Cytosine Nucleoside Analogues Research Articles

Boosting Clear Cell Renal Carcinoma-Specific Drug Discovery Using a Deep Learning Algorithm and Single-Cell Analysis.

Clear cell renal carcinoma (ccRCC), the most common subtype of renal cell carcinoma, has the high heterogeneity of a highly complex tumor microenvironment. Existing clinical intervention strategies, such as target therapy and immunotherapy, have failed to achieve good therapeutic effects. In this article, single-cell transcriptome sequencing (scRNA-seq) data from six patients downloaded from the GEO database were adopted to describe the tumor microenvironment (TME) of ccRCC, including its T cells, tumor-associated macrophages (TAMs), endothelial cells (ECs), and cancer-associated fibroblasts (CAFs). Based on the differential typing of the TME, we identified tumor cell-specific regulatory programs that are mediated by three key transcription factors (TFs), whilst the TF EPAS1/HIF-2α was identified via drug virtual screening through our analysis of ccRCC's protein structure. Then, a combined deep graph neural network and machine learning algorithm were used to select anti-ccRCC compounds from bioactive compound libraries, including the FDA-approved drug library, natural product library, and human endogenous metabolite compound library. Finally, five compounds were obtained, including two FDA-approved drugs (flufenamic acid and fludarabine), one endogenous metabolite, one immunology/inflammation-related compound, and one inhibitor of DNA methyltransferase (N4-methylcytidine, a cytosine nucleoside analogue that, like zebularine, has the mechanism of inhibiting DNA methyltransferase). Based on the tumor microenvironment characteristics of ccRCC, five ccRCC-specific compounds were identified, which would give direction of the clinical treatment for ccRCC patients.

Resistance of Leukemia Cells to 5-Azacytidine: Different Responses to the Same Induction Protocol.

Three AML cell variants (M/A, M/A* from MOLM-13 and S/A from SKM-1) were established for resistance by the same protocol using 5-azacytidine (AZA) as a selection agent. These AZA-resistant variants differ in their responses to other cytosine nucleoside analogs, including 5-aza-2'-deoxycytidine (DAC), as well as in some molecular features. Differences in global DNA methylation, protein levels of DNA methyltransferases, and phosphorylation of histone H2AX were observed in response to AZA and DAC treatment in these cell variants. This could be due to changes in the expression of uridine-cytidine kinases 1 and 2 (UCK1 and UCK2) demonstrated in our cell variants. In the M/A variant that retained sensitivity to DAC, we detected a homozygous point mutation in UCK2 resulting in an amino acid substitution (L220R) that is likely responsible for AZA resistance. Cells administered AZA treatment can switch to de novo synthesis of pyrimidine nucleotides, which could be blocked by inhibition of dihydroorotate dehydrogenase by teriflunomide (TFN). This is shown by the synergistic effect of AZA and TFN in those variants that were cross-resistant to DAC and did not have a mutation in UCK2.

The Methylation Inhibitor 5-Aza-2′-Deoxycytidine Induces Genome-Wide Hypomethylation in Rice

DNA methylation is a conserved epigenetic modification which is vital for regulating gene expression and maintaining genome stability in both mammals and plants. Homozygous mutation of rice methyltransferase 1 (met1) gene can cause host death in rice, making it difficult to obtain plant material needed for hypomethylation research. To circumvent this challenge, the methylation inhibitor, 5-Aza-2′-deoxycytidine (AzaD), is used as a cytosine nucleoside analogue to reduce genome wide hypomethylation and is widely used in hypomethylation research. However, how AzaD affects plant methylation profiles at the genome scale is largely unknown. Here, we treated rice seedlings with AzaD and compared the AzaD treatment with osmet1-2 mutants, illustrating that there are similar CG hypomethylation and distribution throughout the whole genome. Along with global methylation loss class I transposable elements (TEs) which are farther from genes compared with class II TEs, were more significantly activated, and the RNA-directed DNA Methylation (RdDM) pathway was activated in specific genomic regions to compensate for severe CG loss. Overall, our results suggest that AzaD is an effective DNA methylation inhibitor that can influence genome wide methylation and cause a series of epigenetic variations.

A Phase I/II Study of Sapacitabine and Venetoclax in Relapsed/Refractory Acute Myeloid Leukemia

Background: Sapacitabine is a novel, orally bioavailable, cytosine nucleoside analogue with a unique mechanism that generates single-stranded DNA breaks which are converted into double-stranded DNA breaks resulting in cell death. Previous studies of sapacitabine alone and alternating with decitabine (DAC) have demonstrated activity in acute myeloid leukemia (AML). Preclinical studies have suggested synergy with the combination of sapacitabine and a BCL-2 inhibitor. We conducted a study investigating the combination of sapacitabine and venetoclax (VEN) as an entirely oral regimen for patients with relapsed/refractory (R/R) AML.Methods: This is a phase I/II study for patients ≥ 18 years with R/R AML or MDS with blasts ≥10% investigating the safety and efficacy of escalating doses of sapacitabine combined with VEN. Eligibility included ECOG performance status ≤ 2 and adequate organ function. Two cohorts were investigated: Cohort 1 studied sapacitabine given PO BID on D1-5; Cohort 2 studied sapacitabine given PO BID on D1-3 and D8-10 of each cycle, which was defined as 4 weeks. Sapacitabine was escalated from a starting dose of 250mg. VEN was given to a target dose of 400mg PO on D1-14 of each cycle. VEN ramp-up was utilized, starting at 100mg on D1, 200mg on D2, and 400mg on days 3 through 14. Adjustments to the VEN dose were made based on concomitant CYP3A inhibitors.Results: Between 03/2020 and 03/2021, 13 pts were enrolled in the study. Baseline characteristics are shown in table 1. The median age was 70 years (range, 30-82) and 8 pts (62%) were aged ≥ 70 years. One pt (7%%) had favorable risk, 3 (23%) had intermediate risk, and 9 (70%) had adverse risk karyotype; 5 pts (38%) had a complex karyotype. The most common mutations detected by next-generation sequencing were: TET2 (46%), ASXL1 (30%), NRAS (30%), and WT1 (23%%). One patient (8%) had a TP53 mutation. This was a heavily pretreated population, having received a median of 2 prior therapies (1 - 9), including 6 patients (46%) having received prior intensive chemotherapy and 4 patients (30%) with prior allogeneic SCT. Six patients (46%) had received prior VEN-based therapy. Among 13 evaluable patients, there was 1 (8%) complete response with incomplete platelet recovery (CRp). Patients received a median of 1 cycle (1-9) on protocol. Five patients (38%) were able to remain on protocol without a protocol defined response, but with clinical benefit and good tolerability, for ≥2 cycles.With a median follow up of 14.61 months (1-16), the median overall survival (OS) was 3.8 months with a 60-day survival rate of 76% (Figure 1). There was no significant difference in OS between the two dosing schedules. The median OS among patients that had and had not received prior VEN was 3.32 months and 4.5 months, respectively (P=0.25). The patient that achieved a CRi had secondary AML with 7q deletion, an ASXL1 mutation, and only single-agent DAC as prior therapy.The oral chemotherapy combination was well tolerated, with 4- and 8-week mortality of 8% and 15%, respectively. The most common grade 3/4 non-hematologic adverse events regardless of attribution were: febrile neutropenia (31%), cellulitis (23%) pneumonia (15%), hypophosphatemia (8%), hypokalemia (8%). Two patients (15%) had grade 5 sepsis on study in the setting of active AML.Conclusion: In a heavily pretreated population of patients with R/R AML, the combination of sapacitabine and VEN was well tolerated and feasible to be administered as a completely oral, outpatient regimen. Most patients had multiple prior cycles of nucleoside analogue containing regimens and the only notable responder had only prior DAC. Further study of this well-tolerated combination in less heavily pretreated patients may be considered. [Display omitted] DisclosuresKantarjian: Jazz: Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Aptitude Health: Honoraria; NOVA Research: Honoraria; Ascentage: Research Funding; Ipsen Pharmaceuticals: Honoraria; Astra Zeneca: Honoraria; Astellas Health: Honoraria; BMS: Research Funding; Daiichi-Sankyo: Research Funding; KAHR Medical Ltd: Honoraria; Immunogen: Research Funding; AbbVie: Honoraria, Research Funding; Precision Biosciences: Honoraria; Taiho Pharmaceutical Canada: Honoraria. DiNardo: Novartis: Honoraria; Forma: Honoraria, Research Funding; Foghorn: Honoraria, Research Funding; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Agios/Servier: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; ImmuneOnc: Honoraria, Research Funding; AbbVie: Consultancy, Research Funding; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding. Borthakur: GSK: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Protagonist: Consultancy; Takeda: Membership on an entity's Board of Directors or advisory committees; University of Texas MD Anderson Cancer Center: Current Employment; ArgenX: Membership on an entity's Board of Directors or advisory committees; Astex: Research Funding; Ryvu: Research Funding. Daver: ImmunoGen: Consultancy, Research Funding; Trovagene: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; FATE Therapeutics: Research Funding; Gilead Sciences, Inc.: Consultancy, Research Funding; Trillium: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Glycomimetics: Research Funding; Novimmune: Research Funding; Hanmi: Research Funding; Sevier: Consultancy, Research Funding; Novartis: Consultancy; Jazz Pharmaceuticals: Consultancy, Other: Data Monitoring Committee member; Dava Oncology (Arog): Consultancy; Celgene: Consultancy; Syndax: Consultancy; Shattuck Labs: Consultancy; Agios: Consultancy; Kite Pharmaceuticals: Consultancy; SOBI: Consultancy; STAR Therapeutics: Consultancy; Karyopharm: Research Funding; Newave: Research Funding. Pemmaraju: Dan's House of Hope: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Consultancy; ASH Communications Committee: Membership on an entity's Board of Directors or advisory committees; Sager Strong Foundation: Other; Daiichi Sankyo, Inc.: Other, Research Funding; HemOnc Times/Oncology Times: Membership on an entity's Board of Directors or advisory committees; DAVA Oncology: Consultancy; Aptitude Health: Consultancy; Springer Science + Business Media: Other; Roche Diagnostics: Consultancy; Novartis Pharmaceuticals: Consultancy, Other: Research Support, Research Funding; LFB Biotechnologies: Consultancy; ASCO Leukemia Advisory Panel: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy; MustangBio: Consultancy, Other; Cellectis S.A. ADR: Other, Research Funding; Samus: Other, Research Funding; Plexxicon: Other, Research Funding; Affymetrix: Consultancy, Research Funding; Stemline Therapeutics, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; CareDx, Inc.: Consultancy; Abbvie Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Protagonist Therapeutics, Inc.: Consultancy; Clearview Healthcare Partners: Consultancy; Blueprint Medicines: Consultancy; Bristol-Myers Squibb Co.: Consultancy; ImmunoGen, Inc: Consultancy; Pacylex Pharmaceuticals: Consultancy. Alvarado: Sun Pharma: Consultancy, Research Funding; Daiichi-Sankyo: Research Funding; Astex Pharmaceuticals: Research Funding; CytomX Therapeutics: Consultancy; BerGenBio: Research Funding; FibroGen: Research Funding; Jazz Pharmaceuticals: Research Funding; MEI Pharma: Research Funding. Burger: AstraZeneca: Consultancy; Beigene: Research Funding, Speakers Bureau; Pharmacyclics LLC: Consultancy, Other: Travel/Accommodations/Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel/Accommodations/Expenses, Speakers Bureau; Gilead: Consultancy, Other: Travel/Accommodations/Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel/Accommodations/Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel/Accommodations/Expenses, Speakers Bureau. Ravandi: Syros Pharmaceuticals: Consultancy, Honoraria, Research Funding; Jazz: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Taiho: Honoraria, Research Funding; Agios: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; AstraZeneca: Honoraria; Novartis: Honoraria; Xencor: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Prelude: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Research Funding. Kadia: AstraZeneca: Other; Ascentage: Other; Jazz: Consultancy; BMS: Other: Grant/research support; AbbVie: Consultancy, Other: Grant/research support; Aglos: Consultancy; Amgen: Other: Grant/research support; Cure: Speakers Bureau; Dalichi Sankyo: Consultancy; Genfleet: Other; Cellonkos: Other; Sanofi-Aventis: Consultancy; Pulmotech: Other; Novartis: Consultancy; Pfizer: Consultancy, Other; Liberum: Consultancy; Genentech: Consultancy, Other: Grant/research support; Astellas: Other.

FNC inhibits non-small cell lung cancer by activating the mitochondrial apoptosis pathway.

Previously, we published that 4'-azid-2'-deoxy-2'-fluorarabinoside (FNC), a novel cytosine nucleoside analog, has good anti-viral and anti-tumor activity. This study aimed to further explore the role and molecular mechanism of FNC in non-small cell lung cancer (NSCLC). FNC was tested in the NSCLC H460 cell line, the Lewis mouse model, and the H460 cell xenograft model. The effects of FNC were assessed by cell viability, transwell migration, and wound scratch analyses of cell migration and invasion. Apoptosis was assessed by flow cytometry. Proteins expression was assessed by western blot and immunohistochemistry staining (IHC). FNC inhibits the proliferation and metastasis of H460 cells in a time- and dose-dependent manner. FNC treatment showed efficacy and low toxicity in the Lewis mouse lung cancer model as well as in the H460 cell xenograft model. Further, FNC induced H460 cell apoptosis through the activation of the mitochondrial pathway. Notably, FNC inhibited invasion by increasing E-cadherin protein and reducing the protein expression of VEGF, MMP-2, MMP-9, and CD31. FNC inhibits NSCLC by activating the mitochondrial apoptosis pathway and regulating the expressions of multiple proteins related to cell adhesion and invasion, highlighting its potential as an NSCLC therapeutic.

Glycomimetic Antagonist of E-Selectin, GMI-1271, Enhances Therapeutic Activity of the Hypomethylating Agent, 5-Azacitidine, in the KG1 Model of AML

Components of the bone marrow stroma afford a permissive environment for the malignant progression of AML. These stromal components include E-selectin, an adhesion molecule expressed in the bone marrow vasculature. Through the recognition and binding of E-selectin by specific ligands on AML blasts, survival pathways are activated within the blasts leading to the development of/increase in chemoresistance. GMI-1271 is a novel E-selectin antagonist that disrupts this process, down-regulating survival pathways and enhancing chemotherapy response with improved survival in mouse xenograft and syngeneic AML tumor models. GMI-1271 is currently in clinical trials for the treatment of AML in combination with intensive chemotherapy; the FDA has granted Breakthrough Therapy Designation to GMI-1271 for the treatment of adults with relapse/refractory AML. Azacitidine (5-AZC) is a DNA-methyltransferase inhibiting cytosine nucleoside analog that at low doses induces DNA hypomethylation and transcriptional activation, while at higher doses is directly cytotoxic to neoplastic cells including AML blasts. Vidaza (5-AZC, Celgene) is approved in Europe for the treatment of limited populations with AML. In these studies we evaluated the anti-tumor activity of GMI-1271 in combination with 5-AZC in the KG1 AML tumor model to identify if there was potential for therapeutic benefit of the combination.Seven days post i.v. injection of KG1 cells, female NSG mice (n=10/group) were treated with saline; GMI-1271 (40 mg/kg IP QDx14) alone; 5-AZC (5 mg/kg IP Q3Dx5) alone, or the combination of GMI-1271 and 5-AZC. All treatments were well tolerated. The median survival time (MST) of mice treated with 5-AZC was 88 days and statistically different (P<0.005) to groups treated with saline (MST=69.5 days) or GMI-1271 alone (MST=69 days). At study conclusion (Day 110 post tumor injection), 20% of mice treated with 5-AZC remained alive; as expected all mice treated with saline or GMI-1271 alone succumbed to progressive tumor growth. Importantly, the therapeutic activity of 5-AZC was significantly enhanced when combined with GMI-1271 (MST>110 days, P=0.0217 compared to 5-AZC alone) with 70% of mice surviving to study conclusion. These results indicate that E-selectin/AML blast interaction in the KG1 model protects from the anti-tumor activity of 5-AZC and that GMI-1271 can attenuate this protection.To investigate the nature of the observed in vivo activity of GM-1271 and 5-AZC in the KG1 model, in vitro assays of cytotoxicity and E-selectin ligand expression in KG1 and the KG1a variant were assessed. The IC50 of 5-AZC alone or in combination with a saturating dose of GMI-1271 on KG1 cells was 518 and 528 nM, respectively. The IC50 of 5-AZC alone or in combination with GMI-1271 on KG1a cells was 637 and 711 nM, respectively. These results imply that the enhanced anti-tumor activity observed in vivo was not due to a simple shift in the cytotoxicity profile of 5-AZC. KGI and KG1a cells were subsequently cultured for 96 h in a noncytotoxic concentration (100 nM) of 5-AZC and the reactivity of the cells to HECA-452, a surrogate marker of E-selectin ligand, or binding E-selectin was determined by flow cytometry. Treatment with 5-AZC resulted in an increased reactivity of KG1 and KG1a cells to HECA-452 (29 and 27%, respectively compared to nontreated cells) and an increased binding of E-selectin to KG1 cells (32% compared to nontreated cells). Collectively, these results demonstrate that noncytotoxic concentrations of 5-AZC lead to an increased expression of E-selectin ligands on KG1 and KG1a cells.In summary, these studies show that even though the hypomethylating agent 5-AZC improves outcomes with AML, therapeutic potential could be improved. In this regard, 5-AZC at noncytotoxic concentrations promotes the increased expression of E-selectin ligands on AML blasts which may dampen 5-AZC activity. However, attenuation or disruption of E-selectin ligand/E-selectin interactions with GMI-1271 in combination with 5-AZC overcomes this tumor survival pathway with the potential to maximize therapeutic outcome. This work has relevance to patients with AML who do not achieve optimal or long-term response with 5-AZC and other hypomethylating agents. Clinical assessment of GMI-1271 with hypomethylating agents in AML is planned. DisclosuresFogler:GlycoMimetics: Employment, Equity Ownership. Smith:GlycoMimetics: Employment, Equity Ownership. Lee:GlycoMimetics: Employment, Equity Ownership. Magnani:GlycoMimetics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Abstract 2867: Glycomimetic antagonist of E-selectin, GMI-1271, enhances therapeutic activity of the hypomethylating agent 5-azacitidine in the KG1 model of AML

Abstract Binding of AML blasts to E-selectin activates cellular survival pathways leading to chemoresistance. GMI-1271 is a novel E-selectin antagonist that when used in combination with chemotherapy results in improved survival in mouse syngeneic and xenogeneic AML tumor models. GMI-1271 in combination with two chemotherapy regimens is in early clinical trials for the treatment of AML. Azacitidine (5-AC) is a DNA-methyltransferase inhibiting cytosine nucleoside analog that at low doses induces DNA hypomethylation and transcriptional activation, while at higher doses is directly cytotoxic to neoplastic cells including AML blasts. 5-AC is approved in Europe for the treatment of limited populations with AML. We evaluated GMI-1271 in combination with 5-AC in the KG1 AML tumor model to assess the potential for therapeutic benefit of the combination. NSG mice (10/group) received i.v. injections of KG1 cells, and were treated with saline, GMI-1271 alone, 5-AC alone, or the combination of GMI-1271 and 5-AC. The median survival time (MST) of mice treated with 5-AC was 88 days and statistically different (P&amp;lt;0.002) to groups treated with saline (MST=69.5 days) or GMI-1271 alone (MST=69 days). All mice treated with saline or GMI-1271 alone succumbed to progressive tumor growth. At study conclusion (Day 104 post tumor injection) 20% of mice treated with 5-AC remained alive. Importantly, the therapeutic activity of 5-AC was significantly enhanced when combined with GMI-1271 (MST&amp;gt;104 days, P=0.0140 compared to 5-AC alone) with 70% of mice surviving to study conclusion. These results indicate that E-selectin/AML blast interaction in the KG1 model protects from the anti-tumor activity of 5-AC and that GMI-1271 attenuates this protection. To investigate the nature of the observed in vivo activity of GMI-1271 and 5-AC, KG1 cells were cultured for 96 h in a noncytotoxic concentration (100 nM) of 5-AC and the reactivity of the cells to HECA-452 (an antibody that recognizes an E-selectin carbohydrate ligand) and binding to E-selectin were determined by flow cytometry. Treatment with 5-AC resulted in a 28% increase in reactivity of cells to HECA-452 and a 32% increase in binding to E-selectin. Further in vitro assays of static adhesion revealed an increase in the adhesion of 5-AC-treated KG1 cells to E-selectin. Notably, the enhanced adhesion of KG-1 cells to E-selectin was reversed using GMI-1271. Collectively, these results demonstrate that 5-AC can lead to increased expression of E-selectin ligands on AML cells and that the therapeutic potential of 5-AC could be improved by combination with GMI-1271. Citation Format: Theodore A. Smith, William E. Fogler, Ji-Won Lee, John L. Magnani. Glycomimetic antagonist of E-selectin, GMI-1271, enhances therapeutic activity of the hypomethylating agent 5-azacitidine in the KG1 model of AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2867.

Construction of All‐Carbon Quaternary Stereocenters via Asymmetric Cyclopropanations: Synthesis of Chiral Carbocyclic Pyrimidine Nucleosides

AbstractAn efficient route to synthesize chiral carbocyclic pyrimidine nucleoside analogues containing all‐carbon quaternary stereocenters has been established via the asymmetric intermolecular cyclopropanation of N1‐vinylpyrimidines and α‐aryl diazoesters. With 2 mol% of chiral dirhodium (II) carboxylate complex as the catalyst, a variety of chiral carbocyclic cytosine or uracil nucleoside analogues were obtained in good yields (up to 96% yield), high diastereoselectivities (&gt;20:1 dr), and excellent enantioselectivities (up to 99% ee).magnified image

Critical Role of TGF-β and IL-2 Receptor Signaling in Foxp3 Induction by an Inhibitor of DNA Methylation.

Under physiological conditions, CD4+ regulatory T (Treg) cells expressing the transcription factor Foxp3 are generated in the thymus [thymus-derived Foxp3+ Treg (tTregs) cells] and extrathymically at peripheral sites [peripherally induced Foxp3+ Treg (pTreg) cell], and both developmental subsets play non-redundant roles in maintaining self-tolerance throughout life. In addition, a variety of experimental in vitro and in vivo modalities can extrathymically elicit a Foxp3+ Treg cell phenotype in peripheral CD4+Foxp3− T cells, which has attracted much interest as an approach toward cell-based therapy in clinical settings of undesired immune responses. A particularly notable example is the in vitro induction of Foxp3 expression and Treg cell activity (iTreg cells) in initially naive CD4+Foxp3− T cells through T cell receptor (TCR) and IL-2R ligation, in the presence of exogenous TGF-β. Clinical application of Foxp3+ iTreg cells has been hampered by the fact that TGF-β-driven Foxp3 induction is not sufficient to fully recapitulate the epigenetic and transcriptional signature of in vivo induced Foxp3+ tTreg and pTreg cells, which includes the failure to imprint iTreg cells with stable Foxp3 expression. This hurdle can be potentially overcome by pharmacological interference with DNA methyltransferase activity and CpG methylation [e.g., by the cytosine nucleoside analog 5-aza-2′-deoxycytidine (5-aza-dC)] to stabilize TGF-β-induced Foxp3 expression and to promote a Foxp3+ iTreg cell phenotype even in the absence of added TGF-β. However, the molecular mechanisms of 5-aza-dC-mediated Foxp3+ iTreg cell generation have remained incompletely understood. Here, we show that in the absence of exogenously added TGF-β and IL-2, efficient 5-aza-dC-mediated Foxp3+ iTreg cell generation from TCR-stimulated CD4+Foxp3− T cells is critically dependent on TGF-βR and IL-2R signaling and that this process is driven by TGF-β and IL-2, which could either be FCS derived or produced by T cells on TCR stimulation. Overall, these findings contribute to our understanding of the molecular mechanisms underlying the process of Foxp3 induction and may provide a rational basis for generating phenotypically and functionally stable iTreg cells.

In situ evaluation of gemcitabine–DNA interaction using a DNA-electrochemical biosensor

The electrochemical behaviour of the cytosine nucleoside analogue and anti-cancer drug gemcitabine (GEM) was investigated at glassy carbon electrode, using cyclic, differential pulse and square wave voltammetry, in different pH supporting electrolytes, and no electrochemical redox process was observed. The evaluation of the interaction between GEM and DNA in incubated solutions and using the DNA-electrochemical biosensor was studied. The DNA structural modifications and damage were electrochemically detected following the changes in the oxidation peaks of guanosine and adenosine residues and the occurrence of the free guanine residues electrochemical signal. The DNA–GEM interaction mechanism occurred in two sequential steps. The initial process was independent of the DNA sequence and led to the condensation/aggregation of the DNA strands, producing rigid structures, which favoured a second step, in which the guanine hydrogen atoms, participating in the C–G base pair, interacted with the GEM ribose moiety fluorine atoms.

Human cytidine deaminase: A biochemical characterization of its naturally occurring variants

Human cytidine deaminase is an enzyme of the pyrimidine salvage pathways that metabolizes several cytosine nucleoside analogs used as prodrugs in chemotherapy. We carried out a characterization of the cytidine deaminase 79A>C and 208G>A Single Nucleotide Polymorphisms, in order to highlight their functional role and provide data that could help fine-tune the chemotherapic use of cytosine nucleosides in patients carrying the above mentioned SNPs. The 79A>C SNP results in a K27Q change in a protein region not involved in the catalytic event. The 208G>A SNP produces an alanine to threonine substitution (A70T) within the conserved catalytic domain. Q27 variant is endowed with a greater catalytic efficiency toward the natural substrates and the antileukemic agent cytarabine (Ara-C), when compared to K27 variant. Molecular modeling, protein stability experiments and site-directed mutagenesis suggest that K27 variant may have an increased stability with respect to Q27 due to an ionic interaction between a lysine residue at position 27 and a glutamate residue at position 24. The T70 variant has a lower catalytic efficiency toward the analyzed substrates when compared to the A70 variant, suggesting that patients carrying the 208G>A SNP may have a greater exposure to cytosine based pro drugs, with possible toxicity consequences.

Improving outcomes for elderly patients with AML

Available treatments are inadequate for many elderly patients with acute myeloid leukaemia (AML) who are considered by their physician to be unfit for intensive chemotherapy—at least 80% of patients die within 1 year of diagnosis. Findings from cancer registry studies suggest that up to 70% of patients are not offered any treatment other than best supportive care.1 In The Lancet Oncology, Hagop Kantarjian and colleagues report the findings from their randomised phase 2 study of three different dose schedules of the novel oral cytosine nucleoside analogue, sapacitabine, in elderly patients with AML who were either treatment-naive or at first relapse.

Oral sapacitabine for the treatment of acute myeloid leukaemia in elderly patients: a randomised phase 2 study

Available treatments for acute myeloid leukaemia (AML) have limited durable activity and unsatisfactory safety profiles in most elderly patients. We assessed the efficacy and toxicity of sapacitabine, a novel oral cytosine nucleoside analogue, in elderly patients with AML. In this randomised, phase 2 study, we recruited patients with AML who were either treatment naive or at first relapse and who were aged 70 years or older from 12 centres in the USA. We used a computer-generated randomisation sequence to randomly allocate eligible patients to receive one of three schedules of oral sapacitabine (1:1:1; stratified by a history of AML treatment): 200 mg twice a day for 7 days (group A); 300 mg twice a day for 7 days (group B); and 400 mg twice a day for 3 days each week for 2 weeks (group C). All schedules were given in 28 day cycles. To confirm the safety and tolerability of dosing schedules, after 20 patients had been treated in a group we enrolled an expanded cohort of 20-25 patients to that group if at least four patients had achieved complete remission or complete remission with incomplete blood count recovery, and if the 30 day death rate was 20% or less. Our primary endpoint was 1-year overall survival, analysed by intention-to-treat (ie, patients who have received at least one dose of sapacitabine) in those patients who had been randomly allocated to treatment. This trial is registered with ClinicalTrials.gov, number NCT00590187. Between Dec 27, 2007, and April 21, 2009, we enrolled 105 patients: 86 patients were previously untreated and 19 were at first relapse. Of the 60 patients randomly allocated to treatment, 1-year overall survival was 35% (95% CI 16-59) in group A, 10% (2-33) in group B, and 30% (13-54) in group C. 14 (13%) of 105 patients died within 30 days and 27 (26%) died within 60 days. The most common grade 3-4 adverse events were anaemia (eight of 40 patients in group A, 12 of 20 patients in group B, and 15 of 45 patients in group C), neutropenia (14 in group A, 10 in group B, 11 in group C), thrombocytopenia (24 in group A, 12 in group B, and 22 in group C), febrile neutropenia (16 in group A, nine in group B, and 22 in group C), and pneumonia (seven in group A, five in group B, and 10 in group C). The most common grade 5 events were pneumonia (two in group A, one in group B, and three in group C) and sepsis (six in group A, three in group B, and one in group C). Seven deaths were thought to be probably or possibly related to sapacitabine treatment. Sapacitabine seems active and tolerable in elderly patients with AML. The 400 mg dose schedule had the best efficacy profile. Future investigations should aim to combine sapacitabine with other low-intensity therapies in elderly patients with AML. Cyclacel Limited.

Decitabine for the treatment of acute myeloid leukemia (AML): Memorial Sloan-Kettering Cancer Center experience.

6593 Background: Decitabine is a cytosine nucleoside analog that is FDA-approved for previously untreated and treated patients with myelodysplastic syndromes. Recent studies have also demonstrated clinical activity of decitabine in AML as it has been investigated for remission induction (Cashen et al J Clin Oncol, 2010; Blum et al PNAS, 2010), salvage therapy and bridging to hematopoietic cell transplantation (HCT). We conducted a retrospective analysis of the efficacy of decitabine in patients with AML treated at Memorial Sloan-Kettering Cancer Center (MSKCC). Methods: Using the pharmacy database, patients who received decitabine for AML between June 2006 and August 2010 at MSKCC were identified. Patients had to be at least 18 years of age treated for AML with single-agent decitabine at 20 mg/m2 daily for 5 days to be included. The primary endpoint was the rate of complete remission (CR). Secondary endpoints include time to response and median overall survival. Results: Sixty patients with a mean age 61.9 (range 28-80) were treated at MSKCC between the specified time period. Thirteen (22%) patients received decitabine as initial remission induction therapy. Of the 47 (78%) patients that received decitabine as salvage therapy, 14 were post HCT. The average number of prior salvage chemotherapy regimens was 2.5. Decitabine was successfully used as bridge therapy to HCT in 3 patients. The overall response rate was 13% as 7 (12%) patients achieved a CR and 1 patient achieved PR. The mean number of cycles to achieve maximal response was 3.25 cycles. Three of 13 (23%) patients that received decitabine as initial remission induction therapy achieved a CR. Only 4 (8.5%) patients receiving decitabine as salvage therapy achieved a CR. Conclusions: Our results support earlier reports of the promising role of decitabine in the treatment of patients with newly diagnosed AML who are not candidates for intensive chemotherapy. However, the use of decitabine as a salvage regimen needs to be re-evaluated as our results demonstrated a rather poor response rate. Future studies are warranted to better understand the clinical benefit of decitabine in AML, particularly the efficacy of the 10-day regimen and bridge therapy to HCT.

2'-Spirocyclopropyl-carbocyclic Nucleoside as a Novel Scaffold for Potent Anti-HCV Agents

The discovery of 2'-spirocyclopropyl-ribocytidine (J. Med. Chem. 2010, 53, 8150-8160) as a potent inhibitor of RNA synthesis by NS5B (<TEX>$IC_{50}=7.3{\mu}M$</TEX>), the RNA polymerase encoded by hepatitis C Virus (HCV), has led to the synthesis and biological evaluation of several carbocyclic versions of 2'-spiropropyl-nucleosides. The cyclopentenol intermediate 7 was successfully constructed via ring-closing metathesis (RCM) from divinyl 6. Spirocyclopropanation of enone 8 was effected by using (2-chloroethyl)-dimethylsulfonium iodide and potassium tert-butoxide to form the desired intermediate 9. The synthesized nucleoside analogues 21-24 were assayed for their ability to inhibit HCV RNA replication in a subgenomic replicon Huh7 cell line. Among them, the cytosine nucleoside analogue 22 exhibited significant anti-HCV activity (<TEX>$EC_{50}= 8.2{\mu}M$</TEX>).

Novel method of intralesional cidofovir injection into laryngotracheal papillomata

Laryngeal papillomatosis is characterised by multiple papillomata affecting the upper respiratory tract. This condition is difficult to treat due to its recurrent nature. Treatment often involves surgical debulking. A number of non-surgical treatments have been reported. Intralesional cidofovir, a cytosine nucleoside analogue with antiviral activity, has been used in an attempt to manage the condition. We present a novel technique of administering cidofovir in a case of recurrent laryngotracheal papillomata.

Influence of C-5 substituted cytosine and related nucleoside analogs on the formation of benzo[a]pyrene diol epoxide-dG adducts at CG base pairs of DNA.

Endogenous 5-methylcytosine (MeC) residues are found at all CG dinucleotides of the p53 tumor suppressor gene, including the mutational ‘hotspots’ for smoking induced lung cancer. MeC enhances the reactivity of its base paired guanine towards carcinogenic diolepoxide metabolites of polycyclic aromatic hydrocarbons (PAH) present in cigarette smoke. In the present study, the structural basis for these effects was investigated using a series of unnatural nucleoside analogs and a representative PAH diolepoxide, benzo[a]pyrene diolepoxide (BPDE). Synthetic DNA duplexes derived from a frequently mutated region of the p53 gene (5′-CCCGGCACCC GC[15N3,13C1-G]TCCGCG-3′, + strand) were prepared containing [15N3, 13C1]-guanine opposite unsubstituted cytosine, MeC, abasic site, or unnatural nucleobase analogs. Following BPDE treatment and hydrolysis of the modified DNA to 2′-deoxynucleosides, N2-BPDE-dG adducts formed at the [15N3, 13C1]-labeled guanine and elsewhere in the sequence were quantified by mass spectrometry. We found that C-5 alkylcytosines and related structural analogs specifically enhance the reactivity of the base paired guanine towards BPDE and modify the diastereomeric composition of N2-BPDE-dG adducts. Fluorescence and molecular docking studies revealed that 5-alkylcytosines and unnatural nucleobase analogs with extended aromatic systems facilitate the formation of intercalative BPDE–DNA complexes, placing BPDE in a favorable orientation for nucleophilic attack by the N2 position of guanine.

Review of azacitidine trials in Intermediate-2-and High-risk myelodysplastic syndromes

The efficacy of azacitidine, a cytosine nucleoside analog, has been demonstrated in two large phase III trials in myelodysplastic syndromes (MDS) patients, primarily in higher-risk patients. In the Cancer and Leukemia Group B (CALGB) study (lower-and higher-risk MDS patients; n = 191) azacitidine significantly prolonged median time-to-progression to acute myeloid leukemia or death (21 vs 13 months for supportive care; p = 0.007). The AZA-001 trial (Intermediate-2-and High-risk MDS patients; n = 358) showed azacitidine significantly prolonged median overall survival compared with conventional care regimens (24.5 vs 15.0 months; p = 0.0001). These results establish azacitidine as a reference first-line treatment in patients with Intermediate-2-and High-risk MDS who are not immediate candidates for allogeneic stem cell transplantation.

The DNA methyltransferase inhibitors azacitidine, decitabine and zebularine exert differential effects on cancer gene expression in acute myeloid leukemia cells

The three DNA methyltransferase (DNMT)-inhibiting cytosine nucleoside analogues, azacitidine, decitabine and zebularine, which are currently studied as nonintensive therapy for myelodysplastic syndromes and acute myeloid leukemia (AML), differ in structure and metabolism, suggesting that they may have differential molecular activity. We investigated cellular and molecular effects of the three substances relative to cytarabine in Kasumi-1 AML blasts. Under in vitro conditions mimicking those used in clinical trials, the DNMT inhibitors inhibited proliferation and triggered apoptosis but did not induce myeloid differentiation. The DNMT inhibitors showed no interference with cell-cycle progression whereas cytarabine treatment resulted in an S-phase arrest. Quantitative methylation analysis of hypermethylated gene promoters and of genome-wide LINE1 fragments using bisulfite sequencing and MassARRAY suggested that the hypomethylating potency of decitabine was stronger than that of azacitidine; zebularine showed no hypomethylating activity. In a comparative gene expression analysis, we found that the effects of each DNMT inhibitor on gene transcription were surprisingly different, involving several genes relevant to leukemogenesis. In addition, the gene methylation and expression analyses suggested that the effects of DNMT-inhibiting cytosine nucleoside analogues on the cellular transcriptome may, in part, be unrelated to direct promoter DNA hypomethylation, as previously shown by others.

Repair of 2′-C-Cyano-2′-Deoxy-1-β-d-arabino-Pentofuranosylcytosine–Induced DNA Single-Strand Breaks by Transcription-Coupled Nucleotide Excision Repair

The cytosine nucleoside analogue 2'-C-cyano-2'-deoxy-1-beta-d-arabino-pentofuranosylcytosine (CNDAC) causes DNA single-strand breaks after its incorporation into DNA. This investigation sought to determine if DNA excision repair pathways were activated to repair this damage. Neither the base excision repair nor the mismatch repair pathway seemed to be involved. Cells deficient in the CSB protein, which initiates transcription-coupled nucleotide excision repair (NER) pathway (TC-NER), exhibited increased clonogenic sensitivity to CNDAC, whereas cells deficient in XPC, which initiates global genome NER, were slightly resistant relative to wild-type cells. The cells lacking either helicase XPB, which unwinds 5' of the lesion, or endonuclease XPF, which incises 5' to a lesion, exhibited increased clonogenic sensitivity to CNDAC, as did cells lacking the XPF partner protein ERCC1. This sensitization was independent of p53 function. Repletion of XPF restored sensitivity comparable with the wild type. In contrast, cells lacking either XPD, the 3'-helicase, or the 3'-endonuclease XPG were equally as sensitive as wild-type cells. In comparison, cells deficient in XPF were not sensitized to other cytosine nucleoside analogues, troxacitabine and cytarabine. Thus, the single-strand nick caused by CNDAC is recognized and, in part, repaired by the TC-NER pathway. NER proteins that function in the 5' direction relative to the UV-induced lesion also participate in the repair of the CNDAC-induced nick, in contrast to proteins that process on the 3' side of the lesion.