Blockade of BCL-2 proteins efficiently induces apoptosis in progenitor cells of high-risk myelodysplastic syndromes patients AbstractDeregulated 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. IntroductionMyelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem cells mostly observed in older patients.1, 2, 3, 4 Owing to the progressive cytopenias associated with MDS, patients often suffer from fatigue, infections and bleeding. In contrast to the hypocellularity observed in the peripheral blood, bone marrow (BM) cellularity is often normal or even elevated.2 This disparity between BM cellularity and cytopenia has been attributed to an increased level of apoptosis in the BM compartment.2 Interestingly, the level of cell death in BM cells differs substantially between different clinical MDS risk categories.2, 3 This is exemplified by the fact that the level of apoptosis in low-risk MDS is substantially higher than the levels of apoptosis observed in high-risk MDS/secondary acute myeloid leukemia (sAML) or in healthy control BM cells.5, 6, 7, 8, 9, 10, 11 This illustrates that MDS cells acquire an apoptotic resistance upon disease progression, which coincides with the appearance of an elevated number of myeloid blasts and an increased likelihood of disease acceleration into sAML.2, 12Previous work has shown that the balance between pro- and anti-apoptotic BCL-2 family proteins in the BM of MDS patients is deregulated. Using flow cytometry for quantification of protein expression, the ratio between the expression levels of BCL-2 and BCL-XL was compared with the levels of pro-apoptotic BAX and BAD in primary MDS samples. In higher-risk MDS patients, the balance was shifted toward the anti-apoptotic BCL-2 family members. In addition, elevated BCL-2 expression was also observed by immunocytochemistry on BM smears from high-risk MDS patients, which showed significantly more BCL-2-positive cells when directly compared with smears from low-risk MDS patients.13 These findings supported the notion that an acquired apoptotic resistance in the malignant MDS clone contributes to disease progression.3, 5, 6, 7, 8, 9, 10, 11Pro- and anti-apoptotic members of the BCL-2 family tightly control the intrinsic apoptotic signaling pathway. The pro-apoptotic BCL-2 members can be separated into two classes, which include the BAX/BAK-like proteins and the BH3-only proteins. The BAX/BAK-like proteins form pores in the outer mitochondrial membrane to release pro-apoptotic factors such as cytochrome c.14 The BH3-only proteins exhibit their pro-apoptotic function by either sequestering the pro-survival BCL-2 family members away from BAX or BAK or, alternatively, by directly activating them.15, 16 Together, this results in the activation of the caspases-3 and -7 and subsequently in cell death.17 In contrast, pro-survival proteins such as the BCL-2 family members BCL-2, MCL-1 or BCL-XL or, alternatively, the inhibitor of apoptosis proteins, protect cells from apoptosis by blocking the activation of caspases.14, 15, 16, 18The functional role of BCL-2 proteins to apoptotic resistance in MDS can be studied using pro-apoptotic BH3-mimetic compounds. BH3 mimetics bind into the BH3 groove of pro-survival BCL-2 proteins thereby displacing them from their inhibitory binding to BAX or BAK.19, 20, 21, 22 We utilized the BH3 mimetics ABT-737 and the BCL-2-specific compound ABT-199. ABT-737 binds with high affinity (Ki猢?/span>1鈥塶M) to BCL-2, BCL-XL and BCL-W, but not to MCL-1 or A1.19, 20, 21, 22 The BCL-2-selective compound termed ABT-199 has a sub-nanomolar affinity for BCL-2 (Ki 0.010鈥塶M) and binds less avidly to BCL-XL (Ki=48鈥塶M) or BCL-W (Ki=245鈥塶M).18 It has no measurable binding to MCL-1 (Ki 444鈥塶M).23 ABT-199 currently holds great promise for the treatment of B-cell neoplasias24, 25 and initial data also suggest efficacy in de novo AML.26, 27Here, we studied the effect of ABT-737 and ABT-199 on BM cells from a large cohort of primary human MDS patients and compared it with a cohort of healthy age-matched controls. We found that ABT-737 and ABT-199 efficiently killed primary stem/progenitor cells as well as more differentiated BM cells from high-risk MDS/sAML patients, whereas low-risk MDS or healthy controls remained unaffected.Patients and methodsPatient samples and cell linesHuman BM samples were collected according to the institutional guidelines and in concordance with the Declaration of Helsinki. Written informed consent was obtained from each patient. The investigation was approved by the Local Ethics Committee of the University Hospital of the Technical University in Munich. sAML was defined as 猢?/span>20% of blasts in the BM and a history of MDS. All other MDS samples or sAML were classified according to the International Prognostic Scoring System (IPSS), the revised WHO 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 required from patients either before or during treatment and irrespective of the therapeutic regimen. Control samples were obtained from human femoral heads discarded after implantation of total endoprosthesis of the hip joint from 57 hematologically healthy age-matched donors. The embryonic liver-derived stromal cell line EL08-1D2 was used as a stromal support cell line as described previously.29, 30, 31Cell isolation and cultureMononuclear cells from primary human BM samples were isolated via density-gradient centrifugation using the Biocoll Separation Solution (Biochrom AG, Berlin, Germany) following the manufacturer鈥檚 instructions. CD34+ cells were purified via positive selection using the CD34+ MicroBeads kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and purity was confirmed to be at least 95%. BM mononuclear cells (BMMNCs) were cultured at a density of 5 脳 105 cells/ml in serum-free media consisting of Iscove\'s Modified Dulbecco\'s Medium (IMDM) with l-alanyl-l-glutamine (IMDM GlutaMAX) with 20% BIT 9500 serum substitute (1% (w/v) bovine serum albumin, 10鈥壩糶/ml insulin, 200鈥壩糶/ml iron-saturated transferrin; StemCell Technologies, Vancouver, BC, Canada) and enriched with recombinant human stem cell factor (100鈥塶g/ml), FMS-related tyrosine kinase-3 ligand (100鈥塶g/ml), thrombopoetin (10鈥塶g/ml), interleukin-6 (5鈥塶g/ml), interleukin-3 (10鈥塶g/ml; all from R D Systems, Minneapolis, MN, USA), 尾-mercaptoethanol (10鈥壩?span >M; Gibco, Carlsbad, CA, USA) and low-density lipoproteins (4鈥壩糶/ml; Sigma-Aldrich, St Louis, MO, USA). To assess the impact of stromal cells, samples were seeded on the murine embryonic liver-derived cell line EL08-1D2 at a density of 5 脳 105 cells/ml. EL08-1D2 were cultured on 0.1% gelatin-coated 24-well plates in long-term culture medium (StemCell Technologies) with 35% essential medium with l-alanyl-l-glutamine (alpha-MEM GlutaMAX, Gibco), 15% FCS (StemCell Technologies), horse serum (5%; StemCell Technologies), penicillin and streptomycin (1%; Gibco), 尾-mercaptoethanol (10鈥塵M; Merck, Darmstadt, Germany).InhibitorABT-737 (Active Biochem, Maplewood, NJ, USA) and ABT-199 (AbbVie, North Chicago, IL, USA) were dissolved in dimethyl sulfoxide (DMSO) and used in a final concentration of 1鈥壩?span >M or as stated otherwise. DMSO was used at 0.001% as vehicle control.Colony formation assayHematopoietic progenitors were assessed after treatment with ABT-737 (1鈥壩?span >M), ABT-199 (1鈥壩?span >M) or DMSO (0.001%) for 72鈥塰 in cytokine-supplemented, serum-free culture with or without stromal support. 1 脳 104 BMMNCs were plated in duplicates in methylcellulose medium supplemented with an optimal cytokine mix according to the manufacturer\'s protocols (MethoCult H4435 enriched; StemCell Technologies). Numbers of erythroid progenitor colonies (Burst-forming units-erythroid or colony-forming units for the granulocytic-macrophagic lineage, and multi-potential granulocytic-erythroid-macrophagic-megakaryocytic lineage) were assessed after 14 days. Transmitted light photographs were obtained on a Keyence BIOREVO BZ-900 microscope.Flow cytometryBMMNCs were stained with Annexin V-FITC in AnnexinV staining solution (0.1M HEPES/NaOH, pH 7.4, 1.4M NaCl 0.9%, 25鈥塵M CaCl2), followed by staining with fluorescently labeled antibodies against CD34 (clone 4H11), CD45 (clone 2D1) or isotype control (clone P3.6.2.8.1). Dead cells were excluded by 7-aminoactinomycin D (7AAD) staining. For intracellular staining, cells were stained against CD34, followed by fixation in 2% paraformaldehyde, permeabilization using perm/wash buffer (BD Bioscience, Franklin Lakes, NJ, USA) and subsequent staining with fluorescently labeled antibodies against BCL-2 (clone Bcl-2/100, BD Bioscience), BCL-XL (clone 54H11, Cell Signalling, Cambridge, UK), MCL-1 (clone 19C4, WEHI, Melbourne, VIC, Australia) or respective isotype controls (Cat.: 556357, BD Bioscience; clone DA1E, Cell Signaling; clone eBRG1). Dead cells were excluded by Fixable Viability Dye staining. If not otherwise stated, reagents and antibodies were purchased from eBioscience. Flow analysis was performed on a BD FACS Canto II (BD Bioscience) and data were analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA).Gene expression analysisGene expression analysis was performed using the Human Genome U133 Plus 2.0 Array from Affymetrix (Santa Clara, CA, USA). The Affymetrix normalization method was used. All expression measurements of each array are divided by the median (calculated across all calls (present, mixed and absent)) and plotted on a logarithmic scale to normalize the data and show a log2 median-centered intensity blot. BMMNCs of 66 MDS patients (refractory anemia: n=3, refractory anemia with ringed sideroblasts (RARS): n=6, refractory cytopenia with multilinear dysplasia (RCMD): n=7, refractory cytopenia with multilinear dysplasia and ringed sideroblasts (RCMD-RS) n=6, RAEB-1 (refractory anemia with blast excess 5-9%): n=23, RAEB-2 (refractory anemia with blast excess 猢?/span>10%): n=21) and peripheral blood mononuclear cells from 110 healthy controls were analyzed. To test for any significant differences of gene expression between the MDS groups, 鈥榚arly鈥? 鈥榠ntermediate鈥? 鈥榣ate鈥?and healthy control one-way analysis of variances (ANOVA) were used with post-hoc pairwise comparisons in case of significance. All reported P-values are two-sided, with a significance level of 0.05 and have not been adjusted for multiple testing (unpaired Student\'s t-test). Pairwise differences are presented with 95% confidence intervals (CIs). Statistical analyses were performed using SPSS version 19.0.0 (IBM Corporation, Armonk, NY, USA).Statistical analysisTo test for any significant differences in apoptosis induction after inhibitor treatment between the different MDS/sAML risk categories one-way ANOVA was used with post-hoc pairwise comparisons in case of significance. Pairwise differences are presented with 95% CIs. Comparing two samples, the unpaired Student\'s t-test was used to test for any significant differences between treated samples and control. All reported P-values are two-sided, with a significance level of 0.05 and have not been adjusted for multiple testing. For the correlation of protein expression by mean fluorescence intensity and MDS cell survival, the strength of the association between mean fluorescence intensity ratio and viability under inhibitor treatment was calculated by the Spearman rank correlation and the functional relationship was described by linear regression analysis. Statistical analyses were performed using GraphPadPrism version 5.01 (Graphpad Software, Inc., San Diego, CA, USA).ResultsInduction of apoptosis by ABT-737 or ABT-199 efficiently kills high-risk MDS/sAML cells but not low-risk/intermediate-risk MDS cellsDifferential expression of pro-survival BCL-2 family members at distinct clinical stages of MDS have prompted the idea that the clinical progression to higher-risk disease is accompanied by an acquired resistance to apoptosis.2, 9, 13 To test whether differences in expression of critical pro- or anti-apoptotic BCL-2 proteins might explain the differences in apoptotic susceptibility, we analyzed the gene expression data from a large cohort of MDS patient samples and respective controls.32 The gene expression analysis showed a significantly decreased expression of the BCL-2 inhibiting BH3-only protein BIM in all seven BIM probe sets in late stages of MDS (RAEB-2) when compared with early stages of MDS (refractory anemia, RARS, RCMD, RCMD-RS; Supplementary Figure 1). BIM is a pro-apoptotic member of the BCL-2 family that binds and inhibits all pro-survival BCL-2 family members and potently induces apoptosis.15 The reduced expression of pro-apoptotic BIM in higher-risk MDS supports the hypothesis that an acquired resistance to apoptosis develops upon disease progression. Of note, we did not observe clear differences in the expression levels of any of the pro-survival BCL-2 family members, which might be explained by the regulation of these proteins on the transcriptional or post-translational level.33, 34, 35The reduced BIM expression in higher-risk MDS samples suggested that re-activation of apoptosis by BH3 mimetics such as ABT-737 or ABT-199 might be specifically effective in higher-risk MDS samples. To functionally test the apoptotic susceptibility of MDS cells, we obtained BMMNCs from MDS patients, who underwent BM aspiration for diagnosis, disease monitoring or evaluation of treatment response. BM aspirates of 104 patients with MDS and 20 patients with sAML were analyzed. MDS patients were classified according to the WHO classification,36 the IPSS12 or r-WPSS37 scores and the cytogenetic risk score according to Schanz et al.28 (Supplementary Table 1).The clonal hierarchy within cancers often defines a stem cell-like population and a more differentiated cellular population, which has recently also been detected in MDS.38 To differentiate between MDS-propagating cells and a more differentiated cellular subpopulation, we gated on CD34+ cells by flow cytometry, the cellular subpopulation harboring, at least in part, the MDS-propagating cells.9, 10, 38, 39 Of note, all samples were used directly after extraction from the BM to retain optimal survival properties and to prevent apoptosis induction by prior deep-freezing.Consistent with our hypothesis, we observed a dose-dependent induction of cell death in three representative high-risk/sAML samples (Supplementary Figure 2A). Owing to the high level of heterogeneity of human MDS and in accordance with previous publications on myeloid malignancies,40 we found inter-individual differences in drug sensitivity.27 Sample two and three proved to be highly sensitive to ABT-737, with ~50% of cells undergoing cell death at doses as low as 10鈥塶M. Sample one was more resistant showing a drop in viability at higher doses (Supplementary Figure 2A).ABT-737 also showed a time-dependent toxicity mostly inducing cell death within the first 24鈥塰 of treatment but effectively killing all cells after 72鈥塰 in a representative high-risk/sAML sample (Figures 1a and b, high-risk/sAML). Interestingly, stem/progenitor cells from representative low-risk or intermediate-risk MDS patients remained largely unaffected by treatment with ABT-737 (Figures 1a and b, low-risk and intermediate-risk). This supported the notion that ABT-737 was specifically toxic for cells from patients that had experienced disease acceleration, a clinical situation that has previously been associated with an acquired resistance to apoptosis.2, 3Figure 1ABT-737 or ABT-199 kill stem/progenitor cells from high-risk MDS/sAML patients in a time-dependent manner. (a) Viability of BMMNC measured by flow cytometry using Annexin V and 7AAD and gating on CD34+ cells from individual representative MDS patients diagnosed with low-risk, intermediate-risk (Int.-risk) MDS or high-risk MDS/sAML after treatment with ABT-199 (1鈥壩?span >M), ABT-737 (1鈥壩?span >M) or DMSO for the indicated time points. (b) Viability of cells treated with ABT-199 or ABT-737 as in a represented as percentage of viable cells after inhibitor treatment and viable cells after vehicle treatment for the indicated time points. Samples are classified using r-WPSS.Full size imageA similar pattern of cell death induction was observed in samples treated with the BCL-2-selective inhibitor ABT-199 when directly compared with ABT-737. A representative low-risk and intermediate-risk samples remained unaffected by treatment with ABT-199 for 72鈥塰 (Figures 1a and b, low-risk and intermediate-risk), whereas progenitor cells from a high-risk/sAML sample rapidly underwent apoptosis (Figures 1a and b, high-risk/sAML).As previously described, we identified an elevated number of apoptotic cells (7AAD+) in low-risk samples when compared with intermediate-risk samples at baseline (Figure 1a).2, 8, 9, 13 However, because of the variable sample quality, the differences between intermediate-risk and high-risk samples were less prominent (Figure 1a, 0鈥塰). Irrespective of the level of apoptosis at baseline, the response to ABT-199 or ABT-737 treatment was strikingly different between the clinical MDS risk groups.Healthy age-matched hematopoietic stem/progenitor cells remain unaffected by BCL-2 inhibitionIt is important to preserve the healthy remaining hematopoiesis in MDS patients to avoid additional cytopenia. To exclude toxic effects of ABT-737 or ABT-199 on the healthy hematopoiesis, we monitored apoptosis induction in BM hematopoietic cells of healthy, age-matched controls (Supplementary Table 1B). We utilized age-matched controls to mimic the patient population accurately, as the hematopoietic capacity of the BM changes substantially in elderly patients. To obtain control samples, we utilized femoral heads discarded after hip replacement surgery from 57 age-matched donors and purified CD34+ progenitor cells when required (Supplementary Table 1B).In contrast to the toxic effect observed in high-risk MDS cells, ABT-737 (Figures 2a and b) or ABT-199 (Figures 2c and d) did not induce apoptosis in healthy CD34+-purified stem/progenitor cells. Moreover, the bulk of more differentiated BM cells extracted from age-matched controls also remained unaffected by treatment with ABT-737 (Supplementary Figure 2B,C) or treatment with ABT-199 (Supplementary Figure 2D and E). This showed that the healthy hematopoietic compartment of elderly patients remained unaffected by BH3-mimetic compounds and suggested that BH3 mimetics might be safely employed in MDS patients. The data also support the notion that pro-apoptotic cues present specifically in MDS cells from higher-risk MDS patients predispose to apoptosis induction by BH3-mimetic compounds.Figure 2Stem/progenitors from healthy age-matched control bone marrow remain unaffected by BCL-2 family inhibition. (a) BMMNCs of an individual healthy age-matched donor after enrichment for CD34+ cells to a purity of 95% were subjected to ABT-737 (1鈥壩?span >M). Apoptosis was measured after 72鈥塰. (b) Shown is the viability over time of BMMNC from an individual healthy donor after treatment with ABT-737 (1鈥壩?span >M) as in a. Viable cells are represented as percentage of viable cells at the start of the experiment and compared with vehicle control. (c) BMMNCs of an individual healthy age-matched donor treated with ABT-199 (1鈥壩?span >M) as in a. (d) Shown is the viability over time of BMMNC after treatment with ABT-199 (1鈥壩?span >M) as in c. Viable cells are represented as percentage of viable cells at the start of the experiment and compared with vehicle control.Full size imageReactivation of apoptosis overcomes the acquired apoptotic resistance in high-risk MDS/sAMLHigh-risk MDS/sAML cells largely resist spontaneous cell death.2, 5, 6, 7, 8, 9, 10, 11As BH3 mimetics are capable of reactivating mitochondrial apoptosis, we tested whether such compounds override the apoptotic resistance observed in higher-risk MDS using a large cohort of primary patient samples (Supplementary Table 1).22, 41, 42The molecular, cytogenetic and clinical heterogeneity of MDS is exceptionally high, making it difficult to conclude cellular outcomes from only limited patient samples or even cell line models. We therefore tested the induction of apoptosis in primary MDS cells from patients diagnosed with different clinical MDS risk categories after treatment with ABT-737 or ABT-199. We subjected 39 primary BMMNC samples from MDS/sAML patients to ABT-737 treatment and directly compared this cohort with 15 healthy control subjects.When compared with healthy controls, we found that induction of apoptosis was significantly increased in the stem/progenitor population from r-WPSS-classified patients with intermediate-risk MDS, (very) high-risk MDS or sAML (Figure 3a). Of note, the level of apoptosis induction afforded by ABT-737 was similar in all samples within an individual risk category, which pointed toward a risk group-specific effect. This is exemplified by the fact that significant differences in cell death induction were observed even between closely related risk groups such as (very) low-risk and intermediate-risk patients or intermediate-risk and high-risk patients (Figure 3a).Figure 3Toxicity of ABT-737 or ABT-199 treatment correlates with MDS disease progression to elevated clinical risk groups. (a) Viable BMMNCs of 15 healthy donors and 8 patients with r-WPSS-based classification as (very) low-risk MDS, 9 intermediate-risk MDS, 12 (very) high-risk MDS and 10 patients with sAML were treated for 72鈥塰 with ABT-737 (1鈥壩?span >M) or vehicle control (DMSO). One-way ANOVA (P 0.001) and post-hoc pairwise t-tests as shown *P 0.05, **P 0.005 and ***P 0.0005), mean differences and 95% CI are listed in Supplementary Figure 7. (b) Viable BMMNC of 7 healthy donors and 8 patients with r-WPSS-based classification as (very) low-risk MDS, 7 intermediate-risk MDS, 10 (very) high-risk MDS and 5 patients with sAML were treated for 72鈥塰 with ABT-199 (1鈥壩?span >M) or vehicle control (DMSO). One-way ANOVA (P 0.0001) and post-hoc pairwise t-tests as shown (*P 0.05, **P 0.005 and ***P 0.0005). Mean differences and 95% CI are listed in Supplementary Figure 7. (c) BMMNCs from 15 healthy donors, 7 patient with IPSS-based classification as low-risk, 12 patient with intermediate-1 risk, 4 patients with intermediate-2 risk and 1 patient with high-risk according to IPSS were treated with ABT-737 (1鈥壩?span >M) or vehicle control (DMSO). One-way ANOVA (P 0.0001) and post-hoc pairwise t-tests as shown (*P 0.05, **P 0.005 and ***P 0.0005). Mean differences and 95% CI are listed in Supplementary Figure 7. (d) BMMNCs from 7 healthy donors, 5 patient with IPSS-based classification as low-risk, 11 patient with intermediate-1 risk, 4 patients with intermediate-2 risk and 4 patients with high-risk were treated with ABT-199 (1鈥壩?span >M) or vehicle control (DMSO). One-way ANOVA (P 0.0036) and post-hoc pairwise t-tests as shown (*P 0.05, **P 0.005 and ***P 0.0005), mean differences and 95% CI are listed in Supplementary Figure 7. (e) Viable BMMNCs from 15 healthy donors and samples from 11 patients with WHO classification-based RCMD (refractory cytopenia with multilinear dysplasia), 11 patients RAEB-1 (refractory anemia with blast excess 5-9%), 7 patients RAEB-2 (refractory anemia with blast excess 猢?/span>10%) and 10 patients with sAML were treated with ABT-737 (1鈥壩?span >M) or vehicle control (DMSO). One-way ANOVA (P 0.0001) and post-hoc pairwise t-tests as shown (*P 0.05, **P 0.005 and ***P 0.0005), mean differences and 95% CI are listed in Supplementary Figure 7. (f) Viable BMMNCs from 7 healthy donors and samples from 4 patient with WHO classification-based RARS, 8 patient with RCMD, 10 patients RAEB-1, 3 patients RAEB-2 and 5 patients with sAML were treated with ABT-199 (1鈥壩?span >M) or vehicle control (DMSO). One-way ANOVA (P 0.0018) and post-hoc pairwise t-tests as shown (*P 0.05, **P 0.005 and ***P 0.0005), mean differences and 95% CI of differences are listed in Supplementary Figure 7. (g) BMMNCs from 15 healthy donors, 27 patients with good prognosis, 7 patients with intermediate prognosis and 5 patients with very poor prognosis according to the cytogenetic risk score by Schanz et al.28 were treated with ABT-737 (1鈥壩?span >M) or vehicle control (DMSO). One-way ANOVA (P 0.0001) and post-hoc pairwise t-tests as shown (*P 0.05, **P 0.005 and ***P 0.0005), mean differences and 95% CI are listed in Supplementary Figure 7. (h) BMMNCs from 7 healthy donors, 23 patients with good prognosis, 4 patients with intermediate prognosis and 3 patients with (very) poor prognosis according to the cytogenetic risk score by Schanz et al.28 were treated with ABT-199 (1鈥壩?span >M) or vehicle control (DMSO). One-way ANOVA (P 0.0541). Mean differences and 95% CI are listed in Supplementary Figure 7. All panels: each circle represents the ratio between viable cells after inhibitor treatment and viable cells after vehicle treatment for 72鈥塰. Cells were gated for CD34 expression and stained for viability as in Figure 1. Shown is the mean and error bars denote standard deviation.Full size imageIn the 24 primary MDS patient samples that were sufficiently annotated to calculate the IPSS, we found significant differences in cell death induction when comparing healthy control samples to intermediate-risk 1 or intermediate-risk 2 samples. We also observed significant differences in cell death induction when comparing apoptosis in low-risk samples to intermediate-risk 1 samples or intermediate-risk 2 samples (Figure 3c).The same pattern was observed when patients were classified according to the WHO 2008 classification.36 This classification is based primarily on histomorphological parameters and does not include clinical parameters. Significant differences for induction of apoptosis in CD34+ cells were detected between all MDS categories except RAEB-1 vs RAEB-2 (Figure 3e).The substantial heterogeneity of human MDS is partly explained by cytogenetic aberrations that are increasingly used for prognostication. We therefore subjected our cohort to the cytogenetic risk score published by Schanz et al.28 Using this risk score, we tested whether cytogenetic aberrations preclude the treatment effect of ABT-737 or ABT-199. We observed significant differences comparing apoptosis in samples from healthy controls to the level of apoptosis observed in good prognosis samples, intermediate prognosis samples or very poor prognosis samples when treated with ABT-737 (Figure 3g) and a trend toward significant differences in ABT-199-treated samples (one-way ANOVA P=0.0541, Figure 3h).Clinically used scoring systems use composite clinical variables to define different risk categories. To better understand which individual parameter might differentiate between resistant and sensitive MDS samples, we also grouped our cohort by the presence of cytopenia as the sole parameter. In line with our data from IPSS or r-WPSS-grouped samples and in line with the notion that higher-risk samples are more sensitive, we found that samples from patients with two or three cytopenias underwent apoptosis significantly better than healthy controls or patients with only one cytopenia (Supplementary Figure 6). The blast count as single parameter is best represented by the WHO category (Figure 3e).The significant differences observed between lower-risk samples compared with higher-risk samples in all classification systems available emphasized the uniformity of the apoptotic response for each individual risk group. This argues that the level of pro-apoptotic cues (also referred to as 鈥榩riming for cell death鈥? increases as the disease progresses over time, rendering BH3 mimetics effective only in higher-risk samples.To understand whether ABT-737 also influences the cellular survival of more differentiated cellular subpopulations, we measured the induction of apoptosis in the bulk of BMMNC. We found a slightly reduced propensity of ABT-737 to kill mature BMMNC compared with the CD34+ subpopulation. When patients were grouped according to the r-WPSS, significant differences were detected between (very) low/intermediate-risk samples compared with (very) high-risk MDS samples or compared with sAML samples (Supplementary Figure 3A). Similar results were obtained when patients were grouped by the IPSS or the WHO category (Supplementary Figures 3B and 4A). For the cytogenetic risk score of Schanz et al.,28 statistical significant differences were only detected comparing healthy controls to MDS samples (Supplementary Figure 4B).We also measured apoptosis following treatment with the BCL-2 selective compound ABT-199, which is currently in clinical development and holds promise for treatment of various B-cell neoplasias and de novo AML. Elevated apoptosis induction by ABT-199 was observed in stem/progenitor cells from higher-risk samples but not in lower-risk samples and exhibited a similar pattern to that observed after treatment with ABT-737. However, the overall capacity of the BCL-2-selective compound ABT-199 to induce apoptosis was lower compared with ABT-737 in all risk categories (Figures 3b, d, f and h), suggesting some protective effect by pro-survival BCL-xL. Nevertheless, ABT-199 killed r-WPSS-categorized high-risk/sAML samples significantly better than healthy or low-risk samples (Figure 3b).A similar elevation in cell death was observed in high-risk samples compared with healthy controls after ABT-199 treatment when using the IPSS (Figure 3d). High-risk samples (RAEB-1 and sAML) also underwent apoptosis more readily than healthy controls/RARS samples when the WHO classification was utilized (Figure 3f).In summary, the level of apoptosis observed in CD34+ stem/progenitor cells from MDS patients after ABT-737 and to a slightly lower degree also after ABT-199 treatment correlated with an elevated clinical risk category. These data suggested that pro-apoptotic intervention is a powerful tool to overcome apoptotic resistance in high-risk/sAML cells, whereas healthy or low-risk cells remained largely unaffected.ABT-737 or ABT-199 specifically target colony-forming stem/progenitor cells from high-risk MDS/sAML patientsMDS is a clonal disorder driven by the transformation of a hematopoietic stem cell.38, 43 It is therefore critical to target the stem/progenitor compartment to combat the disease. Hence, we interrogated whether ABT-737- or ABT-199-induced killing of CD34+ cells had any impact on the colony-forming capacity. We subjected primary MDS samples to ABT-737 and/or ABT-199 treatment for 72鈥塰 before cultivation in growth factor-supplemented methylcellulose to monitor colony formation. Of note, the cells were treated with the compounds for 72鈥塰 in liquid culture, whereas the cultivation on methylcellulose was carried on for 14 days without the drugs.In line with our fluorescence-activated cell sorting-based data, the number of colonies in healthy controls was not affected by ABT-737 treatment as exemplified by the numbers of individual colony types (Figure 4a). ABT-199 also proved non-toxic to any colony type in healthy age-matched control samples (Figure 4b). Similarly, colony numbers in samples from (very) low-risk (Figures 4a and b, low-risk) or intermediate-risk MDS patients (Figures 4a and b, intermediate-risk) remained largely unaffected by treatment with either compound when compared with vehicle control. In contrast, ABT-737 and ABT-199 treatment substantially decreased colony formation in high-risk MDS/sAML samples (Figures 4a and b). This further supported our previous finding that BH3-mimetic compounds are selectively toxic for progenitor cells from higher-risk MDS/sAML patients.Figure 4BH3 mimetics ABT-737 or ABT-199 kill colony-forming stem/progenitor cells from high-risk MDS/sAML patients but not from healthy or low/intermediate (Int.)-risk patients. (a) BMMNCs (1 脳 104) were plated in growth factors-enriched methylcellulose after 72鈥塰 of treatment with ABT-737 (1鈥壩?span >M). Numbers of colony-forming units (CFU) of multi-potential granulocytic-erythroid-macrophagic-megakaryocytic lineage (CFU-GEMM), CFU-granulocytic-macrophagic lineage (CFU-GM), burst-forming units-erythroid (BFU-E) and total number of colonies were scored after 14 days for the indicated patient samples. Shown is the mean of the total colony numbers as stacked bar chart of the single colony types for the indicated risk groups/sAML. Replicates as shown in the figure. Error bars denote standard deviation. Samples are classified using r-WPSS. P-values as shown in the figure. (b) BMMNCs (1 脳 104) were plated in growth factors-enriched methylcellulose after 72鈥塰 of treatment with ABT-199 (1鈥壩?span >M). Numbers of CFU-GEMM, CFU-GM, BFU-E and total number of colonies were scored after 14 days for the indicated patient samples. Shown is the mean of the total colony numbers as stacked bar chart of the single colony types for the indicated risk groups/sAML. Replicates as indicated in the figure. Error bars denote standard deviation of total colony number. Samples are classified using r-WPSS. P-values as shown in the figure. (c) Shown in the direct comparison in the total number of colonies from two healthy age-matched control samples, three low-risk MDS, three int.-risk MDS, two high-risk MDS and three sAML samples treated in parallel with ABT-737 or ABT-199 as described in a or b, respectively. Experiments were performed in duplicates and error bars denote standard deviation. Samples are classified using r-WPSS. P-values: *P 0.05 and **P 0.005.Full size imageIn addition, we treated a small cohort with both compounds in parallel. We found a striking similarity in the numbers of overall colonies especially in high-risk and sAML samples (Figure 4c). A significant and very similar reduction in the progenitor potential was observed after treatment with either compound.Collectively, these data showed that ABT-737 or ABT-199 targeted the stem/progenitor cell-containing subpopulation in high-risk MDS/sAML but not in lower-risk MDS or healthy controls. Both compounds exhibited a very similar activity in blocking MDS progenitor potential. The lack of toxicity in healthy progenitors supports the notion that this class of compounds might be safely used in MDS patients as it avoids toxic effects on the healthy hematopoiesis.MCL-1 expression conveys resistance against ABT-737 or ABT-199 in MDS cellsWe next sought to define the molecular mechanisms of resistance to ABT-737 or ABT-199. We therefore subjected a cohort of MDS samples to flow cytometry-based intracellular staining for protein expression of BCL-2, BCL-XL and MCL-1. In addition, we treated the same samples with either compound for 72鈥塰 to directly compare cell death induction of each individual sample to the respective protein expression. In line with our data from the liquid culture and the colony formation assays, we found that most low-risk MDS samples remained unaffected by treatment with ABT-737 or ABT-199, whereas most high-risk MDS samples induced apoptosis substantially better (Figure 5). Interestingly, we found that the resistance to cell death induction by ABT-737 or ABT-199 was largely defined by the expression of the pro-survival BCL-2 family member MCL-1 (Figure 5, compared #1 with #9). This is not unexpected since both compounds are unable to inhibit MCL-1. MCL-1 expression was found to be substantially higher in lower-risk MDS samples compared with high-risk samples, indicating that differential expression of this protein likely explains the discrepancy in treatment response to BH3 mimetics in diverse MDS risk groups (Figure 5, compared #1 to #3 with #6 to #8). The critical role of MCL-1 in treatment resistance can also be appreciated in an individual high-risk MDS samples with elevated MCL-1 expression, which proved insensitive to the treatment with either BH3-mimetic despite being collected from a high-risk patient (Figure 5, #9). In addition, we observed that the expression of BCL-XL, albeit lower than MCL-1 expression, was capable of largely blocking cell death induction by ABT-199 but not by ABT-737, which is best exemplified in samples #2 or #5 and corresponds to the previously reported binding affinities of this compound (Figure 5). Of note, expression of BCL-2 was lower compared with the expression of BCL-XL or MCL-1, suggesting that even low levels of this protein might be able to protect MDS cells (Figure 5).Figure 5Elevated protein levels of MCL-1 convey resistance to ABT-737 or ABT-199 in primary MDS samples. Protein expression levels of BCL-2, BCL-xL and MCL-1 were measured by intracellular flow cytometry before treating the primary human MDS cells with ABT-737, ABT-199 or DMSO for 72鈥塰. Shown are isotype control (gray) and expression level of the indicated proteins (red) for each patient sample. The samples are listed from sensitive to resistant according to the viability of MDS BMMNC after 72鈥塰 of ABT-199 treatment. Viability is shown as percentage of viable cells after inhibitor treatment and viable cells after vehicle treatment for 72鈥塰. The risk category was defined using the r-WPSS. Cells were gated for CD34 expression and stained for viability as in Figure 1.Full size imageTo check whether protein expression levels measured by intracellular flow cytometry might serve as biomarkers for the response of MDS samples to BH3 mimetics before treatment, we correlated protein abundance with cell survival. The strength of the association between protein abundance measured by the mean fluorescence intensity and the viability under inhibitor treatment was calculated by the Spearman rank correlation. Subsequently, the functional relationship was described by linear regression analysis (Supplementary Figure 5). This analysis showed that the abundance of BCL-XL and MCL-1 clearly predicted resistance to ABT-199, whereas elevated levels of BCL-2 predicted good responses of ABT-199 (Supplementary Figures 5A and B). For ABT-737, the level of MCL-1 predicted resistance when directly compared with the levels of BCL-2 plus BCL-XL in the same sample (Supplementary Figure 5C). This linear regression analysis supported the notion that the protein levels measured by fluorescence-activated cell sorting are functionally relevant and can therefore be used as biomarkers to predict response patterns against BH3-mimetic compounds.Together, our intracellular flow cytometry data showed that the protein expression level of individual BCL-2 family members define the differential sensitivity of MDS samples to the treatment with ABT-737 or ABT-199.Stromal cells only partially protect high-risk MDS/sAML cells from cell deathMesenchymal stromal cells closely interact with hematopoietic stem/progenitor cells within the BM compartment.44, 45, 46 The interaction is, at least in part, referred to as the hematopoietic niche and supports stem/progenitor cell survival.47, 48 Overcoming the stromal cell protection of MDS cells is therefore considered an important challenge for MDS therapy.49, 50 To understand whether stromal cells protect high-risk MDS/sAML progenitor cells from ABT-737-induced apoptosis, we co-cultured CD34+ BMMNC from MDS patients in the presence of the stromal cell line EL08-1D2. Despite the limitation of using a murine stromal cell line that might protect human MDS cells only partially, EL08-1D2 cells have been shown to support the maintenance of primitive human cobblestone area-forming cells,51 facilitate human embryonic stem cell differentiation into hematopoietic cells and support their survival in vitro.31, 52, 53When comparing the effect of stromal cells in a cohort of high-risk/sAML samples, we observed a marginally protective effect against ABT-737-induced killing (Figure 6a). Similar to our findings from the suspension culture, BMMNCs from nine high-risk MDS/sAML patients co-cultured on stromal cells underwent apoptosis significantly better than six healthy controls co-cultured on stromal cells (Figure 6a).Figure 6Stromal cells only marginally protect high-risk MDS cells against apoptosis induced by ABT-737. (a) CD34+ BMMNCs from six healthy donors and nine patients with high-risk MDS (denoted 鈥榟igh鈥? or sAML were cultured in the presence of the stromal cell line EL08-1D2 and treated with ABT-737 (1鈥壩?span >M) or vehicle control for 72鈥塰. Each circle represents the ratio between viable cells after ABT-737 treatment and viable cells after vehicle treatment for 72鈥塰. Cells were stained for viability as in Figure 1. Shown is the mean and error bars denote standard deviation. Samples are classified using r-WPSS. One-way ANOVA (P 0.001) and post-hoc pairwise t-tests as shown (***P 0.0005). Healthy vs low-risk: mean difference=2.632, 95% CI (-10.01 to 15.28); healthy vs high-risk/sAML: mean difference=38.48, 95% CI (29.06 to 47.90); low-risk vs high-risk/sAML: mean difference=35.85. 95% CI (23.93 to 47.77). (b) CD34+ BMMNCs from two patients with high-risk MDS and one patient with sAML were cultured in the presence or absence of the stromal cell line EL08-1D2 and treated with ABT-737 (1鈥壩?span >M) or vehicle control for 72鈥塰. Each circle represents the ratio between viable cells after ABT-737 treatment and viable cells after vehicle treatment for 72鈥塰. Cells were stained for viability as in Figure 1. No statistical significant difference could be detected comparing the group with stromal support vs without stromal support (P=0.3264 using Student\'s t-test). (c) Viability of EL08-1D2 cells after treatment with ABT-737 (1鈥壩?span >M) is shown for the indicated time points. Viable cells are represented as percentage of viable cells at the start of the experiment and compared with vehicle control. Experiment was performed in triplicates and error bars denote standard deviation.Full size imageWhen comparing the survival on stromal cells with the survival in liquid culture conditions, we found a minor protection of stromal cell culture conditions in high-risk MDS samples (Figure 6a: mean survival high/sAML with stroma=54.98% compared with Figure 3a: mean survival high-risk without stroma=50.32%). However, a partial protection was observed in sAML samples when comparing the survival on stromal cells to liquid culture (Figure 6a: mean survival high/sAML with stroma=54.98% compared with Figure 3a: mean survival sAML without stroma=26.45%).To directly compare the effect of stromal cells, we subjected three high-risk MDS/sAML samples to ABT-737 with or without stromal cell support (Figure 6b). A partial protection by stromal cells was observed in only one sample, whereas the induction of apoptosis proceeded irrespective of stromal cell support in two additional samples (Figure 6b).To exclude any toxicity of ABT-737 directly in stromal cells that might cause the release of danger-associated molecular pattern molecules, which might in turn facilitate cell death in MDS cells, we measured apoptosis specifically in the stromal cells. In line with a cell-intrinsic effect, we did not observe any relevant induction of apoptosis in EL08-1D2 cells alone (Figure 6c).Despite the limited sample size, our data suggest that stromal cells only partially protected MDS cells from ABT-737-induced cell death, implying that the inhibition of BCL-2 proteins primarily executed apoptosis in a cell-intrinsic manner and that niche protection in the BM might not severely obstruct the clinical efficacy of pro-apoptotic compounds in patients.DiscussionDeregulated apoptosis is a key feature of MDS and an acquired apoptotic resistance in the BM compartment has been associated with higher-risk disease.2, 6, 7, 8, 9, 10, 11, 12 This is likely caused by additional molecular and cytogenetic aberrations acquired during the course of the disease, which is characteristic for the transition from lower-risk MDS to higher-risk MDS.2, 12 Our findings show that re-activation of mitochondrial apoptosis using compounds that inhibit the pro-survival members of the BCL-2 family is specifically toxic to cells from higher-risk MDS or sAML patients and supports the notion that these cells experience an elevated level of apoptotic stress.3This feature of high-risk MDS or sAML cells is referred to as elevated priming for mitochondrial apoptosis, which is reminiscent of the level of apoptotic priming previously reported for de novo AML blasts.40, 41, 42 Using a methodology termed BH3 profiling, the therapeutic effect of chemotherapy was shown to correlate with the level of apoptotic priming in leukemic blasts. Poorly primed and chemotherapy-resistant cells showed a striking resistance to apoptosis in response to synthetic BH3 peptides.42 In contrary, highly \'primed鈥?myeloblasts efficiently underwent apoptosis after BH3-peptide treatment, which also correlated with the response of patients to first induction chemotherapy.42 This finding and our data suggest that the resistance to apoptosis imposed by anti-apoptotic BCL-2 family members is a defining hallmark in AML and also in MDS. The killing observed after ABT-737 or ABT-199 treatment primarily in high-risk MDS/sAML supports the notion that the clinical parameter \'high-risk鈥?in MDS might be comparable to the parameter 鈥榤itochondrial priming鈥?in AML.40, 41, 42We validated the correlation between cell death and disease stage in four major MDS risk classification systems. Irrespective of the scoring system used, we found that the clinical category 鈥榟igh-risk MDS鈥?or 鈥榮AML鈥?is a valid parameter to identify patient samples that will likely respond to treatment with BH3 mimetics. This supports the notion that apoptotic priming might serve as a parameter independent of the molecular, cytogenetic or clinical features present in individual patients. In addition, our samples were collected irrespective of any treatment regimen thereby including samples from patients at diagnosis, under first-line treatment or after treatment failure. The largely uniform pattern of response to ABT-199 or ABT-737 indicates that this class of compounds exerted its potency primarily depending on the level of mitochondrial priming but not depending on parameters such as previous treatment regimens.Colony formation showed that the pool of stem/progenitor cells in MDS efficiently induces apoptosis in response to ABT-737 or ABT-199. This is an important feature as it suggests that BH3 mimetics are capable of targeting the MDS cell of origin likely residing within the CD34+ progenitor cell compartment.38 Similar findings were reported in a MDS mouse model, where ABT-737 was capable of prolonging the survival of mice by targeting leukemia-initiating cells and primitive Lin鈭?/sup>/Sca1+/Kit+ cells.54, 55 However, to accurately recapitulate the high level of clinical and molecular heterogeneity of human MDS, it is of critical importance to test the effects of BH3-mimetic compounds on a large cohort or primary patient samples.Similar to previously published data that show MCL-1 expression as the primary resistance mechanism to ABT-737 or ABT-199 treatment in B-cell neoplasia,56 we found that MDS samples that express high levels of MCL-1 were resistance to the treatment with either compound. Most low-risk MDS samples exhibited high MCL-1 levels rendering those samples resistant to the treatment. In line with our viability data from the liquid culture, most high-risk samples showed low MCL-1 levels and accordingly responded better to ABT-737 or ABT-199. Of note, individual resistant high-risk samples exhibited high MCL-1 levels indicating that MCL-1 serves as the primary resistance factor irrespective of the disease risk category. These inter-individual differences in BCL-2 protein expression were also found in AML, where BCL-2 expression varied substantially between samples from a given French-American-British subgroup.27 Given the critical role of MCL-1 as a resistance biomarker, the detection of protein expression by intracellular flow cytometry might serve as a reasonable way of screening patients before BH3-mimetic treatment.Recent data obtained primarily from AML or CML cell lines or primary patient samples suggested that ABT-199 efficiently killed blast cells.26, 57 This finding is represented in our data by the loss of viability observed in sAML samples harboring an elevated number of blasts (Figure 3). This feature of BH3-mimetic together with the previously described amplification of toxicity to azacitidine or mitogen-activated protein/extracellular signal-regulated kinase inhibition supports the notion that pro-apoptotic intervention in MDS alone or in combination should be further evaluated in the clinical setting.26, 27, 58Our data argue that pro-apoptotic drug treatment reduces the disease burden in higher-risk MDS or sAML patients by selectively killing leukemic progenitors as well as blast cells without significantly affecting the healthy progenitor cell population. BH3-mimetic compounds might therefore represent an interesting approach to treat higher-risk MDS patients to delay progression into sAML or to perform a bridging therapy for patients awaiting allogeneic stem cell transplantation, especially since current therapeutic strategies such as azacitidine have an only short response duration in this patient population.59 References1Tefferi A, Vardiman JW . Myelodysplastic syndromes. N Engl J Med 2009; 361: 1872鈥?885.CAS聽 Article聽Google Scholar聽 2Corey SJ, Minden MD, Barber DL, Kantarjian H, Wang JC, Schimmer AD . Myelodysplastic syndromes: the complexity of stem-cell diseases. Nat Rev Cancer 2007; 7: 118鈥?29.CAS聽 Article聽Google Scholar聽 3Raza A, Galili N . The genetic basis of phenotypic heterogeneity in myelodysplastic syndromes. Nat Rev Cancer 2012; 12: 849鈥?59.CAS聽 Article聽Google Scholar聽 4Ma X, Does M, Raza A, Mayne ST . Myelodysplastic syndromes: incidence and survival in the United States. Cancer 2007; 109: 1536鈥?542.Article聽Google Scholar聽 5Shetty V, Hussaini S, Broady-Robinson L, Allampallam K, Mundle S, Borok R et al. Intramedullary apoptosis of hematopoietic cells in myelodysplastic syndrome patients can be massive: apoptotic cells recovered from high-density fraction of bone marrow aspirates. Blood 2000; 96: 1388鈥?392.CAS聽 PubMed聽Google Scholar聽 6Raza A, Gezer S, Mundle S, Gao XZ, Alvi S, Borok R et al. Apoptosis in bone marrow biopsy samples involving stromal and hematopoietic cells in 50 patients with myelodysplastic syndromes. Blood 1995; 86: 268鈥?76.CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 7Raza A, Alvi S, Broady-Robinson L, Showel M, Cartlidge J, Mundle SD et al. Cell cycle kinetic studies in 68 patients with myelodysplastic syndromes following intravenous iodo- and/or bromodeoxyuridine. Exp Hematol 1997; 25: 530鈥?35.CAS聽 PubMed聽Google Scholar聽 8Parker JE, Mufti GJ . Ineffective haemopoiesis and apoptosis in myelodysplastic syndromes. Br J Haematol 1998; 101: 220鈥?30.CAS聽 Article聽Google Scholar聽 9Parker JE, Mufti GJ, Rasool F, Mijovic A, Devereux S, Pagliuca A . The role of apoptosis, proliferation, and the Bcl-2-related proteins in the myelodysplastic syndromes and acute myeloid leukemia secondary to MDS. Blood 2000; 96: 3932鈥?938.CAS聽Google Scholar聽 10Albitar M, Manshouri T, Shen Y, Liu D, Beran M, Kantarjian HM et al. Myelodysplastic syndrome is not merely 鈥榩releukemia鈥? Blood 2002; 100: 791鈥?98.CAS聽 Article聽Google Scholar聽 11Bogdanovic AD, Trpinac DP, Jankovic GM, Bumbasirevic VZ, Obradovic M, Colovic MD . Incidence and role of apoptosis in myelodysplastic syndrome: morphological and ultrastructural assessment. Leukemia 1997; 11: 656鈥?59.CAS聽 Article聽Google Scholar聽 12Greenberg P, Cox C, LeBeau MM, Fenaux P, Morel P, Sanz G et al. International scoring system for evaluating prognosis in myelodysplastic syndromes. Blood 1997; 89: 2079鈥?088.CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 13Invernizzi R, Pecci A, Bellotti L, Ascari E . Expression of p53, bcl-2 and ras oncoproteins and apoptosis levels in acute leukaemias and myelodysplastic syndromes. Leuk Lymphoma 2001; 42: 481鈥?89.CAS聽 Article聽Google Scholar聽 14Strasser A, Cory S, Adams JM . Deciphering the rules of programmed cell death to improve therapy of cancer and other diseases. EMBO J 2011; 30: 3667鈥?683.CAS聽 Article聽Google Scholar聽 15Adams JM, Cory S . The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene 2007; 26: 1324鈥?337.CAS聽 Article聽Google Scholar聽 16Chipuk JE, Moldoveanu T, Llambi F, Parsons MJ, Green DR . The BCL-2 family reunion. Mol Cell 2010; 37: 299鈥?10.CAS聽 Article聽Google Scholar聽 17Pop C, Salvesen GS . Human caspases: activation, specificity, and regulation. J Biol Chem 2009; 284: 21777鈥?1781.CAS聽 Article聽Google Scholar聽 18Davids MS, Letai A . ABT-199: taking dead aim at BCL-2. Cancer Cell 2013; 23: 139鈥?41.CAS聽 Article聽Google Scholar聽 19Oltersdorf T, Elmore SW, Shoemaker AR, Armstrong RC, Augeri DJ, Belli BA et al. An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature 2005; 435: 677鈥?81.CAS聽 Article聽Google Scholar聽 20Letai A . BCL-2: found bound and drugged!. Trends Mol Med 2005; 11: 442鈥?44.CAS聽 Article聽Google Scholar聽 21Cragg MS, Harris C, Strasser A, Scott CL . Unleashing the power of inhibitors of oncogenic kinases through BH3 mimetics. Nat Rev Cancer 2009; 9: 321鈥?26.CAS聽 Article聽Google Scholar聽 22Khaw SL, Huang DC, Roberts AW . Overcoming blocks in apoptosis with BH3-mimetic therapy in haematological malignancies. Pathology 2011; 43: 525鈥?35.CAS聽 Article聽Google Scholar聽 23Souers AJ, Leverson JD, Boghaert ER, Ackler SL, Catron ND, Chen J et al. ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets. Nat Med 2013; 19: 202鈥?08.CAS聽 Article聽Google Scholar聽 24Roberts AW, Seymour JF, Brown JR, Wierda WG, Kipps TJ, Khaw SL et al. Substantial susceptibility of chronic lymphocytic leukemia to BCL2 inhibition: results of a phase I study of navitoclax in patients with relapsed or refractory disease. J Clin Oncol 2012; 30: 488鈥?96.CAS聽 Article聽Google Scholar聽 25Wilson WH, O\'Connor OA, Czuczman MS, LaCasce AS, Gerecitano JF, Leonard JP et al. Navitoclax, a targeted high-affinity inhibitor of BCL-2, in lymphoid malignancies: a phase 1 dose-escalation study of safety, pharmacokinetics, pharmacodynamics, and antitumour activity. Lancet Oncol 2010; 11: 1149鈥?159.CAS聽 Article聽Google Scholar聽 26Pan R, Hogdal LJ, Benito JM, Bucci D, Han L, Borthakur G et al. Selective BCL-2 Inhibition by ABT-199 Causes On-Target Cell Death in Acute Myeloid Leukemia. Cancer Discov 2014; 4: 362鈥?75.CAS聽 Article聽Google Scholar聽 27Bogenberger JM, Kornblau SM, Pierceall WE, Lena R, Chow D, Shi CX et al. BCL-2 family proteins as 5-Azacytidine-sensitizing targets and determinants of response in myeloid malignancies. Leukemia 2014; 28: 1657鈥?665.CAS聽 Article聽Google Scholar聽 28Schanz J, Tuchler H, Sole F, Mallo M, Luno E, Cervera J et al. New comprehensive cytogenetic scoring system for primary myelodysplastic syndromes (MDS) and oligoblastic acute myeloid leukemia after MDS derived from an international database merge. J Clin Oncol 2012; 30: 820鈥?29.Article聽Google Scholar聽 29Oostendorp RA, Medvinsky AJ, Kusadasi N, Nakayama N, Harvey K, Orelio C et al. Embryonal subregion-derived stromal cell lines from novel temperature-sensitive SV40 T antigen transgenic mice support hematopoiesis. J Cell Sci 2002; 115: 2099鈥?108.CAS聽 PubMed聽Google Scholar聽 30Oostendorp RA, Robin C, Steinhoff C, Marz S, Brauer R, Nuber UA et al. Long-term maintenance of hematopoietic stem cells does not require contact with embryo-derived stromal cells in cocultures. Stem Cells 2005; 23: 842鈥?51.CAS聽 Article聽Google Scholar聽 31Ledran MH, Krassowska A, Armstrong L, Dimmick I, Renstrom J, Lang R et al. Efficient hematopoietic differentiation of human embryonic stem cells on stromal cells derived from hematopoietic niches. Cell Stem Cell 2008; 3: 85鈥?8.CAS聽 Article聽Google Scholar聽 32Haferlach T, Kohlmann A, Wieczorek L, Basso G, Kronnie GT, Bene MC et al. Clinical utility of microarray-based gene expression profiling in the diagnosis and subclassification of leukemia: report from the International Microarray Innovations in Leukemia Study Group. J Clin Oncol 2010; 28: 2529鈥?537.CAS聽 Article聽Google Scholar聽 33Zekavati A, Nasir A, Alcaraz A, Aldrovandi M, Marsh P, Norton JD et al. Post-transcriptional regulation of BCL2 mRNA by the RNA-binding protein ZFP36L1 in malignant B cells. PLoS One 2014; 9: e102625.Article聽Google Scholar聽 34Kutuk O, Letai A . Regulation of Bcl-2 family proteins by posttranslational modifications. Curr Mol Med 2008; 8: 102鈥?18.CAS聽 Article聽Google Scholar聽 35Ruvolo PP, Deng X, May WS . Phosphorylation of Bcl2 and regulation of apoptosis. Leukemia 2001; 15: 515鈥?22.CAS聽 Article聽Google Scholar聽 36Vardiman JW . The World Health Organization (WHO) classification of tumors of the hematopoietic and lymphoid tissues: an overview with emphasis on the myeloid neoplasms. Chem Biol Interact 2010; 184: 16鈥?0.CAS聽 Article聽Google Scholar聽 37Malcovati L, Della Porta MG, Strupp C, Ambaglio I, Kuendgen A, Nachtkamp K et al. Impact of the degree of anemia on the outcome of patients with myelodysplastic syndrome and its integration into the WHO classification-based Prognostic Scoring System (WPSS). Haematologica 2011; 96: 1433鈥?440.CAS聽 Article聽Google Scholar聽 38Woll PS, Kjallquist U, Chowdhury O, Doolittle H, Wedge DC, Thongjuea S et al. Myelodysplastic syndromes are propagated by rare and distinct human cancer stem cells in vivo. Cancer Cell 2014; 25: 794鈥?08.CAS聽 Article聽Google Scholar聽 39Pang WW, Pluvinage JV, Price EA, Sridhar K, Arber DA, Greenberg PL et al. Hematopoietic stem cell and progenitor cell mechanisms in myelodysplastic syndromes. Proc Natl Acad Sci USA 2013; 110: 3011鈥?016.CAS聽 Article聽Google Scholar聽 40Konopleva M, Contractor R, Tsao T, Samudio I, Ruvolo PP, Kitada S et al. Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in acute myeloid leukemia. Cancer Cell 2006; 10: 375鈥?88.CAS聽 Article聽Google Scholar聽 41Ni Chonghaile T, Sarosiek KA, Vo TT, Ryan JA, Tammareddi A, Moore Vdel G et al. Pretreatment mitochondrial priming correlates with clinical response to cytotoxic chemotherapy. Science 2011; 334: 1129鈥?133.Article聽Google Scholar聽 42Vo TT, Ryan J, Carrasco R, Neuberg D, Rossi DJ, Stone RM et al. Relative mitochondrial priming of myeloblasts and normal HSCs determines chemotherapeutic success in AML. Cell 2012; 151: 344鈥?55.CAS聽 Article聽Google Scholar聽 43Jaiswal S, Ebert BL . MDS is a stem cell disorder after all. Cancer Cell 2014; 25: 713鈥?14.CAS聽 Article聽Google Scholar聽 44Zhang J, Niu C, Ye L, Huang H, He X, Tong WG et al. Identification of the haematopoietic stem cell niche and control of the niche size. Nature 2003; 425: 836鈥?41.CAS聽 Article聽Google Scholar聽 45Calvi LM, Adams GB, Weibrecht KW, Weber JM, Olson DP, Knight MC et al. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 2003; 425: 841鈥?46.CAS聽 Article聽Google Scholar聽 46Raaijmakers MH, Scadden DT . Evolving concepts on the microenvironmental niche for hematopoietic stem cells. Curr Opin Hematol 2008; 15: 301鈥?06.Article聽Google Scholar聽 47Wilson A, Trumpp A . Bone-marrow haematopoietic-stem-cell niches. Nat Rev Immunol 2006; 6: 93鈥?06.CAS聽 Article聽Google Scholar聽 48Scadden DT . The stem-cell niche as an entity of action. Nature 2006; 441: 1075鈥?079.CAS聽 Article聽Google Scholar聽 49Li X, Deeg HJ . Murine xenogeneic models of myelodysplastic syndrome: an essential role for stroma cells. Exp Hematol 2014; 42: 4鈥?0.CAS聽 Article聽Google Scholar聽 50Raaijmakers MH, Mukherjee S, Guo S, Zhang S, Kobayashi T, Schoonmaker JA et al. Bone progenitor dysfunction induces myelodysplasia and secondary leukaemia. Nature 2010; 464: 852鈥?57.CAS聽 Article聽Google Scholar聽 51Kusadasi N, Oostendorp RA, Koevoet WJ, Dzierzak EA, Ploemacher RE . Stromal cells from murine embryonic aorta-gonad-mesonephros region, liver and gut mesentery expand human umbilical cord blood-derived CAFC(week6) in extended long-term cultures. Leukemia 2002; 16: 1782鈥?790.CAS聽 Article聽Google Scholar聽 52Oostendorp RA, Harvey KN, Kusadasi N, de Bruijn MF, Saris C, Ploemacher RE et al. Stromal cell lines from mouse aorta-gonads-mesonephros subregions are potent supporters of hematopoietic stem cell activity. Blood 2002; 99: 1183鈥?189.CAS聽 Article聽Google Scholar聽 53Parmar A, Marz S, Rushton S, Holzwarth C, Lind K, Kayser S et al. Stromal niche cells protect early leukemic FLT3-ITD+ progenitor cells against first-generation FLT3 tyrosine kinase inhibitors. Cancer Res 2011; 71: 4696鈥?706.CAS聽 Article聽Google Scholar聽 54Omidvar N, Kogan S, Beurlet S, le Pogam C, Janin A, West R et al. BCL-2 and mutant NRAS interact physically and functionally in a mouse model of progressive myelodysplasia. Cancer Res 2007; 67: 11657鈥?1667.CAS聽 Article聽Google Scholar聽 55Beurlet S, Omidvar N, Gorombei P, Krief P, Le Pogam C, Setterblad N et al. BCL-2 inhibition with ABT-737 prolongs survival in an NRAS/BCL-2 mouse model of AML by targeting primitive LSK and progenitor cells. Blood 2013; 122: 2864鈥?876.CAS聽 Article聽Google Scholar聽 56Choudhary GS, Al-Harbi S, Mazumder S, Hill BT, Smith MR, Bodo J et al. MCL-1 and BCL-xL-dependent resistance to the BCL-2 inhibitor ABT-199 can be overcome by preventing PI3K/AKT/mTOR activation in lymphoid malignancies. Cell Death Dis 2015; 6: e1593.CAS聽 Article聽Google Scholar聽 57Mak DH, Wang RY, Schober WD, Konopleva M, Cortes J, Kantarjian H et al. Activation of apoptosis signaling eliminates CD34+ progenitor cells in blast crisis CML independent of response to tyrosine kinase inhibitors. Leukemia 2012; 26: 788鈥?94.CAS聽 Article聽Google Scholar聽 58Konopleva M, Milella M, Ruvolo P, Watts JC, Ricciardi MR, Korchin B et al. MEK inhibition enhances ABT-737-induced leukemia cell apoptosis via prevention of ERK-activated MCL-1 induction and modulation of MCL-1/BIM complex. Leukemia 2012; 26: 778鈥?87.CAS聽 Article聽Google Scholar聽 59Muller-Thomas C, Rudelius M, Rondak IC, Haferlach T, Schanz J, Huberle C et al. Response to azacitidine is independent of p53 expression in higher-risk myelodysplastic syndromes and secondary acute myeloid leukemia. Haematologica 2014; 99: e179鈥揺181.Article聽Google Scholar聽 Download referencesAcknowledgementsWe thank T Haferlach from the Munich Leukemia Laboratory (MLL) for providing gene expression data. We thanks T Haferlach from MLL and M Zingerle from the Gemeinschaftspraxis H盲mato-Onkologie Pasing for providing MDS samples and clinical data and J Tuebel from the Klinik f眉r Orthop盲die und Sportorthop盲die, Klinikum rechts der Isar for technical support. PJJ was supported by a Max Eder-Program grant from the Deutsche Krebshilfe (program #111738), a Human Frontiers Science Program grant (program #RGY0073/2012), a German Jose Carreras Leukemia Foundation grant (DJCLS R 12/22), and a research grant from the Deutsche Forschungsgemeinschaft, Forschergruppe FOR2036 and Novartis for travel support. RAJO was supported by the German Research Foundation (DFG grants OO 8/5, OO 8/9, and FOR 2033)). RAJO and KSG were supported by the German Jose Carreras Leukemia Foundation grant (DJCLS R 11/12). KSG was supported by the German Research Foundation (Go 713/2-1) and the Deutsche Konsortium f眉r Translationale Krebsforschung (DKTK) of the German Cancer Center (DKFZ). We thank Abbvie for supplying ABT-199. Author contributions PJJ conceived and supervised the project, analyzed the data and wrote the manuscript. SJ performed the experiments, analyzed the data and wrote the manuscript. KG provided primary samples and clinical data and gave conceptual advice. VR, JK, UH, OG and CH performed experiments. JS, BS, RB, CM-T, H-JK, RAJO, JR and CP provided primary samples and clinical data, and gave conceptual advice.Author informationAffiliationsIII. Medizinische Klinik f眉r H盲matologie und Internistische Onkologie, Klinikum rechts der Isar, Technische Universit盲t M眉nchen, Munich, GermanyS Jilg,聽V Reidel,聽C M眉ller-Thomas,聽J K枚nig,聽U H枚ckendorf,聽C Huberle,聽H-J Kolb,聽C Peschel,聽R A J Oostendorp,聽K S G枚tze聽 聽P J JostKlinik f眉r Orthop盲die und Sportorthop盲die, Klinikum rechts der Isar, Technische Universit盲t M眉nchen, Munich, GermanyJ Schauwecker聽 聽R BurgkartInstitut f眉r Klinische Chemie und Pathobiochemie, Klinikum rechts der Isar, Technische Universit盲t M眉nchen, Munich, GermanyO Gorka聽 聽J RulandGemeinschaftspraxis H盲mato-Onkologie Pasing, Munich, GermanyB SchmidtAuthorsS JilgView author publicationsYou can also search for this author in PubMed聽Google ScholarV ReidelView author publicationsYou can also search for this author in PubMed聽Google ScholarC M眉ller-ThomasView author publicationsYou can also search for this author in PubMed聽Google ScholarJ K枚nigView author publicationsYou can also search for this author in PubMed聽Google ScholarJ SchauweckerView author publicationsYou can also search for this author in PubMed聽Google ScholarU H枚ckendorfView author publicationsYou can also search for this author in PubMed聽Google ScholarC HuberleView author publicationsYou can also search for this author in PubMed聽Google ScholarO GorkaView author publicationsYou can also search for this author in PubMed聽Google ScholarB SchmidtView author publicationsYou can also search for this author in PubMed聽Google ScholarR BurgkartView author publicationsYou can also search for this author in PubMed聽Google ScholarJ RulandView author publicationsYou can also search for this author in PubMed聽Google ScholarH-J KolbView author publicationsYou can also search for this author in PubMed聽Google ScholarC PeschelView author publicationsYou can also search for this author in PubMed聽Google ScholarR A J OostendorpView author publicationsYou can also search for this author in PubMed聽Google ScholarK S G枚tzeView author publicationsYou can also search for this author in PubMed聽Google ScholarP J JostView author publicationsYou can also search for this author in PubMed聽Google ScholarCorresponding authorCorrespondence to P J Jost.Ethics declarations Competing interests The authors declare no conflict of interest. Additional informationSupplementary Information accompanies this paper on the Leukemia websiteSupplementary information Supplementary Information (PDF 1345 kb)Rights and permissionsReprints and PermissionsAbout this articleCite this articleJilg, S., Reidel, V., M眉ller-Thomas, C. et al. Blockade of BCL-2 proteins efficiently induces apoptosis in progenitor cells of high-risk myelodysplastic syndromes patients. Leukemia 30, 112鈥?23 (2016). https://doi.org/10.1038/leu.2015.179Download citationReceived: 15 January 2015Revised: 24 June 2015Accepted: 25 June 2015Published: 08 July 2015Issue Date: January 2016DOI: https://doi.org/10.1038/leu.2015.179 Stefanie Jilg, Richard T. Hauch, Johanna Kauschinger, Lars Buschhorn, Timo O. Odinius, Veronika Dill, Catharina M眉ller-Thomas, Tobias Herold, Peter M. Prodinger, Burkhard Schmidt, Dirk Hempel, Florian Bassermann, Christian Peschel, Katharina S. G枚tze, Ulrike H枚ckendorf, Torsten Haferlach Philipp J. Jost Experimental Hematology Oncology (2019) Hendrik Folkerts, Albertus T. Wierenga, Fiona A. van den Heuvel, Roy R. Woldhuis, Darlyne S. Kluit, Jennifer Jaques, Jan Jacob Schuringa Edo Vellenga Cell Death Disease (2019) Binu K. Sasi, Claudio Martines, Elena Xerxa, Fabiola Porro, Hilal Kalkan, Rosa Fazio, Sven Turkalj, Engin Bojnik, Beata Pyrzynska, Joanna Stachura, Abdessamad Zerrouqi, Ma艂gorzata Bobrowicz, Magdalena Winiarska, Valdemar Priebe, Francesco Bertoni, Larry Mansouri, Richard Rosenquist Dimitar G. Efremov Leukemia (2019) Eimear O鈥?Reilly, Sukhraj Pal S. Dhami, Denis V. Baev, Csaba Ortutay, Anna Halpin-McCormick, Ruth Morrell, Corrado Santocanale, Afshin Samali, John Quinn, Michael E O鈥橠wyer Eva Szegezdi Scientific Reports (2018) Brett M. Stevens, Nabilah Khan, Angelo D鈥橝lessandro, Travis Nemkov, Amanda Winters, Courtney L. Jones, Wei Zhang, Daniel A. Pollyea Craig T. Jordan Nature Communications (2018)