida³⁴ showed that the injection of vitamin D3 induces an increase in the level of expression of CDA mRNA in tumors. Shord and Patel⁴¹ showed that the administration of paclitaxel (antineoplastic anticancer agent) induces a decrease in CDA mRNA level in cancer cell lines, while not having a significant effect on protein, but it nonetheless resulted in CDA increased activity. In another study, Frese et al.⁴² showed that albumin-conjugated paclitaxel (nab-paclitaxel) reduces CDA protein due to reactive oxygen species (ROS) production, and that CDA levels can be restored when cells are treated with N-acetylcysteine (NAC, a ROS scavenger molecule). This study demonstrates at least in part why cells treated by taxol are more sensitive to gemcitabine.

CDA expression seems to be finely tuned by microRNAs, a class of short, non-coding RNA experts in gene silencing. Along this line, Shao and colleagues⁴³ negatively correlated miR-484 and CDA expression in breast cancer cells; they found that miR-484 directly inhibits CDA translation by targeting CDA 3′ UTR, and relieves tumor cells’ chemoresistance. In another study, Rajabpour et al.⁴⁴ correlated the decrease of miR-608 and the upregulation of CDA protein in pancreatic cancer cells resisting to chemotherapy. Finally, Gil and colleagues45, 46, 47 showed that tumor-associated macrophages induce CDA expression in response to treatment with gemcitabine via miR-365.

Regulation of CDA Protein Activity

Many polymorphisms of the CDA gene have been identified in the population. The work of Kühn et al.⁴⁸ and Laliberté and Momparler,⁴⁹ supplemented by the numerous studies of Vincenzetti et al.,⁵⁰ have highlighted the existence of two major variants of CDA mRNA that differ by codon 27 (79A > C) that alternatively gives a glutamine or a lysine. These two proteins called CDA1 and CDA2, respectively, are capable of forming the different combinations of tetramers, i.e., five enzymatic isoforms that have essentially the same physicochemical and enzymatic properties, as the amino acid is not located in the active site of the enzyme.⁵⁰ According to these authors, the only difference would be in the net charge of each monomer, giving them a different capacity for migration under electrophoresis, with CDA1 being revealed slightly lower than CDA2 on polyacrylamide gel electrophoresis (PAGE).⁵⁰ These polymorphisms are found in the population with variable frequencies according to ethnicity. The CDA2 variant is the one most commonly found in the white population (65%).⁵¹ Koizumi and colleagues⁵² also highlighted another variant in codon 70 (208G > A) that consists of a threonine instead of an alanine, but it is found in only 4.3% of the Japanese population studied. Other studies showed that this variant appears to be mostly found in African and Asian populations.^53,54 The simple nucleotide substitutions (SNPs) 79A > C and 208G > A have been the two most studied polymorphisms.

These works have been supplemented by numerous clinical trials aimed at demonstrating a possible correlation between the numerous variants of CDA and the activity of the enzyme. Indeed, genetic variations can affect the structure of the enzyme, albeit only a few of them have been correlated with a variation in the catalytic activity of the enzyme. In humans, serum CDA activity was measured in a cohort of 150 cancer patients, ranging from 0.6 to more than 18 U/mg, which shows a high degree of inter-individual variability.²⁷

Despite some inconclusive studies and even contradictory results on the correlation between the enzymatic activity of CDA and these SNPs, studies showed that the SNP 208G>A (A70T) results in a lower activity of CDA^30,52 while the 79A > C (K27Q) mutation favors cytidine and deoxycytidine deamination.^55,56 In addition, Vincenzetti and colleagues⁵⁷ identified an activating haplotype of CDA (−451T/−92G/−31Del/79C/435C). These polymorphisms also have repercussions on drug metabolization with a 68% decrease in activity of A70T CDA mutants against Ara-C (cytarabine).⁵² The picture is less clear when it comes to the K27Q mutation. One group showed that recombinant CDA27Q has a 23%–70% decrease in activity for Ara-C⁵⁸ and a 34% decrease in activity for gemcitabine,⁵⁹ while another study showed an increase in activity for Ara-C but a decrease for decitabine for the exact same mutant.⁵⁶ Such discrepancies between CDA activity and the presence of SNPs strongly suggests that gene polymorphisms cannot fully explain inter-patient heterogeneity in serum CDA activity.

Unfortunately, data about CDA activity modulation mechanisms are rare. Along this line, database mining reveals one potential site of phosphorylation on CDA protein on Y79 residue, documented by seven different large-scale studies.⁶⁰ However, no functional studies were further carried out to better understand the consequences of CDA post-translational modification or the mechanism by which CDA is phosphorylated. To date, CDA activity regulation by the nucleotide pool remains to be documented. Although CDA products such as uridine and its derivatives or CMP have the ability to inhibit the enzyme by competing with substrates, their affinity for CDA is very moderate and their concentration in the environment is far too low to exert significant inhibition of the enzyme.⁶¹ However, regulation of CDA expression or activity by the nucleotide pool could be further considered, as this was reported for related salvage enzymes, such as cytosine deaminase (COD)⁶² or adenosine deaminase (ADA), which is regulated by the presence of inosine.⁶³

CDA Inhibitors

Current known pharmacological inhibitors of CDA are based on the same mechanism of action. The conversion of cytidine to uridine by CDA goes through an intermediate reaction resulting from the nucleophilic attack of the water/zinc complex on the C4 of the pyrimidine cycle, following which ammonia is released. By replacing the NH₃ moiety with a proton, for example (as for zebularine, Figure 5), the reaction intermediate is then blocked. The molecule therefore behaves as a powerful competitive inhibitor of the enzyme. Although other CDA inhibitors such as 3,4,5,6-tetrahydrouridine (THU) or 1,3-diazepinone riboside (DR) bear more distant structures (Figure 5), their mechanism of action is also based on blocking the enzyme in an unresolved reaction intermediate state. Vincenzetti and colleagues⁶¹ conducted a screening of CDA inhibitors that were sorted by inhibitory constants (K_is), with best candidates (THU, zebularine, and its fluorinated derivative) with K_i less than or equal to the Michaelis constant (K_m) of CDA for cytidine (i.e., <3.9). In greater details, THU was first identified and purified in 1967 following affinity capture with CDA as a bait,⁶⁴ and it was studied by Cohen and Wolfenden⁶⁵ in 1971 in Escherichia coli to understand its mechanism of action and pharmacokinetics profile. The THU mechanism of action is based on its C4 hydroxyl moiety in the pyrimidic ring, which binds to zinc instead of the water molecule.⁶⁶ However, Manome and colleagues⁶⁷ recently put at stakes the specificity of THU for CDA, as they showed that THU is cytostatic regardless of CDA expression in target cells, strongly suggesting a mechanism of action distinct from CDA inhibition, as THU can impact cell cycle distribution by inhibiting the E2F1 transcription factor.⁶⁷ Since the bioavailability of THU is weak (∼20%),⁶⁸ Tsukamoto and colleagues⁶⁹ synthesized new fluorinated versions of the drug (namely (4R)-2′-deoxy-2′,2′-difluoro-3,4,5,6-tetrahydrouridine), with better oral bioavailability. The 50% inhibitory concentration (IC₅₀) of this new compound is comparable to that of THU (0.4 μM versus 0.34 μM for THU), and it has a better pharmacokinetic profile for CDA inhibition due to its stability. This team therefore suggests that using this compound as an oral complement should improve Ara-C efficacy, yet no clinical trial has been registered to date.

Figure 5.

Schematic Representation of the Natural Substrates, Substrates Analogs Used as Chemotherapies, and Inhibitors of CDA

The CDA inhibitor zebularine was first described by Driscoll and colleagues⁷⁰ in 1980. Zebularine blocks CDA in a tetrahedral intermediate thanks to its proton in C4.¹⁴ The efficacy of this drug was also studied in Escherichia coli to understand its precise mechanism of action and the reaction kinetics.⁷¹ However, zebularine is far from being specific to CDA only, as this drug interacts with cytosine-[C5]-DNA methyltransferases (C5 MTases) to regulate DNA methylation on a large scale.⁷² Diazepinone riboside (DR) was discovered in 1981,⁷³ but its mechanism of action was only elucidated much later. Due to its structure, DR cannot interact with CDA through coordination with the water/zinc complex. Consequently, its mechanism of action is therefore slightly different from the first two above-mentioned inhibitors. Chung et al.¹¹ demonstrated that DR inhibitory activity on CDA rather results from the electrostatic interaction (π-π type) between the double bond of the DR ring and the aromatic ring of the amino acid Phe137 of the catalytic site of the enzyme. However, to date, DR has never been tested in cell cultures. Several groups recently investigated for more stable inhibitors of CDA, such as pseudoisocytidine,⁶¹ often at the expense of specificity or inhibitory potential.

Role of CDA in Physiology and Physiopathology

Animal models for CDA knockout are still cruelly lacking, meaning that the role of CDA during development is unknown to date. Historically, CDA deficiency has been long associated with Bloom syndrome,⁷⁴ due to the pioneering work of Amor-Guéret. Bloom syndrome is a rare autosomal disease caused by mutation of the Bloom (BLM) gene on both alleles that results in high genetic instability.⁷⁵ BLM encodes for a DNA helicase, the main function of which is to maintain genomic integrity.⁷⁶ BLM is relocated to DNA breaks in a complex that also contains RAD51, which reports DNA damages and thus contributes to their management by repair mechanisms. Generally, mutations found in BLM are deletions or base insertions that result in decreased gene transcription or termination of translation due to a nonsense mutation of mRNA.⁷⁶ Therefore, a defect or alteration of this protein induces a malfunction of the homologous recombinant repair system. Spontaneous mutations of somatic cells are thus much more frequent than in the wild-type (WT) BLM genetic background. A systematic feature of genetic instability associated with Bloom syndrome is the presence of segment exchanges between sister chromatids of a chromosome, which has long been a method of diagnosing this disease, now confirmed by sequencing.⁷⁷ As a result, patients with this condition develop congestive redness of the skin, called telangiectatic erythema, as well as growth retardation and other malformations.⁷⁸. These patients are also more predisposed to cancer, in particular hematologic cancers and carcinomas of diverse organs, which considerably reduces their life expectancy.⁷⁴ Amor-Guéret and colleagues⁷⁹ nicely showed that loss of CDA expression is concomitant with BLM mutation. They found that CDA has an important role in maintaining the nucleotide pool to prevent replicative stress that is harmful to the cell. Among the many chromatid segregation defects, chromatin bridge formation (consequences of poor sister chromatid segregation) is directly due to the absence of BLM, while the presence of ultra-fine bridges (UFBs) (which follow incomplete DNA replication when cells enter mitosis) results from an imbalance in the pyrimidine pool due to CDA deregulation.⁸⁰ The mechanism explaining this phenomenon is the accumulation of dCTP due to the absence of CDA, which induces a decrease in the activity of PARP-1, a protein involved in the response to DNA damage.⁸⁰ Witte and colleagues¹⁹ supplemented this hypothesis by showing that the accumulation of intermediate species of pyrimidine catabolism due to CDA invalidation is toxic.

As stated before, a significant functional aspect of CDA is the ability to deaminate synthetic or natural cytidine analogs. This is the reason why numerous studies have been focusing on CDA expression as a marker of cancer chemoresistance since the 1960s, with the emergence of the first anti-metabolite, anticancer analogs of cytidine and deoxycytidine such as Ara-C.⁸¹ Indeed, CDA is recognized as a key chemoresistance factor to cytidine and deoxycytidine analogs such as Ara-C, gemcitabine (dFdC), or decitabine (5-Aza-dC)⁸² in vitro (Figure 5), since it catalyzes their transformation into inactive metabolites.^81,83 Indeed, CDA precedes synthetic metabolites and natural substrates with similar affinities (K_m = 11 μM for Ara-C, K_m = 3.9 μM for cytidine).^61,84 In mice, CDA largely participates in gemcitabine inactivation and clearance.²⁷ In humans, 80% of the intravenous Ara-C bolus is eliminated in the urine, 90% of which is in its uracil form, thus strongly suggesting direct and active “detoxification” by CDA.⁸⁵ Furthermore, a study conducted on 40 patients with pancreatic cancers treated with gemcitabine correlated high activity of CDA (>6 U/mg) with progressive disease and concluded that patients with high CDA activity are 5-fold more likely to progress following gemcitabine-based therapy.⁸⁶ Very recently, commensal CDA was identified as a key player of chemoresistance originating from the tumor microbiome. Geller et al.⁸⁷ identified that 76% of patients with pancreatic cancer are positive for gammaproteobacteria, which possess an orthologous CDA capable of deaminating gemcitabine and so participate in resistance to treatment. Quite the contrary, capecitabine, a synthetic deoxycytidine analog, has to be deaminated to be active. Enforced CDA expression consequently improves capecitabine chemotherapy efficacy in vitro.⁸⁸

Therefore, CDA has an important therapeutic role since its modulation would make it possible to defeat the chemoresistance of some cancers. Alternatively, CDA levels may also predict toxicities associated with chemotherapies; indeed, patients with lower CDA activity than normal treated with chemotherapy (gemcitabine or Ara-C) have serious side effects due to exacerbated toxicity of the treatment.^27,89,90 Along the same line, Ciccolini et al.²⁷ showed that CDA-deficient patients (i.e., with a CDA activity <1.3 U/mg) develop serious adverse effects (grade 3–4) when treated with gemcitabine. Thus, CDA may have a role in genetic diseases and in response to treatment in cancer patients. CDA might be an appealing molecular target, particularly in patients treated with chemotherapy.

Therapeutic Strategies Based on CDA Expression

Given the high heterogeneity of expression of CDA, different therapeutic strategies can be considered depending on the pathological context. Most cancers underexpress CDA compared to healthy tissues.^40,91 Amor-Guéret and colleagues⁴⁰ focused on breast tumors that poorly express CDA due to DNA methylation. They showed that tumors with low CDA expression are more sensitive to aminoflavone derivative AFP464. Tissues that express higher levels of CDA are, however, resistant to these drugs, which would place CDA as a predictive biological marker of response. High-CDA-expressing tumors should be in theory more resistant to cytidine-based therapies. In line with this assumption, several studies combining various chemotherapy and CDA inhibitors have been conducted so far.^68,92, 93, 94, 95 However, only a few clinical trials resulted from these studies. Phase I clinical trials studied the effects of 5-fluoro-2′-deoxcytidine with THU (ClinicalTrials.gov: NCT00359606 and NCT01041443), and two phase II clinical trials were completed recently combining 5-fluoro-2′-deoxcytidine with THU and another combining THU and decitabine with nivolumab (ClinicalTrials.gov: NCT00978250 and NCT02664181), but none of them has shared the results yet. Another interesting hypothesis made by Amit and Gil⁴⁵ could be to target macrophages in order to reduce CDA levels and thus chemosensitize the tumor to gemcitabine. Zauri et al.⁹¹ recently performed an elegant study, focusing on tissues that express high levels of CDA, including pancreatic cancer, in which CDA appears to be expressed mainly in epithelial tumor cells and not in the stroma.⁹⁶ They took advantage of CDA overexpression to metabolize synthetic deoxycytidine analogs to be inserted into DNA. In greater details, overexpression of CDA provokes the deamination of deoxycytidine analogs 2′-deoxy-5-(hydroxymethyl)cytidine (5hmdC) and 5-formyl-2′deoxycytidine (5fdC) into 5hmdU and 5fdU, respectively, which are then phosphorylated and incorporated into DNA, triggering cell cycle arrest and cell death,⁹¹ due to activation of surveillance factors such as uracil glycosylase, which causes multiple DNA breaks.⁹⁷ In normal cells, these nucleotides are not integrated into DNA due to the presence of cytidine monophosphate kinase 1 (CMPK1), involved in nucleoside recycling and metabolism pathways, which acts as barrier to protect the genome. These observations clearly provide a therapeutic opportunity to treat cancers that overexpress CDA.

Another interesting issue is that CDA, through induction of chemoresistance, also protects tissues from the toxic effect of such intravenous drugs throughout the body, as severe toxicity is observed in rapidly proliferating tissues with low levels of CDA.^27,89,90 A therapeutic strategy developed by Moritz and colleagues^98,99 consists in overexpressing CDA in these tissues using gene transfer, to protect them from side effects. This group demonstrated the interest of such an approach in acute leukemia treated with Ara-C post-transplantation to protect the healthy marrow.

Lastly, stepping back from cancer management, CDA can also be also considered as a target per se. Indeed, CDA, as well as other enzymes from the pyrimidine salvage pathway, may represent interesting targets for killing pathogens such as trypanosomiasis (flagellated protozoa causing, for example, sleeping sickness or leishmaniasis) that do not produce some enzymes in the de novo pathway and are therefore dependent on CDA for their nucleotide synthesis.¹⁰⁰

Conclusion

CDA is historically known to be involved in the salvage of pyrimidines by recycling free cytidines and deoxycytidines into uridines and deoxyuridines, respectively, for DNA and RNA synthesis. This enzyme is well conserved among species, highlighting the importance of this pathway during evolution. In Bloom syndrome, CDA defects induce replicative stress. In cancer, CDA detoxifies cytidine and deoxycytidine analog-based therapies such as gemcitabine and Ara-C and can be considered as a key player in tumor resistance to treatment. Taking advantage or modulating CDA activity in patients is within reach and might constitute a promising strategy in cancer therapy. Alternatively, CDA can be heterogeneously expressed in tumors, regardless of chemotherapeutic treatment. This advocates for a better understanding of the molecular mechanisms involved in CDA regulation of expression, but it also suggests a new and undiscovered role of CDA in oncogenesis. Thus, better understanding the role and function of CDA may not only help overcome chemoresistance and improve the management of patients with cancer, notably pancreatic cancer, it may also reveal the unforeseen role of CDA during carcinogenesis to help develop new therapeutic strategies.

Author Contributions

A.F. and P.C. conducted the bibliographic search and wrote the paper.

Conflicts of Interest

The authors declare no competing interests.