Adult T-cell leukemia-lymphoma acquires resistance to DNA demethylating agents through dysregulation of enzymes involved in pyrimidine metabolism.

Adult T-cell leukemia-lymphoma (ATL) is an aggressive neoplasm derived from T-cells transformed by human T-cell lymphotropic virus-1 (HTLV-1). Recently, we reported that regional DNA hypermethylation in HTLV-1-infected T-cells reflects the disease status of ATL and the anti-ATL effects of DNA demethylating agents, including azacitidine (AZA), decitabine (DAC) and a new DAC prodrug, OR-2100 (OR21), which we developed. Here, to better understand the mechanisms underlying drug resistance, we generated AZA-, DAC- and OR21-resistant (AZA-R, DAC-R and OR21-R, respectively) cells from the ATL cell line TL-Om1 and the HTLV-1-infected cell line MT-2 via long-term drug exposure. The efficacy of OR21 was almost the same as that of DAC, indicating that the pharmacodynamics of OR21 were due to release of DAC from OR21. Resistant cells did not show cellular responses observed in parental cells induced by treatment with drugs, including growth suppression, depletion of DNA methyltransferase DNMT1 and DNA hypomethylation. We also found that reduced expression of deoxycytidine kinase (DCK) correlated with lower susceptibility to DAC/OR21 and that reduced expression of uridine cytidine kinase2 (UCK2) correlated with reduced susceptibility to AZA. DCK and UCK2 catalyze phosphorylation of DAC and AZA, respectively; reconstitution of expression reversed the resistant phenotypes. A large homozygous deletion in DCK and a homozygous splice donor site mutation in UCK2 were identified in DAC-R TL-Om1 and AZA-R TL-Om1, respectively. Both genomic mutations might lead to loss of protein expression. Thus, inactivation of UCK2 and DCK might be a putative cause of phenotypes that are resistant to AZA and DAC/OR21, respectively.

Hypoxia-induced deoxycytidine kinase contributes to epithelial proliferation in pulmonary fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a deadly lung disease with few therapeutic options. Apoptosis of alveolar epithelial cells, followed by abnormal tissue repair characterized by hyperplastic epithelial cell formation, is a pathogenic process that contributes to the progression of pulmonary fibrosis. However, the signaling pathways responsible for increased proliferation of epithelial cells remain poorly understood. OBJECTIVES: To investigate the role of deoxycytidine kinase (DCK), an important enzyme for the salvage of deoxynucleotides, in the progression of pulmonary fibrosis. METHODS: DCK expression was examined in the lungs of patients with IPF and mice exposed to bleomycin. The regulation of DCK expression by hypoxia was studied in vitro and the importance of DCK in experimental pulmonary fibrosis was examined using a DCK inhibitor and alveolar epithelial cell-specific knockout mice. MEASUREMENTS AND MAIN RESULTS: DCK was elevated in hyperplastic alveolar epithelial cells of patients with IPF and in mice exposed to bleomycin. Increased DCK was localized to cells associated with hypoxia, and hypoxia directly induced DCK in alveolar epithelial cells in vitro. Hypoxia-induced DCK expression was abolished by silencing hypoxia-inducible factor 1α and treatment of bleomycin-exposed mice with a DCK inhibitor attenuated pulmonary fibrosis in association with decreased epithelial cell proliferation. Furthermore, DCK expression, and proliferation of epithelial cells and pulmonary fibrosis was attenuated in mice with conditional deletion of hypoxia-inducible factor 1α in the alveolar epithelium. CONCLUSIONS: Our findings suggest that the induction of DCK after hypoxia plays a role in the progression of pulmonary fibrosis by contributing to alveolar epithelial cell proliferation.

Deoxycytidine kinase regulates the G2/M checkpoint through interaction with cyclin-dependent kinase 1 in response to DNA damage.

Deoxycytidine kinase (dCK) is a rate limiting enzyme critical for phosphorylation of endogenous deoxynucleosides for DNA synthesis and exogenous nucleoside analogues for anticancer and antiviral drug actions. dCK is activated in response to DNA damage; however, how it functions in the DNA damage response is largely unknown. Here, we report that dCK is required for the G2/M checkpoint in response to DNA damage induced by ionizing radiation (IR). We demonstrate that the ataxia-telangiectasia-mutated (ATM) kinase phosphorylates dCK on Serine 74 to activate it in response to DNA damage. We further demonstrate that Serine 74 phosphorylation is required for initiation of the G2/M checkpoint. Using mass spectrometry, we identified a protein complex associated with dCK in response to DNA damage. We demonstrate that dCK interacts with cyclin-dependent kinase 1 (Cdk1) after IR and that the interaction inhibits Cdk1 activity both in vitro and in vivo. Together, our results highlight the novel function of dCK and provide molecular insights into the G2/M checkpoint regulation in response to DNA damage.

Requirement for deoxycytidine kinase in T and B lymphocyte development.

Deoxycytidine kinase (dCK) is a rate-limiting enzyme in deoxyribonucleoside salvage, a metabolic pathway that recycles products of DNA degradation. dCK phosphorylates and therefore activates nucleoside analog prodrugs frequently used in cancer, autoimmunity, and viral infections. In contrast to its well established therapeutic relevance, the biological function of dCK remains enigmatic. Highest levels of dCK expression are found in thymus and bone marrow, indicating a possible role in lymphopoiesis. To test this hypothesis we generated and analyzed dCK knockout (KO) mice. dCK inactivation selectively and profoundly affected T and B cell development. A 90-fold decrease in thymic cellularity was observed in the dCK KO mice relative to wild-type littermates. Lymphocyte numbers in the dCK KO mice were 5- to 13-fold below normal values. The severe impact of dCK inactivation on lymphopoiesis was unexpected given that nucleoside salvage has been thought to play a limited, "fine-tuning" role in regulating deoxyribonucleotide triphosphate pools produced by the de novo pathway. The dCK KO phenotype challenges this view and indicates that, in contrast to the great majority of other somatic cells, normal lymphocyte development critically requires the deoxyribonucleoside salvage pathway.

Development of gene therapy in association with clinically used cytotoxic deoxynucleoside analogues.

The clinical use of cytotoxic deoxynucleoside analogues is often limited by resistance mechanisms due to enzymatic deficiency, or high toxicity in nontumor tissues. To improve the use of these drugs, gene therapy approaches have been proposed and studied, associating clinically used deoxynucleoside analogues such as araC and gemcitabine and suicide genes or myeloprotective genes. In this review, we provide an update of recent results in this area, with particular emphasis on human deoxycytidine kinase, the deoxyribonucleoside kinase from Drosophila melanogaster, purine nucleoside phosphorylase from Escherichia coli, and human cytidine deaminase. Data from literature clearly show the feasibility of these systems, and clinical trials are warranted to conclude on their use in the treatment of cancer patients.

Study of the efficacy of a pronucleotide of 2-chloro-2'-deoxyadenosine in deoxycytidine kinase-deficient lymphoma cells.

2-Chloro-2 '-deoxyadenosine (CdA, cladribine) is a nucleoside analogue (NA) used for the treatment of lymphoproliferative disorders. Phosphorylation of the drug to CdAMP by deoxycytidine kinase (dCK) and its subsequent conversion to CdATP is essential for its efficacy. DCK deficiency is a common mechanism of resistance to NA, which could be overcome by the pronucleotide approach. The latter consists of using the nucleoside monophosphate conjugated to a lipophilic group enabling CdAMP to enter the cells by passive diffusion. In this study, we show that cycloSaligenyl-2-chloro-2 '-deoxyadenosine monophosphate (cycloSal-CdAMP) is 10-fold more potent that CdA in a dCK-deficient lymphoma cell line. These results suggest that the use of cycloSal-nucleotides could be a strategy to counteract resistance caused by dCK deficiency.

Potential mechanisms of resistance to cytarabine in AML patients.

To determine whether the human equilibrative nucleoside transporter 1 (hENT1), deoxycytidine kinase (dCK), cytoplasmic 5'-nucleotidase (5NT), cytidine deaminase (CDD), topoisomerase I (TOPO I) and topoisomerase II alpha (TOPO II) are involved in clinical resistance to cytarabine (ara-C), we analyzed the level of expression of these parameters by reverse transcriptase polymerase chain reaction (rt-PCR), at diagnosis in the blast cells of 77 acute myeloid leukemia (AML) patients treated with ara-C, including 31 for whom samples were collected at first relapse. By univariate and/or multivariate analyses, patients with expression of 5NT or hENT1 deficiency at diagnosis had significantly shorter disease-free survival (DFS) and overall survival (OS). These results suggest that expression of 5NT and reduced hENT1 in leukemic blasts at diagnosis are correlated with clinical outcome and may play a role in resistance mechanisms to ara-C in patients with AML.

New targets for pyrimidine antimetabolites for the treatment of solid tumours. 2: Deoxycytidine kinase.

Deoxycytidine kinase is an enzyme required for the activation of, for example, cytarabine, the most widely used agent for the chemotherapy of haematological malignancies. However, deoxycytidine kinase also plays an important role in the activation of several new agents used in the treatment of leukaemia, such as cladribine. Recently, a new cytidine analogue, gemcitabine, has shown impressive activity as a single agent against several solid malignancies (ovarian cancer, non-small cell lung cancer), demonstrating that in solid tumours deoxycytidine kinase can be an important target for the activation of antimetabolites. Studies on the regulation of deoxycytidine kinase have shown that the enzyme has a complicated regulation (feedback inhibition by the product and regulation by ribonucleotides). Modulation of deoxycytidine kinase activity has already been shown to be an effective way to improve the effect of cytarabine and will probably be a target for new therapies.

Regulation of purine deoxynucleoside phosphorylation by deoxycytidine kinase from human leukemic blast cells.

The kinetics and regulation of nucleoside phosphorylation by highly purified human deoxycytidine kinase from leukemic lymphoblasts were studied. The phosphorylation of purine nucleosides by this enzyme showed sensitivity to the endogenous inhibitors dCTP and UDP three times greater than the phosphorylation of dCyd. Examination of nucleotide pools in human T and B lymphoblasts disclosed that the levels of dCTP and UDP in these cells were sufficient to regulate kinase activity. The enhanced sensitivity of the kinase to dCTP and UDP was related to its reduced ability to interact with purine nucleosides. Comparison of the phosphorylation kinetics for pyrimidine and purine dideoxynucleosides used in antiviral therapy showed that the purine nucleosides were at least 50-fold less efficient as enzyme substrates. These results suggest that the phosphorylation of pharmacologically active purine nucleosides by deoxycytidine kinase is regulated by cellular nucleotide pools.

Deoxyribonucleoside triphosphate metabolism and the mammalian cell cycle. Effects of hydroxyurea on mutant and wild-type mouse S49 T-lymphoma cells.

DNA precursor synthesis can be blocked specifically by the drug hydroxyurea (HU) which has therefore been used for anticancer therapy. High concentrations of HU, however, affect other processes than DNA synthesis; nevertheless, most studies on the biological action of HU have been made with concentrations at least one order of magnitude higher than those needed for cell-growth inhibition. In this study we characterized the effects of low concentrations of HU (i.e. concentrations leading to 50% inhibition of cell growth in 72 h) on cell cycle kinetics and nucleotide pools in mouse S49 cells with various defined alterations in DNA precursor synthesis. The effect of 50 microM HU on deoxyribonucleoside triphosphate pools was a 2-3-fold decrease in the dATP and dGTP pools, with no change in the dCTP pool and a certain increase in the dTTP pool. Addition of deoxycytidine or thymidine led to a partial reversal of the growth inhibition and cell-cycle perturbation caused by HU, and was accompanied by an increased level of the deoxyribonucleoside triphosphates. Addition of purine deoxyribonucleoside gave no protection, indicating that salvage of these nucleosides could not supply precursors for DNA synthesis in T-lymphoma cells. We observed a higher sensitivity to HU of cells lacking purine nucleoside phosphorylase or with a ribonucleotide reductase with altered allosteric regulation. Cells lacking thymidine kinase or deoxycytidine kinase were just as sensitive as wild-type cells.

Role of deoxycytidine kinase in the inhibitory activity of 5-substituted 2'-deoxycytidines and cytosine arabinosides on tumor cell growth.

A number of 5-substituted derivatives of dCyd and 1-beta-D-arabinofuranosylcytosine (araC) have been evaluated for their inhibitory effects on the growth of three murine leukemia cell lines (L1210/0, L1210/BdUrd, and L1210/araC). The L1210/BdUrd and L1210/araC cell lines were selected from the parental L1210/0 cell line by their ability to grow at high concentrations of 5-bromo-2'-deoxyuridine and araC, respectively; the L1210/BdUrd cell line was deficient in dThd kinase activity, whereas the L1210/araC cell was deficient in dCyd kinase activity. The most effective inhibitors of L1210/0 cell proliferation were 5-fluoro-dCyd, araC, and 5-fluoro-araC. Their 50% inhibitory dose fell within the 0.001-0.015 micrograms/ml range. The 5-substituted araC analogues were much less inhibitory for L1210/araC cells but equally inhibitory for L1210/BdUrd as for the parental L1210/0 cell line. The role of dCyd kinase in the antitumor activity of the dCyd and araC analogues was further assessed by kinetic studies with dCyd kinase extracted from L1210/0 cells. All dCyd and araC analogues caused a competitive inhibition of dCyd kinase, the most potent inhibitor being 5-fluoro-dCyd (Ki/Km value 0.24). The Km of dCyd kinase from L1210/0 cells for dCyd was 23.1 microM as compared with 50 microM for araC. These values were increased to 53 and 182 microM, respectively, for the dCyd kinase isolated from L1210/araC cells.