Cytidine Deaminase Overexpression Research Articles

Cytidine Deaminase Enhances Liver Cancer Invasion by Modulating Epithelial-Mesenchymal Transition via NFκB Signaling

BackgroundCancer metastasis is the leading cause of cancer-related deaths, underscoring the importance of understanding its underlying mechanisms. Hepatocellular carcinoma (HCC), a highly malignant type of cancer, was selected as our research model. Material and methodsWe aimed to develop high-metastatic cell lines using in vitro and in vivo selection strategies and identify critical metastasis-related genes through microarray analyses by comparing them with parental cells. ResultsOur results showed that the high-metastatic cell lines exhibited significantly stronger invasion abilities than parental cells. Microarray analyses identified cytidine deaminase (CDA), a gene associated with systemic chemotherapy resistance, as one of the overexpressed genes in the high-metastatic cells. Data analysis from The Cancer Genome Atlas Program revealed that while CDA is downregulated in HCC, patients with high CDA expression tend to have poorer prognoses. Cell models confirmed that CDA overexpression enhances cell migration and invasion, whereas CDA knockdown inhibits these abilities. Investigating the key molecules involved in the epithelial-mesenchymal transition (EMT), we found that CDA overexpression increases the expression of fascin, N-cadherin, β-catenin, and snail while decreasing E-cadherin expression. Conversely, CDA knockdown produced opposite results. Additionally, we discovered that CDA regulates NF-κB signaling, which controls the expression of N-cadherin, thereby promoting the invasion capability of HCC cells. ConclusionsWe isolated highly metastatic cells and identified potential HCC metastasis-related genes. CDA promotes cell invasion by regulating EMT through the NF-κB pathway. Future studies are warranted to explore the potential of CDA as a biomarker for prognosis and therapeutic decision-making.

Nucleotide metabolism in cancer cells fuels a UDP-driven macrophage cross-talk, promoting immunosuppression and immunotherapy resistance

Many individuals with cancer are resistant to immunotherapies. Here, we identify the gene encoding the pyrimidine salvage pathway enzyme cytidine deaminase (CDA) among the top upregulated metabolic genes in several immunotherapy-resistant tumors. We show that CDA in cancer cells contributes to the uridine diphosphate (UDP) pool. Extracellular UDP hijacks immunosuppressive tumor-associated macrophages (TAMs) through its receptor P2Y6. Pharmacologic or genetic inhibition of CDA in cancer cells (or P2Y6 in TAMs) disrupts TAM-mediated immunosuppression, promoting cytotoxic T cell entry and susceptibility to anti-programmed cell death protein 1 (anti-PD-1) treatment in resistant pancreatic ductal adenocarcinoma (PDAC) and melanoma models. Conversely, CDA overexpression in CDA-depleted PDACs or anti-PD-1-responsive colorectal tumors or systemic UDP administration (re)establishes resistance. In individuals with PDAC, high CDA levels in cancer cells correlate with increased TAMs, lower cytotoxic T cells and possibly anti-PD-1 resistance. In a pan-cancer single-cell atlas, CDAhigh cancer cells match with T cell cytotoxicity dysfunction and P2RY6high TAMs. Overall, we suggest CDA and P2Y6 as potential targets for cancer immunotherapy.

Activation-induced cytidine deaminase overexpression in double-hit lymphoma: potential target for novel anticancer therapy

Activation-induced cytidine deaminase (AID) is one kind of the mutant enzymes, which target regulating the immunoglobulin (Ig) gene in Burkitt’s lymphoma to initiate class switch recombination (CSR), resulting in c-Myc chromosomal translocation. However, it is not clear that whether AID induces c-Myc/IgH translocation in double-hit lymphoma (DHL) with c-Myc gene translocation. In this study, the AID in DHL tissues and classical diffuse large b-cell lymphoma (DLBCL) tissues were compared. The results suggested that AID is of important value in predicting DHL, stronger CSR of AID was observed in DHL patients, which exhibited AID overexpression and c-Myc gene translocation of DHL after CSR induction. It is concluded that AID directly induces CSR in DHL and may result in c-Myc gene translocation. Targeting AID may be a good treatment regimen for DHL.

Abstract 4576: Oncolytic vaccinia virus TG6010 expressing cytidine deaminase induces DNA damage and shows potent anti-tumor effects

Abstract The recombinant Vaccinia virus Copenhagen strain deleted in Thymidine Kinase and Ribonucleotide Reductase (VVCOPTK−RR−) is a potent and versatile oncolytic platform that has demonstrated strong activity in various preclinical models. The deletion of such VV genes inhibits viral replication in normal cells, while retaining therapeutic replication in tumor cells. TG6002, a VVCOPTK−RR− expressing the suicide gene FCU1, is under investigation in Phase I trials in patients with advanced gastrointestinal tumors. We have developed a new product based on the VVCOPTK−RR− vector, named TG6010, expressing the human cytidine deaminase (CDA) that efficiently catalyzes the deamination of cytidine and deoxycytidine to uridine and deoxyuridine, respectively. The tumor specific expression of CDA by the VV leads to a depletion of cytidine and deoxycytidine. This cytidine/deoxycytidine depletion resulting from CDA overexpression, activated a DNA damage response highlighted by an induction of H2AX phosphorylation. Next, to validate the potential therapeutic use of TG6010, we analyzed the effects of the virus on human xenograft tumors implanted in mice. We observed, after systemic injection of TG6010, high expression of CDA in the tumors with a significant increase in DNA damage in vivo as revealed by the γH2AX foci assay. In addition, intraperitoneal administration of TG6010 in a xenograft model of human ovarian carcinomatosis resulted in significantly prolonged animal survival. In conclusion, the oncolytic vaccinia virus expressing the cytidine deaminase (TG6010) has shown potent anti-tumor effects. Mechanistically, due to the cytidine deaminase overexpression, we observed induction of a DNA damage response. These are data encouraging of clinical development for this highly potent oncolytic Vaccinia virus TG6010 against ovarian cancer. Citation Format: Johann Foloppe, Juliette Kempf, Christelle Pichon, Annie Findeli, Isabelle Farine, Julie Hortelano, Baptiste Moreau, Michèle Klein, Sandrine Cochin, Renée Brandely, Véronique Koerper, Nathalie Silvestre, Christelle Machon, Jérôme Guitton, Eric Quéméneur, Philippe Erbs. Oncolytic vaccinia virus TG6010 expressing cytidine deaminase induces DNA damage and shows potent anti-tumor effects [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4576.

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

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.

5-Fluorotroxacitabine Displays Potent Anti-Leukemic Effects and Circumvents Resistance to Ara-C

The cytotoxic nucleoside cytarabine (Ara-C) is a cornerstone of AML induction and consolidation therapies, but drug resistance contributes to disease relapse. Among clinically relevant mechanisms of Ara-C resistance is the over-expression of cytidine deaminase (CDA). CDA degrades Ara-C through deamination into inactive metabolites and its increased expression in AML cells results in drug resistance. Therefore, nucleoside analogues that are not degraded by CDA may be new therapeutic agents for this disease.Through our efforts to develop novel nucleoside analogues that overcome mechanisms of resistance to Ara-C, we identified 5-fluorotroxacitabine (5FTRX), a chain-terminating cytidine-based L-nucleoside. First, we tested whether 5FTRX was a substrate of CDA by evaluating its cytotoxicity in HEK-293 cells over-expressing CDA. Compared to wild type cells, Ara-C was 6-fold less active in cells over-expressing CDA (IC50 wild type HEK293: 3.7µM (95% CI: 3.2-4.2uM) vs IC50 CDA over-expression: 25.6uM (95% CI 21.6-30.3uM), consistent with degradation of Ara-C by CDA. In contrast, cells over-expressing CDA were more sensitive to 5FTRX, compared to wild type cells (IC50 wild type HEK293: 5.5uM (95% CI 4.7-6.3uM) vs IC50 CDA over-expression: 0.4uM (95% CI 0.4-0.5uM), potentially due to depletion of endogenous nucleotide pools by increased CDA. Thus, 5FTRX is not a substrate of CDA.We then treated 6 AML cell lines for 72 hours with increasing concentrations of 5FTRX and then measured cell growth and viability using the MTS assay. 5FTRX reduced the growth of 5 of 6 tested AML cell lines, with mean IC50 values (n= > 3) of 92nM (TEX), 130nM (KG1a), 150nM (MV4-11), 410nM (NB4), and 250nM (OCI-AML2). In contrast, K562 cells that have mutant p53 were resistant with an IC50 >50,000nM. The K562 cells were also resistant to Ara-C. 5FTRX reduced the clonogenic growth of primary AML samples (n=2) with a >90% reduction in growth at 50nM, demonstrating that 5FTRX targets leukemia initiating cells. To test whether 5FTRX induced DNA damage in AML cells, we measured changes in phosphorylated H2AX (pH2AX) after 5FTRX treatment. In the tested cell lines (OCI-AML2, TEX, NB4 and MV4-11 cells), 5FTRX increased pH2AX at concentrations associated with loss of viability.Finally, we evaluated the efficacy and toxicity of 5FTRX in mouse models of AML. 5FTRX displayed robust and dose-dependent inhibition of MV4-11 and OCI-AML2 tumors in mouse xenograft models, with complete tumor regressions and long-lasting tumor growth delays (>20 days at 100mg/kg in MV4-11 and >30 days in OCI-AML2) after a single cycle of 5 days of once-daily drug treatment, with no changes in body weight or behavior. The efficacy of 5FTRX was superior to Ara-C dosed for daily for 10 days at its MTD, 60 mg/kg (35%TGI, no regressions). MV4-11 tumors treated with 5FTRX displayed induction of pH2AX, reduction in proliferation (BrdU incorporation) and induction of necrosis, consistent with the mode of action and dramatic tumor regressions. The anti-leukemic effects of 5FTRX were further evaluated in mice engrafted intrafemorally with primary AML cells. 5FTRX (100 mg/kg i.p, x 5 days) reduced primary AML engraftment >95% compared to controls without toxicity (Fig 1).In summary, 5FTRX was identified as a potent inhibitor of AML cell proliferation in vitro and in vivo. In contrast to Ara-C, 5FTRX was not a substrate for CDA. Further development of analogues of 5FTRX is ongoing using protide prodrugs of the 5FTRX monophosphate to further increase potency and evade additional resistance mechanisms. Taken together, our findings support the further development of 5FTRX-based therapies for the treatment of AML, including AML patients with reduced sensitivity to Ara-C through high CDA expression.#AB and TPS contributed equally to this work.#ADS and MA contributed equally to this work. [Display omitted] DisclosuresRizoska:Medivir AB: Employment, Equity Ownership. Rydergård:Medivir AB: Employment, Equity Ownership. Kylefjord:Medivir AB: Employment, Equity Ownership. Rraklli:Medivir AB: Employment. Eneroth:Medivir AB: Employment, Equity Ownership. Pinho:Medivir AB: Employment, Equity Ownership. Norin:Medivir AB: Employment, Equity Ownership. Bylund:Medivir AB: Employment, Equity Ownership. Moses:Medivir AB: Employment, Equity Ownership. Bethell:Medivir AB: Employment, Equity Ownership. Targett-Adams:Medivir AB: Employment, Equity Ownership. Schimmer:Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Otsuka Pharmaceuticals: Consultancy; Medivir AB: Research Funding. Albertella:Medivir AB: Employment, Equity Ownership.

CDA directs metabolism of epigenetic nucleosides revealing a therapeutic window in cancer.

SummaryCells require nucleotides to support DNA replication and to repair damaged DNA. In addition to de novo synthesis, cells recycle nucleotides from the DNA of dying cells or from cellular material ingested through the diet. Salvaged nucleosides come with the complication that they can contain epigenetic modifications. Since epigenetic inheritance of DNA methylation mainly relies on copying of the modification pattern from parental strands1-3, random incorporation of pre-modified bases during replication could have profound implications for epigenome fidelity and yield adverse cellular phenotypes. Although the salvage mechanism of 5-methyl-2′deoxycytidine (5mdC) has been investigated before4-6, currently it remains unknown how cells deal with the recently identified oxidised forms of 5mdC – 5-hydroxymethyl-2′deoxycytidine (5hmdC), 5-formy-2′deoxycytidine (5fdC) and 5-carboxyl-2′deoxycytidine (5cadC)7-10. Here we demonstrate that enzymes of the nucleotide salvage pathway display substrate selectivity, effectively protecting newly synthesized DNA from the incorporation of epigenetically modified forms of cytosine. Thus cell lines and animals can tolerate high doses of these modified cytidines without any deleterious effects on physiology. Interestingly, by screening cancer cell lines for growth defects following exposure to 5hmdC, we unexpectedly identify a subset of cell lines where 5hmdC or 5fdC administration leads to cell lethality. Using genomic approaches we discover that the susceptible cell lines overexpress cytidine deaminase (CDA). CDA converts 5hmdC and 5fdC into variants of uridine that are incorporated into DNA, resulting in accumulation of DNA damage and ultimately, cell death. Our observations extend current knowledge of the nucleotide salvage pathway by revealing the metabolism of oxidised epigenetic bases, and suggest a therapeutic option for cancers, such as pancreatic cancer, that have CDA overexpression and are resistant to treatment with other cytidine analogues11.

Myeloprotection by Cytidine Deaminase Gene Transfer in Antileukemic Therapy

Gene transfer of drug resistance (CTX-R) genes can be used to protect the hematopoietic system from the toxicity of anticancer chemotherapy and this concept recently has been proven by overexpression of a mutant O6-methylguaninemethyltransferase in the hematopoietic system of glioblastoma patients treated with temozolomide. Given its protection capacity against such relevant drugs as cytosine arabinoside (ara-C), gemcitabine, decitabine, or azacytidine and the highly hematopoiesis-specific toxicity profile of several of these agents, cytidine deaminase (CDD) represents another interesting candidate CTX-R gene and our group recently has established the myeloprotective capacity of CDD gene transfer in a number of murine transplant studies. Clinically, CDD overexpression appears particularly suited to optimize treatment strategies for acute leukemias and myelodysplasias given the efficacy of ara-C (and to a lesser degree decitabine and azacytidine) in these disease entities. This article will review the current state of the art with regard to CDD gene transfer and point out potential scenarios for a clinical application of this strategy. In addition, risks and potential side effects associated with this approach as well as strategies to overcome these problems will be highlighted.

Cytidine Deaminase Genetic Variants Influence RNA Expression and Cytarabine Cytotoxicity in Acute Myeloid Leukemia

Cytidine deaminase (CDA) irreversibly deaminates cytarabine (Ara-C), a key component of acute myeloid leukemia (AML) induction and consolidation therapy. CDA overexpression results in Ara-C resistance, while decreased expression is associated with toxicity. We evaluated factors influencing variation in CDA mRNA expression in adult AML patients and normal controls, and how they contributed to Ara-C cytotoxicity in AML cells. CDA mRNA expression in 100 de novo AML patients and 36 normal controls were determined using quantitative reverse-transcriptase PCR. Genetic variants in the CDA gene were screened by direct sequencing. IC₅₀ of Ara-C was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. CDA RNA expression as well as Ara-C IC₅₀ showed wide variation in AML samples and normal controls. Fourteen sequence variants were identified, three of which (-33delC, intron 2 TCAT repeat and the 3´untranslated region 816delC variants) showed significant association with RNA expression and the nonsynonymous coding variant 79A>C was associated with Ara-C cytotoxicity. CDA genetic variants explain the variation in RNA expression and may be candidates for individualizing Ara-C therapy.

Gene transfer of cytidine deaminase protects myelopoiesis from cytidine analogs in an in vivo murine transplant model

AbstractHematopoietic stem cell gene transfer of the drug-resistance gene cytidine deaminase (CDD) protecting cells from the cytotoxic cytidine analogs cytarabine and gemcitabine was investigated in a murine transplant model. Following transplantation of CDD-transduced cells and cytarabine application (500 mg/kg; days 1-4; intraperitoneally) significant myeloprotection was demonstrated with nadir counts of peripheral blood granulocytes and thrombocytes of 2.9 ± 0.6/nL versus 0.7 ± 0.1/nL (P < .001) and 509 ± 147/nL versus 80 ± 9/nL (P = .008), respectively (CDD versus control). Protection also was observed from otherwise lethal gemcitabine treatment (250 mg/kg; days 1-3). Stable levels of gene-marked cells in primary and secondary recipients were demonstrated for up to 9 months, and whereas CDD overexpression clearly reduced B- and T-lymphocyte numbers, no major toxicity was observed in the myeloid compartment. Despite the profound myeloprotective properties, however, CDD overexpression did not allow for pharmacologic enrichment of transduced hematopoiesis in our model. Thus, in summary, our data establish CDD as a drug-resistance gene highly suitable for myeloprotective purposes, which, given the lack of selection observed in our hands, might best be used in combination with selectable drugresistance genes such as MGMT (P140K) or MDR1.

Resistance to cytarabine and gemcitabine and in vitro selection of transduced cells after retroviral expression of cytidine deaminase in human hematopoietic progenitor cells

Overexpression of the detoxifying enzyme cytidine deaminase (CDD) renders normal and leukemic hematopoietic cells resistant to cytarabine (1-beta-D-arabinofuranosylcytosine), and studies on murine cells have suggested transgenic CDD overexpression as a way to reduce the substantial myelotoxicity induced by the deoxycytidine analogs cytarabine and gemcitabine (2',2'-difluorodeoxycytidine). We now have investigated CDD (over-)expression in the human hematopoietic system. Retroviral gene transfer significantly increased the resistance of CDD-transduced cord blood and peripheral blood-derived progenitor cells for doses ranging from 20-100 nM cytarabine and 8-10 nM gemcitabine. Protection was observed for progenitors of erythroid as well as myeloid differentiation, though the degree of protection varied for individual drugs. In addition, significant selection of CDD-transduced cells was obtained after a 4-day culture in 30-100 nM cytarabine. Thus, our data demonstrate that overexpression of CDD cDNA results in significant protection of human progenitors from cytarabine- as well as gemcitabine-induced toxicity, and allows in vitro selection of transduced cells. This strongly argues for a potential therapeutic role of CDD gene transfer in conjunction with dose-intensive cytarabine- or gemcitabine-containing chemotherapy regimen.

Gene Transfer of Cytidine Deaminase (CDD) Protects from Cytidine Analogs in an In Vivo Murine Transplant Model.

In vitro gene transfer of CDD protects murine and human hematopoietic cells from cytarabine (araC) and gemcitabine induced toxicity. Thus CDD represents a drug resistance gene with considerable clinical potential. We now have examined CDD gene transfer in a bone marrow transplant model with C57Bl/6 mice using the retroviral vectors SF91-CDD-IRES-EGFP and SFβ1-EGFP (control).Protection from araC was investigated in three experiments. Gene transfer efficiency was analysed by EGFP expression in the peripheral blood after hematopoietic reconstitution. In these experiments 42±4%, 31±1% and 69±2% EGFP+ granulocytes and 9±1%, 10±1% and 36±3% EGFP+ lymphocytes were detected following SF91-CDD-IRES-EGFP transduction (n=5–6 for each experiment). After araC application (500mg/kg; 1xdaily; d1-4; i.p.) substantial protection of granulocytes and thrombocytes was observed. Following the first treatment cycle nadir counts [x103/μl] were 2.9±0.6 vs. 0.7±0.1, 1.1±0.2 vs. 0.5±0.1 and 2.3±0.3 vs. 0.7±0.2 for granulocytes and 510±148 vs. 80±9, 165±48 vs. 70±42 and 728±133 vs. 85±22 for thrombocytes, respectively (SF91-CDD-IRES-EGFP vs. control; all differences significant at p<0.01 for granulocytes and p<0.05 for thrombocytes). Similar protection was noted during repetitive araC treatment cycles. However, no significant enrichment of CDD transduced cells following araC application was seen and lack of selection was confirmed in two additional experiments utilizing low efficiency gene transfer (<5% and <12% EGFP+ granulocytes or lymphocytes throughout the experiment). No major toxicity of CDD-expression in hematopoietic cells was noted and steady levels of gene marked cells were seen for up to six months in primary and at 5 weeks in secondary recipients. Substantial protection of hematopoiesis by CDD overexpression also was observed for gemcitabine. Mice receiving CDD transduced cells and exposed to 250mg/kg gemcitabine (1xdaily i.p., d1-2), showed granulocyte and thrombocyte nadirs [x103/μl] of 2.4±0.6 vs. 1.4±0.2 and 416±62 vs. 277±97 (SF91-CDD-IRES-EGFP vs. control). When a third day of gemcitabine treatment was added, all mice in the control group died within 4 days while animals receiving CDD-transduced bone marrow survived with nadir counts [x103/μl] of 0.8±0.1 granulocytes and 1130±292 thrombocytes [n=3]. Again, no enrichment of transduced cells was observed.In summary, our data demonstrate efficient protection from araC and gemcitabine hematotoxicity by CDD overexpression. This protective effect was on progenitor rather than stem cells, as no selection of CDD-expressing cells was demonstrated. Thus, a promising approach to utilize CDD clinically may be the combination with a selectable drug resistance gene such as MGMT or MDR-1 to allow enrichment of transduced cells while at the same time protecting against additional drugs.

The role of cytidine deaminase and GATA1 mutations in the increased cytosine arabinoside sensitivity of Down syndrome myeloblasts and leukemia cell lines.

Myeloblasts from Down syndrome (DS) children with acute myeloid leukemia (AML) are significantly more sensitive in vitro to 1-beta-D-arabinofuranosylcytosine (ara-C) and generate higher 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP) than non-DS AML myeloblasts. Semiquantitative reverse transcription-PCR analyses demonstrated that transcripts for cytidine deaminase (CDA) were 2.7-fold lower in DS than for non-DS myeloblasts. In contrast, transcripts of cystathionine-beta-synthase and deoxycytidine kinase were a median 12.5- and 2.6-fold higher in DS compared with non-DS myeloblasts. The ratio of deoxycytidine kinase/CDA transcripts significantly correlated with ara-C sensitivities and ara-CTP generation. In clinically relevant AML cell line models, high cystathionine-beta-synthase transcripts in DS CMK cells were accompanied by 10-fold greater ara-C sensitivity and 2.4-fold higher levels of ara-CTP compared with non-DS CMS cells. Overexpression of CDA in non-DS THP-1 cells was associated with a 100-fold decreased ara-C sensitivity and 40-fold decreased ara-CTP generation. THP-1 cells secreted CDA into the incubation media and converted extracellular ara-C completely to 1-beta-D-arabinofuranosyluracil within 30 min. Rapid amplification of 5'-cDNA ends (5'-RACE) and reverse transcription-PCR assays identified short- (sf) and long-form (lf) CDA transcripts in THP-1 cells with different 5' untranslated regions and translational start sites; however, only the latter resulted in the active CDA. Although 5' flanking sequences for both CDA transcripts exhibited promoter activity in reporter gene assays, activity for the CDAlf was low. The presence of several GATA1 binding sites in the CDAsf promoter and the uniform detection of GATA1 mutations in DS megakaryocytic leukemia suggested the potential role of GATA1 in regulating CDA transcription and the CDAsf promoter acting as an enhancer. Transfection of GATA1 into Drosophila Mel-2 cells stimulated the CDAlf promoter in a dose-dependent fashion. Additional identification of the mechanisms of differential expression of genes encoding enzymes involved in ara-C metabolism between DS and non-DS myeloblasts may lead to improvements in AML therapy.