Original Article | Published:

Molecular Targets for Therapy

AMPK inhibition enhances apoptosis in MLL-rearranged pediatric B-acute lymphoblastic leukemia cells

Leukemia volume 27, pages 10191027 (2013) | Download Citation

Abstract

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.

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 α-catalytic subunit, a β-subunit important both for complex formation and glycogen binding, and a γ-regulatory subunit, which binds AMP.3 AMPK activity is enhanced by phosphorylation of the α-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 adiponectin6 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, autophagy 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 osteosarcoma15 and in anti-apoptotic effects, observed in multiple myeloma,16 prostate cancer17 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 AG, Berlin, Germany) with 10% fetal calf serum (FCS), glutamine (2 mM/l; GIBCO, Invitrogen Life Technologies, Carlsbad, CA, USA), penicillin (100 U/ml; GIBCO) and streptomycin (100 μg/ml; GIBCO), and maintained at 37 °C in a humidified atmosphere with 5% CO2.

Cells were treated with compound C (Calbiochem, Darmstadt, Germany) dissolved in dimethylsulphoxide (DMSO) at different times and concentrations, or with DMSO alone.

MTT assay

Cell proliferation was assessed by MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay after treatment. Equal concentrations of cells were plated in triplicate in a 96-well plate and incubated with 10 μl MTT (Sigma-Aldrich, St Louis, MO, USA) for 4 h. Absorbance was measured at 560 nm using Victor3TM 1420 Multilabel Counter (PerkinElmer, Waltham, MA, USA). The growth inhibition50 (GI50=compound concentration required to inhibit cell proliferation by 50%) was calculated by plotting the data as a logarithmic function of (x) when viability was 50%. DMSO-treated cells viability was set to 100%.

Cytofluorimetric assays

Apoptosis was determined using the Annexin-V–FLUOS staining kit (Roche, Basel, Switzerland), following the manufacturer’s instructions. Samples were analyzed by flow cytometric analysis (Cytomics FC500, Beckman Coulter, Fullerton, CA, USA). DMSO-treated cells viability was set to 100%. The lethal concentration50 (LC50=compound concentration required to induce cell mortality by 50%) was calculated by plotting the data as a logarithmic function of (x) when viability was 50%.

The mitochondrial membrane potential (ψmt) was measured with the lipophilic cation 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine (JC-1, Molecular Probes, Invitrogen Life Technologies), as already described21 in DMSO alone and compound C (8 μM, 48 h–LC50)-treated cells.

Mitochondrial membrane depolarization is associated with mitochondrial production of reactive oxygen species (ROS). The production of ROS was measured in DMSO alone and compound C (LC50 compound C 8 μM, 48 h) treated cells by flow cytometry using hydroethidine (Molecular Probes, Invitrogen Life Technologies) 2.5 μM.21

Caspase-3 activation was evaluated by flow cytometry using a human active caspase-3 fragment antibody conjugated with fluorescein isothiocyanate (BD Biosciences, Franklin Lakes, NJ, USA). Briefly, DMSO alone and compound C (8 μM, 48 h) treated cells were collected by centrifugation and resuspended in Cytofix (BD Pharmingen, BD Biosciences) buffer for 20 min, washed with Perm/Wash (BD Pharmingen, BD Biosciences), and then incubated for 30 min with the antibody. Cells were then washed and analyzed by flow cytometry.

Lentiviral vector-mediated transduction of shRNA in leukemia cells

The lentiviral plasmids containing AMPKα1 short hairpin RNA (shRNA) expression cassette or an un-relevant shRNA sequence were purchased from Sigma-Aldrich. The lentiviral vector stocks were generated by a transient three-plasmid vector-packaging system.22 One microgram of p24 equivalent of lentiviral vector-containing supernatant was used to transduce 1 × 106 target cells in 35-mm diameter Petri dish. After 6–9 h at 37 °C, the supernatant was replaced with complete medium. Evaluation of AMPKα1 silencing, through Sybr Green real-time quantitative PCR and western blot experiments, and quantification of apoptosis, through Annexin V–propidium iodide (PI) staining, were carried out 72–96 h after transduction.

Real-time quantitative PCR

Total RNA was isolated and retrotranscribed as previously described.23 Reactions were performed in a total volume of 20 μl, containing 1 μl of complementary DNA, 10 μl of SYBR Green (Invitrogen Life Technologies), and 150 nM forward and reverse primers, on an ABI Prism 7900HT Fast Real Time PCR system (Applied Biosystems, Foster City, CA, USA) with the annealing temperature set at 60 °C for all tested genes. Relative quantification was done using the ΔΔCt method, normalizing to GUS mRNA. Primers used for real-time quantitative PCR analysis: AMPKα1-fwd: 5′-GGAGCCTTGATGTGGTAGGA-3′; AMPKα1-rev: 5′-GTTTCATCCAGCCTTCCATTC-3′; AMPKα2-fwd 5′-ACCAGCTTGCAGTGGCTTAT-3′; AMPKα2-rev: 5′-CAGTGCATCCAATGGACATC-3′; GUS-fwd: 5′-GAAAATATGTGGTTGGAGAGCTCATT-3′; GUS-rev: 5′-CGGAGTGAAGATCCCCTTTTTA-3′.

Western blotting

The following antibodies and final concentrations were used for western blotting. Primary antibodies: anti-phospho-AMPKα T172 (1:500), anti-AMPKα (23A3) (1:1000), anti-AMPKα1 (1:1000), anti-phospho-AMPKß S108 (1:1000), anti-phospho-BCL-2 S70 (1:500), anti-β-ACTIN (1:10 000; all from Cell Signaling Technology, Inc., Danvers, MA, USA), anti-phospho-endothelial nitric oxide synthase/NOS III S116 (1:1000; Millipore, Billerica, MA, USA). Secondary antibodies: horseradish peroxidase-goat anti-rabbit and anti-mouse immunoglobulin G-conjugate (Zymed Laboratories, Inc., South San Francisco, CA, USA; 1:50 000). Total cell lysates were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing conditions, and transferred to a nitrocellulose sheet (Hybond-P, GE Healthcare, Chalfont, St Giles, UK) following standard methods. Membranes were saturated for 3 h with 2% Amersham ECL Advance Blocking Reagent (GE Healthcare), primary antibodies were incubated overnight at 4 °C and secondary antibodies for 1 h at room temperature. The immunoreactivity was determined by an enhanced chemiluminescent reaction (Amersham ECL ADVANCE Western Blotting Detection Kit, GE Healthcare). For the stripping, membranes were incubated with Restore PLUS Western Blot Stripping Buffer (Pierce, Rockford, IL, USA) following the manufacturer’s instructions and then washed in T-phosphate-buffered saline 1 × and resaturated.

To perform densitometric analysis shown in Figure 2c, western blot images were analyzed by ImageJ software (NIH, Bethesda, MA, USA). The intensity of the bands was normalized to the intensity of the bands corresponding to the β-ACTIN protein. Control cells (shRNA neg) ratio was set as 1.

Cell cycle analysis

Cells were synchronized 24 h before treatment by starvation without FBS. Compound C was then added 6 h after the end of starvation. DMSO alone and compound C (8 μM, 24 h) treated cells were centrifuged, fixed with ice-cold ethanol (70%), treated with lysis buffer containing RNAseA (Qiagen, Hilden, Germany) and PI. Samples were analyzed on a Cytomic FC500 flow cytometer (Beckman Coulter), and cell cycle analyses were performed using Multicycle Wincycle software (Phoenix Flow Systems, San Diego, CA, USA).

Immunofluorescence

Compound C (8 μM) and DMSO-treated human MLL-rearranged BCP-ALL cell lines and one primary BCP-ALL cell culture were placed on the slide by cytospin and then fixed with formaldehyde (4%) for 15 min. Slides were treated with NH4Cl (Sigma-Aldrich) 50 mM for 15 min to reduce background. Cells were permeabilized with 0.1% Triton X-100/phosphate-buffered saline 1 × for 3 min and incubated first with blocking buffer (5% bovine serum albumin in phosphate-buffered saline 1 × ) and then with primary antibody (1:100) diluted in blocking buffer overnight at 4 °C. Cells were then incubated for 1 h with species-specific secondary antibodies conjugated to Alexa-fluor 488 or 594 (1:1000–1:2000; Invitrogen Life Technologies), and for 10 min with 4,6-diamidino-2-phenylindole (1:10 000; Sigma-Aldrich). Primary antibodies: cytochrome c, cleaved poly(ADP-ribose) polymerase (PARP) (Asp214) and BCL-XL (from Cell Signaling Technology, Inc.), caspase-7 active and caspase-9 active (from GeneTex Inc., Irvine, CA, USA). Images of decorated cells were acquired by the videoconfocal system ViCo microscope (Nikon Eclipse 80i, Nikon, Japan). Images were acquired with ImageProPlus (Media Cybernetics, Rockville, MD, USA) and collected at magnification × 60.

Primary leukemia cell cultures

BCP-ALL patient samples were obtained after informed consent following the tenets of the Declaration of Helsinki. The study was approved by the ethical committee board of the University of Padova, the Padova Academic Hospital and the Italian Association of Pediatric Onco-Hematology (AIEOP). Diagnosis was made according to standard cytomorphology, cytochemistry and immunophenotypic criteria.24 All analyzed BCP-ALL samples were obtained at the time of diagnosis before treatment, after Ficoll–Hypaque (Pharmacia, Uppsala, Sweden) separation of mononuclear cells. Mononuclear cells were frozen as viable cells in FCS and 10% DMSO and stored in liquid nitrogen. The percentage of CD19+ cells ranged from 84 to 96%. We included in the study five patients positive for the 11q23 MLL-rearrangement MLL-AF4 and four patients negative for the 11q23 MLL-rearrangement and the chromosomal translocations t(12;21) TEL-AML1, t(9;22) BCR-ABL and t(1;19) E2A-PBX1. Chromosomal translocation analyses were performed for standard diagnostic procedures.

Human bone marrow-derived mesenchimal stromal cells (MSCs) immortalized by telomerase reverse transcriptase transduction were kindly donated by Dr Dario Campana (Yong Loo Lin School of Medicine, National University of Singapore, Singapore).25 MSCs were maintained in RPMI 1640 medium supplemented with 10% FCS, glutamine (2 mM/l), penicillin (100 U/ml), streptomycin (100 μg/ml) and 10−6M hydrocortisone (Sigma-Aldrich). MSCs were seeded in 24-well flat-bottomed plates (Costar Corning, Cambridge, MA, USA) and grown until confluence. To prepare cultures of primary samples, culture media was removed and adherent cells were washed seven times with AIM-V tissue culture medium (Invitrogen Life Technologies) with 10% FCS. Leukemic cells were then resuspended in AIM-V medium with 10% FCS, and 1 × 105 leukemic cells were placed on the MSCs layer in each well. Compound C was added 24 h after seeding at different concentrations (1, 5 and 8 μM). Quantification of apoptosis was carried out 24 and 48 h after treatment, downregulation of the AMPK pathway was tested after 48 h.

Combined drugs analysis

To test potential synergistic, additive or antagonistic effects of the combination of AMPK inhibition and drugs commonly used in ALL treatment, we performed MTT experiments as follows.

SEM, RS4;11 and ALL-PO cells were treated for 48 h with the different chemotherapeutics cytarabine (Aractyn, Pfizer, New York, NY, USA; 0.001–100 μM), daunorubicin (Pfizer; 0.0001–10 μM), vincristine (Pfizer; 0.0001–1 μM), dexamethasone (Sigma-Aldrich; 0.0001–10 μM) and L-asparaginase (Kidrolase, Eusa Pharma, Jazz Pharmaceuticals plc, Dublin, Ireland; 0.001–100 IU/ml). Compound C was added to drug solutions at fixed combination ratios. Cell viability was determined after 48 h of treatment. To determine the synergistic, additive or antagonistic effects of the drug combinations, we used CalcuSyn software (version 2.0, Biosoft, Cambridge, UK), which is based on the method of the combination index (CI) of Chou and Talalay.26 Synergy, additivity and antagonism were defined by a CI<1, CI=1 or CI>1, respectively.

Results

AMPK inhibition decreases proliferation and survival in MLL-rearranged cells

To study the functional role of AMPK in the survival of MLL-rearranged cells, we used five BCP-ALL cell lines, three with MLL gene rearrangements (RS4;11, SEM and ALL-PO) and two without (MHH-CALL-2 and MHH-CALL-4). As shown in Figure 1a, AMPK was activated in MLL-rearranged cells, similarly to what was observed in primary cells from patients.19 By contrast, AMPK activation was not detected in MHH-CALL-2 and MHH-CALL-4 (Figure 1a).

Figure 1
Figure 1

AMPK inhibition induces a decrease in cell proliferation and survival. (a) AMPK pathway activation status in BCP-ALL cell lines. MHH-CALL-2 and MHH-CALL-4 are non-translocated cell lines, RS4;11, SEM and ALL-PO carry the (4;11) MLL-AF4 translocation. (b) Cell proliferation rates were determined through MTT assay after treatment with compound C at different times and concentrations. Here the 48 h are shown. DMSO-treated cells viability was set to 100%. GI50=compound concentration required to inhibit cell proliferation by 50%. (c) Cell viability was determined by flow cytometry with Annexin V–PI staining after treatment with compound C at different times and concentrations. Here the 10 μM 48 h results are shown. DMSO-treated cells viability was set to 100%. Results represent the mean of three independent experiments±s.e.m. LC50=compound concentration required to induce cell mortality by 50%. eNOS, endothelial nitric oxide synthase.

We treated the five BCP-ALL cell lines with the AMPK inhibitor compound C. The capability of compound C to inhibit the AMPK pathway in SEM and RS4;11 cells was previously demonstrated.19 Downregulation of the AMPK pathway in ALL-PO cells is shown in Supplementary Figure S1. Cells were exposed to the inhibitor for different time periods (6–96 h) at concentrations ranging from 0.001 to 100 μM. The three MLL-rearranged cell lines were much more sensitive to AMPK inhibition than the two non-rearranged ones. The GI50 measured by MTT assay was 0.16 μM for SEM, 3.2 μM for RS4;11 and 0.06 μM for ALL-PO cells, whereas it resulted to be 25.7 μM and 19.1 μM for the non-translocated cell lines (Figure 1b). Supplementary Figure S2a shows the results obtained at all tested times and concentrations. MLL-rearranged cell lines exposed to the inhibitor also underwent significantly more apoptosis, as measured with Annexin V and PI staining. LC50 was 7.5 μM for SEM, 8.5 μM for RS4;11 and 9 μM for ALL-PO, whereas it was 37.5 μM for MHH-CALL-2 and 31.4 μM for MHH-CALL-4 (Figure 1c, for more detailed results please see Supplementary Figure S2b). Therefore, activation of the AMPK pathway appears to be essential for sustaining the survival of MLL-rearranged cells.

To validate the association between AMPK inhibition and apoptosis, we suppressed AMPKα1 expression using two different shRNAs (shAMPKalpha1 1 and 2) in SEM, ALL-PO and MHH-CALL-2 cells. AMPKα1 mRNA expression was efficiently reduced after lentiviral vector transduction in the three cell lines (Figure 2a, control AMPKα mRNA expression was set to 100%). In line with the previous results, SEM and ALL-PO cells underwent more apoptosis than MHH-CALL-2 cells (Figure 2b, control cell viability was set to 100%; t-test, SEM vs MHH-CALL-2 P=0.004 and P=0.0003 for shAMPKalpha1 1 and 2, respectively, ALL-PO vs MHH-CALL-2 P=0.006 and P=0.0001). AMPKα1 protein expression was also reduced in MLL-rearranged and non-translocated silenced cells, but the downregulation of the downstream pathway was observed only in SEM and ALL-PO cells (Figure 2c).

Figure 2
Figure 2

AMPK-specific silencing brings to the same effects of compound C treatment. (a) Inhibition of AMPKα1 mRNA expression in SEM, ALL-PO and MHH-CALL-2 cells with both shAMPKalpha1 1 and 2. AMPKα1 mRNA expression in control cells (transfected with un-relevant shRNA sequence) was set to 100%. Results represent mean±s.e.m. of three experiments. mRNA expression levels were measured through Sybr Green real-time quantitative PCR. (b) Cell viability after AMPKα1 silencing in SEM, ALL-PO and MHH-CALL-2 cells was evaluated through Annexin V–PI staining. Control cells viability was set to 100%. Results represent mean±s.e.m. of three experiments. Silenced MLL-rearranged SEM and ALL-PO cells undergo more apoptosis than non-translocated MHH-CALL-2 cells (**P<0.01, ***P<0.001). (c) Inhibition of AMPKα1 protein levels and decrease in the downstream AMPK pathway activation was examined using western blot in control and silenced SEM, ALL-PO and MHH-CALL-2 cells. Representative results are shown with densitometric analysis results indicated under gel images.

shRNA silencing has effects on MLL-rearranged cells survival similar to compound C, therefore what we observed after compound C treatment in these cells can be mainly attributed to AMPK deactivation. These experiments also demonstrated that the specific silencing of AMPK has no consequences on non-translocated cells survival, meaning that the apoptosis we previously observed with 30–40 μM of compound C probably derived from off-target effects because of high concentration of the inhibitor. For this reason, to investigate cell cycle modifications and apoptosis driven by AMPK inhibition, we performed the following experiments only on MLL-rearranged cells.

Compound C treatment induces cell cycle alterations

Representative cell cycle histograms based on flow cytometric analyses of MLL-rearranged cells exposed to compound C are shown in Figure 3. After 24 h of compound C treatment (8 μM), an accumulation in G2/M phase was observed in SEM and RS4;11 cell lines. SEM cells in G2/M phase increased from about 13 up to 34%, whereas cells in G1 decreased from about 72 to 56% and cells in S phase from about 15 down to 10% (Figure 3a). Similarly, after treatment RS4;11 cells in G2/M phase increased from about 17 up to 38%, whereas cells in G1 decreased from about 53 to 43% and cells in S phase from about 30 down to 19% (Figure 3b). Differently, in ALL-PO cells an accumulation in G1 was observed after AMPK inhibition. The percentage of cells in G1 phase increased from about 48 up to 91%, whereas G2/M and S phase cells decreased from about 19 to 5% and from 33 to 4%, respectively (Figure 3c).

Figure 3
Figure 3

Cell cycle alterations after AMPK inhibition. Results are shown in (a) for SEM cells, (b) for RS4;11 cells and (c) for ALL-PO cells. DMSO and CC (8 μM, 24 h) samples were analyzed by flow cytometry and cell cycle analyses were performed using Multicycle Wincycle software. Representative histograms are shown. The percentage of each phase of the cell cycle (G1, S, G2/M) was calculated. Results come from three independent experiments.

Compound C induces apoptosis through the mitochondrial pathway

Next, we examined whether apoptosis occurs through the extrinsic or the intrinsic pathway. For this purpose, we treated MLL-rearranged cell lines SEM, RS4;11 and ALL-PO with 8 μM compound C for 24, 48 and 72 h. Results are resumed in Figure 4a (SEM cells), Figure 4b (RS4;11 cells) and Figure 4c (ALL-PO cells).

Figure 4
Figure 4

Compound C (CC)-mediated cell death follows the mitochondrial pathway (I). Results are shown in (a) for SEM cells, (b) for RS4;11 cells and (c) for ALL-PO cells. In the left panels, depolarization of the mitochondrial transmembrane potential, monitored by the fluorescent dye JC-1, in MLL-rearranged cells treated with CC C (8 μM, 48 h) is reported. The method is based on the ability of this fluorescent probe to enter selectively into the mitochondria, and its color changes reversibly from green to orange as membrane potential increases. This property is due to the reversible formation of JC-1 aggregates on membrane polarization. Aggregation causes a shift in the emitted light from 530 nm (emission by JC-1 monomers) to 590 nm (emission by JC-1 aggregates) following excitation at 490 nm. In middle panels, shown are the mitochondrial production of reactive oxygen species in MLL-rearranged cells treated with CC (8 μM, 48 h). The fluorescence indicator hydroethidine (HE), that is oxidized by superoxide anion into ethidium ion, which emits red fluorescence, was measured. Gray: DMSO-treated cells, red: CC-treated cells. In the right panels, shown are the cleavage of caspase-3 measured by flow cytometry in MLL-rearranged cells after CC (8 μM, 48 h) treatment. Gray: DMSO-treated cells, blue: CC-treated cells.

Both FAS and FAS-L were present at low levels in control and DMSO-treated cells and did not increase after AMPK inhibition (data not shown). We also examined if exposure to compound C for 24 or 48 h could induce BCP-ALL cell differentiation using a panel of B-lymphocyte differentiation markers including CD58/10/45/19/34/20; and no change in marker expression was detected (data not shown). Thus, apoptosis induced by compound C does not involve FAS and AMPK inhibition does not promote B-cell differentiation.

Apoptotic stimuli may alter the mitochondrial transmembrane potential Ψmt monitored by the fluorescent dye JC-1. JC-1 fluorescence shifted in MLL-rearranged cells exposed to compound C for 48 h, indicating depolarization of mitochondrial membrane potential (Figure 4, left panels). As mitochondrial membrane depolarization is associated with mitochondrial production of ROS, we investigated whether ROS production was increased after compound C treatment. We used the fluorescent indicator hydroethidine that is oxidized by superoxide anion into ethidium ion, which emits red fluorescence; superoxide is produced by mitochondria when cytochrome c is released. As shown in Figure 4 middle panels, exposure to compound C for 48 h induced ROS production in treated MLL-rearranged cells, and we also observed the activation of caspase-3 (Figure 4 right panels) and decreased levels of the anti-apoptotic protein BCL-XL (Figure 5). This leads to mitochondrial outer membrane permeabilization through BAX/BAK action, with the consequent release of cytochrome c into the cytoplasm, a necessary event for downstream caspases activation. As shown in Figure 5, the release of cytochrome c in the cytoplasm of SEM, RS4;11 and ALL-PO cells after 48 h of AMPK inhibition is particularly evident. Release of cytochrome c results in activation of caspase-9 through self-cleavage at Asp315. Cleaved caspase-9 further processes other caspase members, including caspase-3 and caspase-7 that are effector caspases responsible for the proteolytic cleavage of many key proteins such as PARP. When cleaved, PARP is no longer able to initiate DNA repair and cells undergo apoptosis. In treated MLL-rearranged cells we observed at 48 h the activation of caspase-7 and caspase-9, and at 72 h the cleavage of PARP (Figure 5). Finally, after 72 h of AMPK inhibition we also observed an increase in chromosomal DNA fragmentation into multiples of the 180-bp nucleosomal unit (Supplementary Figure S3).

Figure 5
Figure 5

Compound C (CC)-mediated cell death follows the mitochondrial pathway (II). Apoptotic proteins expression was determined by immunofluorescence in MLL-rearranged cells after DMSO or CC (8 μM, 48 and 72 h) treatment. Panel shows pseudo-color merged images of cell lines decorated with 4,6-diamidino-2-phenylindole (DAPI), Alexa-fluor 488 and 594. Scale bar 5 μm.

AMPK inhibition induces cell death in primary leukemia cultures

We treated nine primary BCP-ALL cultures (five with MLL gene rearrangements and four non-translocated) with DMSO alone or compound C. Cells with MLL rearrangements underwent apoptosis when exposed to low concentrations (1 and 5 μM) of compound C more extensively than cells lacking MLL gene rearrangements (t-test, 1 μM 24 h P=0.005, 5 μM 24 h P=0.007, 1 μM 48 h P=0.03, 5 μM 48 h P=0.01; Figure 6a). We also show that AMPK and its downstream targets were dephosphorylated in two MLL-rearranged samples after 48 h of treatment with 5 μM of compound C (Figure 6b). Moreover we observed, as for MLL-rearranged cell lines, the decrease in BCL-XL, the release of cytochrome c and the cleavage of caspase-7, caspase-9 and PARP (Figure 6c).

Figure 6
Figure 6

AMPK inhibition induces apoptosis in primary leukemia cultures. (a) Primary cells from nine patients (five MLL-rearranged and four non-translocated) were cultured on MSCs layer and treated with compound C (CC, 1–5–8 μM) for 24 and 48 h. Apoptosis was determined by flow cytometry with Annexin V–PI staining. DMSO-treated cells viability was set to 100%. Results represent mean±s.e.m. MLL-rearranged primary cells undergo more apoptosis than the non-translocated ones at 24 and 48 h after treatment with 1 and 5 μM CC. (b) Inhibition of the AMPK pathway activation was analyzed by western blot in control and CC (5 μM, 48 h) treated patients’cells. Representative results of two MLL-rearranged patients are shown. (c) Apoptotic proteins expression was determined by immunofluorescence after CC (8 μM, 48 and 72 h) treatment in one MLL-rearranged primary culture. Panel shows pseudo-color merged confocal images of cells decorated with 4,6-diamidino-2-phenylindole (DAPI), Alexa-fluor 488 and 594. Scale bar 5 μm.

AMPK inhibition synergizes with drugs used in ALL chemotherapy

MLL-rearranged SEM, RS4;11 and ALL-PO cells were exposed to five drugs, namely daunorubicin, vincristine, cytarabine, dexamethasone and L-asparaginase in the presence or absence of the AMPK inhibitor compound C. The complete dose-response curves are shown in Supplementary Figures S4a (SEM), b (RS4;11) and c (ALL-PO). We estimated the concentration of each drug that caused 50% of growth inhibition with or without the compound C added at fixed combination ratio as indicated in Table 1 and searched for synergistic interactions. As can be observed, the addition of compound C significantly lowered the GI50 of all drugs in ALL-PO cells, whereas in RS4;11 we did not observe any variation of the efficacy of vincristine and dexamethasone. The addition of compound C failed to increase the efficacy of dexamethasone also in SEM cells. For the other conditions, the calculated combination indexes showed values <1, indicating that compound C strongly synergizes with these chemotherapeutic drugs, and at the same time suggesting the potential synergistic effect of AMPK inhibition in combination with chemotherapeutic drugs with different mechanisms of action in the treatment of MLL-rearranged BCP-ALL.

Table 1: Treatment of MLL-rearranged BCP-ALL cell lines with compound C and drugs used in ALL chemotherapy

Discussion

In this study, we report that the activation of the serine/threonine kinase AMPK is a key factor for the survival of BCP-ALL MLL-rearranged cells. Inhibition of AMPK activity caused a decrease in cell proliferation and survival both in cell lines and primary cultures, and it synergistically enhanced the antiproliferative activity of ALL chemotherapeutic drugs, thus pointing out this cellular fuel sensor as a promising new therapeutic target.

AMPK has been frequently proposed as a potential therapeutic target in cancer because of its position downstream of tumor-suppressor LKB1 and its known role in the regulation of cell proliferation, cell cycle progression and autophagy via mTORC1, p53 and p27.11 Its activation through Metformin or AICAR (5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide) treatment can suppress cell proliferation and induce apoptosis in many cell types, that is, melanoma,27 glioblastoma,28 breast cancer29 and renal cell carcinoma.30 Two studies31, 32 showed that AMPK activation by AICAR (5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide) can induce apoptosis in some BCP-ALL cell lines, but MLL-rearranged cells were not included in these studies.

Interestingly, there is emerging evidence that AMPK activation has a dual role in cancer depending on alterations of interconnected signaling pathways, thus its effects are likely to be cell-type and tissue specific. Recent papers report that AMPK is hyperactivated in some cancers and increased cell death is observed after inhibition of its activity. Compound C treatment or AMPK depletion by small interfering RNA induce growth arrest and apoptosis in human prostate cancer,17 multiple myeloma,16 human and rat glioma18 and pheochromocytoma cell lines.33 AMPK activity was also reported to be higher in some cancer cell lines such as OVCAR3 and A431 than in primary keratinocytes.34 Moreover, increased expression of LKB1 and AMPK was observed in UVB (Ultraviolet B)-induced murine basal cell carcinoma.35

These findings, together with our previous observations that the AMPK pathway is hyperactivated in MLL-rearranged BCP-ALL cells and that the AMPK inhibitor compound C induces dephosphorylation of AMPK and its downstream targets,19 prompted us to study the effects of AMPK inhibition on the survival of MLL-rearranged blast cells. We first used the commercially available AMPK inhibitor compound C to treat MLL-rearranged and non-translocated cell lines. MLL-rearranged cells show decrease in proliferation and survival at really low concentrations of compound C with respect to non-translocated cells, with very different levels of GI50 and LC50. The specific silencing of AMPK by two different shRNA induced a decrease in cell survival similarly to compound C treatment, thus what we observed after compound C treatment can be mainly attributed to AMPK inhibition. Of note, in non-translocated cells the activation of the pathway downstream AMPK is not modified by specific silencing, confirming our idea that this pathway is specific for MLL-rearranged cells. These results prove that AMPK activation has a very important role in supporting the survival of MLL-rearranged cells and can be specifically targeted in order to induce cell death. Of note, we treated with compound C also the acute monocytic leukemia cell line carrying the t(4;11) MV4;11. These cells showed, as MLL-rearranged BCP-ALL cells, very low levels of GI50 (0.62 μM) and LC50 (0,77 μM) after 48 h of treatment (data not shown). We then described that compound C-induced apoptosis follows the mitochondrial pathway. We observed mitochondrial membrane depolarization, ROS production, cytochrome C release and activation of caspases followed by PARP cleavage. We previously reported that compound C treatment also brings to a reduction of BCL-2 S70 levels.19 Compound C-induced apoptosis was already shown to be related to ROS production and BCL-2 downregulation also in multiple myeloma16 and in glioma cells,18 thus supporting our findings. Of note, not only cell lines but also MLL-rearranged primary leukemia cells responded to AMPK inhibition with compound C following the mitochondrial apoptotic pathway. MLL-rearranged BCP-ALL primary cultures underwent much more apoptosis than those lacking MLL rearrangements after exposure to low concentrations of compound C, indicating that AMPK is critical for cell survival also in patients’ primary cells.

Interestingly, apoptosis occurred after an initial cell cycle block at the G2/M phase in SEM and RS4;11 cells. In agreement with these findings, inhibition of the AMPK pathway in glioma cells18 also brings to G2/M arrest. In addition, in Drosophila melanogaster the activation of the LKB1-AMPK pathway was demonstrated to be strictly required for an accurate mitosis and chromosome segregation.36, 37 Further investigation will be performed in order to understand the mechanisms underlying the G2/M cell cycle block in these cells. On the other hand, ALL-PO cells showed an accumulation in G1 phase after treatment. Very little is known about this cell line, thus further studies, that is, cell cycle proteins mutation status, will be required in order to explain their different cell cycle variation in response to AMPK inhibition. Nevertheless, a decrease in S phase, which reflects a proliferation decrease is a common signature after compound C treatment.

We also investigated the effects of the simultaneous treatment of SEM, RS4;11 and ALL-PO cells with compound C and vincristine, daunorubicin, cytarabine, dexamethasone and L-asparaginase. Our results show that AMPK inhibition enhanced the chemotherapeutic effects of these drugs significantly lowering their GI50 values in most of the evaluated conditions. These results suggest that AMPK inhibition could augment the effects of conventional chemotherapy for MLL-rearranged BCP-ALL and pointed towards the potential clinical utility of AMPK inhibitors, although further studies are needed to better understand the molecular mechanism(s) involved in the synergistic effect.

In conclusion, this study provides new insights into the role of AMPK in cancer and, in particular, in BCP-ALL. AMPK activation is required for MLL-rearranged cell survival and its inhibition is able to induce cell death. For these reasons, AMPK could be considered as a new drug target in MLL-rearranged leukemias and kinase inhibitors targeting AMPK should be further studied to make new therapeutic options available for this high-risk form of leukemia.

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Acknowledgements

We thank Dr E Giarin for helping us with the BioBank and Dr R Bortolozzi for figures editing. We are also grateful to Professor D Campana for providing MSCs and constructive comments on the manuscript. This work was supported by grants from the Istituto Superiore di Sanità (Italy/USA program), the Fondazione Città della Speranza, the Associazione Italiana per la Ricerca sul Cancro, the Ministero della Salute (Ricerca Finalizzata 2006—Programma Integrato Oncologia) to GB. Progetto d'Ateneo—Bando 2010 to S.I. and Progetto d'Eccellenza Fondazione CARIPARO to SI and G. teK.

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Affiliations

  1. Oncohematology Laboratory, Department of Woman and Child Health, University of Padova, Padova, Italy

    • B Accordi
    • , L Galla
    • , G Milani
    • , V Serafin
    • , V Lissandron
    • , G Viola
    • , G te Kronnie
    •  & G Basso
  2. Immunology and Diagnostic Molecular Oncology, Istituto Oncologico Veneto IRCCS, Padova, Italy

    • M Curtarello
    •  & S Indraccolo
  3. Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy

    • R De Maria
  4. Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA, USA

    • E F Petricoin 3rd
    •  & L A Liotta

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Competing interests

BA, LL, EP, and GB are co-inventors on pending patent applications that cover findings within this paper and the authors could receive royalties as a consequence. These applications have been licensed to Theranostics Health, Inc., and LL and EP are equity shareholders. The other authors declare no competing financial interests.

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Correspondence to B Accordi.

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https://doi.org/10.1038/leu.2012.338

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

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