ORIGINAL ARTICLE AMPK inhibition enhances apoptosis in MLL-rearranged pediatric B-acute lymphoblastic leukemia cells B Accordi 1 , L Galla 1 , G Milani 1 , M Curtarello 2 , V Serafin 1 , V Lissandron 1 , G Viola 1 , G te Kronnie 1 , R De Maria 3 , EF Petricoin 3rd 4 , LA Liotta 4 , S Indraccolo 2 and G Basso 1 The serine/threonine kinase AMP-activated protein kinase (AMPK) and its downstream effectors, including endothelial nitric oxide synthase and BCL-2, are hyperactivated in B-cell precursor-acute lymphoblastic leukemia (BCP-ALL) cells with MLL gene rearrangements. We investigated the role of activated AMPK in supporting leukemic cell survival and evaluated AMPK as a potential drug target. Exposure of leukemic cells to the commercial AMPK inhibitor compound C resulted in massive apoptosis only in cells with MLL gene rearrangements. These results were confirmed by targeting AMPK with specific short hairpin RNAs. Compound C-induced apoptosis was associated with mitochondrial membrane depolarization, reactive oxygen species production, cytochrome c release and caspases cleavage, indicating intrinsic apoptosis pathway activation. Treatment with low concentrations of compound C resulted in a strong antileukemic activity, together with cytochrome c release and cleavage of caspases and poly(ADP-ribose) polymerase, also in MLL-rearranged primary BCP-ALL samples. Moreover, AMPK inhibition in MLL-rearranged cell lines synergistically enhanced the antiproliferative effects of vincristine, daunorubicin, cytarabine, dexamethasone and L-asparaginase in most of the evaluated conditions. Taken together, these results indicate that the activation of the AMPK pathway directly contributes to the survival of MLL-rearranged BCP-ALL cells and AMPK inhibitors could represent a new therapeutic strategy for this high-risk leukemia. Leukemia (2013) 27, 1019–1027; doi:10.1038/leu.2012.338 Keywords: acute lymphoblastic leukemia; MLL rearrangements; AMPK; apoptosis INTRODUCTION AMP-activated protein kinase (AMPK) is a serine/threonine kinase that acts as a cellular fuel sensor activated under conditions of ATP depletion and elevated AMP levels such as heat-shock, nutrient deprivation, hypoxia and other metabolic or environmental stresses. 1,2 AMPK is an heterotrimeric complex composed of an a-catalytic subunit, a b-subunit important both for complex formation and glycogen binding, and a g-regulatory subunit, which binds AMP. 3 AMPK activity is enhanced by phosphorylation of the a-subunit at threonine 172 by LKB1, a serine/threonine kinase encoded by the tumor-suppressor gene STK11, which is mutated in patients with Peutz–Jeghers syndrome. 4,5 In addition, AMPK can also be activated by several hormones and cytokines such as adiponectin 6 and leptin. 7 The physiological role ascribed to AMPK is the inactivation of ATP-consuming metabolic processes, including fatty acid, cholesterol and protein synthesis, and the activation of ATP-generating pathways such as glycolysis and fatty acid oxidation. 8,9 This is initially accomplished by direct phosphorylation of key metabolic enzymes, followed by effects on gene expression. Although AMPK is traditionally regarded as a sensor of cellular energy status and a regulator of metabolism, recently it has been reported to be involved in the regulation of several biological processes including cell growth, proliferation, apoptosis, autop- hagy and cell polarity. 10,11 In cancer, the role of AMPK is not yet fully understood and data reported in literature so far are contradictory. The effects of AMPK activation are determined by the cell type investigated, depending on signaling alterations in related pathways. AMPK activation results in pro-apoptotic effects reported in acute myeloid leukemia, 12 ovarian cancer, 13 astrocytoma, 14 and osteosarcoma 15 and in anti-apoptotic effects, observed in multiple myeloma, 16 prostate cancer 17 and glioma. 18 We previously found that pediatric B-cell precursor-acute lymphoblastic leukemia (BCP-ALL) patients with rearrangements of the MLL gene display the hyperactivation of a signal transduction pathway that leads from phosphorylation of LKB1 and AMPK to phosphorylation of BCL-2, through downstream endothelial nitric oxide synthase activation. 19 In this study, we assessed whether this hyperactivation supports the survival of MLL-rearranged BCP-ALL cells, and whether its inhibition affects leukemic cell growth and drug resistance. MATERIALS AND METHODS Cell lines and culture Human leukemia cell lines SEM, RS4;11, MHH-CALL-2 and MHH-CALL-4 were purchased from DSMZ German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). Human leukemia cell line ALL-PO was kindly donated by Professor Andrea Biondi (University of Milano-Bicocca, Monza, Italy). 20 SEM, RS4;11 and ALL-PO cell lines were derived from BCP- ALLs carrying the t(4;11) MLL-AF4 translocation. MHH-CALL-2 and MHH- CALL-4 cell lines were derived from BCP-ALLs without recurrent chromosomal translocations. Cells were cultured in RPMI 1640 (Biochrom 1 Oncohematology Laboratory, Department of Woman and Child Health, University of Padova, Padova, Italy; 2 Immunology and Diagnostic Molecular Oncology, Istituto Oncologico Veneto IRCCS, Padova, Italy; 3 Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanita `, Rome, Italy and 4 Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA, USA. Correspondence: Dr B Accordi, Dipartimento di Salute della Donna e del Bambino—SDB, Universita ` degli Studi di Padova, via Giustiniani 3, 35128 Padova, Italy. E-mail: benedetta.accordi@unipd.it Received 30 April 2012; revised 30 October 2012; accepted 8 November 2012; accepted article preview online 21 November 2012; advance online publication, 11 December 2012 Leukemia (2013) 27, 1019–1027 & 2013 Macmillan Publishers Limited All rights reserved 0887-6924/13 www.nature.com/leu