dCK Expression Research Articles

Antineoplastic Activity of Sodium Caseinate in a Cytarabine-Resistant Mouse Acute Myeloid Leukemia Cell Line.

Acute myeloid leukemia (AML) is a hematological neoplasm of rapid and progressive onset, and is the most common form of leukemia in adults. Chemoresistance to conventional treatments such as cytarabine (Ara-C) and daunorubicin is a main cause of relapse, recurrence, metastasis, and high mortality in AML patients. It is known that sodium caseinate (SC), a salt derived from casein, a milk protein, inhibits growth and induces apoptosis in acute myeloid leukemia cells but not in normal hematopoietic cells. However, it is unknown whether SC retains its antileukemic effect in cytarabine-resistant AML cell lines. To evaluate the antineoplastic effect of SC in cytarabine-resistant leukemia models. The SC inhibits the growth and induces apoptosis in parental WEHI-3 AML cells. Here, we generated two cytarabine-resistant sublines, WEHI-CR25 and WEHI-CR50, which exhibit 6- and 16-fold increased resistance to cytarabine, respectively, compared to the parental WEHI-3 cells. Thus, these sublines mimic a chemoresistant model. We demonstrate that WEHI-CR25 and WEHI-CR50 cells retain sensitivity to SC, similar to parental WEHI-3 cells. This sensitivity results in inhibited cell proliferation, induced apoptosis, and increased expression of ENT1 and dCK, molecules involved in the entry and metabolism of Ara-C, while decreasing MDR1 expression. Additionally, we observed that SC prolonged the survival of WEHI-CR50 tumor-bearing mice, despite their resistance to Ara-C. This is the first evidence that SC, a milk protein, may inhibit proliferation and induce apoptosis in cytarabine-resistant cells.

Hyperthermia improves gemcitabine sensitivity of pancreatic cancer cells by suppressing the EFNA4/β-catenin axis and activating dCK

BackgroundPreviously, our investigations have underscored the potential of hyperthermia to improve the therapeutic efficacy of gemcitabine (GEM) in pancreatic cancer (PC). Nonetheless, the precise underlying mechanisms remain elusive. MethodsWe engineered two GEM-resistant PC cell lines (BxPC-3/GEM and PANC-1/GEM) and treated them with GEM alongside hyperthermia. The impact of hyperthermia on the therapeutic potency of GEM was ascertained through MTT assay, assessment of the concentration of its active metabolite dFdCTP, and evaluation of deoxycytidine kinase (dCK) activity. Lentivirus-mediated dCK silencing was further employed to validate its involvement in mediating the GEM-sensitizing effect of hyperthermia. The mechanism underlying hyperthermia-mediated dCK activation was explored using bioinformatics analyses. The interplay between hyperthermia and the ephrin A4 (EFNA4)/β-catenin/dCK axis was investigated, and their roles in GEM resistance was further explored via the establishment of xenograft tumor models in nude mice. ResultsHyperthermia restored dCK expression in GEM-resistant cell lines, concurrently enhancing GEM sensitivity and fostering DNA damage and cell death. These observed effects were negated by dCK silencing. Regarding the mechanism, hyperthermia activated dCK by downregulating EFNA4 expression and mitigating β-catenin activation. Overexpression of EFNA4 activated the β-catenin while suppressing dCK, thus diminishing cellular GEM sensitivity—a phenomenon remediated by the β-catenin antagonist MSAB. Consistently, in vivo, hyperthermia augmented the therapeutic efficacy of GEM on xenograft tumors through modulation of the ephrin A4/β-catenin/dCK axis. ConclusionThis study delineates the role of hyperthermia in enhancing GEM sensitivity of PC cells, primarily mediated through the suppression of the EFNA4/β-catenin axis and activation of dCK.

Role of Intracellular Drug Disposition in the Response of Acute Myeloid Leukemia to Cytarabine and Idarubicin Induction Chemotherapy.

Despite its often low efficacy and high toxicity, the standard treatment for acute myeloid leukemia (AML) is induction chemotherapy with cytarabine and idarubicin. Here, we have investigated the role of transporters and drug-metabolizing enzymes in this poor outcome. The expression levels (RT-qPCR) of potentially responsible genes in blasts collected at diagnosis were related to the subsequent response to two-cycle induction chemotherapy. The high expression of uptake carriers (ENT2), export ATP-binding cassette (ABC) pumps (MDR1), and enzymes (DCK, 5-NT, and CDA) in the blasts was associated with a lower response. Moreover, the sensitivity to cytarabine in AML cell lines was associated with ENT2 expression, whereas the expression of ABC pumps and enzymes was reduced. No ability of any AML cell line to export idarubicin through the ABC pumps, MDR1 and MRP, was found. The exposure of AML cells to cytarabine or idarubicin upregulated the detoxifying enzymes (5-NT and DCK). In AML patients, 5-NT and DCK expression was associated with the lack of response to induction chemotherapy (high sensitivity and specificity). In conclusion, in the blasts of AML patients, the reduction of the intracellular concentration of the active metabolite of cytarabine, mainly due to the increased expression of inactivating enzymes, can determine the response to induction chemotherapy.

Human equilibrative nucleoside transporter 1 (hENT1) and deoxycytidine kinase (DCK) are predictive biomarkers for Gemcitabine-based chemoradiotherapy in resected pancreatic cancer patients: a retrospective cohort study

Background and Aim Pancreatic cancer is reported to be one of the cancer-related deaths all over the world. Gemcitabine has been identified as one of the important therapies of malignant diseases. Gemcitabine cellular uptake is allowed by the transporter protein, human equilibrative nucleoside transporter 1 (hENT1). Gemcitabine is phosphorylated by deoxycytidine kinase (dCK) to its active metabolites intracellularly. This study aimed to determine retrospectively the correlation between hENT1 and dCK expression by immunohistochemistry and the overall survival outcome in pancreatic cancer patients who underwent resection and received gemcitabine chemotherapy postoperatively. Materials and methods The immunohistochemical analysis was accomplished on samples from 140 patients, who underwent pancreatic cancer resection and was treated by gemcitabine. Immunostaining was done by two expert pathologists and both are blind to patient’s characteristics. The intensity of staining of the tumor determines the scoring for hENT1 and dCK. Results Of the 140 tissue samples, 73 (52.1%) had detectable hENT1 cell membrane immunostaining and 67 samples (47.8%) without detectable hENT1. Patients who showed no significant hENT1 immunostaining had a median survival of 6 months, while patients who showed significant hENT1 staining had a median survival longer by 17 months. Of the 140 tissue samples, 52 (37.1%) had detectable dCK cytoplasmic immunostaining and 88 samples (62.9%) without detectable dCK. Patients who showed no significant dCK immunostaining had a median survival of 6.57 months, while patients who showed significant dCK staining had a median survival longer by 19 months. Conclusion In patients who received gemcitabine after pancreatic resection, a high expression of hENT1 and dCK are significantly correlated with a longer survival. In addition, in patients who underwent pancreatic cancer resection, immunohistochemical analysis of hENT1 and dCK are considered significant predictive markers to choose patients responding to gemcitabine therapy.

Efficient Knock-out of Serine Protease Cathepsin G Enhances Chemosensitivity to Cytarabine By Modulating the Cytarabine Metabolic Pathway Genes in Acute Myeloid Leukemia

Relapse and refractoriness to chemotherapy may exist prior to the exposure to chemotherapeutic agents or can develop and increase during the treatment leading to acquired resistance. Despite considerable progress in developing new therapeutic agents to be administered singly or in combination with the old regimen, drug resistance poses a serious concern preventing adequate response to Acute Myeloid Leukemia (AML) treatment with cytarabine (AraC) and daunorubicin (DNR). Differentially expression analysis of the RNA sequencing data between the naive and the AraC-resistant human monocytic MLL-AF9 cell line THP1 revealed cathepsin G (CTSG) as one of the top hits among the upregulated genes with Log2Fc 4.2. This was further validated in the AML cell line by q-RT-PCR with a fold change ~5 and immunoblot, confirming increased CTSG expression in THP1 Ara-C resistant cell line. Although the role of (CTSG) in host defence, granulopoiesis has been intensively investigated, its role in acquired cytarabine resistance has not been explored yet. To investigate the functional role of CTSG in the acquired resistant model, we performed CRISPR/Cas9-based editing of CTSG in THP1 Ara-C resistant cell line using dual gRNAs, which resulted in a robust deletion of the targeted region and complete loss of protein expression as measured by immunoblotting. CTSG KO cells showed a reduced cell proliferation rate with a significant reduction at 72hrs (compared to the scrambled gRNA (ScrG) transduced cells doubling time CTSG KO=30.3 vs ScrG=22.39, p= 0.0049). We also checked the sensitivity of the CTSG KO cells to AraC and other chemo drugs (DNR, ATO and Venetoclax). In vitro cytotoxicity assay (72hrs) for the CTSG KO cells treated with AraC showed sensitivity to Ara-C with a reduction in IC50 as low as the parental IC50 (IC50 ScrG: 1457 µM; IC50 CTSG KO: 38.13 µM; IC50 Parental: 56.6 µM). CTSG KO cells also sensitized the cells to DNR (IC50 ScrG: 288nM vs and CTSG KO: 58nM but not to ATO and venetoclax. Further, we measured the intracellular DNR by flow cytometry in the CTSG KO cells. There was a considerable increase in the intracellular DNR at the end of the treatment compared to the ScrG cells (~1.5-fold, p <0.0001). Our results show that CTSG KO re-sensitized the resistant cells to cytarabine and showed significant sensitivity to DNR. To further understand the molecular mechanism contributing to the increased sensitivity of the CTSG KO cells to AraC, we checked our basal RNA sequencing data for the expression of AraC metabolizing genes. The THP1 Ara-C R cell line showed decreased expression of DCK and increased expression of NT5C2 and CDA compared to the parental cell line. We checked the RNA expression of Ara-C metabolizing genes post CTSG KO by RT-q PCR. There was a 6-fold increase in DCK and a 5-fold decrease in NT5C2 with a further increase in SAMHD1 and CDA genes with no changes in the RRM1 and ENT1 genes, suggesting that CTSG KO increased the sensitivity to by rewiring the AraC metabolic pathway. Further studies are ongoing to assess the in-vivo efficacy of molecular and pharmacological inhibition of CTSG in reversing AraC resistance in the AML mouse model.

ET-7 Roles for hENT1 and dCK in gemcitabine sensitivity and malignancy of meningioma

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.

ET-6 Gemcitabine radiosensitization primes irradiated malignant meningioma cells for senolytic elimination by navitoclax

BACKGROUND: Malignant meningioma is an aggressive tumor that requires adjuvant radiotherapy after surgery, yet there has been no standard systemic therapy established so far. We have demonstrated that malignant meningioma cells are exquisitely sensitive to gemcitabine due to their increased expression of hENT1 and dCK, which play critical roles in the intracellular transport and activation of gemcitabine, respectively (Takeda et al. Oncotarget 8:90996, 2017; Yamamoto et al., Neuro-Oncol 23:945, 2021). Significantly, in support of our findings, the efficacy and safety of gemcitabine have recently been documented in a small case series of patients with recurrent meningiomas, which has further led to a phase 2 clinical trial to evaluate the efficacy of gemcitabine in recurrent high-grade meningiomas (Khaddar et al., South Asian J Cancer 9:261, 2020). Besides its efficacy as a single agent, gemcitabine reportedly has a radiosensitizing effect in pancreatic cancer. However, it remains unknown whether or how gemcitabine interacts with ionizing radiation (IR) in malignant meningioma cells. METHODS: We examined radiosensitization effects of gemcitabine using malignant meningioma cell lines and xenografts (s.c. and i.c.) and explored the underlying mechanisms. RESULTS: Gemcitabine sensitized malignant meningioma cells remarkably to IR through the induction of senescence both in vitro and in vivo. Gemcitabine augmented the intracellular production of reactive oxygen species (ROS) by IR, which, together with cell growth suppression/senescence induced by this combination, was inhibited by N-acetyl-cysteine, suggesting a pivotal role for ROS in these combinatorial effects. Navitoclax, a senolytic drug, further enhanced the effects of the combination of gemcitabine and IR in vitro and in vivo by strongly inducing apoptotic cell death in senescent cells. CONCLUSION: These results suggest that gemcitabine is not only a promising radiosensitizer for malignant meningioma but also creates in combination with IR a therapeutic vulnerability of senescent meningioma cells to senolytics. (submitted for publication)

Differences between intrinsic and acquired nucleoside analogue resistance in acute myeloid leukaemia cells

BackgroundSAMHD1 mediates resistance to anti-cancer nucleoside analogues, including cytarabine, decitabine, and nelarabine that are commonly used for the treatment of leukaemia, through cleavage of their triphosphorylated forms. Hence, SAMHD1 inhibitors are promising candidates for the sensitisation of leukaemia cells to nucleoside analogue-based therapy. Here, we investigated the effects of the cytosine analogue CNDAC, which has been proposed to be a SAMHD1 inhibitor, in the context of SAMHD1.MethodsCNDAC was tested in 13 acute myeloid leukaemia (AML) cell lines, in 26 acute lymphoblastic leukaemia (ALL) cell lines, ten AML sublines adapted to various antileukaemic drugs, 24 single cell-derived clonal AML sublines, and primary leukaemic blasts from 24 AML patients. Moreover, 24 CNDAC-resistant sublines of the AML cell lines HL-60 and PL-21 were established. The SAMHD1 gene was disrupted using CRISPR/Cas9 and SAMHD1 depleted using RNAi, and the viral Vpx protein. Forced DCK expression was achieved by lentiviral transduction. SAMHD1 promoter methylation was determined by PCR after treatment of genomic DNA with the methylation-sensitive HpaII endonuclease. Nucleoside (analogue) triphosphate levels were determined by LC-MS/MS. CNDAC interaction with SAMHD1 was analysed by an enzymatic assay and by crystallisation.ResultsAlthough the cytosine analogue CNDAC was anticipated to inhibit SAMHD1, SAMHD1 mediated intrinsic CNDAC resistance in leukaemia cells. Accordingly, SAMHD1 depletion increased CNDAC triphosphate (CNDAC-TP) levels and CNDAC toxicity. Enzymatic assays and crystallisation studies confirmed CNDAC-TP to be a SAMHD1 substrate. In 24 CNDAC-adapted acute myeloid leukaemia (AML) sublines, resistance was driven by DCK (catalyses initial nucleoside phosphorylation) loss. CNDAC-adapted sublines displayed cross-resistance only to other DCK substrates (e.g. cytarabine, decitabine). Cell lines adapted to drugs not affected by DCK or SAMHD1 remained CNDAC sensitive. In cytarabine-adapted AML cells, increased SAMHD1 and reduced DCK levels contributed to cytarabine and CNDAC resistance.ConclusionIntrinsic and acquired resistance to CNDAC and related nucleoside analogues are driven by different mechanisms. The lack of cross-resistance between SAMHD1/ DCK substrates and non-substrates provides scope for next-line therapies after treatment failure.

Different Susceptibility of T and B Cells to Cladribine Depends On Their Levels of Deoxycytidine Kinase Activity Linked to Activation Status

Deoxycytidine kinase (dCK) and 5’ deoxynucleotidase (NT5C2) are involved in metabolism of cladribine (2CdA), the immunomodulatory drug for multiple sclerosis; by mediating phosphorylation (activation) or phosphorolysis (deactivation) of 2CdA, respectively, these enzymes promote or prevent its accumulation in the cell, which leads to cell death. In particular, lymphocytes which present with a high intracellular dCK/NT5C2 ratio are more sensitive to 2CdA than other immune cells. We aim at determining if the expression of these enzymes and/or their activity differ in specific progenitor and mature immune cells and are influenced by cellular activation and/or exposure to 2CdA. Flow cytometry analysis showed no difference in dCK/NT5C2 ratio in progenitor and mature immune cells. 2CdA induced apoptosis in stimulated T and B cells and unstimulated B cells. dCK expression was enhanced by 2CdA at mRNA and protein levels in activated T cells and mRNA level in activated B cells. dCK activity, measured through an in-house luminescence release enzyme assay was higher in activated T and B cells, and such an increase was abrogated in activated B cells, but not T cells, upon exposure to 2CdA. These results reveal an important relationship between dCK activity and the effect of 2CdA on B and T cells, according to their activation status. Further study is warranted to evaluate whether dCK activity could, in the future, be a suitable predictive biomarker of lymphocyte response to 2CdA.Graphical

Roles for hENT1 and dCK in gemcitabine sensitivity and malignancy of meningioma.

BackgroundHigh-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.MethodsWe 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.ResultshENT1 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.ConclusionsThe 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.

Drug-repositioning screening identified fludarabine and risedronic acid as potential therapeutic compounds for malignant pleural mesothelioma

SummaryObjectives Malignant pleural mesothelioma (MPM) is an occupational disease mainly due to asbestos exposure. Effective therapies for MPM are lacking, making this tumour type a fatal disease. Materials and Methods In order to meet this need and in view of a future “drug repositioning” approach, here we screened five MPM (Mero-14, Mero-25, IST-Mes2, NCI-H28 and MSTO-211H) and one SV40-immortalized mesothelial cell line (MeT-5A) as a non-malignant model, with a library of 1170 FDA-approved drugs. Results Among several potential compounds, we found that fludarabine (F-araA) and, to a lesser extent, risedronic acid (RIS) were cytotoxic in MPM cells, in comparison to the non-malignant Met-5A cells. In particular, F-araA reduced the proliferation and the colony formation ability of the MPM malignant cells, in comparison to the non-malignant control cells, as demonstrated by proliferation and colony formation assays, in addition to measurement of the phospho-ERK/total-ERK ratio. We have shown that the response to F-araA was not dependent upon the expression of DCK and NT5E enzymes, nor upon their functional polymorphisms (rs11544786 and rs2295890, respectively). Conclusion This drug repositioning screening approach has identified that F-araA could be therapeutically active against MPM cells, in addition to other tumour types, by inhibiting STAT1 expression and nucleic acids synthesis. Further experiments are required to fully investigate this.

Decreased drug resistance of bladder cancer using phytochemicals treatment.

The aim of the study is to investigate the ability of phytochemicals to overcome the multiple drug resistance (MDR) of bladder cancer. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the cytotoxic sensitivity of T24-GCB cells, a GCB resistant cell line, to different phytochemicals, including capsaicin, quercetin, curcumin, and resveratrol, and their combination with gemcitabine. Western blot analysis was used to detect the expression of membranous ABCC2 and metabolic proteins, DCK, TK1, and TK2 in tumor cells. Animal models were used to confirm the treatment efficacy of phytochemicals in combination with gemcitabine to bladder cancer. The observed/expected ratio of cytotoxicity analysis revealed that capsaicin has synergistic effect with gemcitabine to T24-GCB cells in a dose-dependent pattern. Quercetin, curcumin, and resveratrol have additive effect with gemcitabine to T24-GCB cells. Capsaicin and quercetin alone and combination with gemcitabine decreased the expression of ABCC2 and DCK and TKs, in T24-GCB cells. On the contrary, resveratrol and curcumin alone and combination with gemcitabine increased the expression of ABCC2 but decreased cytoplasmic kinases simultaneously. In xenografted subcutaneous tumor model on nude mice, combination treatment of capsaicin and gemcitabine demonstrated the highest tumor suppression effect when compared to capsaicin or gemcitabine treatment alone. The MDR of bladder cancer is closely related to membranous ABCC2, cytoplasmic DCK, and TKs expression. Capsaicin owns the strongest synergistic cytotoxic effect of gemcitabine to T24-GCB cells. This combination regimen may provide as an adjunctive treatment for overcoming MDR in bladder cancer.

Cell- and Polymerase-Selective Metabolic Labeling of Cellular RNA with 2′-Azidocytidine

Metabolic labeling of cellular RNA is a powerful approach to investigate RNA biology. In addition to revealing whole transcriptome dynamics, targeted labeling strategies can be used to study individual RNA subpopulations within complex systems. Here, we describe a strategy for cell- and polymerase-selective RNA labeling with 2'-azidocytidine (2'-AzCyd), a modified nucleoside amenable to bioorthogonal labeling with SPAAC chemistry. In contrast to 2'-OH-containing pyrimidine ribonucleosides, which rely upon uridine-cytidine kinase 2 (UCK2) for activation, 2'-AzCyd is phosphorylated by deoxycytidine kinase (dCK), and we find that expression of dCK mediates cell-selective 2'-AzCyd labeling. Further, 2'-AzCyd is primarily incorporated into rRNA and displays low cytotoxicity and high labeling efficiency. We apply our system to analyze the turnover of rRNA during ribophagy induced by oxidative stress or mTOR inhibition to show that 28S and 18S rRNAs undergo accelerated degradation. Taken together, our work provides a general approach for studying dynamic RNA behavior with cell and polymerase specificity and reveals fundamental insights into nucleotide and nucleic acid metabolism.

Targeting Casein Kinase 1 Delta Sensitizes Pancreatic and Bladder Cancer Cells to Gemcitabine Treatment by Upregulating Deoxycytidine Kinase.

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.

DCK is a promising prognostic biomarker and correlated with immune infiltrates in hepatocellular carcinoma

BackgroundDeoxycytidine kinase (DCK), an enzyme in the nucleoside biosynthetic pathway, can affect the development of immune cells. However, the relationships between the expression of DCK, patient prognosis, and tumor-infiltrating immune cells (TIICs) in hepatocellular carcinoma (HCC) are still unclear.MethodsThe expression of DCK in HCC was analyzed through the Oncomine and Tumor Immune Estimation Resource (TIMER) databases. The impact of DCK on clinical prognosis was investigated via the Kaplan-Meier plotter and verified in the Gene Expression Profiling Interactive Analysis (GEPIA) databases. The interrelationships between DCK expression and TIICs in HCC were analyzed by the TIMER database. Additionally, the relationship between DCK expression and immune cell gene markers was calculated through TIMER and GEPIA databases.ResultsCompared with the adjacent normal tissues, high expression of DCK was observed in HCC tissues. Also, the higher expression of DCK was correlated to poorer prognosis in HCC patients, and it was associated with decreased survival in those with early stage and grade. Moreover, DCK expression was positively correlated with TIICs, including CD4+ and CD8+ T cells, B cells, monocytes, tumor-associated macrophages (TAMs), M1 and M2 macrophages, neutrophils, natural killer cells, and dendritic cells. Specifically, DCK expression levels were significantly associated with diverse immune gene marker sets, including those of Tregs and exhausted T cells.ConclusionThese findings suggest that DCK expression is correlated with patient outcomes and tumor infiltration cell levels in HCC patients. Additionally, the increased level of DCK was associated with marker genes of Tregs and exhaustion-related inhibitory receptors, suggesting the potential role of DCK in immunosuppression and immune escape. These findings suggest that DCK can function as a potential novel prognostic biomarker and reflect the immune infiltration status in HCC patients.

Human equilibrative nucleoside transporter-1 (hENT1) and ribonucleotide reductase regulatory subunit M1 (RRM1) expression; do they have survival impact to pancreatic cancer?

Backgrounds/AimsGemcitabine is still one of adjuvant options in chemotherapeutic agent for pancreatic ductal adenocarcinoma (PDAC). Integral membrane transporter protein and intracellular enzymes including human equilibrative nucleoside transporter 1 (hENT1), deoxycytidine kinase (dCK), ribonucleotide reductase (RR) M1, and M2 are known as important factors for chemosensitivity of gemcitabine. We aimed to investigate the correlation between these key molecules and 5-year actual survival in PDAC patients.MethodsThe expression of intratumoral hENT1, dCK, RRM1, and RRM2 was assessed immunohistochemically in 160 PDAC patients underwent surgical resection. Association between clininopathologic factors, immunohistochemical results, and overall survival were analyzed.ResultsAdjuvant chemotherapy including concurrent chemoradiotherapy was not associated with overall survival (HR, 0.92; 95% CI, 0.65-1.31; p=0.658). High hENT1 expression group did not show statistical survival difference, compared with all others (HR, 1.16; 95% CI, 0.82-1.65, p=0.396). Gemcitabine therapy and high hENT1 group was compared with all other patients, and no difference in overall survival was identified (HR, 0.99; 95% CI, 0.68-1.42; p=0.940). And, gemcitabine therapy and high hENT1 group did not differ statistically from gemcitabine therapy and low hENT1 expression (HR, 0.92; 95% CI, 0.55-1.56; p=0.764). The intensity of dCK, RRM1, and RRM2 expression was not associated with overall survival (p=0.413, p=0.138 and p=0.061) in univariate analysis.ConclusionsThe expression of hENT1, dCK, RRM1 and RRM2 may not be associated with overall survival for patients with pancreatic cancer on gemcitabine adjuvant therapy. These proteins and other factors that may interact with or confound these results should be investigated in the near future.

Abstract C58: p53/p16-independent cytoreduction of chemoresistant pancreatic adenocarcinoma by metabolically optimized epigenetic therapy

Abstract Although several reasons for chemorefractoriness of pancreatic ductal adenocarcinoma (PDAC) response have been proposed, a well-established basis is genetic inactivation of the p53/p16 apoptosis axis. Therefore, there is a need for p53/p16-independent PDAC treatments. The key epigenetic regulator DNA methyltransferase 1 (DNMT1) has been scientifically validated as a molecular target for p53-independent cytoreduction of pancreatic cancer, e.g., by activating terminal epithelial programs. Moreover, the pyrimidine nucleoside analog prodrugs decitabine (Dec) or 5-azacytidine (5Aza) are metabolically processed into a nucleotide analog that depletes DNMT1. Motivated by the scientific data, several clinical trials evaluating Dec, 5Aza, or analogs to treat PDAC have been completed. However, results from these trials have been discouraging and contradict encouraging preclinical results. To model the clinical situation, we evaluated Dec and 5Aza efficacy in gemcitabine-resistant PDAC in vitro and in mice. Gemcitabine-resistant PDAC cells were cross-resistant to Dec but not 5Aza. Measurement of the pyrimidine metabolism enzymes needed for prodrug conversion demonstrated the reason: DCK that activates gemcitabine and Dec was suppressed, but UCK2 that activates 5Aza was preserved and upregulated. Also, the catabolic enzyme cytidine deaminase (CDA), which rapidly catabolizes Dec and 5Aza into inactive uridine counterparts, was upregulated. Importantly, these shifts in pyrimidine metabolism that were observed in established gemcitabine-resistant cells were rapidly recapitulated (within hours/days) upon exposure of naïve PDAC cells to Dec or 5Aza, although 5Aza exposure immediately upregulated DCK, whereas Dec exposure immediately upregulated UCK2. Measurements of nucleotide levels by LC-MS/MS indicated that these contrasting shifts were caused by opposite effects of Dec and 5Aza on dCTP levels. These observations suggested potential solutions to overcome resistance: We evaluated alternating Dec with 5Aza, to exploit their mutual priming effects on DCK and UCK2 expression, and the addition of tetrahydrouridine (THU) to inhibit the catabolism of Dec and 5Aza by CDA. This optimized regimen was very efficacious in a murine xenograft model of gemcitabine-resistant PDAC (median vehicle control tumor measurements 972 mm3(range 726-1267.5); median THU-Dec/THU-5Aza 16 mm3 (range 0-87.5); P&amp;lt;0.00001). Terminal epithelial-differentiation of gemcitabine-resistant PDAC cells was confirmed by a significant increase in pancreatic epithelial markers PTF1A, MIST1, and N-cadherin, while apoptosis markers were not induced. In sum, the pyrimidine metabolism network, which senses and regulates nucleotide amounts, automatically dampens Dec (and gemcitabine) and 5Aza activity. Nevertheless, these reactions can be anticipated and exploited instead to extend and deepen DNMT1 depletion and noncytotoxic tumor cytoreduction. Citation Format: Rita Tohme, Xiaorong Gu, Asmaa El Desoky, Caroline Schuerger, Daniel Lindner, Davendra Sohal, Yogen Saunthararajah. p53/p16-independent cytoreduction of chemoresistant pancreatic adenocarcinoma by metabolically optimized epigenetic therapy [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C58.

Feedback Responses of the Pyrimidine Metabolism Network Mediate Resistance to Decitabine and 5-Azacytidine

There is a need to understand and counter mechanisms-of-resistance to decitabine (Dec) and 5-azacytidine (5Aza), the only agents approved to treat all subtypes of myelodysplastic syndromes (MDS). Both Dec and 5Aza are pro-drugs, and here, in vitro, in vivo and patient data show that while processing the pro-drugs, pyrimidine metabolism is itself altered, dampening pro-drug conversion and causing resistance. Anticipation of these adaptive responses of the pyrimidine metabolism network enables their exploitation instead. Dec and 5Aza are processed by pyrimidine metabolism into a nucleotide analog that depletes DNA methyltransferase 1 (DNMT1). We found that DNMT1 protein, although substantially depleted (~50%) in patients' bone marrows at time-of-response, rebounded at time-of-relapse (Fig 1A). The pyrimidine metabolism enzymes DCK and UCK2 rate-limit cellular uptakes of Dec and 5Aza, respectively. At relapse on Dec (n=13), DCK was halved vs patients' baseline bone marrows (Fig 1B). At relapse on 5Aza (n=14), it was UCK2 that was halved (Fig 1B). Simultaneous with DCK or UCK2 suppression, however, the other enzyme (UCK2 or DCK) was upregulated up to 40-fold (Fig 1B). Seesaw DCK and UCK2 expression was observed also in DCK knock-out (KO) and UCK2-KO AML cells (HAP1): DCK-KO cells upregulated UCK2, resisted Dec, but were more sensitive to 5Aza. UCK2-KO cells upregulated DCK, resisted 5Aza, but were more sensitive to Dec (concentrations for 50% growth inhibition [GI50] Dec: HAP1 wildtype 3mM, DCK-KO 12mM, UCK2-KO 0.1mM; 5Aza: wildtype 4mM, DCK-KO 2mM, UCK2-KO 15mM). These compensatory upregulations of UCK2 or DCK were adaptive to DCK- or UCK2-KO-induced decreases in dCTP or dTTP respectively. We thus examined if Dec or 5Aza also perturbed dCTP/dTTP amounts to trigger adaptive metabolic shifts. Dec decreased dTTP and increased dCTP, while 5Aza decreased dCTP, 24 hours after their addition to AML cells (THP1, OCI-AML3, MOLM13, K562)(Fig 1C). A single dose of Dec or 5Aza was also sufficient to upregulate UCK2 or DCK &amp;gt;2-fold, and the catabolic enzyme CDA &amp;gt;4-fold, within 72 hours (Fig 1D). Protein levels tracked the mRNA changes. Exponentially proliferating Dec or 5Aza-resisting AML cells emerged within 35 days in presence of pro-drug (5 AML cell lines) and displayed the same metabolic reconfigurations seen acutely. We then used patient-derived xenotransplant (PDX) models of chemorefractory AML to identify methods to counter this automatic metabolic adaptation or 'auto-resistance'. The Dec and 5Aza doses used (subcutaneous Dec 0.2 mpk or 5Aza 2 mpk) were chosen to deplete DNMT1 from marrow without cytotoxicity, demonstrated by flow cytometry for DNMT1 and gH2AX. We found (i) Dec timed to avoid DCK troughs (D1 &amp; 2 each week) was superior to Dec timed to coincide with DCK troughs (D1 &amp; 4 each week)(median survival 74 vs 62 days [vehicle 40 days], Log-rank p=0.006); (ii) Adding a CDA inhibitor (intraperitoneal tetrahydrouridine 4-10 mpk) to halved doses of Dec or 5Aza (0.1 mpk, 1 mpk respectively, to avoid cytotoxicity) was superior to no CDA inhibitor + Dec 0.2 mpk or 5Aza 2 mpk (median survival 180 vs 115 days [vehicle 50 days], Log-rank p=0.004), and (iii) Alternating Dec and 5Aza timed to peaks of mutual priming (e.g., D1 &amp; 4 each week) was superior to Dec alone or 5Aza alone, or Dec/5Aza alternating every month or Dec/5Aza given together - the regimen incorporating these lessons extended survival by several months vs vehicle (median survival 223 vs 50 days, Log-rank p=0.003). Eventual AML relapse/progression on this therapy was again caused by AML cells in which DNMT1 was not depleted (Fig 1E), because of even larger increases in CDA and upregulation of de novo pyrimidine synthesis. We are thus evaluating higher CDA-inhibitor doses (40 mpk) and incorporation of non-cytotoxic doses of a de novo pyrimidine synthesis inhibitor. In sum, the pyrimidine metabolism network responds automatically to Dec or 5Aza perturbation with adaptive shifts that decrease pro-drug conversion into the DNMT1-depleting nucleotide. These metabolic shifts can be anticipated (Fig 1F) and exploited, using simple, clinically viable treatment modifications. Disclosures Maciejewski: Novartis: Consultancy; Alexion: Consultancy. Saunthararajah:EpiDestiny: Consultancy, Equity Ownership, Patents &amp; Royalties; Novo Nordisk: Consultancy.

Arginine Starvation and Docetaxel Induce c-Myc-Driven hENT1 Surface Expression to Overcome Gemcitabine Resistance in ASS1-Negative Tumors.

The response to acute and long-term arginine starvation results in a conditional adaptive metabolic reprogramming that can be harnessed for therapeutic opportunities in ASS1-negative tumors. Here, we investigate the underlying biology of priming ASS1- tumors with arginine deiminase (ADI-PEG20) before treatment with gemcitabine (GEM) and docetaxel (DTX) in sarcoma, pancreatic cancer, and melanoma cell lines. ASS1- tumor cell lines were treated to create LTAT (long-term ADI treated) cell lines (ASS1+) and used for drug combination studies. Protein expression of ASS1, dCK, RRM2, E2F1, c-MYC, and hENT1 was measured. c-MYC activity was determined, live-cell immunofluorescent studies for hENT1, uptake assays of FITC-cytosine probe, and rescue studies with a c-MYC inhibitor were all determined in the presence or absence of the ADI-PEG20:GEM:DTX. In examining modulations within the pyrimidine pathway, we identified that the addition of DTX to cells treated with ADI-PEG20 resulted in translocation of stabilized c-Myc to the nucleus. This resulted in an increase of hENT1 cell-surface expression and rendered the cells susceptible to GEM. In vivo studies demonstrate that the combination of ADI-PEG20:GEM:DTX was optimal for tumor growth inhibition, providing the preclinical mechanism and justification for the ongoing clinical trial of ADI-PEG20, GEM, and DTX in sarcoma. The priming of tumors with ADI-PEG20 and DTX results in the stabilization of c-MYC potentiating the effect of GEM treatment via an increase in hENT1 expression. This finding is applicable to ASS1-deficient cancers that are currently treated with GEM.

Bioavailability and cytosolic kinases modulate response to deoxynucleoside therapy in TK2 deficiency

BackgroundTK2 is a nuclear gene encoding the mitochondrial matrix protein thymidine kinase 2 (TK2), a critical enzyme in the mitochondrial nucleotide salvage pathway. Deficiency of TK2 activity causes mitochondrial DNA (mtDNA) depletion, which in humans manifests predominantly as a mitochondrial myopathy with onset typically in infancy and childhood. We previously showed that oral treatment of the Tk2 H126N knock-in mouse model (Tk2−/−) with the TK2 substrates, deoxycytidine (dCtd) and thymidine (dThd), delayed disease onset and prolonged median survival by 3-fold. Nevertheless, dCtd + dThd treated Tk2−/− mice showed mtDNA depletion in brain as early as postnatal day 13 and in virtually all other tissues at age 29 days. MethodsTo enhance mechanistic understanding and efficacy of dCtd + dThd therapy, we studied the bioavailability of dCtd and dThd in various tissues as well as levels of the cytosolic enzymes, TK1 and dCK that convert the deoxynucleosides into dCMP and dTMP. FindingsParenteral treatment relative to oral treatment produced higher levels of dCtd and dThd and improved mtDNA levels in liver and heart, but did not ameliorate molecular defects in brain or prolong survival. Down-regulation of TK1 correlated with temporal- and tissue-specificity of response to dCtd + dThd. Finally, we observed in human infant and adult muscle expression of TK1 and dCK, which account for the long-term efficacy to dCtd + dThd therapy in TK2 deficient patients. InterpretationsThese data indicate that the cytosolic pyrimidine salvage pathway enzymes TK1 and dCK are critical for therapeutic efficacy of deoxynucleoside therapy for Tk2 deficiency. FundNational Institutes of Health P01HD32062.