Antineoplastic activity of ouabain and pyrithione zinc in acute ... - Nature

Oncogene (2012) 31, 3536–3546

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ORIGINAL ARTICLE

Antineoplastic activity of ouabain and pyrithione zinc in acute myeloid leukemia M Tailler1,2,3, L Senovilla1,2,3, E Lainey1,2,3, S The´pot1,3,4, D Me´tivier1,2,3, M Se´bert1,3,4, V Baud5,6,7, K Billot5,6,7, P Fenaux1,3,4, L Galluzzi1,2,3, S Boehrer1,3,4, G Kroemer1,7,8,9,10,11 and O Kepp1,2,3,11 1

INSERM, U848, Villejuif, France; 2Institut Gustave Roussy, Villejuif, France; 3Universite´, Paris Sud/Paris XI, Le Kremlin Biceˆtre, France; 4Service d’He´matologie Clinique, Hoˆpital Avicenne, AP-HP/Universite´ Paris XIII, Bobigny, France; 5INSERM, U1016, Institut Cochin, Paris, France; 6CNRS, UMR8104, Paris, France; 7Universite´ Paris Descartes, Paris, France; 8Metabolomics Platform, Institut Gustave Roussy, Villejuif, France; 9Centre de Recherche des Cordeliers, Paris, France and 10Poˆle de Biologie, Hoˆpital Europeén Georges Pompidou, AP-HP, Paris, France

Despite recent progress in the treatment of acute myeloid leukemia (AML), the prognosis of this rather heterogeneous disease remains poor and novel chemotherapeutics that specifically target leukemic cells must be developed. To address this need at the preclinical level, we implemented a high content imaging-based screen for the identification of small agents that induce AML cell death in vitro. Among a panel of 1040 Food and Drug Administration-approved agents, we identified pyrithione zinc (PZ) and ouabain (OUA) as potential antileukemic compounds. Both PZ and OUA efficiently induced cell death associated with apoptotic chromatin condensation and inhibition of nuclear factor-jB survival signaling, leading to reduced expression of antiapoptotic proteins, in several AML cell lines. PZ- and OUA-induced cell death was associated with the permeabilization of the outer mitochondrial membrane and led to the release of cytochrome c followed by caspase activation. Both PZ and OUA exerted significant anticancer effects in vivo, on human AML cells xenografts as well as ex vivo, on CD34 þ (but not CD34) malignant myeloblasts from AML patients. Altogether, our results suggest that PZ and OUA may exhibit antileukemic effects by inducing the apoptotic demise of AML cells. Oncogene (2012) 31, 3536–3546; doi:10.1038/onc.2011.521; published online 21 November 2011 Keywords: apoptosis; necrosis; myeloblasts; HL-60; HCS

Introduction Acute myeloid leukemia (AML) constitutes a heterogeneous group of diseases that are commonly treated with conventional chemotherapeutics such as cytarabine Correspondence: Dr G Kroemer or Dr O Kepp, INSERM U848, Institut Gustave Roussy, PR1, 114 rue Edouard Vaillant, Villejuif, Val de Marne F-94805, France. E-mail: [email protected] or [email protected] 11 These authors share senior co-authorship. Received 18 July 2011; revised 1 October 2011; accepted 12 October 2011; published online 21 November 2011

(ara-C), daunorubicin or idarubicin. In the M3 subtype of AML, also known as acute promyelocytic leukemia, these treatments are combined with differentiationinducing agents including all-trans-retinoic acid and arsenic acid (Burnett et al., 2011). In spite of some success of allogenic stem cell transplantation and novel immunotherapies (with antibodies or the combination of histamine dihydrochloride and interleukin-2) (Smits et al., 2011), AML prognosis remains relatively poor, underscoring the need for novel therapeutic options. Experimental AML treatments include demethylating agents, such as azacytidine and decitabine (Burnett et al., 2011), epithelial growth factor receptor inhibitors, such as gefitinib and erlotinib (which both appear to act through off-target effects) (Stegmaier et al., 2005; Boehrer et al., 2008; Hahn et al., 2009), as well as inhibitors that specifically target oncogenic kinases that are overactivated in a subset of AML, like FLT3 and KIT (Burnett et al., 2011). Several high-throughput screening approaches have been undertaken to identify novel antileukemic agents for the treatment of AML. One of these strategies consists of culturing AML cells in the presence of compounds from chemical libraries and measuring cytotoxic effects by the decline of metabolic activity, by performing cell death assays (for instance with cytofluorometry) (Edwards et al., 2009; Kepp et al., 2011) or by assessing cellular differentiation, which also constitutes a therapeutic endpoint (Wald et al., 2008). Encouraged by these approaches, we decided to test 1040 Food and Drug Administration (FDA)-approved agents for their possible antileukemic effects. To this aim, we developed a high content imaging-based highthroughput screening system that assesses nuclear fragmentation and membrane integrity in promyelocytic leukemia HL-60 cells, as an indication for drug-induced apoptosis and necrosis, respectively. Here, we report the discovery of two potential antileukemic agents, namely pyrithione zinc (PZ, which obtained FDA approval for external use, for instance as an antiseborrheic agent) and ouabain (OUA, which has been used in the past for the treatment of cardiac insufficiency). Both of these agents exhibited cytotoxic effects against various AML cell

Ouabain and pyrithione zinc for AML treatment M Tailler et al

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lines in vitro, were efficient in vivo to inhibit the growth of human leukemic cells xenotransplanted in immunodeficient mice, and selectively killed malignant blasts from AML patients ex vivo.

Results Identification of OUA and PZ as potential antileukemic agents To set up a screening platform for the identification of antileukemic agents, we exposed AML HL-60 cells to the pan-tyrosine kinase inhibitor staurosporine, which is a quintessential inducer of caspase-dependent apoptosis (Weil et al., 1996). Cell cultures were performed in Vshaped 96-well plates, followed by co-staining with Hoechst 33342 (to visualize nuclear morphology) and a vital dye (which only incorporates into dead cells owing to permeabilized plasma membranes) (Galluzzi et al., 2009). Cells were then transferred to and immobilized on poly-L-lysine-coated flat-bottomed 96-well imaging plates, and subjected to robotized epifluorescence microscopy and automatic image analysis (Figure 1a). Staurosporin induced a time- and dose-dependent reduction in nuclear area that was inhibited by the broad-spectrum caspase inhibitor Z-VAD-fmk (Figures 1b and c). Based on this system, we screened a library of 1040 FDA-approved drugs (final concentration ¼ 1 mM) to identify agents that induce apoptosis (Figure 1d) yet fail to induce necrosis (Figure 1e) in HL-60 cells. Hits were identified upon Z-score analysis and validated manually by microscopic re-analysis. Among the 40 most efficient inducers of nuclear fragmentation (Supplementary Table S1), those that induced necrosis and/or are already known for their anticancer activity were excluded. We decided to followup cardiac glycosides (digoxin, digitoxin, lanatoside C, OUA, sanguinarine), as well as PZ, an agent that is FDA approved for external use as an antibacterial, antifungal and antiseborrheic. Validation experiments revealed that OUA and PZ are highly efficient in inducing multiple hallmarks of apoptosis in a timeand dose-dependent fashion: nuclear fragmentation (Figures 1f and g), reduction in nuclear DNA content, which is indicative of the activation of nucleases (Figures 1h and i), and loss of clonogenic potential (Figures 1j and k). These pro-apoptotic effects of OUA and PZ were observed in HL-60 cells (Figure 1) as well as in three additional AML cell lines, MV4-11, MOLM13 and KG-1 (Figure 2), pointing to the broad antileukemic potential of these compounds. Mechanism of cell death induction by OUA and PZ OUA and PZ failed to induce DNA damage foci in preapoptotic nuclei, as determined by immunofluorescence detection of histone H2AX phosphorylated on Ser139 (gH2AX, Figures 3a and b). Moreover, neither OUA nor PZ induced antigenic (CD11b) or morphological signs of differentiation (Supplementary Figure S1). However, submicromolar concentrations of OUA and PZ sig-

nificantly reduced the percentage of HL-60 cells exhibiting the nuclear factor (NF)-kB subunit RelA/ p65 in the nucleus (Figures 3c and d) and decreased the abundance of antiapoptotic NF-kB target gene products including the mitochondrial membrane-stabilizing proteins Bcl-XL, Bcl2L10, Mcl-1 and A1 as well as the caspase inhibitor XIAP (Figure 3e, Supplementary Figure S2A). Both OUA and PZ induced a dosedependent reduction in the mitochondrial transmembrane potential (Dcm)—a sign of imminent cell death— and the loss of plasma membrane integrity, as assessed by co-staining with the Dcm-sensitive cationic fluorochrome DiOC6(3) and the vital dye propidium iodide (PI), respectively (Figures 4a and b). Of note, neither OUA nor PZ were able to cause increment in the percentage of HL60 cells succumbing to bortezomib, a proteasome inhibitor that blocks NF-kB activation (Supplementary Figure S2B), indicating that OUA and PZ trigger cell death by partially inhibiting NF-kB. Immunofluorescence microscopy revealed that OUA and PZ cause the redistribution of cytochrome c from mitochondria to the cytoplasm (Figures 4c and d), which is indicative of another apoptosis-related phenomenon, mitochondrial outer membrane permeabilization. OUA and PZ-induced mitochondrial outer membrane permeabilization was followed by the activation of a caspase-like proteolytic activity in all four AML cell lines investigated in this respect (Figure 5a). Accordingly, OUA and PZ reduced the abundance of the inactive form of caspase-3 (pro-caspase-3), followed by the cleavage of the caspase-3 substrate poly(ADPribose) polymerase 1 (Figure 5b). Inhibition of caspases with Z-VAD-fmk abolished nuclear fragmentation induced by either OUA or PZ, underscoring that this phenomenon occurs in a caspase-dependent fashion (Figure 5c). In contrast, addition of the antioxidant Nacetyl cysteine, the nitric oxide synthase inhibitor Nmethyl-L-arginine acetate, or the chemical chaperone 4phenylbutyrate failed to inhibit OUA- or PZ-induced apoptosis (Supplementary Figure S3). The BH3 mimetic ABT737 (which neutralizes both Bcl-2 and Bcl-XL) synergized with OUA but not with PZ for the killing of HL-60 cells (Supplementary Figure S4A). Only additive effects were measured when conventional anti-AML agents (such as cytarabine, etoposide and daunorubicin) were combined with either OUA or PZ (Supplementary Figure S4B). Altogether, these results suggest that OUA and PZ induce apoptosis through a mechanism that does not involve DNA damage, differentiation or cell cycle arrest (Figures 1i and j), occurs independently of reactive oxygen species and protein misfolding, but involves mitochondrial dysfunction and caspase activation. In vivo and ex vivo antileukemic activity of OUA and PZ To determine the possible antineoplastic action of OUA and PZ at the preclinical level, immunodeficient nu/nu mice were injected subcutaneously with HL-60 cells and mice were treated with systemic (intraperitoneal) injections of OUA or PZ when subcutaneous tumors became palpable. At doses that did not cause any overt Oncogene


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signs of toxicity nor any significant weight reduction (Supplementary Figure S5), OUA and PZ strongly reduced the growth of AML-derived xenografts, and had a near-to-complete tumoristatic effect (Figures 6a and b). Similarly the splenomegaly and splenic infiltration caused by intravenously injected human AML cells was reduced by both OUA and PZ (Figures 6c and d). Next, we applied the robotized fluorescence microscopic system that we developed (Figure 1) to malignant blasts from AML patients (Supplementary Table S2, Supplementary Figure S6). Peripheral blood mononuclear cells from four untreated patients undergoing a blast crisis (470% circulating blasts, most of which

CD34 þ ) exhibited signs of apoptosis in response to OUA and PZ (Figure 7a). When CD34 þ circulating blasts from AML patients were purified by immunoselection, such malignant cells systematically exhibited a higher degree of responsiveness to OUA- or PZ-induced apoptosis than their CD34 counterparts, as determined on six additional AML patients (Figures 7b and c). Notably, both CD34 þ and CD34 cells obtained from a healthy donor failed to die in response to the same doses of OUA and PZ that caused consistent levels of cell death among malignant cell populations (Supplementary Figure S7). Altogether, these preclinical data suggest that both OUA and PZ have a therapeutic effect on AML cells in vivo and ex vivo.

Figure 2 Antineoplastic effects of PZ and OUA on AML cell lines in vitro. (a–c) MV4-11, MOLM-13 and KG-1 AML cells were treated with 1 mM PZ or OUA for 18 h (a, b) or the indicated time (c) and then either co-stained with Hoechst 33342 and the Live/Dead vital dye for the assessment of nuclear fragmentation (a, b) or subjected to the colorimetric quantification of cell proliferation. Panel a depicts representative images. Scale bar, 10 mm. Panels b and c report quantitative data (means±s.e.m.; n ¼ 3; * Po0.05).

Figure 1 Microscopic screening platform for the identification of antileukemic agents. (a) Schematic representation of the screening protocol. (b) Representative micrographs of human AML HL-60 cells left untreated or treated for 8 h with 1 mM staurosporine (STS) alone or combined with 50 mM Z-VAD-fmk, followed by staining and imaging as depicted in a. Scale bar, 30 mm. (c) HL-60 cells were treated with 1 mM or the indicated concentration of STS (alone or combined with 50 mM Z-VAD-fmk) for the indicated time or 8 h, followed by the automated assessment of nuclear area, as in a. Results are reported as means±s.e.m. (d, e) HL-60 cells were treated with the compounds from the US Drug library (final concentration ¼ 1 mM) for 18 h, followed by staining and automated assessment of nuclear area as in a. Results are reported as Z-scores for nuclear fragmentation and vital dye uptake. Representative pictures of apoptotic (d) and necrotic (e) cells are depicted. Scale bar, 10 mm. (f, g) HL-60 cells were treated for 9 or 18 h with the indicated concentration of PZ or OUA (1 mM unless otherwise specified) and cell death induction was assessed by automated microscopy. Panels f and g report representative images and quantitative data (means±s.e.m.; n ¼ 3), respectively. Scale bar ¼ 30 mm. (h, i) Cell cycle distribution of HL-60 cells treated for 18 h with the indicated dose of PZ or OUA (1 mM unless otherwise specified). Results from one representative experiment are reported. (j, k) Clonogenic survival of HL-60 cells treated for 18 h with the indicated concentration of PZ or OUA. Panels j and k report representative images and quantitative data (means±s.e.m.; n ¼ 3), respectively. Scale bar, 5 mm. Oncogene


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Figure 3 Inhibition of NF-kB-dependent survival signaling (a, b) HL-60 cells were treated for 4 h with 1 mM PZ, 1 mM OUA or 50 mM cisplatin (CDDP), and the DNA damage response was evaluated by immunofluorescence microscopy for the detection of phosphorylated histone H2AX (gH2AX) nuclear foci. Panels a and b report representative images and quantitative data (means±s.e.m.; n ¼ 3; * Po0.05, NS ¼ not significant), respectively. Scale bar, 2 mm. (c, d) HL-60 cells were treated for 4 h with the indicated concentration of PZ or OUA (1 mM unless otherwise specified) or 5 ng/ml tumor necrosis factor a (TNFa) and then processed for the automated detection of the RelA/p65 NF-kB subunit. Panels c and d report representative images and quantitative data (means±s.e.m.; n ¼ 3), respectively. Scale bar, 2 mm. (e) HL-60 cells were treated for the indicated time with the indicated concentration of PZ or OUA (alone or in combination with 50 mM Z-VAD-fmk), followed by immunoblotting for the detection of IkBa, XIAP and Bcl-XL levels. Actin abundance was monitored to ensure equal lane loading.

Discussion In this work, we describe a strategy for the identification of antileukemic agents that is based on the high content detection (by fluorescence microscopy and automated image analysis) of apoptosis-related changes in human leukemic cells exposed to a battery of pharmacological compounds. This system was suitable to screen more than 1000 compounds in one single experiment and might be easily scaled up for high-throughput purposes, in particular by using 1536-well plates and an entirely automated handling of reagents, cells and microscopy. Among the collection of FDA-approved pharmacological agents, we identified two putative antileukemic agents for the experimental treatment of AML, namely OUA and PZ. OUA is a cardiac glycoside that has been used in the past for the treatment of cardiac insufficiency. Cardiac glycosides are believed to be selectively cytotoxic for tumors because malignant cells express high levels of Na þ /K þ ATPase a-isoforms (such as a3), which are susceptible to inhibition (Newman et al., 2008). Similar to other cardiac glycosides, OUA binds reversibly to the Oncogene

a-subunit of the Na þ /K þ ATPase, leading to a rise in intracellular Na þ and Ca2 þ levels and to the induction of cell death by a variety of mechanisms (Lopez-Lazaro, 2007; Newman et al., 2008). Such mechanisms have been widely studied in solid tumors but most of them are unlikely to be relevant for AML cells. These include the interference with epidermal growth factor receptor signaling (as epithelial growth factor receptor is not expressed in AML cells) (Kometiani et al., 2005; Boehrer et al., 2008; Hahn et al., 2009), the inhibition of topoisomerases (contrasting with the fact that OUA failed to induce a DNA damage response, Figure 3) (Bielawski et al., 2006), the hyperproduction of reactive oxygen species (contrasting with the observation that OUA-induced cell death was not inhibited by antioxidants, Supplementary Figure S3) (Newman et al., 2006), the inhibition of protein synthesis (which has never been documented in leukemia cell lines) (Perne et al., 2009; Hallbook et al., 2011) or the blockage of the p53 system (Wang et al., 2009). More plausibly, inhibition of the Na þ /K þ ATPase may initiate mitochondrial cell death (Yin et al., 2009), in line with the fact that OUA induces two pathognomonic signs of


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Figure 4 PZ and OUA induce AML cell death by triggering the intrinsic pathway apoptosis pathway. (a, b) Cytofluorometric quantification of HL-60 cell death following treatment with the indicated concentration (1 mM unless otherwise specified) of PZ or OUA for the indicated time (18 h when not specified). Panel a reports representative dot plots. In b, white and black columns illustrate the percentage of dying (PI DiOC6(3)low) and dead (PI þ ) cells, respectively (means±s.e.m.; n ¼ 3). (c, d) Release of cytochrome c from the mitochondria of HL-60 cells treated as in a, b. Panel c illustrates representative images. Scale bar, 5 mm. In d, quantitative data are reported (means±s.e.m.; n ¼ 3).

mitochondrial derangement, namely Dcm dissipation and mitochondrial outer membrane permeabilization (Figure 4). Another cardiac glycoside, UNBS1450, kills human leukemic monocyte lymphoma U937 cells while inhibiting NF-kB-mediated gene transactivation (Juncker et al., 2011), suggesting that NF-kB inhibition may be involved in cell killing by this class of agents. However, OUA differs from bufalin, another cardiac glycosides (Zhang et al., 1992), in thus far that it fails to induce the differentiation of AML cells (Figure 3). Altogether these observations suggest a hypothetical scenario whereby OUA would kill AML cells while inhibiting NF-kBdependent survival signaling and hence stimulating an ABT737-amplifiable mitochondrial pathway of apoptosis. PZ is widely used as an antifungal and antibacterial agent in clinical antiseptic products and over-thecounter antimicrobials. Its dermatological applications include the treatment of dandruff and seborrhoeic

dermatitis, and evidence suggests PZ might be beneficial for the treatment of psoriasis, eczema, ringworm, skin mycoses and atopic dermatitis. PZ reportedly perturbs zinc homeostasis (Lamore and Wondrak, 2011), and induces a DNA-damage response followed by lethal activation of poly(ADP-ribose)polymerase (Lamore et al., 2009). In AML cells, however, PZ failed to induce DNA damage (Figure 3). Importantly, we found that ZnCl2 was far less toxic than PZ (Supplementary Figure S8), ruling out that the antileukemic properties of PZ might be mediated by Zn2 þ ions. PZ has also been shown to activate voltage-gated KCNQ potassium channels (Klionsky et al., 2008), which have major roles in the central nervous system and cardiomyocytes, yet lack any known function in hematological cells. Thus, the precise antileukemic mode of action of PZ remains elusive. Nonetheless, our results suggest that PZ inhibits NF-kB pro-survival Oncogene


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Figure 5 PZ and OUA induce caspase-dependent apoptosis. (a) The indicated AML cell lines (HL-60 cells unless otherwise specified) were treated for 18 h with 1 mM PZ or OUA, alone or in combination with 50 mM Z-VAD-fmk, and then processed for the cytofluorometric detection of caspase-3/7 activation (a), subjected to immunoblotting for the detection of poly(ADP-ribose) polymerase 1 cleavage and caspase-3 proteolytic maturation (b), or analyzed for nuclear fragmentation (c). In a and c, quantitative data are reported upon normalization to untreated cells (means±s.e.m.; n ¼ 3). In b, actin levels were monitored to ensure equal loading of lanes.

signaling and stimulates the mitochondrial pathway of apoptosis. As OUA, PZ did not exhibit any synergistic antileukemic effect with conventional chemotherapeutics, suggesting that it might be used as a single agent. Of note, OUA and PZ were highly efficient in inhibiting the growth of human AML cells xenotransplanted in immunodeficient mice, yet neither of these compounds exerted significant toxicity on the host, as determined by the absence of weight loss and treatmentrelated mortality. Moreover, both agents were able to kill primary leukemic cells from AML patients ex vivo. These pre-clinical results point to OUA and PZ as promising antileukemic agents whose activity should be evaluated in prospective clinical studies.

Materials and methods Chemicals, cell lines and culture conditions 4-phenyl butyric acid, all-trans-retinoic acid, cytarabine, daunorubicin, etoposide, N-acetyl-cysteine, N-methyl-L-argiOncogene

nine acetate (L-NMMA) and OUA, PZ and staurosporine were purchased from Sigma (St Louis, MO, USA); benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (Z-VADfmk) from Bachem (Bubendorf, Switzerland) and ABT737 from Merck (Darmstadt, Germany). Solvent cytotoxicity was ruled out by appropriate controls throughout all experiments. Human promyelocytic leukemia HL-60 and human AML MV4-11, KG-1 and MOLM-13 cells were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany). Cell lines were cultured at 37 1C under 5% CO2 atmosphere in RPMI 1640 medium supplemented with 10% fetal calf serum. Patient-derived primary blasts (see ex vivo samples section) were maintained in Iscove’s-modified Dulbecco’s medium (IMDM) supplemented with 1% L-glutamine, 100 units/ml penicillin-streptomycin, 20% BIT 9500 serum substitute (200 mg/ml transferrin, 10 mg/ ml insulin, 2% fetal calf serum; from StemCell Technologies, Grenoble, France), 100 ng/ml FLT3 ligand (Miltenyi Biotec, Bergisch Gladbach, Germany), 50 ng/ml stem cell factor (Miltenyi Biotec), 10 ng/ml interleukin-3, 10 ng/ml interleukin-6 and 50 ng/ml thrombopoietin (all purchased from Peprotech, Neuilly-sur-Seine, France). Unless otherwise specified, cells were seeded at concentrations ranging from 1.0 to 1.5 105 cells/ml. Unless otherwise indicated, media and


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Figure 6 PZ and OUA are effective against xenografted tumors in vivo. (a, b) HL-60 (1 106) cells were subcutaneously xenografted into nude mice. Upon appearance of palpable tumors, mice were injected intraperitoneally every 2 days with 5 mg/kg PZ or 2 mg/kg OUA, and tumor size was monitored for the following 40 days. Panel a shows representative mice at the end of the experiment, whereas in panel b growth curves are illustrated (means±s.e.m.; n ¼ 5 mice per group; * Po0.05; ** Po0.01). Spleen size in representative animals is shown in the inset. Scale bar, 1 cm. (c, d) Nude mice were intravenously given 1 106 HL-60 cells and, 20 days later, treated with intraperitoneal PZ or OUA as in a, b. Ten days after the beginning to treatment splenocytes were collected and checked for the presence of human HLA-expressing infiltrating cells by fluorocytometry. Representative histograms and quantitative data (means±s.e.m.; n ¼ 3; ** Po0.01) are reported in c, d, respectively.

Figure 7 Effect of PZ and OUA on primary blasts from AML patients. (a, b) CD34 þ and CD34 cells from AML patients were treated for 18 h with the indicated concentration of PZ or OUA (1 mM unless otherwise specified) and then co-stained with Hoechst 33 342 and the Live/Dead vital dye for the assessment of nuclear fragmentation and plasma membrane integrity by automated microscopy. Panel a shows representative pictures. Scale bar, 20 mm. In b and c, quantitative results are illustrated (means±s.e.m.; n ¼ 3; * Po0.05, N.S. not significant). Oncogene


3544 supplements for cell culture were purchased from GibcoInvitrogen (Carlsbad, CA, USA) and plasticware was obtained from Corning (Amsterdam, The Netherlands). Ex vivo samples and CD34 þ cell purification Patient samples were assessed upon informed consent, in line with the Declaration of Helsinki. AML and MDS were diagnosed by morphological examination of peripheral blood and bone marrow, according to the WHO and FAB classification. Cytogenetic analyses were performed according to conventional methods. Upon isolation of bone marrow mononuclear cells on a Ficoll-Paque PLUS density gradient (Amersham Biosciences, Sunnyvale, CA, USA), CD34 þ cells were positively purified by means of the MiniMacs system (Miltenyi Biotec), following the manufacturer’s instructions. Proliferation assays Cell viability was assessed by a colorimetric method, as previously described (de La Motte Rouge et al., 2007). Briefly, 2 105 cells were seeded in 96-well plate wells and treated with the indicated drugs (dose range ¼ 10 nM–10 mM) for either 24 or 48 h, followed by the assessment of the conversion of the colorless tetrazolium salt WST-1 (4-[3-(4-iodophenyl)-2-(4nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) to formazan. To this aim, the WST-1 Cell Proliferation Reagent (Roche Applied Science, Penzberg, Germany) was employed following the manufacturer’s instructions, and absorbance at 490 nm was monitored in a FLUOStar Optima multi-label reader (BMG Labtech, Offenburg, Germany). As recommended by (Monks et al., 1991) endpoint WST-1 conversion (an indirect indicator of cell number, in most cases) (Galluzzi et al., 2009) was normalized to that recorded when drugs were added (time 0). Clonogenic survival assays HL-60 cells (2 103) were incubated with the indicated concentrations of PZ or OUA for 18 h and then transferred to methylcellulose matrix-coated (1.5% w/v; Sigma) 100 mm dishes and cultured for up to 14 days under normal conditions. Finally, dishes were photographed under a GBox imaging system (Syngene, Cambridge, UK) and colonies made of more than B50 cells were quantified with the GeneTool software (Syngene). Caspase activation assay Caspase-3/7 activity was quantified by means of the Apo-ONE caspase-3/7 kit (Promega, Madison, WI, USA), according to the manufactures’ instructions. Briefly, 2 104 HL-60 cells were treated with 1 mM PZ or OUA for 12 h and then the caspase 3/7-mediated conversion of the non-fluorescent substrate Z-DEVD-R110 to fluorescent rhodamine 110 was assessed with appropriate excitation and emission filters (499 and 520 nm, respectively) on a FLUOStar Optima multi-label reader. Cytofluorometric detection of apoptosis and cell cycle distribution Apoptosis-related parameters and cell cycle distribution were quantified as previously reported (Metivier et al., 1998; Castedo et al., 2002; Galluzzi et al., 2007, 2009). Briefly, for cell cycle determinations, 5 105 cells were collected, washed once with ice-cold phosphate-buffered saline and permeabilized with 100 ml of 0.5% Triton x-100. Thereafter, cells were stained with 50 mg/ml PI in 0.1% D-glucose (w/v in phosphatebuffered saline) in the presence of 20 mg/ml (w/v) RNAse A. Oncogene

To quantify apoptosis-associated parameters, living cells were co-stained with 40 nM 3,30 dihexiloxalocarbocyanine iodide (DiOC6(3), from Molecular Probes-Invitrogen, Eugene, OR, USA), which measures mitochondrial transmembrane potential (Dcm), and 1 mg/ml PI, which identifies cells with ruptured plasma membrane. Cytofluorometric acquisitions were performed on a FACScan cytofluorometer (BD Biosciences, San Jose, CA, USA) equipped with a 70 mm nozzle. First-line statistical analysis was performed by using the CellQuest software (BD Biosciences) upon gating on the events characterized by normal forward scatter and side scatter parameters. Evaluation of differentiation The morphological assessment of differentiation was carried out upon May–Gruenwald–Giemsa staining of cytospins, as previously described (Stegmaier et al., 2005; Chan and Pilichowska, 2007; Boehrer et al., 2008). Briefly, slides were fixed in methanol for 15 min, stained for 5 min in May– Gruenwald solution and for 10 min in Giemsa solution (both from RAL Diagnostics, Paris, France), rinsed with phosphatebuffered saline (pH ¼ 6.8) and air dried. Signs of differentiation (decrease of cytoplasmic basophilia and nuclear/cytoplasmic ratio, and appearance of granulation, lobulation of the nucleus) were assessed in at least 100 cells per condition. To assess differentiation by cytofluorometry, cells were harvested, washed and stained with an allophycocyanin-conjugated antiCD11b antibody (clone D12, Becton Dickinson) and PI, as described previously (Boehrer et al., 2008). To block unspecific binding to human Fc receptor-expressing cells, samples were pre-incubated for 30 min with the FcR blocking reagent (Miltenyi Biotec). Analysis was carried out on living cells, upon gating on forward and side scatter parameters followed by exclusion of PI-positive cells. An isotypic mouse IgG2aallophycocyanin (BD Biosciences) was used to determine threshold levels. Cells incubated with 1 mM all-trans-retinoic acid for the indicated times served as positive controls for differentiation. Immunofluorescence microscopy Immunofluorescence microscopy studies were performed as previously reported (Vitale et al., 2007). Briefly, cells 1 105 were allowed to adhere on poly-L-lysine-coated coverslips (Sigma) and fixed with 4% paraformaldehyde for 20 min at room temperature. Cells were then permeabilized with 0.1% SDS for 10 min, washed in phosphate-buffered saline, and stained with antibodies specific for p65 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or phosphorylated H2AX (Ser 139, Millipore, Billerica, MA, USA). Slides were then incubated with the appropriate Alexa Fluor 488 conjugates (Molecular Probes-Invitrogen) in the presence of 10 mg/ml Hoechst 33342 (Molecular Probes-Invitrogen) for nuclear counterstaining (allowing for the discernment of chromatin condensation). A minimum of 200 cells per condition were examined by means of a Zeiss Observer.Z1 microscope (Carl Zeiss MicroImaging, Goettingen, Germany) equipped with a 40 objective. Cytochrome c release To measure the release of cytochrome c, we employed the InnoCyte cytofluorometric cytochrome c release kit (Merck), following the manufacturer’s instructions with slight modifications. Upon staining (including nuclear counterstaining with 10 mg/ml Hoechst 33342, cells were cytospun for 5 min at 280 g in a Shandon Cytospin 4 centrifuge (Thermo Electron, Pittsburg, PA, USA) onto poly-L-lysine-coated coverslips and


3545 observed with a Zeiss Observer.Z1 microscope. Alternatively, cells were transferred into poly-L-lysine pre-treated Black/ Clear 96-well Imaging Plates (BD Biosciences), centrifuged (5 min, 350 g) and analyzed using a BD pathway 855 automated microscope and the Attovision software v. 6.1 (both from BD Biosciences). Immunoblotting Immunoblotting was performed following standard procedures (Hoffmann et al., 2008; Tufi et al., 2008; Vicencio et al., 2009). Briefly, 40 mg of proteins were separated according to molecular weight on NuPAGE Novex Bis-Tris 4–12% pre-cast gels (Invitrogen) and electrotransferred to Immobilon polyvinylidene difluoride membranes (Millipore). Unspecific binding sites were blocked by incubating the membranes for 1 h in 0.05% Tween 20 (v/v in TBS) supplemented with 5% w/v bovine serum albumin (Euromedex, Souffelweyersheim, France). Thereafter, membranes were probed with antibodies specific for A1, Bcl-XL, Bcl2L10, caspase 3, IkBa, Mcl-1, poly(ADP-ribose) polymerase 1, or XIAP (all from Cell Signaling Technology, Boston, MA, USA). Primary antibodies were revealed with suitable immunoglobulin G conjugated to horseradish peroxidase (Southern Biotech, Birmingham, AL, USA), followed by chemiluminescence detection with the SuperSignal West Pico reagent and CL-XPosure X-ray films (both from Thermo Scientific-Pierce, Rockford, IL, USA). A primary antibody that specifically recognizes b-actin (Millipore) was used to ensure equal lane loading. High-throughput screening and automated image analysis HL-60 cells (2 104) were seeded in V-shaped 96-well plates and treated for 18 h with compounds from the US Drugs collection (Microsource Discovery Systems, Gaylordsville, CT, USA) at a final concentration of 1 mM. Cells were co-stained with 10 mg/ml Hoechst 33342 and the Live/Dead fixable staining solution (Invitrogen), spun into poly-L-lysine pretreated Black/Clear 96-well Imaging Plates for 5 min at 350 g and fixed with 4% paraformaldehyde for 20 min. Four view fields per well were acquired by means of a BD Pathway 855 automated microscope equipped with PhotoFluor II light sources (Burlington, VT, USA) and a robotized Twister II plate handler (Caliper Life Sciences, Hopkinton, MA, USA) using a 20 objective (Olympus, Center Valley, PA, USA) and analyzed with the Attovision software. Images were segmented and analyzed for nuclear area and Live/Dead dye uptake. Animal experiments and tissue processing All animal experiments were approved by the local Ethics Committee (CEEA IRCIV/IGR n126, registered with the French Ministry of Research), and were in compliance with 63/ 2010/EU directive from the European Parliament. Athymic nude mice (Charles River Laboratory, Wilmington, NC, USA) were housed in a temperature-controlled environment with 12 h light/dark cycles and received food and water ad libitum. Mice were injected subcutaneously or intravenously with 2 106 HL-60 cells, resulting in the formation of local tumors

or blood infiltration, respectively. Twenty days after xenografts, mice were injected twice a week intraperitioneal with 5 mg/kg PZ or 2 mg/kg OUA for the following 40 days. In this period, tumor growth and body weight were monitored three times a week. At the end of the experiment (or earlier if tumors exceeded 20% body mass), mice were anesthetized and killed according to the FELASA (Federation of European Laboratory Animal Science Associations) guidelines. At this stage, spleens were collected, photographed and the tissue was homogenized by means of mechanical disruption in a Precellys 24 (Bertin technologies, Montigny-le-Bretonneux, France) followed by Ficoll-Paque PLUS density centrifugation. Peripheral blood mononuclear cells have been collected and stained with fluorescein isothiocyanate-coupled HLA class1 antibody (Sigma Aldrich) for 30 min on ice followed by addition of PI, for dead cell exclusion, before the flow cytometric acquisitions performed on a FACScan cytofluorometer (BD Biosciences). Statistical procedures Unless otherwise indicated, experiments were performed in triplicates and independently repeated at least three times. Low-throughput data were analyzed using Microsoft Excel (Microsoft, Redmond, WA, USA) and statistical significance was assessed by means of two-tailed Student’s t-tests. Results from one representative experiment are reported (means±s.e.m., * Po0.05). High-throughput screening results were mined and statistically evaluated using the GraphPad Prism software (GraphPad Software Inc., La Jolla, CA, USA). To take into account inter-plate variations, data were normalized by Z-scoring.

Conflict of interest The authors declare no conflict of interest.

Acknowledgements This work is supported by grants to GK from the Ligue Nationale contre le Cancer (Equipes labelliseé), Agence Nationale pour la Recherche (ANR), European Commission (Active p53, Apo-Sys, ChemoRes, ApopTrain), Fondation pour la Recherche Me´dicale (FRM), Institut National du Cancer (INCa), Cance´ropoˆle Ile-de-France, Fondation Bettencourt-Schueller and the LabEx Onco-Immunology; to VB from Agence Nationale pour la Recherche, Association pour la Recherche sur le Cancer, Belgian InterUniversity Attraction Pole and Universite´ Paris Descartes. MT is supported by the Ligue Nationale contre le Cancer. LG is funded by Apo-Sys; KB is supported by Agence Nationale pour la Recherche. Author contributions: MT, LS, EL, ST, DM, MS, VB, KB and OK performed the experiments; MT, LS LG and OK analyzed results and made the figures; PF, SB, LG, OK and GK designed the research and wrote the paper.

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

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