Constitutive activation of the ATM/BRCA1 pathway prevents DNA damage-induced apoptosis in 5-azacytidine-resistant cell lines Satoshi Imanishi a, *, Tomohiro Umezu a,b , Kazushige Ohtsuki a,c , Chiaki Kobayashi c , Kazuma Ohyashiki c , Junko H. Ohyashiki a a Institute for Medical Science, Tokyo Medical University, 6-7-1 Nishi-shinjuku, Shinjuku, Tokyo 160-0023, Japan b Department of Molecular Science, Tokyo Medical University, 6-7-1 Nishi-shinjuku, Shinjuku, Tokyo 160-0023, Japan c First Department of Internal Medicine, Tokyo Medical University, 6-7-1 Nishi-shinjuku, Shinjuku, Tokyo 160-0023, Japan 1. Introduction 5-Azacytidine (AZA) is one of the most effective DNA demethylating agents used in cancer treatment. It is used for the treatment of patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) in the USA, Europe, and Japan, among others, and yields a 40–60% response rate in these diseases [1]. However, some patients treated with AZA develop resistance against AZA after various treatment durations [2,3]. The prognosis of MDS patients after AZA treatment failure is poor, with a median overall survival time of 5.6 months [4]. AZA exerts its anti-tumor effects via its incorporation into RNA and DNA [5]. Induction of the differentiation of malignant cells might also be involved in the anti-tumor effects of AZA [6]. AZA taken up by cells is phosphorylated to azacytidine monophosphate (azaCMP) by uridine–cytidine kinase 2 (UCK2), and approximately 80–90% of azaCMP is subsequently phosphorylated to azacytidine triphosphate (azaCTP), which is incorporated into RNA. AzaCTP incorporated into RNA then promotes the degradation of RNA and inhibits protein synthesis [5]. Biochemical Pharmacology 89 (2014) 361–369 A R T I C L E I N F O Article history: Received 27 January 2014 Accepted 17 March 2014 Available online 26 March 2014 Chemical compounds studied in this article: 5-Azacytidine (PubChem CID: 9444) 2-desoxy-5-azacytidine (PubChem CID: 451668) 3-deazauridine (PubChem CID: 3000824) RG108 (PubChem CID: 702558) caffeine (PubChem CID: 2519) KU55933 (PubChem CID: 5278396) mitomycin C (PubChem CID: 5746) etoposide (PubChem CID: 36462) cisplatin (PubChem CID: 441203) Keywords: 5-Azacytidine DNA demethylation DNA damage A B S T R A C T 5-Azacytidine (AZA) exerts its anti-tumor effects by exerting cytotoxicity via its incorporation into RNA and DNA, which causes the reactivation of aberrantly silenced growth-regulatory genes by promoter demethylation, as well as DNA damage. AZA is used for patients with myelodysplastic syndrome and acute myeloid leukemia. However, some patients demonstrate resistance to AZA, the mechanisms of which are not fully elucidated. We therefore sought to better characterize the molecular mechanism of AZA resistance using an in vitro model of AZA resistance. We established AZA-resistant cell lines by exposing the human leukemia cell lines U937 and HL-60 to clinical concentrations of AZA, and characterized these cells. AZA-resistant cells showed a down-regulation of the DNMT3A protein, in correlation with their marked genome-wide DNA hypomethylation. Furthermore, genes involved in pyrimidine metabolism were down-regulated in both AZA-resistant cell lines; AZA sensitivity was restored by inhibition of CTP synthase. Of note is that the DNA damage response pathway is constitutively activated in the AZA-resistant cell lines, but not in the parental cell lines. Inhibition of the DNA damage response pathway canceled the AZA resistance, in association with an increase in apoptotic cells. We found that the molecular mechanism underlying AZA resistance involves pyrimidine metabolism and the DNA damage response through ATM kinase. This study therefore sheds light on the mechanisms underlying AZA resistance, and will enable better understanding of AZA resistance in patients undergoing AZA treatment. ß 2014 Elsevier Inc. All rights reserved. Abbreviations: AZA, 5-azacytidine; MDS, myelodysplastic syndromes; AML, acute myeloid leukemia; azaCMP, azacytidine monophosphate; azaCTP, azacytidine triphosphate; DNMT, DNA methyltransferase; DAC, 2-desoxy-AZA; CDKN, cyclin dependent kinase inhibitor; TP53, tumor protein p53; PTEN, phosphatase and tensin homolog; WT1, Wilms tumor 1; RB1, retinoblastoma 1; POLR2B, polymerase (RNA) II (DNA directed) polypeptide B; UCK2, uridine–cytidine kinase 2; CDA, cytidine deaminase; AK3, adenylate kinase 3; POL II, RNA polymerase II; CTP, cytidine triphosphate; UTP, uridine triphosphate; 3-DU, 3-deazauridine; MMC, mitomycin C; phosphorylated proteins were indicated as, p-protein symbol. * Corresponding author at: 6-7-1 Nishi-shinjuku, Shinjuku, Tokyo 160-0023, Japan. Tel.: +81 3 3342 6111; fax: +81 3 3345 0185. E-mail address: s-ima@tokyo-med.ac.jp (S. Imanishi). Contents lists available at ScienceDirect Biochemical Pharmacology jo u rn al h om epag e: ww w.els evier.c o m/lo cat e/bio c hem p har m http://dx.doi.org/10.1016/j.bcp.2014.03.008 0006-2952/ß 2014 Elsevier Inc. All rights reserved.