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Nucleosides, nucleotides, and their analogues are an important class of molecules that are used as substrates in research of enzymes and nucleic acid, or as antiviral and antineoplastic agents. Nucleoside phosphorylation is usually achieved with chemical methods; however, enzymatic phosphorylation is a viable alternative. Here, we present a chemoenzymatic synthesis of modified cytidine monophosphates, where a chemical synthesis of novel N 4 -modified cytidines is followed by an enzymatic phosphorylation of the nucleosides by nucleoside kinases. To enlarge the substrate scope, multiple mutant variants of Drosophila melanogaster deoxynucleoside kinase ( Dm dNK) (EC:2.7.1.145) and Bacillus subtilis deoxycytidine kinase ( Bs dCK) (EC:2.7.1.74) have been created and tested. It has been determined that certain point mutations in the active sites of the kinases alter their substrate specificities noticeably and allow phosphorylation of compounds that had been otherwise not phosphorylated by the wild-type Dm dNK or Bs dCK.

Anticancer nucleosides are effective against solid tumors and hematologic malignancies, but typically are prone to nucleoside metabolism resistance mechanisms. Using a nucleoside-specific multiplexed high-throughput screening approach, we discovered 4'-ethynyl-2'-deoxycytidine (EdC) as a third-generation anticancer nucleoside prodrug with preferential activity against diffuse large B-cell lymphoma (DLBCL) and acute lymphoblastic leukemia (ALL). EdC requires deoxycytidine kinase (DCK) phosphorylation for its activity and induces replication fork arrest and accumulation of cells in S-phase, indicating it acts as a chain terminator. A 2.1Å cocrystal structure of DCK bound to EdC and UDP reveals how the rigid 4'-alkyne of EdC fits within the active site of DCK. Remarkably, EdC was resistant to cytidine deamination and SAMHD1 metabolism mechanisms and exhibited higher potency against ALL compared with FDA-approved nelarabine. Finally, EdC was highly effective against DLBCL tumors and B-ALL in vivo. These data characterize EdC as a preclinical nucleoside prodrug candidate for DLBCL and ALL., (©2023 American Association for Cancer Research.)

Pancreatic ductal adenocarcinoma (PDAC) is considered one of the most lethal forms of cancer. Although in the last decade, an increase in 5-year patient survival has been observed, the mortality rate remains high. As a first-line treatment for PDAC, gemcitabine alone or in combination (gemcitabine plus paclitaxel) has been used; however, drug resistance to this regimen is a growing issue. In our previous study, we reported MYC/glutamine dependency as a therapeutic target in gemcitabine-resistant PDAC secondary to deoxycytidine kinase (DCK) inactivation. Moreover, enrichment of oxidative phosphorylation (OXPHOS)-associated genes was a common property shared by PDAC cell lines, and patient clinical samples coupled with low DCK expression was also demonstrated, which implicates DCK in cancer metabolism. In this article, we reveal that the expression of most genes encoding mitochondrial complexes is remarkably upregulated in PDAC patients with low DCK expression. The DCK-knockout (DCK KO) CFPAC-1 PDAC cell line model reiterated this observation. Particularly, OXPHOS was functionally enhanced in DCK KO cells as shown by a higher oxygen consumption rate and mitochondrial ATP production. Electron microscopic observations revealed abnormal mitochondrial morphology in DCK KO cells. Furthermore, DCK inactivation exhibited reactive oxygen species (ROS) reduction accompanied with ROS-scavenging gene activation, such as SOD1 and SOD2. SOD2 inhibition in DCK KO cells clearly induced cell growth suppression. In combination with increased anti-apoptotic gene BCL2 expression in DCK KO cells, we finally reveal that venetoclax and a mitochondrial complex I inhibitor are therapeutically efficacious for DCK-inactivated CFPAC-1 cells in in vitro and xenograft models. Hence, our work provides insight into inhibition of mitochondrial metabolism as a novel therapeutic approach to overcome DCK inactivation-mediated gemcitabine resistance in PDAC patient treatment., (© 2024. The Author(s).)

Deoxycytidine analogues (dCas) are widely used for the treatment of malignant diseases. They are commonly inactivated by cytidine deaminase (CDD), or by deoxycytidine monophosphate deaminase (dCMP deaminase). Additional metabolic pathways, such as phosphorylation, can substantially contribute to their (in)activation. Here, a new technique for the analysis of these pathways in cells is described. It is based on the use of 5-ethynyl 2'-deoxycytidine (EdC) and its conversion to 5-ethynyl 2'-deoxyuridine (EdU). Its use was tested for the estimation of the role of CDD and dCMP deaminase in five cancer and four non-cancer cell lines. The technique provides the possibility to address the aggregated impact of cytidine transporters, CDD, dCMP deaminase, and deoxycytidine kinase on EdC metabolism. Using this technique, we developed a quick and cheap method for the identification of cell lines exhibiting a lack of CDD activity. The data showed that in contrast to the cancer cells, all the non-cancer cells used in the study exhibited low, if any, CDD content and their cytidine deaminase activity can be exclusively attributed to dCMP deaminase. The technique also confirmed the importance of deoxycytidine kinase for dCas metabolism and indicated that dCMP deaminase can be fundamental in dCas deamination as well as CDD. Moreover, the described technique provides the possibility to perform the simultaneous testing of cytotoxicity and DNA replication activity., (© 2023. The Author(s).)

The aryl hydrocarbon receptor (AHR) is a transcription factor that is commonly upregulated in pancreatic ductal adenocarcinoma (PDAC). AHR hinders the shuttling of human antigen R (ELAVL1) from the nucleus to the cytoplasm, where it stabilises its target messenger RNAs (mRNAs) and enhances protein expression. Among these target mRNAs are those induced by gemcitabine. Increased AHR expression leads to the sequestration of ELAVL1 in the nucleus, resulting in chemoresistance. This study aimed to investigate the interaction between AHR and ELAVL1 in the pathogenesis of PDAC in vitro. AHR and ELAVL1 genes were silenced by siRNA transfection. The RNA and protein were extracted for quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot (WB) analysis. Direct binding between the ELAVL1 protein and AHR mRNA was examined through immunoprecipitation (IP) assay. Cell viability, clonogenicity, and migration assays were performed. Our study revealed that both AHR and ELAVL1 inter-regulate each other, while also having a role in cell proliferation, migration, and chemoresistance in PDAC cell lines. Notably, both proteins function through distinct mechanisms. The silencing of ELAVL1 disrupts the stability of its target mRNAs, resulting in the decreased expression of numerous cytoprotective proteins. In contrast, the silencing of AHR diminishes cell migration and proliferation and enhances cell sensitivity to gemcitabine through the AHR-ELAVL1-deoxycytidine kinase (DCK) molecular pathway. In conclusion, AHR and ELAVL1 interaction can form a negative feedback loop. By inhibiting AHR expression, PDAC cells become more susceptible to gemcitabine through the ELAVL1-DCK pathway.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most life-threatening malignancies. Although the deoxycytidine analog gemcitabine has been used as the first-line treatment for PDAC, the primary clinical challenge arises because of an eventual acquisition of resistance. Therefore, it is crucial to elucidate the mechanisms underlying gemcitabine resistance to improve treatment efficacy. To investigate potential genes whose inactivation confers gemcitabine resistance, we performed CRISPR knockout (KO) library screening. We found that deoxycytidine kinase (DCK) deficiency is the primary mechanism of gemcitabine resistance, and the inactivation of CRYBA2, DMBX1, CROT, and CD36 slightly conferred gemcitabine resistance. In particular, gene expression analysis revealed that DCK KO cells displayed a significant enrichment of genes associated with MYC targets, folate/one-carbon metabolism and glutamine metabolism pathways. Evidently, chemically targeting each of these pathways significantly reduced the survival of DCK KO cells. Moreover, the pathways enriched in DCK KO cells represented a trend similar to those in PDAC cell lines and samples of patients with PDAC with low DCK expression. We further observed that short-term treatment of parental CFPAC-1 cells with gemcitabine induces the expression of several genes, which promote synthesis and transport of glutamine in a dose-dependent manner, which suggests glutamine availability as a potential mechanism of escaping drug toxicity in an initial response for survival. Thus, our findings provide insights into novel therapeutic approaches for gemcitabine-resistant PDAC and emphasize the involvement of glutamine metabolism in drug-tolerant persister cells., Implications: Our study revealed the key pathways involved in gemcitabine resistance in PDAC, thus providing potential therapeutic strategies., (©2023 American Association for Cancer Research.)

BRCA2 is a well-established cancer driver in several human malignancies. While the remarkable success of PARP inhibitors proved the clinical potential of targeting BRCA deficiencies, the emergence of resistance mechanisms underscores the importance of seeking novel Synthetic Lethal (SL) targets for future drug development efforts. In this work, we performed a BRCA2-centric SL screen with a collection of plant-derived compounds from South America. We identified the steroidal alkaloid Solanocapsine as a selective SL inducer, and we were able to substantially increase its potency by deriving multiple analogs. The use of two complementary chemoproteomic approaches led to the identification of the nucleotide salvage pathway enzyme deoxycytidine kinase (dCK) as Solanocapsine's target responsible for its BRCA2-linked SL induction. Additional confirmatory evidence was obtained by using the highly specific dCK inhibitor (DI-87), which induces SL in multiple BRCA2-deficient and KO contexts. Interestingly, dCK-induced SL is mechanistically different from the one induced by PARP inhibitors. dCK inhibition generates substantially lower levels of DNA damage, and cytotoxic phenotypes are associated exclusively with mitosis, thus suggesting that the fine-tuning of nucleotide supply in mitosis is critical for the survival of BRCA2-deficient cells. Moreover, by using a xenograft model of contralateral tumors, we show that dCK impairment suffices to trigger SL in-vivo. Taken together, our findings unveil dCK as a promising new target for BRCA2-deficient cancers, thus setting the ground for future therapeutic alternatives to PARP inhibitors., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Hepatocellular carcinoma (HCC) originates from the hepatocytes and accounts for 90% of liver cancer. The study intends to identify novel prognostic biomarkers for predicting the prognosis of HCC patients based on TCGA and GSE14520 cohorts., Methods: Differential analysis was employed to obtain the DEGs (Differentially Expressed Genes) of the TCGA-LIHC-TPM cohort. The lasso regression analysis was applied to build the prognosis model through using the TCGA cohort as the training group and the GSE14520 cohort as the testing group. Next, based on the prognosis model, we performed the following analyses: the survival analysis, the independent prognosis analysis, the clinical feature analysis, the mutation analysis, the immune cell infiltration analysis, the tumor microenvironment analysis, and the drug sensitivity analysis. Finally, the survival time of HCC patients was predicted by constructing nomograms., Results: Through the lasso regression analysis, we obtained a prognosis model of ten genes including BIRC5 (baculoviral IAP repeat containing 5), CDK4 (cyclin-dependent kinase 4), DCK (deoxycytidine kinase), HSPA4 (heat shock protein family A member 4), HSP90AA1 (heat shock protein 90 α family class A member 1), PSMD2 (Proteasome 26S Subunit Ubiquitin Receptor, Non-ATPase 2), IL1RN (interleukin 1 receptor antagonist), PGF (placental growth factor), SPP1 (secreted phosphoprotein 1), and STC2 (stanniocalcin 2). First, we found that the risk score is an independent prognosis factor and is related to the clinical features of HCC patients, covering AFP (α-fetoprotein) and stage. Second, we observed that the p53 mutation was the most obvious mutation between the high-risk and low-risk groups. Third, we also discovered that the risk score is related to some immune cells, covering B cells, T cells, dendritic, macrophages, neutrophils, etc. Fourth, the high-risk group possesses a lower TIDE score, a higher expression of immune checkpoints, and higher ESTIMATE score. Finally, nomograms include the clinical features and risk signatures, displaying the clinical utility of the signature in the survival prediction of HCC patients., Conclusions: Through the comprehensive analysis, we constructed an immune-related prognosis model to predict the survival of HCC patients. In addition to predicting the survival time of HCC patients, this model significantly correlates with the tumor microenvironment. Furthermore, we concluded that these ten immune-related genes ( BIRC5 , CDK4 , DCK , HSPA4 , HSP90AA1 , PSMD2 , IL1RN , PGF , SPP1 , and STC2 ) serve as novel targets for antitumor immunity. Therefore, this study plays a significant role in exploring the clinical application of immune-related genes.

Purpose: Drug resistance is a serious problem in leukemia therapy. A novel purine nucleoside analogue, nelarabine, is available for the treatment of children with T cell acute lymphoblastic leukemia. We investigated the mechanisms of drug resistance to nelarabine., Methods: Nelarabine-resistant cells were selected by stepwise and continuous exposure to nelarabine using the limiting dilution method in human B and T cell lymphoblastic leukemia cell lines. Expression analysis was performed using real-time polymerase chain reaction, and epigenetic analysis was performed using methylation-specific polymerase chain reaction and chromatin immunoprecipitation., Results: The RNA expression level for deoxycytidine kinase (dCK) was decreased in nelarabine-resistant leukemia cells. There were no differences between the parental and nelarabine-resistant leukemia cells in the methylation status of the promoter region of the dCK gene. In the chromatin immune precipitation assay, decreased acetylation of histones H3 and H4 bound to the dCK promoter was seen in the nelarabine-resistant cells when compared to the parental cells. Furthermore, treatment with a novel histone deacetylase inhibitor, vorinostat, promoted the cytotoxic effect of nelarabine along with increased expression of the dCK gene, and it increased acetylation of both histones H3 and H4 bound to the dCK promoter in nelarabine-resistant leukemia cells. The combination index showed that the effect of nelarabine and vorinostat was synergistic., Conclusion: This study reports that nelarabine with vorinostat can promote cytotoxicity in nelarabine-resistant leukemia cells through epigenetic mechanisms., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Cladribine is a synthetic deoxyadenosine analogue with demonstrated efficacy in patients with relapsing-remitting multiple sclerosis (MS). The main mechanism of action described for cladribine is the induction of a cytotoxic effect on lymphocytes, leading to a long-term depletion of peripheral T and B cells. Besides lymphocyte toxicity, the mode of action may include immunomodulatory mechanisms affecting other cells of the immune system. In order to induce its beneficial effects, cladribine is phosphorylated inside the cell by deoxycytidine kinase (DCK) to its active form. However, the mechanism of action of cladribine may also include immunomodulatory pathways independent of DCK activation. This in vitro study was designed to explore the impact of cladribine on peripheral blood mononuclear cells (PBMC) subsets, and to assess whether the immunomodulatory mechanisms induced by cladribine depend on the activation of the molecule. To this end, we obtained PBMCs from healthy donors and MS patients and performed proliferation, apoptosis and activation assays with clinically relevant concentrations of cladribine in DCK-dependent and -independent conditions. We also evaluated the effect of cladribine on myeloid lineage-derived cells, monocytes and dendritic cells (DCs). Cladribine decreased proliferation and increased apoptosis of lymphocyte subsets after prodrug activation via DCK. In contrast, cladribine induced a decrease in immune cell activation through both DCK-dependent and -independent pathways (not requiring prodrug activation). Regarding monocytes and DCs, cladribine induced cytotoxicity and impaired the activation of classical monocytes, but had no effect on DC maturation. Taken together, these data indicate that cladribine, in addition to its cytotoxic function, can mediate immunomodulation in different immune cell populations, by regulating their proliferation, maturation and activation.

Background: High-grade meningiomas are aggressive tumors with high morbidity and mortality rates that frequently recur even after surgery and adjuvant radiotherapy. However, limited information is currently available on the biology of these tumors, and no alternative adjuvant treatment options exist. Although we previously demonstrated that high-grade meningioma cells were highly sensitive to gemcitabine in vitro and in vivo, the underlying molecular mechanisms remain unknown., Methods: We examined the roles of hENT1 (human equilibrative nucleoside transporter 1) and dCK (deoxycytidine kinase) in the gemcitabine sensitivity and growth of meningioma cells in vitro. Tissue samples from meningiomas (26 WHO grade I and 21 WHO grade II/III meningiomas) were immunohistochemically analyzed for hENT1 and dCK as well as for Ki-67 as a marker of proliferative activity., Results: hENT1 and dCK, which play critical roles in the intracellular transport and activation of gemcitabine, respectively, were responsible for the high gemcitabine sensitivity of high-grade meningioma cells and were strongly expressed in high-grade meningiomas. hENT1 expression was required for the proliferation and survival of high-grade meningioma cells and dCK expression. Furthermore, high hENT1 and dCK expression levels correlated with stronger tumor cell proliferative activity and shorter survival in meningioma patients., Conclusions: The present results suggest that hENT1 is a key molecular factor influencing the growth capacity and gemcitabine sensitivity of meningioma cells and also that hENT1, together with dCK, may be a viable prognostic marker for meningioma patients as well as a predictive marker of their responses to gemcitabine., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

Resistance to cytarabine is an important cause of therapy failure in persons with acute myeloid leukemia (AML). Deoxycytidine kinase, encoded by DCK, catalyzes phosphorylation of cytarabine to cytarabine monophosphate, a necessary step for eventual incorporation of cytarabine triphosphate into DNA and for clinical efficacy. Whether DCK mutations make AML cells resistant to cytarabine is controversial. We studied DCK mutations and messenger RNA (mRNA) concentrations in leukemia cells from 10 subjects with AML who received cytarabine-based therapy and relapsed and in 2 artificially induced cytarabine-resistant AML cell lines. DCK mutations were detected in 4 subjects with AML relapsing after achieving a complete remission and receiving high-dose cytarabine postremission therapy. Most mutations were in exons 4-6 and were not present before therapy. DCK was also mutated in cytarabine-resistant but not parental AML cell lines. DCK mRNA concentrations were significantly decreased in cytarabine-resistant K562 and SHI-1 cells compared with cytarabine-sensitive parental cells. Mutation frequency of DCK and mRNA concentration did not correlate with the extent of cytarabine resistance indicating other factors operate. Overexpression of wild-type DCK restored cytarabine sensitivity to previously resistant leukemia cell lines. Our data contribute to the understanding of cytarabine resistance in persons with AML., (© 2021 S. Karger AG, Basel.)

Background: Several biomarkers of gemcitabine effectiveness have been studied in cancers, but less so in hepatocellular carcinoma (HCC), which is identified as the fifth most common cancer worldwide. Investigation of human equilibrative nucleoside transporter-1 (HENT-1) and deoxycytidine kinase (DCK), genes involved in gemcitabine uptake and metabolism, can be beneficial in the selection of potential cancer patients who could be responding to the treatment., Aim: To study HENT-1 and DCK gene expression in HCC patients with different protocols of treatment., Methods: Using real-time PCR, we analyzed expression levels of HENT-1 and DCK genes from peripheral blood samples of 109 patients (20 controls & 89 HCC patients) between March 2015 and March 2017. All the 89 HCC patients received the antioxidants selenium (Se) and vitamin E (Vit.E) either alone (45 patients) or in combination with gemcitabine (24 patients) or radiofrequency ablation (RFA) (20 patients)., Results: There was a significant increase in HENT-1 expression levels in HCC patients treated with Se and Vit.E alone as compared to controls (P ˂ .0001), while there was no significant difference between HCC patients treated with gemcitabine or RFA as compared to controls. In contrast, expression of DCK was significantly increased in all groups of HCC patients as compared to controls (P ˂ .0001)., Conclusions: HENT-1 and DCK mRNA expressions are important markers of HCC and for GEM effect and GEM sensitivity in patients with HCC. This could be beneficial in the selection of HCC patients sensitive to gemcitabine to avoid subjecting resistant patients to unnecessary chemotherapy., (© 2020 The Authors. Journal of Clinical Laboratory Analysis Published by Wiley Periodicals LLC.)

Although gemcitabine is the cornerstone of care for pancreatic ductal adenocarcinoma (PDA), patients lack durable responses and relapse is inevitable. While the underlying mechanisms leading to gemcitabine resistance are likely to be multifactorial, there is a strong association between activating gemcitabine metabolism pathways and clinical outcome. This study evaluated casein kinase 1 delta (CK1δ) as a potential therapeutic target for PDA and bladder cancer, in which CK1δ is frequently overexpressed. We assessed the antitumor effects of genetically silencing or pharmacologically inhibiting CK1δ using our in-house CK1δ small-molecule inhibitor SR-3029, either alone or in combination with gemcitabine, on the proliferation and survival of pancreatic and bladder cancer cell lines and orthotopic mouse models. Genetic studies confirmed that silencing CK1δ or treatment with SR-3029 induced a significant upregulation of deoxycytidine kinase (dCK), a rate-limiting enzyme in gemcitabine metabolite activation. The combination of SR-3029 with gemcitabine induced synergistic antiproliferative activity and enhanced apoptosis in both pancreatic and bladder cancer cells. Furthermore, in an orthotopic pancreatic tumor model, we observed improved efficacy with combination treatment concomitant with increased dCK expression. This study demonstrates that CK1δ plays a role in gemcitabine metabolism, and that the combination of CK1δ inhibition with gemcitabine holds promise as a future therapeutic option for metastatic PDA as well as other cancers with upregulated CK1δ expression., (©2020 American Association for Cancer Research.)

Dexamethasone (DEX) is often used in the initial treatment of leukemia. Earlier we demonstrated that DEX decreased the activity of deoxycytidine kinase (dCK) which is essential for the activation of cytarabine (ara-C). Therefore we investigated the effect of DEX on the in vivo sensitivity of acute myeloid leukemia (AML) to ara-C and another deoxycytidine analog, gemcitabine, in the Brown Norway Myeloid Leukemia (BNML) rat model for AML, and its ara-C resistant variant B-araC, in relation to the effects on dCK activity.The antileukemic effect was evaluated as survival of the rats, while dCK activity was measured in leukemic spleen (completely consisting of BNML cells) with liver as representative normal tissue, 24 hr after treatment with ara-C or DEX with radioactive deoxycytidine (CdR) as a substrate.Treatment with ara-C increased life-span of BNML by 200%, which was not affected by DEX. Gemcitabine was ineffective. In the liver of BNML bearing rats DEX decreased dCK activity 33%, while ara-C increased dCK activity slightly (to 129%), but in the combination of ara-C/DEX dCK activity was also decreased. In the livers of Bara-C bearing rats dCK was 2.7-fold higher compared to BNML rats, which was increased 179% in the gemcitabine-DEX treated rats. In BNML leukemic spleens DEX decreased dCK activity 41% and gem/dex 46%, but ara-C increased dCK activity to 123%, but in the combination this effect was neutralized. In Bara-C spleens only ara-C/dex decreased dCK activity (32%).In conclusion; in an AML rat model DEX did not affect the antileukemic effect of ara-C, nor the dCK activity.

Thymidine kinase 1 (TK1) and deoxycytidine kinase (dCK) are required for the activation of thymidine and deoxycytidine analogs used in antiviral and anticancer therapies. Many anticancer drugs cause oxidative stress, and the rise of GSSG and other reactive oxygen species may lead to alteration in gene expression, protein, nucleic acids and lipid modifications. Here, we investigated the effects of oxidative stress and nucleoside analog on the expression and activity of TK1 and dCK. Treatment with GSSG resulted in glutathionylation of dCK and dGK but not TK1 and Dm-dNK, and glutathionylation led to increased dCK activity but decreased dGK activity. Treatment with hydrogen peroxide resulted in induction of TK1, however, the TK1 activity did not correlate with TK1 protein levels, indicating that TK1 protein was inactive. The cellular expression of dCK, however, was reduced but dCK activity was not affected at concentration ≤ 4 mM. Treatment with TFT or 5FdU resulted in downregulation of both TK1 and dCK. However, araC and dFdC treatment led to increased dCK protein but decreased dCK activity. In contrast, both TK1 protein and activity were upregulated after araC and dFdC treatment. Doxorubicin treatment led to upregulation of the TK1 but downregulation of dCK. In conclusion TK1 and dCK expression and activity are apparently affected by oxidative stress and treatment by nucleoside analogs. These results demonstrate the pharmacokinetic importance of characterizing the expression and activity of TK1 and dCK during chemotherapy with thymidine and deoxycytidine analogs in order to optimize their efficacy.

Background: The biological function of deoxycytidine kinase in tumor is not yet clear, and there are a few studies relating to the correlation of deoxycytidine kinase gene with the occurrence and development of liver cancer., Methods: The messenger RNA expression of deoxycytidine kinase was analyzed with the use of the UALCAN and GEPIA database. Moreover, we assessed the function of deoxycytidine kinase on clinical prognosis with Kaplan-Meier plotter database. The relationship between deoxycytidine kinase and cancer immune infiltrates was investigated via Tumor Immune Estimation Resource site. Furthermore, Tumor Immune Estimation Resource was also used to evaluate the correlations between the expression of deoxycytidine kinase and gene marker sets of immune infiltrates., Results: The deoxycytidine kinase messenger RNA level significantly upregulated in patients with liver cancer compared to normal liver samples. Moreover, the increased expression of deoxycytidine kinase messenger RNA was closely associated with reduced overall survival and disease-free survival in all liver cancers. In addition, deoxycytidine kinase expression displayed a strong correlation with infiltrating levels of macrophages, neutrophils, and dendritic cells in liver cancer, and deoxycytidine kinase expression was positively correlated with diverse immune marker sets in liver cancer., Conclusions: All the above findings suggested that increased expression of deoxycytidine kinase was significantly related to unfavorable prognosis in patients with liver cancer. And deoxycytidine kinase is correlated with immune infiltrating levels, including those of B cells, macrophages, neutrophils, and dendritic cells in patients with liver cancer. These findings suggest that deoxycytidine kinase can be used as a prognostic biomarker for determining prognosis and immune infiltration in liver cancer. And deoxycytidine kinase is a potential target for liver cancer therapy, and these preliminary findings require further study to determine whether deoxycytidine kinase-targeting reagents might be developed for clinical application in liver cancer.

Knowledge about synthetic lethality can be applied to enhance the efficacy of anticancer therapies in individual patients harboring genetic alterations in their cancer that specifically render it vulnerable. We investigated the potential for high-resolution phenomic analysis in yeast to predict such genetic vulnerabilities by systematic, comprehensive, and quantitative assessment of drug-gene interaction for gemcitabine and cytarabine, substrates of deoxycytidine kinase that have similar molecular structures yet distinct antitumor efficacy. Human deoxycytidine kinase (dCK) was conditionally expressed in the S accharomyces cerevisiae genomic library of knockout and knockdown (YKO/KD) strains , to globally and quantitatively characterize differential drug-gene interaction for gemcitabine and cytarabine. Pathway enrichment analysis revealed that autophagy, histone modification, chromatin remodeling, and apoptosis-related processes influence gemcitabine specifically, while drug-gene interaction specific to cytarabine was less enriched in gene ontology. Processes having influence over both drugs were DNA repair and integrity checkpoints and vesicle transport and fusion. Non-gene ontology (GO)-enriched genes were also informative. Yeast phenomic and cancer cell line pharmacogenomics data were integrated to identify yeast-human homologs with correlated differential gene expression and drug efficacy, thus providing a unique resource to predict whether differential gene expression observed in cancer genetic profiles are causal in tumor-specific responses to cytotoxic agents., Competing Interests: JLH has ownership in Spectrum PhenomX, LLC, a company that was formed to commercialize Q-HTCP technology. The authors declare no other competing interests.

This study aimed to investigate whether the anti-tumor effect of gemcitabine (GEM) in non-small-cell lung cancer (NSCLC) treatment was affected by Danggui Buxue decoction (DBD), and explore the potential mechanisms. The combined use of GEM and DBD showed an enhanced tumor growth inhibition effect in a murine Lewis lung carcinoma (LLC) model. LC-MS/MS results showed that the pharmacokinetic behaviors of a GEM active metabolite, gemcitabine triphosphate (dFdCTP), were found to be altered remarkably in the peripheral blood mononuclear cells (PBMC) of DBD co-administration rats. In addition, after co-administration of DBD with GEM, Western Blot and qPCR results confirmed that the expression of deoxycytidine kinase (dCK) in tumor tissues of LLC-bearing mice were markedly increased. DBD co-administration also reversed the upregulation of P-glycoprotein (P-gp) in tumor tissues induced by GEM. Moreover, DBD could notably up-regulate the IL-12p70 and GM-CSF expression in mice serum, suggesting potential immunomodulatory activities in tumor-bearing mice. Meanwhile, DBD inhibited the P-gp efflux activity in A549 cells. Therefore, the regulation of dCK and P-gp played important roles in the alternation of GEM pharmacokinetics and the enhancement of the anti-tumor effect of GEM. DBD being a potential dCK promoter could work as an adjuvant agent to boost the anticancer effect of GEM.

Gemcitabine is a fluoropyrimidine analogue that is used as a mainstay of chemotherapy treatment for pancreatic and ovarian cancers, amongst others. Despite its widespread use, gemcitabine achieves responses in less than 10% of patients with metastatic pancreatic cancer and has a very limited impact on overall survival due to intrinsic and acquired resistance. NUC-1031 (Acelarin), a phosphoramidate transformation of gemcitabine, was the first anti-cancer ProTide to enter the clinic. We find it displays important in vitro cytotoxicity differences to gemcitabine, and a genome-wide CRISPR/Cas9 genetic screening approach identified only the pyrimidine metabolism pathway as modifying cancer cell sensitivity to NUC-1031. Low deoxycytidine kinase expression in tumour biopsies from patients treated with gemcitabine, assessed by immunostaining and image analysis, correlates with a poor prognosis, but there is no such correlation in tumour biopsies from a Phase I cohort treated with NUC-1031.

Acute myeloid leukemia (AML) patients display dismal prognosis due to high prevalence of refractory and relapsed disease resulting from chemoresistance. Treatment protocols, primarily based on the anchor drug Cytarabine, remained chiefly unchanged in the past 50 years with no standardized salvage regimens. Herein we aimed at exploring potential pre-clinical treatment strategies to surmount Cytarabine resistance in human AML cells. We established Cytarabine-resistant sublines derived from human leukemia K562 and Kasumi cells, and characterized the expression of Cytarabine-related genes using real-time PCR and Western blot analyses to uncover the mechanisms underlying their Cytarabine resistance. This was followed by growth inhibition assays and isobologram analyses testing the sublines' sensitivity to the clinically approved drugs hydroxyurea (HU) and azidothymidine (AZT), compared to their parental cells. All Cytarabine-resistant sublines lost deoxycytidine kinase (dCK) expression, rendering them refractory to Cytarabine. Loss of dCK function involved dCK gene deletions and/or a novel frameshift mutation leading to dCK transcript degradation via nonsense-mediated decay. Cytarabine-resistant sublines displayed hypersensitivity to HU and AZT compared to parental cells; HU and AZT combinations exhibited a marked synergistic growth inhibition effect on leukemic cells, which was intensified upon acquisition of Cytarabine-resistance. In contrast, HU and AZT combination showed an antagonistic effect in non-malignant cells. Finally, HU and AZT synergism was demonstrated on peripheral blood specimens from AML patients. These findings identify a promising HU and AZT combination for the possible future treatment of relapsed and refractory AML, while sparing normal tissues from untoward toxicity.

Deoxycytidine kinase (DCK) is a key enzyme for the activation of a broad spectrum of nucleoside-based chemotherapy drugs (e.g., gemcitabine); low DCK activity is one of the most important causes of cancer drug-resistance. Noninvasive imaging methods that can quantify DCK activity are invaluable for assessing tumor resistance and predicting treatment efficacy. Here we developed a "natural" MRI approach to detect DCK activity using its natural substrate deoxycytidine (dC) as the imaging probe, which can be detected directly by chemical exchange saturation transfer (CEST) MRI without any synthetic labeling. CEST MRI contrast of dC and its phosphorylated form, dCTP, successfully discriminated DCK activity in two mouse leukemia cell lines with different DCK expression. This dC-enhanced CEST MRI in xenograft leukemic cancer mouse models demonstrated that DCK(+) tumors have a distinctive dynamic CEST contrast enhancement and a significantly higher CEST contrast than DCK(-) tumors (AUC 0-60 min = 0.47 ± 0.25 and 0.20 ± 0.13, respectively; P = 0.026, paired Student t test, n = 4) at 1 hour after the injection of dC. dC-enhanced CEST contrast also correlated well with tumor responses to gemcitabine treatment. This study demonstrates a novel MR molecular imaging approach for predicting cancer resistance using natural, nonradioactive, nonmetallic, and clinically available agents. This method has great potential for pursuing personalized chemotherapy by stratifying patients with different DCK activity. SIGNIFICANCE: A new molecular MRI method that detects deoxycytidine kinase activity using its natural substrate deoxycytidine has great translational potential for clinical assessment of tumor resistance and prediction of treatment efficacy., (©2019 American Association for Cancer Research.)

Pancreatic ductal adenocarcinoma (PDAC) develops extrinsic- and intrinsic-resistant phenotypes to prevent chemotherapies from entering into the cells by promoting desmoplastic reactions (DR) and metabolic malfunctions of the drugs. It is well established that these responses are also associated with pancreatic cancer cells' gemcitabine resistance. However, the mechanism by which these resistant pathways function in the pancreatic cancer cells remains poorly understood. In these studies, we show that CYR61/CCN1 signaling plays a vital role in making pancreatic cancer cells resistant to gemcitabine i n vitro and also in a tumor xenograft model. We proved that the catastrophic effect of gemcitabine could significantly be increased in gemcitabine-resistant PDAC cells when CYR61/CCN1 is depleted, while this effect can be suppressed in gemcitabine-sensitive neoplastic cells by treating them with CYR61/CCN1 recombinant protein. Ironically, nontransformed pancreatic cells, which are sensitive to gemcitabine, cannot be resistant to gemcitabine by CYR61/CCN1 protein treatment, showing a unique feature of CYR61/CCN signaling that only influences PDAC cells to become resistant. Furthermore, we demonstrated that CYR61/CCN1 suppresses the expression of the gemcitabine-activating enzyme deoxycytidine kinase (dCK) while it induces the expression of a DR-promoting factor CTGF (connective tissue growth factor) in pancreatic cancer cells in vitro and in vivo Thus, the previously described mechanisms (dCK and CTGF pathways) for gemcitabine resistance may be two novel targets for CYR61/CCN1 to protect pancreatic cancer cells from gemcitabine. Collectively, these studies reveal a novel paradigm in which CYR61/CCN1regulates both extrinsic and intrinsic gemcitabine resistance in PDAC cells by employing unique signaling pathways., (©2019 American Association for Cancer Research.)

Suicide transgenes encode proteins that are either capable of activating specific prodrugs into cytotoxic antimetabolites that can trigger cancer cell apoptosis or are capable of directly inducing apoptosis. Suicide gene therapy of cancer (SGTC) involves the targeted or localized delivery of suicide transgene sequences into tumor cells by means of various gene delivery vehicles. SGTC that operates via the potentiation of small-molecule pharmacologic agents can elicit the elimination of cancer cells within a tumor beyond only those cells successfully transduced. Such "bystander effects ", typically mediated by the spread of activated cytotoxic antimetabolites from the transduced cells expressing the suicide transgene to adjacent cells in the tumor, can lead to a significant reduction of the tumor mass without the requirement of transduction of a high percentage of cells within the tumor. The spread of activated cytotoxic molecules to adjacent cells is mediated primarily by diffusion and normally involves gap junctional intercellular communications (GJIC). We have developed a novel SGTC system based on viral vector-mediated delivery of an engineered variant of human deoxycytidine kinase (dCK), which is capable of phosphorylating uridine- and thymidine-based nucleoside analogues that are not substrates for wild-type dCK, such as bromovinyl deoxyuridine (BVdU) and L-deoxythymidine (LdT). Since our dCK-based SGTC system is capable of mediating strong bystander cell killing, it holds promise for clinical translation. In this chapter, we detail the key procedures for the preparation of recombinant lentivectors for the delivery of engineered dCK, transduction of tumor cells, and evaluation of bystander cell killing effects in vitro and in vivo.

Objectives: Decreased deoxycytidine kinase (dCK) expression is a reported indicator of gemcitabine efficacy in pancreatic cancer, due to the impact of this kinase on gemcitabine metabolism. The transcription factor NF-E2 p45-related factor 2 (NRF2, also called Nfe2l2), a master regulator of redox homoeostasis, has been reported to tightly control the expression of numerous ROS-detoxification genes and participates in drug resistance. However, the contribution of dCK to the NRF2 signalling axis has seldom been discussed and needs investigation., Materials and Methods: By overexpressing dCK in pancreatic cancer cells, we assessed the impact of dCK on NRF2 transcriptional activity. Furthermore, we measured the impact of dCK expression on the intracellular redox balance and reactive oxygen species (ROS) production. By utilizing immunohistochemical staining and tissues from pancreatic cancer patients, we assessed the correlation between dCK and NRF2 expression. Through proliferation and metastasis assays, we examined the impact of dCK expression on cell proliferation and metastasis., Results: dCK negatively regulates NRF2 transcriptional activity, leading to the decreased expression of ARE-driven antioxidant genes. In addition, dCK negatively regulates intracellular redox homoeostasis and ROS production. Negative correlations between dCK and NRF2 levels in pancreatic cancer cell lines and patient samples were observed. In vitro cell line studies suggested that dCK negatively regulated proliferation and metastasis., Conclusion: Decreased dCK expression promotes NRF2-driven antioxidant transcription, which further enhances gemcitabine treatment resistance, forming a feedback loop., (© 2018 The Authors Cell Proliferation Published by John Wiley & Sons Ltd.)

Background: The mechanism of action of oral cladribine, recently licensed for relapsing multiple sclerosis, is unknown., Objective: To determine whether cladribine depletes memory B cells consistent with our recent hypothesis that effective, disease-modifying treatments act by physical/functional depletion of memory B cells., Methods: A cross-sectional study examined 40 people with multiple sclerosis at the end of the first cycle of alemtuzumab or injectable cladribine. The relative proportions and absolute numbers of peripheral blood B lymphocyte subsets were measured using flow cytometry. Cell-subtype expression of genes involved in cladribine metabolism was examined from data in public repositories., Results: Cladribine markedly depleted class-switched and unswitched memory B cells to levels comparable with alemtuzumab, but without the associated initial lymphopenia. CD3 + T cell depletion was modest. The mRNA expression of metabolism genes varied between lymphocyte subsets. A high ratio of deoxycytidine kinase to group I cytosolic 5' nucleotidase expression was present in B cells and was particularly high in mature, memory and notably germinal centre B cells, but not plasma cells., Conclusions: Selective B cell cytotoxicity coupled with slow repopulation kinetics results in long-term, memory B cell depletion by cladribine. These may offer a new target, possibly with potential biomarker activity, for future drug development.

Nucleoside analogs with 2'-modified sugar moieties are often used to improve the RNA target affinity and nuclease resistance of therapeutic oligonucleotides in preclinical and clinical development. Despite their enhanced nuclease resistance, oligonucleotides could slowly degrade releasing nucleoside analogs that have the potential to become phosphorylated and incorporated into cellular DNA and RNA. For the first time, the phosphorylation and DNA/RNA incorporation of 2'-O-(2-methoxyethyl) (2'-O-MOE) nucleoside analogs have been investigated. Using liquid chromatography/tandem mass spectrometry, we showed that enzymes in the nucleotide salvage pathway including deoxycytidine kinase (dCK) and thymidine kinase (TK1) displayed poor reactivity toward 2'-O-MOE nucleoside analogs. On the other hand, 2'-fluoro (F) nucleosides, regardless of the nucleobase, were efficiently phosphorylated to their monophosphate forms by dCK and TK1. Consistent with their efficient phosphorylation by dCK and TK1, 2'-F nucleoside analogs were incorporated into cellular DNA and RNA while no incorporation was detected with 2'-O-MOE nucleoside analogs. In conclusion, these data suggest that the inability of dCK and TK1 to create the monophosphates of 2'-O-MOE nucleoside analogs reduces the risk of their incorporation into cellular DNA and RNA.

Background: The addition of high-dose cytarabine to the treatment of mantle cell lymphoma (MCL) has significantly prolonged survival of patients, but relapses are common and are normally associated with increased resistance. To elucidate the mechanisms responsible for cytarabine resistance, and to create a tool for drug discovery investigations, we established a unique and molecularly reproducible cytarabine resistant model from the Z138 MCL cell line., Methods: Effects of different substances on cytarabine-sensitive and resistant cells were evaluated by assessment of cell proliferation using [methyl-14C]-thymidine incorporation and molecular changes were investigated by protein and gene expression analyses., Results: Gene expression profiling revealed that major transcriptional changes occur during the initial phase of adaptation to cellular growth in cytarabine containing media, and only few key genes, including SPIB, are deregulated upon the later development of resistance. Resistance was shown to be mediated by down-regulation of the deoxycytidine kinase (dCK) protein, responsible for activation of nucleoside analogue prodrugs. This key event, emphasized by cross-resistance to other nucleoside analogues, did not only effect resistance but also levels of SPIB and NF-κB, as assessed through forced overexpression in resistant cells. Thus, for the first time we show that regulation of drug resistance through prevention of conversion of pro-drug into active drug are closely linked to increased proliferation and resistance to apoptosis in MCL. Using drug libraries, we identify several substances with growth reducing effect on cytarabine resistant cells. We further hypothesized that co-treatment with bortezomib could prevent resistance development. This was confirmed and show that the dCK levels are retained upon co-treatment, indicating a clinical use for bortezomib treatment in combination with cytarabine to avoid development of resistance. The possibility to predict cytarabine resistance in diagnostic samples was assessed, but analysis show that a majority of patients have moderate to high expression of dCK at diagnosis, corresponding well to the initial clinical response to cytarabine treatment., Conclusion: We show that cytarabine resistance potentially can be avoided or at least delayed through co-treatment with bortezomib, and that down-regulation of dCK and up-regulation of SPIB and NF-κB are the main molecular events driving cytarabine resistance development.

Deoxycytidine kinase (dCK) is a rate limiting enzyme critical for the phosphorylation of endogenous deoxynucleosides and for the anti‑tumor activity of many nucleoside analogs. dCK is activated in response to ionizing radiation (IR) and it is required for the G2/M checkpoint induced by IR. However, whether dCK plays a role in radiation-induced autophagy and apoptosis is less clear. In this study, we reported that dCK decreased IR-induced total cell death and apoptosis, and increased IR-induced autophagy in SKBR3 and MDA‑MB‑231 breast cancer cell lines. A molecular switch exists between apoptosis and autophagy. We further demonstrated that serine 74 phosphorylation was required for the regulation of autophagy. In dCK wild‑type (WT) or dCK S74E (mutant) MDA‑MB‑231 cell models, the expression levels of phospho-Akt, phospho-mammalian target of rapamycin (mTOR) and phospho-P70S6K significantly decreased following exposure to IR. Moreover, the ratio of Bcl‑2/Beclin1 (BECN1) significantly decreased in the S74E mutant cells; however, no change was observed in the ratio of Bcl‑2/BAX. Taken together, our findings indicate that phosphorylated and activated dCK inhibits IR-induced total cell death and apoptosis, and promotes IR-induced autophagy through the mTOR pathway and by inhibiting the binding of Bcl‑2 protein to BECN1.

5-Fluorouracil (5-FU) is a chemotherapeutic agent used to treat a variety of gastric cancers including oesophageal squamous cell carcinoma (OSCC), for which the 5-year mortality rate exceeds 85%. Our study investigated the effects of metformin, an antidiabetic drug with established anti-cancer activity, in combination with 5-FU as a novel chemotherapy strategy, using the OSCC cell lines, WHCO1 and WHCO5. Our results indicate that metformin treatment induces significant resistance to 5-FU in WHCO1 and WHCO5 cells, by more than five- and sixfolds, respectively, as assessed by MTT assay. We show that this is due to global alterations in nucleotide metabolism, including elevated expression of thymidylate synthase and thymidine kinase 1 (established 5-FU resistance mechanisms), which likely result in an increase in intracellular dTTP pools and a "dilution" of 5-FU anabolites. Metformin treatment also increases deoxycytidine kinase (dCK) expression and, as the chemotherapeutic agent gemcitabine relies on dCK for its efficient activity, we speculated that metformin would enhance the sensitivity of OSCC cells to gemcitabine. Indeed we show that metformin pre-treatment greatly increases gemcitabine toxicity and DNA fragmentation in comparison to gemcitabine alone. Taken together, our findings show that metformin alters nucleotide metabolism in OSCC cells and while responsible for inducing resistance to 5-FU, it conversely increases sensitivity to gemcitabine, thereby highlighting metformin and gemcitabine as a potentially novel combination therapy for OSCC., (© 2017 Wiley Periodicals, Inc.)

Decitabine (DAC), a nucleoside-related DNA methylation inhibitor, is taken up into cancer cells via equilibrative nucleoside transporter 1 (ENT1), and is then monophosphorylated by deoxycytidine kinase (dCK). In the present study, we examined the contribution of dCK to the uptake of DAC in HCT116 colon cancer cells. Irinotecan and etoposide inhibited the uptake of [ 3 H]-uridine and [ 3 H]-DAC at 10 s and 5 min, while cytarabine and gemcitabine only inhibited that of [ 3 H]-DAC at 5 min. Irinotecan and etoposide inhibited [ 3 H]-DAC uptake in negative control small interfering RNA (siRNA)- or dCK siRNA-transfected cells at 10 s, whereas cytarabine and gemcitabine did not. Cytarabine and gemcitabine inhibited DAC monophosphate generation by the cytosolic proteins of HCT116 cells and recombinant human dCK protein, assessed using polyethylenimine cellulose thin-layered chromatography. Simulations using simple kinetic models showed that apparent DAC uptake in dCK and ENT1 siRNA-treated cells was attributed to its conversion to monophosphates or a decrease in the cellular flux, respectively, and that the apparent uptake of DAC in dCK-knockdown and ENT1-knockdown cells was similar at longer times, but differed at a very short time. These results suggest that the apparent uptake of DAC is affected by ENT1 and dCK in HCT116 cells., (Copyright © 2017 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Masitinib, a highly selective protein kinase inhibitor, can sensitise gemcitabine-refractory cancer cell lines when used in combination with gemcitabine. Here we report a reverse proteomic approach that identifies the target responsible for this sensitisation: the deoxycytidine kinase (dCK). Masitinib, as well as other protein kinase inhibitors, such as imatinib, interact with dCK and provoke an unforeseen conformational-dependent activation of this nucleoside kinase, modulating phosphorylation of nucleoside analogue drugs. This phenomenon leads to an increase of prodrug phosphorylation of most of the chemotherapeutic drugs activated by this nucleoside kinase. The unforeseen dual activity of protein kinase inhibition/nucleoside kinase activation could be of great therapeutic benefit, through either reducing toxicity of therapeutic agents by maintaining effectiveness at lower doses or by counteracting drug resistance initiated via down modulation of dCK target.

Leukemia cells rely on two nucleotide biosynthetic pathways, de novo and salvage, to produce dNTPs for DNA replication. Here, using metabolomic, proteomic, and phosphoproteomic approaches, we show that inhibition of the replication stress sensing kinase ataxia telangiectasia and Rad3-related protein (ATR) reduces the output of both de novo and salvage pathways by regulating the activity of their respective rate-limiting enzymes, ribonucleotide reductase (RNR) and deoxycytidine kinase (dCK), via distinct molecular mechanisms. Quantification of nucleotide biosynthesis in ATR-inhibited acute lymphoblastic leukemia (ALL) cells reveals substantial remaining de novo and salvage activities, and could not eliminate the disease in vivo. However, targeting these remaining activities with RNR and dCK inhibitors triggers lethal replication stress in vitro and long-term disease-free survival in mice with B-ALL, without detectable toxicity. Thus the functional interplay between alternative nucleotide biosynthetic routes and ATR provides therapeutic opportunities in leukemia and potentially other cancers.Leukemic cells depend on the nucleotide synthesis pathway to proliferate. Here the authors use metabolomics and proteomics to show that inhibition of ATR reduced the activity of these pathways thus providing a valuable therapeutic target in leukemia.

We previously showed that gemcitabine resistance in pancreatic cancer chemotherapy correlates with suppressed expression of deoxycytidine kinase (dCK), which catalyzes the rate-limiting step of gemcitabine activation. The purpose of the present study was to find a drug that might be useful to enhance the cytotoxicity of gemcitabine by increasing dCK expression in gemcitabine-resistant human pancreatic cancer cell line AsPC-1. Screening of 40 prescription drugs identified 35 with no intrinsic cytotoxicity towards AsPC-1 cells. When AsPC-1 cells were pre-incubated with these drugs and then incubated with gemcitabine, we found that all-trans retinoic acid (ATRA) significantly decreased the viability by 28% compared with that of non-treated cells. Luciferase assay showed that ATRA transactivated the DCK promoter in AsPC-1 cells by about 2-fold compared with the untreated control, and an increase of dCK protein expression was confirmed by immunoblotting. ATRA decreased the half-maximal inhibitory concentration (IC 50 ) of gemcitabine by 2.8-fold (ATRA-non-treated cells, 28.8nM; ATRA-treated cells, 10.0nM). The ATRA concentration of 0.03μM was sufficient to enhance gemcitabine cytotoxicity, and the effect was well maintained in the concentration range from 0.03 to 50μM. These results indicate that ATRA enhances gemcitabine cytotoxicity by increasing dCK expression in gemcitabine-resistant human pancreatic cancer cells., (Copyright © 2017 Elsevier B.V. All rights reserved.)

18 F-clofarabine, a nucleotide purine analog, is a substrate for deoxycytidine kinase (dCK), a key enzyme in the deoxyribonucleoside salvage pathway. 18 F-clofarabine might be used to measure dCK expression and thus serve as a predictive biomarker for tumor responses to dCK-dependent prodrugs or small-molecule dCK inhibitors, respectively. As a prerequisite for clinical translation, we determined the human whole-body and organ dosimetry of 18 F-clofarabine. Methods: Five healthy volunteers were injected intravenously with 232.4 ± 1.5 MBq of 18 F-clofarabine. Immediately after tracer injection, a dynamic scan of the entire chest was acquired for 30 min. This was followed by 3 static whole-body scans at 45, 90, and 135 min after tracer injection. Regions of interest were drawn around multiple organs on the CT scan and copied to the PET scans. Organ activity was determined and absorbed dose was estimated with OLINDA/EXM software. Results: The urinary bladder (critical organ), liver, kidney, and spleen exhibited the highest uptake. For an activity of 250 MBq, the absorbed doses in the bladder, liver, kidney, and spleen were 58.5, 6.6, 6.3, and 4.3 mGy, respectively. The average effective dose coefficient was 5.1 mSv. Conclusion: Our results hint that 18 F-clofarabine can be used safely in humans to measure tissue dCK expression. Future studies will determine whether 18 F-clofarabine may serve as a predictive biomarker for responses to dCK-dependent prodrugs or small-molecule dCK inhibitors., (© 2017 by the Society of Nuclear Medicine and Molecular Imaging.)

Human respiratory syncytial virus (RSV) is a negative-sense RNA virus and a significant cause of respiratory infection in infants and the elderly. No effective vaccines or antiviral therapies are available for the treatment of RSV. ALS-8176 is a first-in-class nucleoside prodrug inhibitor of RSV replication currently under clinical evaluation. ALS-8112, the parent molecule of ALS-8176, undergoes intracellular phosphorylation, yielding the active 5'-triphosphate metabolite. The host kinases responsible for this conversion are not known. Therefore, elucidation of the ALS-8112 activation pathway is key to further understanding its conversion mechanism, particularly given its potent antiviral effects. Here, we have identified the activation pathway of ALS-8112 and show it is unlike other antiviral cytidine analogs. The first step, driven by deoxycytidine kinase (dCK), is highly efficient, while the second step limits the formation of the active 5'-triphosphate species. ALS-8112 is a 2'- and 4'-modified nucleoside analog, prompting us to investigate dCK recognition of other 2'- and 4'-modified nucleosides. Our biochemical approach along with computational modeling contributes to an enhanced structure-activity profile for dCK. These results highlight an exciting potential to optimize nucleoside analogs based on the second activation step and increased attention toward nucleoside diphosphate and triphosphate prodrugs in drug discovery.

Enzymatic activity from tumor and adjacent normal tissue of 200 patients involving deoxycytidine kinase (dCK), uridine/cytidine kinase (U/CK), cytidine deaminase (CD) and deoxycytidylate deaminase (dCMPD) was quantified. Patients with brain (17), colon (24), and breast (30) tumors, 53, 67, and 73%, respectively, had an elevated T/N value (Specific Activity of tumor/ Specific Activity of normal tissue) involving dCK and dCMPD suggesting chemotherapy with 5-fluorodeoxycytidine (5-FdC) alone or in combination with thymidine plus deoxytetrahydrouridine, or with the radiosensitizer, 5-chlorodeoxycytidine (5-CldC) plus tetrahydrouridine (H4U). Among patients with colon (19) and pancreatic tumors (40), 53 and 68 %, respectively, displayed T/N values >4 for CD suggesting chemotherapy with 5-FdC, 4-N-methylamino-5-FdC, 5-trifluoromethyldeoxycytidine and radiosensitization with 5- CldC, 4-N-methylamino-5-CldC, 5-iododeoxycytidine and 5-bromodeoxycytidine. The percent of patients with tumors with a T/N value >4 for U/CK in lung (72), colon (23) and breast (28) was 47, 61 and 68, respectively, suggesting zebularine (plus thymidine) treatment for tumors involving gene silencing. Evidence is presented that the 4-N-alkylamino-dC substituted nucleosides and those with large 5-substitutions are activated only via CD to thymidine kinase (TK) using end-points of cytotoxicity and/or radiosensitization: H4U, the inhibitor of CD is an antagonist, cells with low CD or no TK are resistant to the analogs, the end points are indifferent to the dCK status of cells, they are poor substrates for dCK and good substrates for CD, whereas 5-FdC and 5-CldC are good substrates for both enzymes. The analogs present opportunities for Collateral Sensitivity for 5-azacytidine and gemcitabine resistant tumors.

Deoxycytidine kinase (dCK) is a key enzyme in deoxyribonucleoside salvage and the anti-tumor activity for many nucleoside analogs. dCK is activated in response to ionizing radiation (IR)-induced DNA damage and it is phosphorylated on Serine 74 by the Ataxia-Telangiectasia Mutated (ATM) kinase in order to activate the cell cycle G2/M checkpoint. However, whether dCK plays a role in radiation-induced cell death is less clear. In this study, we genetically modified dCK expression by knocking down or expressing a WT (wild-type), S74A (abrogates phosphorylation) and S74E (mimics phosphorylation) of dCK. We found that dCK could decrease IR-induced total cell death and apoptosis. Moreover, dCK increased IR-induced autophagy and dCK-S74 is required for it. Western blotting showed that the ratio of phospho-Akt/Akt, phospho-mTOR/mTOR, phospho-P70S6K/P70S6K significantly decreased in dCK-WT and dCK-S74E cells than that in dCK-S74A cells following IR treatment. Reciprocal experiment by co-immunoprecipitation showed that mTOR can interact with wild-type dCK. IR increased polyploidy and decreased G2/M arrest in dCK knock-down cells as compared with control cells. Taken together, phosphorylated and activated dCK can inhibit IR-induced cell death including apoptosis and mitotic catastrophe, and promote IR-induced autophagy through PI3K/Akt/mTOR pathway., Competing Interests: The authors declare no conflict of interest.

Objectives: Leukaemia initiating cells reside within specialised niches in the bone marrow where they undergo complex interactions with different stromal cell types. The bone marrow niche is characterised by a low oxygen content resulting in high expression of hypoxia-inducible factor 1 α in leukaemic cells conferring a negative prognosis to patients with acute myeloid leukaemia (AML)., Methods and Results: In the current study, we investigated the impact of hypoxic vs. normoxic conditions on the sensitivity of AML cell lines and primary AML blasts to cytarabine. AML cells cultured under 6% oxygen were significantly more resistant against cytarabine compared to cells cultured under normoxic conditions in proliferation and colony-formation assays. Interestingly upon cultivation under hypoxia, the expression of the cytarabine-activating enzyme deoxycytidine kinase was downregulated in all analysed AML cell lines and primary AML samples representing a possible mechanism for resistance to chemotherapy. Furthermore, the downregulation of deoxycytidine kinase could be associated with hypoxia-inducible factor 1 α as treatment with its inhibitor BAY87-2243 hampered the downregulation of deoxycytidine kinase expression under hypoxic conditions., Conclusions: In conclusion, our data reveal that hypoxia-induced downregulation of deoxycytidine kinase represents one stroma-cell-independent mechanism of drug resistance to cytarabine in acute myeloid leukaemia., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

This retrospective study aimed to investigate the role that an RNA-binding protein, HuR, plays in the response of high-grade serous ovarian tumors to chemotherapeutics. We immunohistochemically stained sections of 31 surgically-debulked chemo-naïve ovarian tumors for HuR and scored the degree of HuR cytoplasmic staining. We found no correlation between HuR intracellular localization in tumor sections and progression free survival (PFS) of these patients, 29 of whom underwent second-line gemcitabine/platin combination therapy for recurrent disease. Ribonucleoprotein immunoprecipitation (RNP-IP) analysis of ovarian cancer cells in culture showed that cytoplasmic HuR increases deoxycytidine kinase (dCK), a metabolic enzyme that activates gemcitabine. The effects of carboplatin treatment on HuR and WEE1 (a mitotic inhibitor) expression, and on cell cycle kinetics, were also examined. Treatment of ovarian cancer cells with carboplatin results in increased HuR cytoplasmic expression and elevated WEE1 expression, arresting cell cycle G2/M transition. This may explain why HuR cytoplasmic localization in chemo-naïve tumors is not predictive of therapeutic response and PFS following second-line gemcitabine/platin combination therapy. These results suggest treatment of recurrent ovarian tumors with a combination of gemcitabine, carboplatin, and a WEE1 inhibitor may be potentially advantageous as compared to current clinical practices., Competing Interests: The authors declare no conflicts of interest.

Deoxycytidine kinase (dCK), a rate-limiting enzyme in the cytosolic deoxyribonucleoside (dN) salvage pathway, is an important therapeutic and positron emission tomography (PET) imaging target in cancer. PET probes for dCK have been developed and are effective in mice but have suboptimal specificity and sensitivity in humans. To identify a more suitable probe for clinical dCK PET imaging, we compared the selectivity of two candidate compounds-[(18)F]Clofarabine; 2-chloro-2'-deoxy-2'-[(18)F]fluoro-9-β-d-arabinofuranosyl-adenine ([(18)F]CFA) and 2'-deoxy-2'-[(18)F]fluoro-9-β-d-arabinofuranosyl-guanine ([(18)F]F-AraG)-for dCK and deoxyguanosine kinase (dGK), a dCK-related mitochondrial enzyme. We demonstrate that, in the tracer concentration range used for PET imaging, [(18)F]CFA is primarily a substrate for dCK, with minimal cross-reactivity. In contrast, [(18)F]F-AraG is a better substrate for dGK than for dCK. [(18)F]CFA accumulation in leukemia cells correlated with dCK expression and was abrogated by treatment with a dCK inhibitor. Although [(18)F]CFA uptake was reduced by deoxycytidine (dC) competition, this inhibition required high dC concentrations present in murine, but not human, plasma. Expression of cytidine deaminase, a dC-catabolizing enzyme, in leukemia cells both in cell culture and in mice reduced the competition between dC and [(18)F]CFA, leading to increased dCK-dependent probe accumulation. First-in-human, to our knowledge, [(18)F]CFA PET/CT studies showed probe accumulation in tissues with high dCK expression: e.g., hematopoietic bone marrow and secondary lymphoid organs. The selectivity of [(18)F]CFA for dCK and its favorable biodistribution in humans justify further studies to validate [(18)F]CFA PET as a new cancer biomarker for treatment stratification and monitoring.

The aim of this study was to investigate the relationship of deoxycytidine kinase (dCK) protein expression and gene single-nucleotide polymorphisms to gemcitabine chemosensitivity in patients with pancreatic ductal adenocarcinoma (PDAC).In total, 54 patients with resectable PDAC, who received postoperative gemcitabine-based therapy, were enrolled in this study, from January 2011 to April 2013. The dCK protein expression was measured retrospectively by immunohistochemistry. Furthermore, 5 single-nucleotide polymorphisms (C1205T, A9846G, A70G, C356G, and C364T) of the dCK gene were detected in PDAC cells by PCR amplification and sequencing.The dCK protein expression was found to be negatively correlated with age (P = 0.006), but correlated positively with overall survival (OS) (P = 0.000) and disease-free survival (DFS) (P = 0.003). The A9846G AA genotype in the dCK gene was significantly associated with reduced mortality compared with AG and GG genotypes. The OS and DFS were longer in patients with the A9846G AA genotype than the AG and GG genotypes. In univariate and multivariate analyses, we found that the dCK protein expression and A9846G genotype were significant predictors of both OS and DFS.Our study suggests that the dCK protein expression and A9846G genotype may act as prognostic biomarkers in identifying patients who are likely to benefit from postoperative gemcitabine therapy in PDAC., Competing Interests: The authors have no conflicts of interest to disclose.

Unlike for other digestive cancer entities, chemotherapy, radiotherapy, and targeted therapies have, so far, largely failed to improve patient survival in pancreatic adenocarcinoma (PDAC), which remains the fourth leading cause of cancer-related death in Europe and the United States. In this context, gene therapy may offer a new avenue for patients with PDAC. In this review, we explore the research currently ongoing in French laboratories aimed at defeating PDAC using nonviral therapeutic gene delivery, targeted transgene expression, or oncolytic virotherapy that recently or will soon bridge the gap between experimental models of cancer and clinical trials. These studies are likely to change clinical practice or thinking about PDAC management, as they represent a major advance not only for PDAC but may also significantly influence the field of gene-based molecular treatment of cancer.

Deoxycytidine kinase (dCK) (EC 2.7.1.74) is a key enzyme for salvage of deoxynucleosides and activation of numerous anticancer and antiviral nucleoside analogs. dCK activity is enhanced in response to several genotoxic treatments, which has been correlated with an increase of dCK phosphorylation at Ser-74. ATM was recently identified as the kinase responsible for Ser-74 phosphorylation and dCK activation after ionizing radiation (IR). Here, we investigated the role of ATM and the related kinase ATR in dCK activation induced by other types of DNA damage. Using ATM-deficient cells or the ATM inhibitor KU-60019, we found that ATM was not required for dCK activation caused by UV light, aphidicolin, cladribine, and unexpectedly also IR. On the other hand, the selective ATR inhibitor VE-821 significantly reduced up-regulation of dCK activity induced by these genotoxic agents, though not IR, and also down-regulated basal dCK activity. A role for ATR in the control of dCK activity was confirmed by using ATR siRNA and ATR-Seckel cells. ATR was also found to directly phosphorylate dCK at Ser-74 in vitro. Further studies revealed that ATR, which is also activated in response to IR, although later than ATM, was responsible for IR-induced dCK activation in ATM-deficient cells or in the presence of KU-60019. Overall, our results demonstrate that ATR controls basal dCK activity and dCK activation in response to replication stress and indicate that ATR can activate dCK after IR if ATM is lacking or inhibited., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Cytarabine (Ara-C) is the primary drug in different treatment schemas for acute myeloid leukemia (AML) and requires the human equilibrative nucleoside transporter (hENT1) to enter cells. The deoxycytidine kinase (dCK) enzyme limits its activation rate. Therefore, decreased expression levels of these genes may influence the response rate to this drug., Methods: AML patients without previous treatment were enrolled. The expression of hENT1 and dCK genes was analyzed using RT-PCR. Clinical parameters were registered. All patients received Ara-C + doxorubicin as an induction regimen (7 + 3 schema). Descriptive statistics were used to analyze data. Uni- and multivariate analyses were performed to determine factors that influenced response and survival., Results: Twenty-eight patients were included from January 2011 until December 2012. Median age was 36.5 years. All patients had an adequate performance status (43% with ECOG 1 and 57% with ECOG 2). Cytogenetic risk was considered unfavorable in 54% of the patients. Complete response was achieved in 53.8%. Cox regression analysis showed that a higher hENT1 expression level was the only factor that influenced response and survival., Conclusions: These results highly suggest that the pharmacogenetic analyses of Ara-C influx may be decisive in AML patients., (© 2016 S. Karger AG, Basel.)

Background: Putative biomarkers of gemcitabine response have been extensively studied in pancreatic cancer, but less so in other types of periampullary adenocarcinoma. The most studied biomarker is human equilibrative nucleoside transporter 1 (hENT1), and the activating enzyme deoxycytidine kinase (dCK) has also been linked to treatment response. The RNA-binding protein human antigen R (HuR) has been demonstrated to confer increased dCK levels in vitro and to predict gemcitabine response in vivo. Here, we investigated the prognostic impact of hENT1, dCK and HuR in pancreatobiliary (PB) and intestinal (I) type periampullary cancers, respectively., Material and Methods: Immunohistochemical expression of hENT1, dCK and HuR was evaluated in tissue microarrays with all primary tumours and 103 paired lymph node metastases from a consecutive retrospective cohort of 175 patients with resected periampullary adenocarcinomas., Results: In patients with PB-type tumours, neither hENT1 nor dCK expression was prognostic. A high HuR cytoplasmic/nuclear ratio was associated with a significantly reduced five-year overall survival (OS) in patients receiving adjuvant gemcitabine (HR 2.07, 95% CI 1.03-4.17) but not in untreated patients (pinteraction = 0.028). In patients with I-type tumours receiving adjuvant chemotherapy, high dCK expression was significantly associated with a prolonged recurrence-free survival (RFS) (HR 0.09, 95% CI 0.01-0.73, pinteraction = 0.023). Furthermore, HuR expression was associated with a prolonged OS and RFS in unadjusted but not in adjusted analysis and hENT1 expression was an independent predictor of a prolonged RFS (HR 0.24, 95% CI 0.10-0.59), regardless of adjuvant treatment., Conclusion: hENT1 expression is a favourable prognostic factor in I-type, but not in PB-type tumours. High dCK expression is a favourable prognostic factor in patients with I-type tumours receiving adjuvant treatment and a high cytoplasmic/nuclear HuR ratio is a negative prognostic factor in gemcitabine-treated PB-type tumours. Morphological subtype should always be considered in biomarker studies on periampullary cancer.

The purpose of the present study is to identify the determinant(s) of gemcitabine (dFdC)-sensitivity in pancreatic cancer tissues of patients treated with dFdC alone and in pancreatic cancer cell lines exposed to dFdC in vitro. Protein expression levels of 12 enzymes and 13 transporters potentially involved in transport and metabolism of dFdC in pancreatic cancer cell lines and tissues were quantified by means of our LC-MS/MS-based quantitative targeted proteomics technology. Protein expression levels of deoxycytidine kinase (dCK), uridine monophosphate-cytidine monophosphate (UMP-CMP) kinase, cytosolic nucleotidase III (cN-III), and equilibrative nucleoside transporter 1 (ENT1) were significantly correlated with IC50 or 1/IC50 in five cell lines with different sensitivities to dFdC (p < 0.05). Expression levels of the selected proteins in pancreatic cancer tissues of 10 patients with different progression-free survival (PFS) (49-955 days) were quantified, and their relationship with PFS was examined. Only the protein expression level of dCK was significantly correlated with PFS (p < 0.05). Multiple regression analysis was also performed, and combinations of ENT1, UMP-CMP kinase, CTPS1, and dCK were highly correlated with PFS. Our results indicate that the protein expression level of dCK in pancreatic cancer tissue is a good predictor of PFS, and thus dCK may be the best biomarker of dFdC sensitivity in pancreatic cancer patients treated with dFdC, although other proteins would also contribute to dFdC-sensitivity at the cellular level in vivo and in vitro.

Nucleoside metabolism enzymes are determinants of chemotherapeutic drug activity. The nucleoside salvage enzyme deoxycytidine kinase (dCK) activates gemcitabine (2', 2'-difluoro-2'-deoxycytidine) and is negatively regulated by deoxycytidine triphosphate (dCTP). Reduction of dCTP in tumor cells could, therefore, enhance gemcitabine activity. Mitochondrial thymidine kinase 2 (TK2) phosphorylates deoxycytidine to generate dCTP. We hypothesized that: (1) TK2 modulates human tumor cell sensitivity to gemcitabine, and (2) antisense knockdown of TK2 would decrease dCTP and increase dCK activity and gemcitabine activation. siRNA downregulation of TK2 sensitized MCF7 and HeLa cells (high and moderate TK2) but not A549 cells (low TK2) to gemcitabine. Combined treatment with TK2 siRNA and gemcitabine increased dCK. We also hypothesized that TK2 siRNA-induced drug sensitization results in mitochondrial damage that enhances gemcitabine effectiveness. TK2 siRNA and gemcitabine decreased mitochondrial redox status, DNA content, and activity. This is the first demonstration of a direct role for TK2 in gemcitabine resistance, or any independent role in cancer drug resistance, and further distinguishes TK2 function from that of other dTMP-producing enzymes [cytosolic TK1 and thymidylate synthase (TS)]. siRNA knockdown of TK1 and/or TS did not sensitize cancer cells to gemcitabine indicating that, among the 3 enzymes, only TK2 is a candidate therapeutic target for combination with gemcitabine.

The anti-tumor activity of gemcitabine (GEM) has been clinically proven in several solid tumors, including pancreatic cancer, biliary tract cancer, urinary bladder cancer, and non-small cell lung cancer. However, problems remain with issues such as acquisition of chemoresistance against GEM. GEM is activated after phosphorylation by deoxycytidine kinase (DCK) inside of the cell; thus, DCK inactivation is one of the important mechanisms for acquisition of GEM resistance. We previously investigated the DCK gene in multiple GEM resistant cancer cell lines and identified frequent inactivating mutations. In this study, we identified two crucial genetic alteration in DCK. (1) A total deletion of DCK in RTGBC1-TKB, an acquired GEM resistant cell line derived from a gall bladder cancer cell line TGBC1-TKB. (2) An E197K missense alteration of DCK in MKN28, a gastric cancer cell line; its acquired GEM resistant cancer cell line, RMKN28, showed a loss of the normal E197 allele. We introduced either normal DCK or altered DCK&#95;E197K into RMKN28 and proved that only the introduction of normal DCK restored GEM sensitivity. Furthermore, we analyzed 104 healthy volunteers and found that none of them carried the same base substitution observed in MKN28. These results strongly suggest that (1) the E197K alteration in DCK causes inactivation of DCK, and that (2) loss of the normal E197 allele is the crucial mechanism in acquisition of GEM resistance in RMKN28., (Copyright © 2015 Elsevier Inc. All rights reserved.)