Low dose cytarabine induces differentiation of AML cells by activating checkpoint kinase 1

INTRODUCTION: Lack of differentiation is a hallmark of acute myeloid leukemia (AML), making differentiation therapy a promising treatment strategy. Several novel differentiation targets have been recently identified, including dihydroorotate dehydrogenase (DHODH), one of the key enzymes in de novo pyrimidine synthesis. Our recent studies demonstrated that pyrimidine synthesis inhibitors 5- aminoimidazole-4- carboxamide ribonucleoside (AICAr), a UMP synthase inhibitor, and brequinar, a DHODH inhibitor, induce differentiation of leukemia cells by activating the ataxia telangiectasia and RAD3related (ATR)/checkpoint kinase 1 (Chk1) 29 Systems approches in cacer DNA-damage signaling pathway via pyrimidine depletion. Cytarabine (cytosine arabinoside or AraC) has been the mainstay of the standard first-line induction therapy in adults with AML for the last several decades, as well as one of the main medications used in low-dose regimens administered to patients unfit for intensive chemotherapy. Apart from its cytotoxic effects, monocytic or granulocytic differentiation in response to cytarabine have been confirmed in several AML cell lines. However, the mechanism responsible for cytarabine-induced differentiation remained incompletely understood. Cytarabine interference with the process of DNA synthesis leads to DNA damage by staling replication forks, and ATR/Chk1 pathway is known to promote survival by signaling replication stress. In this study, we tested the role of Chk1 in differentiation of AML cells induced by cytarabine and compared the effects of cytarabine with the effects of AICAr and brequinar. MATERIALS AND METHODS: Human AML cell lines U937 and THP-1 were purchased from ECACC (UK) and DSMZ (Germany). Non adherent mononuclear cells from bone marrow of five AML patients were obtained by density gradient separation, purified by overnight adherence to plastic, and seeded in liquid medium containing 50 ng/mL interleukin-3, interleukin-6, stem cell factor and FLT3-ligand. Cells were collected after obtaining patients’ written informed consent in accordance with the Declaration of Helsinki and approval obtained by the University Hospital Centre Zagreb Institutional ethic committee. Cells were incubated in the presence of cytarabine (10, 100, 1000 nM), AICAr (0.2 mM), brequinar (0.5 µM), and pharmacological inhibitors of ATR/Chk1, Torin2 (10 nM, 100 nM) or VE-821 (2 µM, 10 µM). The number of viable cells was determined by trypan blue exclusion. The expression of differentiation markers, as well as DNA content for cell cycle analysis were determined by flow cytometry (Attune, Life Technologies) and FlowJo software. siRNA transfections were performed using siRNA targeting Chk1 (Dharmacon) and NeonTM transfection system (Invitrogen). Total cell lysates were analyzed for the level of Chk1, Ser-345-p-Chk1, Tyr-15-p-CDC2 and β actin by Western blot. Statistical analysis was performed using Student’s t-test (p < 0.05). RESULTS: The effects of cytarabine on cell viability and the expression of differentiation markers in monocytic leukemia cells are similar to the effects of AICAr and brequinar. Cytarabine dose-dependently decreases cell number and induces expression of differentiation markers CD11b and CD64 in AML cell lines. Western blot analysis revealed the increase in the level of Ser-345-phosphorylated Chk1, a marker of Chk1 activation, in response to cytarabine. Cytarabine, AICAr and brequinar increased the level of inhibitory Tyr-15- phosphorylation of cyclin-dependent kinase 1 (Cdk1, CDC2), suggesting that CDC2 inactivation is an important downstream target of Chk1 in cell cycle restriction. Pharmacological inhibition of ATR/Chk1 pathway by Torin2 and VE821 reduced differentiative effects and cell cycle arrest in response to cytarabine, AICAr and brequinar. Moreover, transfection of U937 cells with siRNA targeting Chk1 reduced differentiative effects of all agents tested. In addition, cytarabine dose- dependently induced differentiation in two primary AML samples that were responsive to pyrimidine synthesis inhibitors. CONCLUSION: Low dose cytarabine-mediated differentiation of AML cells occurs through the activation of DNA damage checkpoint kinase Chk1.