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Blockade of BCL-2 Proteins Efficiently Induces Apoptosis in Progenitor

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Blockade of BCL-2 Proteins Efficiently Induces Apoptosis in Progenitor Leukemia (2016) 30, 112–123 © 2016 Macmillan Publishers Limited All rights reserved 0887-6924/16 www.nature.com/leu ORIGINAL ARTICLE Blockade of BCL-2 proteins efficiently induces apoptosis in progenitor cells of high-risk myelodysplastic syndromes patients S Jilg1, V Reidel1, C Müller-Thomas1, J König1, J Schauwecker2, U Höckendorf1, C Huberle1, O Gorka3, B Schmidt4, R Burgkart2, J Ruland3, H-J Kolb1, C Peschel1, RAJ Oostendorp1, KS Götze1 and PJ Jost1 Deregulated apoptosis is an identifying feature of myelodysplastic syndromes (MDS). Whereas apoptosis is increased in the bone marrow (BM) of low-risk MDS patients, progression to high-risk MDS correlates with an acquired resistance to apoptosis and an aberrant expression of BCL-2 proteins. To overcome the acquired apoptotic resistance in high-risk MDS, we investigated the induction of apoptosis by inhibition of pro-survival BCL-2 proteins using the BCL-2/-XL/-W inhibitor ABT-737 or the BCL-2-selective inhibitor ABT-199. We characterized a cohort of 124 primary human BM samples from MDS/secondary acute myeloid leukemia (sAML) patients and 57 healthy, age-matched controls. Inhibition of anti-apoptotic BCL-2 proteins was specifically toxic for BM cells from high-risk MDS and sAML patients, whereas low-risk MDS or healthy controls remained unaffected. Notably, ABT-737 or ABT-199 treatment was capable of targeting the MDS stem/progenitor compartment in high-risk MDS/sAML samples as shown by the reduction in CD34+ cells and the decreased colony-forming capacity. Elevated expression of MCL-1 conveyed resistance against both compounds. Protection by stromal cells only partially inhibited induction of apoptosis. Collectively, our data show that the apoptotic resistance observed in high-risk MDS/sAML cells can be overcome by the ABT-737 or ABT-199 treatment and implies that BH3 mimetics might delay disease progression in higher-risk MDS or sAML patients. Leukemia (2016) 30, 112–123; doi:10.1038/leu.2015.179 INTRODUCTION positive cells when directly compared with smears from low-risk 13 Myelodysplastic syndromes (MDS) are clonal disorders of hema- MDS patients. These findings supported the notion that an topoietic stem cells mostly observed in older patients.1–4 Owing to acquired apoptotic resistance in the malignant MDS clone 3,5–11 the progressive cytopenias associated with MDS, patients often contributes to disease progression. suffer from fatigue, infections and bleeding. In contrast to the Pro- and anti-apoptotic members of the BCL-2 family tightly hypocellularity observed in the peripheral blood, bone marrow control the intrinsic apoptotic signaling pathway. The pro- (BM) cellularity is often normal or even elevated.2 This disparity apoptotic BCL-2 members can be separated into two classes, between BM cellularity and cytopenia has been attributed to an which include the BAX/BAK-like proteins and the BH3-only increased level of apoptosis in the BM compartment.2 Interest- proteins. The BAX/BAK-like proteins form pores in the outer ingly, the level of cell death in BM cells differs substantially mitochondrial membrane to release pro-apoptotic factors such as 14 between different clinical MDS risk categories.2,3 This is exempli- cytochrome c. The BH3-only proteins exhibit their pro-apoptotic fied by the fact that the level of apoptosis in low-risk MDS is function by either sequestering the pro-survival BCL-2 family substantially higher than the levels of apoptosis observed in high- members away from BAX or BAK or, alternatively, by directly risk MDS/secondary acute myeloid leukemia (sAML) or in healthy activating them.15,16 Together, this results in the activation of the control BM cells.5–11 This illustrates that MDS cells acquire an caspases-3 and -7 and subsequently in cell death.17 In contrast, apoptotic resistance upon disease progression, which coincides pro-survival proteins such as the BCL-2 family members BCL-2, with the appearance of an elevated number of myeloid blasts and MCL-1 or BCL-XL or, alternatively, the inhibitor of apoptosis an increased likelihood of disease acceleration into sAML.2,12 proteins, protect cells from apoptosis by blocking the activation of – Previous work has shown that the balance between pro- and caspases.14 16,18 anti-apoptotic BCL-2 family proteins in the BM of MDS patients is The functional role of BCL-2 proteins to apoptotic resistance in deregulated. Using flow cytometry for quantification of protein MDS can be studied using pro-apoptotic BH3-mimetic com- expression, the ratio between the expression levels of BCL-2 pounds. BH3 mimetics bind into the BH3 groove of pro-survival and BCL-XL was compared with the levels of pro-apoptotic BAX BCL-2 proteins thereby displacing them from their inhibitory – and BAD in primary MDS samples. In higher-risk MDS patients, binding to BAX or BAK.19 22 We utilized the BH3 mimetics ABT-737 the balance was shifted toward the anti-apoptotic BCL-2 and the BCL-2-specific compound ABT-199. ABT-737 binds with family members. In addition, elevated BCL-2 expression was high affinity (Ki ⩽ 1nM) to BCL-2, BCL-XL and BCL-W, but not to also observed by immunocytochemistry on BM smears from MCL-1 or A1.19–22 The BCL-2-selective compound termed ABT-199 high-risk MDS patients, which showed significantly more BCL-2- has a sub-nanomolar affinity for BCL-2 (Ki o0.010 nM) and binds 1III. Medizinische Klinik für Hämatologie und Internistische Onkologie, Klinikum rechts der Isar, Technische Universität München, Munich, Germany; 2Klinik für Orthopädie und Sportorthopädie, Klinikum rechts der Isar, Technische Universität München, Munich, Germany; 3Institut für Klinische Chemie und Pathobiochemie, Klinikum rechts der Isar, Technische Universität München, Munich, Germany and 4Gemeinschaftspraxis Hämato-Onkologie Pasing, Munich, Germany. Correspondence: Dr PJ Jost, III. Medical Department for Hematology and Oncology, Klinikum rechts der Isar Technical University of Munich, Ismaninger Strasse 22, Munich 81675, Germany. E-mail: [email protected] Received 15 January 2015; revised 24 June 2015; accepted 25 June 2015; accepted article preview online 8 July 2015; advance online publication, 28 July 2015 Blockade of BCL-2 induces apoptosis in high-risk MDS patients S Jilg et al 113 18 less avidly to BCL-XL (Ki =48nM) or BCL-W (Ki = 245 nM). It has no Numbers of erythroid progenitor colonies (Burst-forming units-erythroid 23 measurable binding to MCL-1 (Ki4444 nM). ABT-199 currently or colony-forming units for the granulocytic-macrophagic lineage, holds great promise for the treatment of B-cell neoplasias24,25 and and multi-potential granulocytic-erythroid-macrophagic-megakaryocytic initial data also suggest efficacy in de novo AML.26,27 lineage) were assessed after 14 days. Transmitted light photographs were Here, we studied the effect of ABT-737 and ABT-199 on BM cells obtained on a Keyence BIOREVO BZ-900 microscope. from a large cohort of primary human MDS patients and compared it with a cohort of healthy age-matched controls. We Flow cytometry found that ABT-737 and ABT-199 efficiently killed primary stem/ BMMNCs were stained with Annexin V-FITC in AnnexinV staining solution progenitor cells as well as more differentiated BM cells from high- (0.1M HEPES/NaOH, pH 7.4, 1.4M NaCl 0.9%, 25 mM CaCl2), followed by risk MDS/sAML patients, whereas low-risk MDS or healthy controls staining with fluorescently labeled antibodies against CD34 (clone 4H11), remained unaffected. CD45 (clone 2D1) or isotype control (clone P3.6.2.8.1). Dead cells were excluded by 7-aminoactinomycin D (7AAD) staining. For intra- cellular staining, cells were stained against CD34, followed by fixation in PATIENTS AND METHODS 2% paraformaldehyde, permeabilization using perm/wash buffer (BD Bioscience, Franklin Lakes, NJ, USA) and subsequent staining with Patient samples and cell lines fluorescently labeled antibodies against BCL-2 (clone Bcl-2/100, BD Human BM samples were collected according to the institutional guide- Bioscience), BCL-XL (clone 54H11, Cell Signalling, Cambridge, UK), MCL-1 lines and in concordance with the Declaration of Helsinki. Written informed (clone 19C4, WEHI, Melbourne, VIC, Australia) or respective isotype controls consent was obtained from each patient. The investigation was approved (Cat.: 556357, BD Bioscience; clone DA1E, Cell Signaling; clone eBRG1). by the Local Ethics Committee of the University Hospital of the Technical Dead cells were excluded by Fixable Viability Dye staining. If not otherwise University in Munich. sAML was defined as ⩾ 20% of blasts in the BM and a stated, reagents and antibodies were purchased from eBioscience. Flow history of MDS. All other MDS samples or sAML were classified according to analysis was performed on a BD FACS Canto II (BD Bioscience) and data the International Prognostic Scoring System (IPSS), the revised WHO were analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA). classification-based Prognostic Scoring System (r-WPSS), the World Health Organization (WHO) 2008 classification or the cytogenetic risk score according to Schanz et al.28 Samples were obtained when clinically Gene expression analysis required from patients either before or during treatment and irrespective Gene expression analysis was performed using the Human Genome U133 of the therapeutic regimen. Control samples were obtained from human Plus 2.0 Array from Affymetrix (Santa Clara, CA, USA). The Affymetrix femoral heads discarded after implantation of total endoprosthesis of the normalization method was used. All expression measurements of each hip joint from 57 hematologically healthy age-matched donors. The array are divided by the median (calculated across all calls (present, mixed embryonic liver-derived stromal cell line EL08-1D2 was used as a stromal and absent)) and plotted on a logarithmic scale to normalize the data and support cell line as described previously.29–31 show a log2 median-centered intensity blot.
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