5-Azacytidine (5-azaC) is an azanucleo-side approved for myelodysplastic syndrome. Approximately 80%-90% of 5-azaC is believed to be incorporated into RNA, which disrupts nucleic acid and protein metabolism leading to apoptosis. A smaller fraction (10%-20%) of 5-azaC inhibits DNA methylation and synthesis through conversion to decitabine triphos-phate and subsequent DNA incorporation. However, its precise mechanism of action remains unclear. Ribonucleotide reductase (RR) is a highly regulated enzyme comprising 2 subunits, RRM1 and RRM2, that provides the deoxyribonucle-otides required for DNA synthesis/repair. In the present study, we found for the first time that 5-azaC is a potent inhibitor of RRM2 in leukemia cell lines, in a mouse model, and in BM mononuclear cells from acute myeloid leukemia (AML) patients. 5-azaC-induced RRM2 gene expression inhibition involves its direct RNA incorporation and an attenuated RRM2 mRNA stability. Therefore, 5-azaC causes a major perturbation of deoxyribonucleotide pools. We also demonstrate herein that the initial RR-mediated 5-azaC conversion to decitabine is terminated through its own inhibition. In conclusion, we identify RRM2 as a novel molecular target of 5-azaC in AML. Our findings provide a basis for its more widespread clinical use either alone or in combination. (Blood. 2012;119(22):5229-5238) Introduction 5-Azacytidine (5-azaC; Vidaza) is a pyrimidine analog that was first synthesized 45 years ago and later found to be a natural product from Streptoverticillium ladakanus. 1-3 Currently, 5-azaC is approved by the US Food and Drug Administration (FDA) for the treatment of myelodysplastic syndrome (MDS), but it has also been widely used for the treatment of acute myeloid leukemia (AML). 4 Despite decades of efforts made to delineate the mechanisms of the antileukemia activity for this compound in terms of its interference with RNA and DNA metabolism, 5 the precise basis of its clinical efficacy remains to be elucidated fully. 6 After cell uptake, 5-azaC is first anabolized to its nucleoside monophosphate, 5-aza-CMP, by uridine-cytidine kinase (UCK) and is eventually phosphorylated by diphosphate kinase to its triphosphate metabolite, 5-aza-CTP, which is thought to be incorporated into RNA. 7,8 Approximately 80%-90% of 5-azaC is processed through the entire sequence and incorporated into RNA. 7 This ultimately disrupts mRNA and protein metabolism, leading to apoptosis. 4,6 A small fraction (10%-20%) of the 5-azaC disphos-phate intermediate product 5-aza-CDP is instead converted to 5-aza-dCDP by ribonucleotide reductase (RR), followed by further phosphorylation to 5-azaC-dCTP (5-aza-2'-deoxycyridine triphos-phate; DAC-TP). 4,7 DAC-TP is incorporated into DNA and cova-lently binds DNA methyltransferases (DNMTs), thereby inhibiting the activity of these proteins. It has been recognized that DNMTs add methyl groups to the cytosine in the context of the CpG dinucleotides in promoter region thereby promoting gene hyper-methylation and epigenetic silencing. Thus 5-azaC has be...

Hypermethylation of 5Ј-cytosine-guanosine islands of tumor suppressor genes resulting in their silencing has been proposed to be a hallmark of various tumors. Modulation of DNA methylation with DNA methylation inhibitors has been shown to result in cancer cell differentiation or apoptosis and represents a novel strategy for chemotherapy. Currently, effective DNA methylation inhibitors are mainly limited to decitabine and 5-azacytidine, which still show unfavorable toxicity profiles in the clinical setting. Thus, discovery and development of novel hypomethylating agents, with a more favorable toxicity profile, is essential to broaden the spectrum of epigenetic therapy. Parthenolide, the principal bioactive sesquiterpene lactone of feverfew, has been shown to alkylate Cys 38 of p65 to inhibit nuclear factor-B activation and exhibit anti-tumor activity in human malignancies. In this article, we report that parthenolide 1) inhibits DNA methyltransferase 1 (DNMT1) with an IC 50 of 3.5 M, possibly through alkylation of the proximal thiolate of Cys 1226 of the catalytic domain by its ␥-methylene lactone, and 2) down-regulates DNMT1 expression possibly associated with its SubG 1 cell-cycle arrest or the interruption of transcriptional factor Sp1 binding to the promoter of DNMT1. These dual functions of parthenolide result in the observed in vitro and in vivo global DNA hypomethylation. Furthermore, parthenolide has been shown to reactivate tumor suppressor HIN-1 gene in vitro possibly associated with its promoter hypomethylation. Hence, our study established parthenolide as an effective DNA methylation inhibitor, representing a novel prototype for DNMT1 inhibitor discovery and development from natural structural-diversified sesquiterpene lactones.DNA methylation of cytosine residues in the context of the sequence 5Ј-cytosine-guanosine (CpG) in gene promoter regions is an epigenetic mechanism that controls gene transcription, genome stability, and genetic imprinting (Robertson, 2005). This process is regulated by DNA methyltransferases (DNMT1, DNMT3a, and DNMT3b) in the presence of S-adenosyl-methionine (SAM) that serves as a methyl donor for methylation of cytosine residues at the C-5 position to yield 5-methylcytosine (Robertson, 2005). Aberrant hypermethylation of promoter CpG-rich regions (Ͼ55% CG content, the so-called CpG islands) of tumor suppressor genes (TSGs) results in transcriptional silencing in a variety of solid tumors and blood cancers (Yoo and Jones, 2006). In vitro and in vivo treatment with DNA methyl-

We observed that decitabine and valproic acid are an effective combination in reactivating hypermethylated genes as demonstrated by re-expressing fetal hemoglobin. This combination in patients with advanced stage IV NSCLC, however, is limited by unacceptable neurological toxicity at a relatively low dosage. Combining hypomethylating agents with alternative HDAC inhibitors that lack the toxicity of VPA should be explored further.

Purpose-To simultaneously quantify intracellular nucleoside triphosphate (NTP) and deoxynucleoside triphosphate (dNTP) pools and to assess their changes produced by interfering with ribonucleotide reductase (RNR) expression in leukemia cells. Methods-A HPLC-MS/MS system was used to quantify intracellular NTP and dNTP pools.Results-The assay was linear between 50 nM, the lower limit of quantification (LLOQ), and 10 μMin cell lysate. The within-day coefficients of variation (CVs, n=5) were found to be 12.0-18.0% at the LLOQ and 3.0-9.0% between 500 and 5,000 nM for dNTPs and 8.0-15.0% and 2.0-6.0% for NTPs. The between-day CVs (n=5) were 9.0-13.0% and 3.0-11.0% for dNTPs and 9.0-13.0% and 3.0-6.0% for NTPs. The within-day accuracy values were 93.0-119.0% for both NTPs and dNTPs. ATP overlapped with dGTP and they were analyzed as a composite. This method was applied to measure basal intracellular dNTPs/NTPs in five leukemia cell lines exposed to the RNR antisense GTI-2040. Following drug treatment, dCTP and dATP levels were found to decrease significantly in MV4-11 and K562 cells. Additionally, perturbation of dNTP/NTP levels in bone marrow sample of a patient treated with GTI-2040 was detected.Conclusions-This method provides a practical tool to measure intracellular dNTP/NTP levels in cells and clinical samples.

Carbohydrate moiety is found in many anticancer nature products. To explore the carbohydrate moiety of daunorubicin in enhancing anticancer efficacy, several daunorubicin derivatives bearing disaccharide (1-8) have been synthesized. Their cytotoxicities were tested in leukemia K562 and colon cancer SW620 cells. Topoisomerase II (topo II) poisoning was performed with the in vivo complex of topoisomerase bioassay. In both cell lines, compounds with various terminal 2,6-dideoxy sugars (compounds 1, 3, 5, and 8) showed 30- to 60-fold higher anticancer activity than compounds with 2-deoxy- or 6-deoxy sugar (compounds 6 and 7). Compounds with an alpha-linkage between two sugar units (compound 3) showed 35-fold higher anticancer activity than compounds with a beta-linkage (compound 4). In addition, the anticancer activities of these compounds correlated with their ability to target topo II mediated genomic DNA damage in vivo. Compounds 1 and 3 with 2,6-dideoxy sugars produced more covalent topo-DNA complex than compounds with 2-deoxy sugar (6) and 6-deoxy sugar (7). Compounds with an alpha-configuration of terminal 2,6-dideoxy sugar (compounds 1 and 3) showed higher topo II poisoning than their counterparts with the beta-configuration (compounds 2 and 4). These results indicate that sugar moieties in daunorubicin play a significant role in its anticancer activity and topo II inhibition. The second sugar of disaccharide daunorubicin should possess 2,6-dideoxy with alpha-linkage to the first sugar to exhibit better anticancer activity.