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BCL-2 family proteins as 5-Azacytidine-sensitizing targets and determinants of response in myeloid malignancies

BCL-2 family proteins as 5-Azacytidine-sensitizing targets and determinants of response in... Leukemia (2014) 28, 1657–1665 OPEN & 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14 www.nature.com/leu ORIGINAL ARTICLE BCL-2 family proteins as 5-Azacytidine-sensitizing targets and determinants of response in myeloid malignancies 1 2 3 3 4 1 1 1 4 4,6 JM Bogenberger , SM Kornblau , WE Pierceall , R Lena , D Chow , C-X Shi , J Mantei , G Ahmann , IM Gonzales , A Choudhary , 5 1 1 1 2 2 3 1 4 4 1 R Valdez , J Camoriano , V Fauble , RE Tiedemann , YH Qiu , KR Coombes , M Cardone , E Braggio , H Yin , DO Azorsa , RA Mesa , 1 1 AK Stewart and R Tibes Synergistic molecular vulnerabilities enhancing hypomethylating agents in myeloid malignancies have remained elusive. RNA-interference drug modifier screens identified antiapoptotic BCL-2 family members as potent 5-Azacytidine-sensitizing targets. In further dissecting BCL-X , BCL-2 and MCL-1 contribution to 5-Azacytidine activity, siRNA silencing of BCL-X and MCL-1, but not L L BCL-2, exhibited variable synergy with 5-Azacytidine in vitro. The BCL-X , BCL-2 and BCL-w inhibitor ABT-737 sensitized most cell lines more potently compared with the selective BCL-2 inhibitor ABT-199, which synergized with 5-Azacytidine mostly at higher doses. Ex vivo, ABT-737 enhanced 5-Azacytidine activity across primary AML, MDS and MPN specimens. Protein levels of BCL-X , BCL-2 and MCL-1 in 577 AML patient samples showed overlapping expression across AML FAB subtypes and heterogeneous expression within subtypes, further supporting a concept of dual/multiple BCL-2 family member targeting consistent with RNAi and pharmacologic results. Consequently, silencing of MCL-1 and BCL-X increased the activity of ABT-199. Functional interrogation of BCL-2 family proteins by BH3 profiling performed on patient samples significantly discriminated clinical response versus resistance to 5-Azacytidine-based therapies. On the basis of these results, we propose a clinical trial of navitoclax (clinical-grade ABT-737) combined with 5-Azacytidine in myeloid malignancies, as well as to prospectively validate BH3 profiling in predicting 5-Azacytidine response. Leukemia (2014) 28, 1657–1665; doi:10.1038/leu.2014.44 Keywords: 5-Azacytidine; BH3 profiling; myeloid malignancies INTRODUCTION secondary RNAi screen results indicated that inhibition of antiapoptotic BCL-2 family proteins constitutes an important Conventional therapies for poor-risk, elderly and secondary acute concept for modulating 5-Aza antileukemic activity. Antiapoptotic myeloid leukemia (AML), relapsed/refractory high-risk myelodys- BCL-2 family members have been proposed as targets in myeloid plastic syndrome (MDS) and accelerated and transformed malignancies, particularly MCL-1, BCL-2 and BCL-X , and (leukemic blast phase) myeloproliferative neoplasm (MPN) have therapeutic agents for these targets are currently in preclinical limited benefit. Response and long-term outcome with cytotoxic or clinical development. However, there is no direct comparison of chemotherapies are poor. Hypomethylating agents, 5-Azacytidine these antiapoptotic BCL-2 family members as targets in myeloid (5-Aza) and decitabine, demonstrate substantial single-agent 3,4 5 malignancies. Therefore, we focused on further studies to dissect activity in B25–50% of MDS and MPN, and are active in 6–8 how inhibition of BCL-X , BCL-2 and MCL-1, with siRNA and B15–25% of AML. Although these single-agent activities are 9,10 pharmacological inhibitors, modulates the antileukemic activity of encouraging, substantial room for improvement remains. The 5-Aza. These functional studies are complemented by proteomic development of rational combinations with hypomethylating data analyzing BCL-X , BCL-2 and MCL-1 in 577 primary AML agents has been hampered by a lack of precise mechanistic specimens, demonstrating that expression of antiapoptotic family understanding. Furthermore, although promising predictive 11–13 members is heterogeneous within AML FAB subtypes and biomarkers for 5-Aza have recently been reported, there is overlapping across subtypes, suggesting functional redundancy. no clinically validated assay or biomarker predicting response to We present additional evidence that the overall balance of BCL-2 hypomethylating agents. Therefore, we sought to identify family proteins, assessed with a functional BH3-profiling assay, therapeutically exploitable molecular vulnerabilities to design rational combinations with 5-Aza, and, in parallel, to develop predicts clinical response to 5-Aza. The results presented herein biomarkers of response to 5-Aza. have important implications for the design of clinical trials with As a mechanistically unbiased starting point to identify targets BCL-2 family-targeting agents in myeloid malignancies, the future that modulate the antileukemic activity of 5-Aza, we performed development of novel BCL-2 family-targeted agents with distinct replicate RNA-interference (RNAi) sensitizer screens of 861 kinase, inhibitory profiles and the clinical application of 5-Aza in myeloid cell cycle, apoptosis and other cancer-associated genes in myeloid malignancies through the potential development of a predictive cell lines in combination with 5-Aza treatment. Primary and biomarker of response to 5-Aza. 1 2 Department of Hematology and Oncology, Mayo Clinic, Scottsdale, AZ, USA; Section of Molecular Hematology and Therapy, Department of Leukemia, The University of Texas 3 4 M.D. Anderson Cancer Center, Houston, TX, USA; Eutropics Pharmaceuticals, Cambridge, MA, USA; Translational Genomics Research Institute, Phoenix, AZ, USA and Department of Lab Medicine and Pathology, Mayo Clinic, Scottsdale, AZ, USA. Correspondence: Dr R Tibes, Department of Hematology and Oncology, Mayo Clinic, 13400 East Shea Boulevard, Scottsdale, AZ 85259, USA. E-mail: tibes.raoul@mayo.edu Current address: Ashish Choudhary, Asuragen Inc., Austin, TX, USA. Roger Tiedemann, Ontario Cancer Institute, University of Toronto, Toronto, ON, Canada. Received 25 September 2013; revised 21 December 2013; accepted 2 January 2014; accepted article preview online 23 January 2014; advance online publication, 14 February 2014 5-Azacytidine sensitizers and response predictor JM Bogenberger et al MATERIALS AND METHODS AML specimens suspended in 1% FBS, 2 mM EDTA-PBS were stained with primary antibodies CD45-V450 (BD Biosciences, Franklin Lakes, NJ, USA), Cells, culture conditions and reagents CD3-Biotin (BD Biosciences) and CD20-Biotin (eBiosciences, San Diego, CA, Cell lines were obtained from ATCC or DSMZ, and MDS-L was kindly USA), and secondary antibody Streptavidin-APC (BD Biosciences). Speci- provided by Professor Kaoru Tohyama (Kawasaki Medical School, Kurashiki, 14 mens were then permeabilized with digitonin (Sigma-Aldrich) and Japan). Primary specimens were obtained in accordance with incubated with JC-1 mitochondrial dye (Enzo Life Sciences, Farmingdale, Institutional Review Board-approved protocols, separated by Ficoll NY, USA) and peptides (BIM 100mM, BIM 0.1mM, PUMA 100mM, PUMA 10mM, gradient centrifugation and/or treated with ACK lysis buffer before short- NOXA 100mM, BAD 100mM, BMF 100mM, HRK 100mM or PUMA2A 100mM)or term ex vivo culture. All cells were cultured in RPMI-1640 with 10% FBS, with dimethyl sulfoxide (DMSO (1%) or carbonyl cyanide m-chlorophenyl 2mML-glutamine, 100 IU/ml penicillin and 100mg/ml streptomycin hydrazone (CCCP (10mM)) at room temperature. Samples were run in (Invitrogen, Carlsbad, CA, USA) at 37 1C/5% CO . 5-Azacytidine was duplicate except in cases where insufficient viable cells were available. obtained from Sigma-Aldrich (St Louis, MO, USA), ABT-737 from Samples were analyzed on a FACS CantoII (BD Biosciences) using the BD ChemieTek (Indianapolis, IN, USA), and ABT-199 from SeleckChem FACS Diva software (BD Biosciences). The blast population was identified as (Houston, TX, USA). CD45 dim, CD3 and CD20 negative. Intensely stained CD45 cells (mature 20,21 lymphocytes) were excluded from analyses as described previously. siRNA drug-dose-response assays The quantifiable propensity of a pro-apoptotic peptide to induce mitochondrial depolarization relative to an uncoupling reagent control is Selected candidates from 5-Aza RNAi sensitizer screens were validated with siRNA drug-dose-response (siDDR) assays as described previously for referred to as percent priming. For the blast population, this was calculated using the median signal intensity of the PE channel normalized for DMSO primary RNAi screens. All siRNA were obtained from Qiagen, except for as background (negative control), and CCCP served as 100% priming two additional validation BCL-2 siRNA sequences, IDs: s224526 and (positive control). For calculation of % priming, the following formula is s194310 (Silencer Select, Ambion, Carlsbad, CA, USA). Four different utilized. siRNA sequences for each selected target, nonsilencing negative control siRNA, universal lethal positive control siRNA and buffer-transfection Peptide CCCP reagent were included on each 384-well siDDR assay plate. % priming ¼ 1 100 DMSO CCCP Drug-dose-response experiments and CalcuSyn analysis For ABT-737 and ABT-199 combination experiments with 5-Aza, com- Statistical Analysis pounds were added simultaneously and relative cell number was BH3-profiling biomarkers were analyzed by testing the association determined at 96 h with CTG. Prism Version 5.03 software (Prism Software between the biomarker status (% priming) and responder or nonresponder Corporation, Irvine, CA, USA) was used to calculate 5-Aza EC values at classification. Univariate comparisons were made using the Mann–Whitney various concentrations of ABT-737 and ABT-199. Synergy was assessed by test; all reported P-values are two sided. The predictive ability of markers calculating combination index values with CalcuSyn Version 2.1 software was assessed using the area under the curve (AUC) statistic. Analyses were (Biosoft, Cambridge, UK) according to the Chow and Talalay model. performed using SAS software, version 9.2 (SAS Institute Inc., Cary, NC, USA), R version 2.14.2 (R Core Team) and/or Graphpad Prism version 5.04 Cleaved caspase-3 analysis (Prism Software Corporation). Cells were processed according to the Cell Signaling Technology protocol. TF-1 cells were treated for 24 h with 625 nM ABT-737 before addition of RESULTS 1.0mM 5-Aza and fixed at 72 h total. HL-60 was dosed with 500 nM ABT-737 simultaneously with 1.0mM 5-Aza, before fixation at 8, 24 and 48 h. Cells Knockdown of antiapoptotic BCL-2 family proteins results in were incubated for 1 h with cleaved caspase-3 (Asp175)-Alexa Fluor 488 differential effects on viability and 5-Azacytidine sensitization antibody conjugate (Cell Signaling Technology, Danvers, MA, USA) at 1:50 Preliminary RNAi drug modifier screens targeting genes capable of dilution. Fluorescence intensity was measured on a CyAn flow cytometer sensitizing AML-derived cell lines TF-1 and ML-2 to 5-Aza (Beckman Coulter, Pasadena, CA, USA) and data analyzed with Summit identified BCL-2 family members as potential targets. Validation Version 4.3 software (DAKO, Carpinteria, CA, USA). studies confirmed that RNAi-mediated knockdown of BCL-X resulted in three- to fourfold 5-Aza sensitization in TF-1 (data not Protein expression/reverse phase protein array (RPPA) shown). To further explore the role of BCL-2 family members as Proteomic profiling was performed on primary AML specimens using therapeutic targets in myeloid malignancies, we examined the 17,18 validated methods described previously. Primary specimens were role of three major antiapoptotic BCL-2 family proteins, BCL-2, printed in five serial dilutions onto slides with normalization and MCL-1 and BCL-X , and tested their impact on cell proliferation expression controls. Slides were probed with validated primary and sensitivity to 5-Aza. siRNA silencing of BCL-X or MCL-1 antibodies (Cell Signaling Technology; Epitomics, Burlingame, CA, USA) L effectively reduced viability in most AML cell lines examined, at 1:500 dilution and secondary antibody to amplify the signal at 1:15 000 dilution. Stained slides were analyzed using Vigene Tech Microvigene whereas BCL-2 siRNA had less of an effect (Table 1a). In regards to Version 3.4 software (Carlisle, MA, USA) to produce quantified data as 5-Aza sensitization, silencing of BCL-X potently sensitized previously described. erythroid cells TF-1 and HEL (Supplementary Figure 1A), while MCL-1 sensitized more broadly across the AML cells examined mRNA expression from public data sets (Table 1b). Growth inhibitory effects of siRNA silencing alone generally paralleled the effects of siRNA sensitization to 5-Aza. Data from public data sets GEO accession numbers GSE19429, GSE6891, GSE12417 were MAS5 transformed using Expression Console Software Effective siRNA silencing was demonstrated for BCL-X by (Affymetrix, Santa Clara, CA, USA) and subsequently median normalized. reduction in protein levels in cells that were sensitized (TF-1) or The number of cases were as follows: CD34þ (17), MDS (50), M0 (16), M1 were not sensitized (THP-1) (Supplementary Figures 1B and C), (95), M2 (104), M3 (23), M4 (23), M5 (104), M6 (6). The ANOVA test statistical showing that differential 5-Aza sensitization was not due to analysis was performed across all groups, thus the P-value corresponds to differences in knockdown. simultaneous analysis of all French-American-British (FAB) classifications. The Valk and Metzeler2 data sets were obtained from Oncomine. ABT-737 synergizes with 5-Azacytidine more potently than ABT-199 in myeloid cell lines BH3-profiling assay Two therapeutic agents, ABT-263 and ABT-199, directly targeting Viably frozen patient specimens (N¼ 22) consisted of 11 bone marrow and antiapoptotic BCL-2 family members by acting as BH3-domain 11 peripheral blood mononuclear cells. For in vitro experiments, identical mimetics are currently undergoing clinical testing. Thus far, cell line passages were used for BH3-profiling assays and 5-Aza drug-dose- ABT-263 and ABT-199 have been tested primarily for the response experiments, performed simultaneously with the same lot of freshly prepared 5-Aza. treatment of solid tumors and lymphoid malignancies, and their Leukemia (2014) 1657 – 1665 & 2014 Macmillan Publishers Limited 5-Azacytidine sensitizers and response predictor JM Bogenberger et al 22–24 efficacy in myeloid malignancies remains to be determined. sensitization to 5-Aza was mediated by enhancing apoptosis ABT-263 (navitoclax), an orally available analog and the clinical (Supplementary Figures 2C and D). We conclude that, although grade compound of the experimental tool compound ABT-737 BCL-2 inhibition with ABT-199 is effective in myeloid malignancies, with a nearly identical binding profile, inhibits BCL-X , BCL-2 and in most cell lines simultaneous targeting of BCL-X , BCL-2 and Bcl- L L 25,26 BCL-w with K values o1nM. Because of the on-target effects w with ABT-737 is more potent than targeting BCL-2 alone, and in of ABT-263 on BCL-X , a megakaryocytic lineage gene, ABT-263 combination with 5-Aza. induces thrombocytopenia. Recently ABT-199, a more selective inhibitor of BCL-2 that does not inhibit BCL-X at low-to-moderate RNAi silencing of antiapoptotic BCL-2 family proteins in concentrations, has shown promising clinical responses in combination with ABT-199 or ABT-737 lymphoid malignancies, without some of the clinical toxicities of Next we aimed to explore the individual contribution of inhibiting 28,29 ABT-263, particularly thrombocytopenia. BCL-X or MCL-1 in combination with BCL-2. Thus, BCL-X or MCL-1 L L To determine which agent is more potent in myeloid was silenced by siRNA and ABT-199 activity subsequently malignancies, we assessed single-agent activity and 5-Aza assessed. BCL-X or MCL-1 silencing strongly potentiated ABT- sensitization with ABT-737 (the tool compound of ABT-263) versus 199 as shown by average ABT-199 EC fold-shifts (Table 2). MCL-1 ABT-199 across a spectrum of genomically heterogeneous AML has been shown to confer resistance to ABT-737. Conversely, cell lines. ABT-737 exhibited lower single-agent EC values inhibition of MCL-1 enhances ABT-737 sensitivity of several tumor (median 0.14mM for ABT-737 versus 4.3mM for ABT-199) 31 types; however, this has not been shown for myeloid cells. (Figure 1), and resulted in greater 5-Aza sensitization, as Indeed, siRNA silencing of MCL-1 strongly sensitized to ABT-737 determined by EC fold-enhancement and Combination Index (Supplementary Figure 3). These data confirm BCL-X and MCL-1 synergy with CalcuSyn (Figures 2a–c and Supplementary as critical targets in myeloid malignancies, and inhibiting either Figures 2A and B). Generally, higher concentrations of ABT-199, enhances effects of BCL-2 inhibition. than ABT-737, were required to enhance 5-Aza activity (Figure 2b). Moreover, ABT-737 exhibited dose-dependent sensitization in all Protein and mRNA expression of antiapoptotic BCL-2 family AML cells, whereas ABT-199 sensitization was not dose-dependent members in primary AML specimens except in MDS-L (Supplementary Figure 2A). ABT-737 also resulted To assess putative correlations between BCL-2 family expression in greater synergy than ABT-199 in most cell lines, as determined levels and pharmacological/functional siRNA data, we measured by lower combination index values and/or greater Fractional Effect the expression of BCL-X , BCL-2 and MCL-1 in 577 primary AML by CalcuSyn analysis, which again were seen at lower doses of samples using a reverse phase protein array (RPPA) proteomic ABT-737 than ABT-199 (low nM versus low mM concentrations, approach. There was no strong association between BCL-X , BCL-2 respectively) (Supplementary Figure 2B). As expected, ABT-737 or MCL-1 expression and cytogenetic-molecular features (data not shown), consistent with our previous report. However, when analyzed by AML FAB classification, BCL-X was highest in M6, Table 1a. In vitro % viability reduction by siRNA alone whereas BCL-2 was highest in M1 and M0 (Figure 3). Median BCL-2 levels were higher in normal CD34 -selected hematopoietic Cell line siRNA progenitor cells as compared with all FAB classifications except M0, M1 and M4 EOS. In contrast, median BCL-X and MCL-1 levels BCL-2 BCL-X MCL-1 þ were lower in normal CD34 progenitor cells as compared with the majority of FAB classifications. MCL-1 expression was more SET-2 0–2 39–52 22–65 uniformly distributed across FAB classifications than BCL-X or TF-1 0–4 11–71 0–17 BCL-2. There were often strong differences in expression levels of HEL 0–1 21–67 14–39 BCL-X , BCL-2 and MCL-1 within a given FAB subgroup, indicating THP-1 0–21 11–32 33–87 OCI-AML3 0–20 66–71 49–99 significant patient-to-patient variability. Similar trends were ML-2 0–4 11–38 18–35 confirmed by mRNA expression in AML data sets reanalyzed and in public databases (Supplementary Figures 4A–C). Antiapoptotic BCL-2 family siRNA drug-dose-response assays with 5-Azacytidine in myeloid cells in vitro. Cell lines are listed in the left-most column. The BCL-2 family member silenced by siRNA is shown as the 5-Azacytidine and ABT-737 exhibit synergy ex vivo across a heading for each column. The range of % viability reduction due to the spectrum of myeloid malignancies: AML, MDS and MPNs given siRNA alone (without 5-Aza) is shown (0¼ no affect and 100¼ To demonstrate that inhibition of antiapoptotic BCL-2 family maximal reduction in viability). proteins is a potent 5-Aza sensitization strategy for AML and Table 1b. In vitro 5-Azacytidine EC fold-shift enhancement by siRNA Cell line siRNA BCL-2 BCL-X MCL-1 ± ± ± SET-2 1.0 0.0 (P¼ 0.20) 1.4 0.2 (P¼ 0.052) 1.1 0.1 (P¼ 0.14) ± ± ± TF-1 1.2 0.1 (P¼ 0.011) 3.4 0.7 (P¼ 3.5e–5) 1.6 0.1 (P¼ 0.061) ± ± ± HEL 1.2 0.1 (P¼ 0.38) 4.4 1.8 (P¼ 0.031) 3.0 0.4 (P¼ 0.086) ± ± ± THP-1 1.0 0.1 (P¼ 0.82) 1.1 0.2 (P¼ 0.079) 1.2 0.0 (P¼ 0.076) ± ± ± OCI-AML3 1.2 0.5 (P¼ 0.61)  1.1 0.2 (P¼ 0.016) 2.2 0.8 (P¼ 0.056) ± ± ± ML-2 1.0 0.0 (P¼ 0.35) 1.1 0.1 (P¼ 0.026) 1.4 0.0 (P¼ 0.024) Anti-apoptotic BCL-2 family siRNA drug-dose-response assays with 5-Azacytidine in myeloid cells in vitro. Cell lines are listed in the left-most column. The BCL-2 family member silenced by siRNA is shown as the heading for each column. 5-Aza EC fold-shifts are listed with P values associated with EC fold-shift 50 50 measurements averaged for the different siRNA sequences against each BCL-2 family member. ‘ ’ denotes antagonistic fold-shift. & 2014 Macmillan Publishers Limited Leukemia (2014) 1657 – 1665 5-Azacytidine sensitizers and response predictor JM Bogenberger et al myeloid malignancies including MDS and MPNs, we evaluated the secondary AML (prior MDS or PMF) (Figure 4 and Table 3). We combination of ABT-737 with 5-Aza in short-term ex vivo cultures. reasoned that the combination would be particularly effective in ABT-737 sensitizes primary specimens from de novo AML and MPNs, given evidence for BCL-X as a lineage-specific proto- 32,33 oncogene, and its importance in erythroid progenitors. Indeed, primary samples from MDS and MPN patients were sensitive to the combination, exhibiting strong synergy (Figure 4 and Table 3). BH3 profiling discriminates clinical response to 5-Azacytdine- based therapies Although there are protein expression differences for BCL-X ,BCL-2 and MCL-1 by RPPA, within and between AML FAB subtypes, we did not find a strong correlation with clinical response to 5-Aza-based therapies (data not shown). This may be expected considering possible mechanistic distinctions and functional redundancies amongst the different antiapoptotic BCL-2 family members exhibiting overlapping expression patterns, as well as the distinct, yet overlapping binding affinities between anti- and proapoptotic 36,37 proteins. Thus, we aimed to functionally interrogate the overall balance of pro- and antiapoptotic BCL-2 family proteins, a cellular- Figure 1. Single-agent BCL-2 family inhibitor activity in AML cell molecular characteristic described as ‘primedness’, by BH3 lines. Seven AML-derived cells lines (TF-1, HEL, THP-1, U937, ML-2, profiling. This assay uses peptides derived from proapoptotic HL-60 and MDS-L) are plotted for ABT-737, whereas 11 are plotted BH3-only proteins and determines the degree to which these for ABT-199 activity (UKE-1, SET-2, M07e, TF-1, HEL, THP-1, ML-2, OCI- peptides induce apoptosis. As proof-of-concept in vitro,several AML3, OCI-AML2, HL-60 and MDS-L). Six cell lines were tested with both compounds (TF-1, HEL, THP-1, ML-2, HL-60 and MDS-L). BH3-profiling metrics significantly correlated with 5-Aza sensitivity ABT-737 ABT-199 12.0 10.1 10.0 8.0 8.0 6.0 4.8 3.8 3.4 3.3 4.0 4 30 2.9 2.1 2.0 1.8 1.6 2.0 0.0 -2.0 SET-2 M07e TF-1 THP-1 ML-2 HL-60 MDS-L -1.1 -1.1 Cell Lines ABT-737 ABT-199 2000 2000 2000 261 261 78 80 SET-2 M07e TF-1 THP-1 ML-2 HL-60 MDS-L Cell Lines Cell Line: SET-2 M07e TF-1 THP-1 ML-2 HL-60 MDS-L ABT Compound: 737 199 737 199 737 199 737 199 737 199 737 199 737 199 CI Value* 0.81 1.25 0.18 0.17 0.50 1.10 0.05 0.09 0.09 0.90 0.30 0.44 0.51 0.48 Figure 2. ABT-737 compared with ABT-199 in vitro synergy with 5-Azacytidine. (a) Maximal 5-Aza EC fold-shifts for ABT-737 and ABT-199 are shown side-by-side for each cell line. These EC fold-shifts are a ‘one-sided’ measurement of 5-Aza enhancement. (b) Corresponding ABT-737 or ABT-199 doses at which maximal 5-Aza EC fold-shifts occurred are shown side-by-side. (c) *CalcuSyn Combination Index (CI) values corresponding to the greatest synergy (thus doses shown in b) nearest to the 5-Aza EC dose are listed. It is important to note that CI values are a ‘two-sided’ measurement of drug synergy for two specific doses, thus a single CI value is not a universal characteristic of the interaction between two drugs because interactions can be dose dependent. See Supplementary Figure 2B for an extensive data set of CI values across multiple dose combinations plotted against the corresponding Fractional Effect, where 0¼ no effect and 1.0¼ maximal effect. CI values o0.8 indicate synergy, whereas CI values 41.1 indicate antagonism. Leukemia (2014) 1657 – 1665 & 2014 Macmillan Publishers Limited nM Dose of ABT Compounds At Maximal 5-Aza EC Fold- Enhancement Maximal 5-Aza EC Fold-Shift 5-Azacytidine sensitizers and response predictor JM Bogenberger et al (Supplementary Figure 5). To correlate clinical responses to 5-Aza underlying mechanisms of many therapeutic agents, including the 39,40 with BH3 profiling, specimens from 22 AML, MDS and MDS/MPN hypomethylating agent 5-Aza, are still poorly understood. This overlap patients treated with 5-Aza-based regimens, and for whom lack of mechanistic understanding has impeded development of clinical outcome was available, were assayed (Supplementary novel rational combinations. We aimed to circumvent these Table 1). Clinically, the best BH3 metric comprised combined values challenges by employing a previously successful RNAi sensitizer from NOXA and BIM peptides, which discriminated clinical screening approach as a mechanistically unbiased strategy to responses, defined as achieving a response (sensitive) versus a discover genes that modulate sensitivity to 5-Aza. Primary and patient being resistance/refractory, with statistical significance secondary RNAi screens indicated that inhibition of antiapoptotic (Mann–Whitney two-tailed P¼ 0.001) and an area under the BCL-2 family proteins constitutes an important concept for receiver operating characteristic curve of 0.950 (Figure 5). sensitizing myeloid cells to 5-Aza. Direct comparison of BCL-X , MCL-1 and BCL-2 inhibition by siRNA and pharmacologically suggests that, although inhibition of individual antiapoptotic BCL-2 proteins (that is, BCL-2 with DISCUSSION ABT-199) can be effective alone or in combination with 5-Aza, Therapeutic progress in the treatment of myeloid malignancies inhibiting more than one BCL-2 member simultaneously (that is, has been slow. Despite increased molecular knowledge, the BCL-X , BCL-2 and BCL-w with ABT-737, or siRNA against MCL-1 or BCL-X combined with ABT-199) has more potent antileukemic activity. Importantly, dual or multiple BCL-2 family targeting sensitizes to 5-Aza across a broader range of AML cell lines. Table 2. In vitro ABT-199 EC fold-shift enhancement by siRNA Proteomic data from 577 primary AML samples showed over- lapping expression of BCL-X , MCL-1 and BCL-2 across samples for Cell line siRNA the FAB subtypes. This overlap of expression, together with siRNA and pharmacological data, suggests functional redundancy BCL-X MCL-1 between BCL-X , BCL-2 and MCL-1 in myeloid malignancies. Thus, ± ± SET-2 1.2 0.1 (P¼ 0.032) 1.5 0.3 (P¼ 0.049) we conclude that two or more antiapoptotic BCL-2 family ± ± TF-1 1.6 0.2 (P¼ 0.0073) 2.3 1.0 (P¼ 0.076) members determine the apoptotic threshold and response to ± ± THP-1 4.2 2.8 (P¼ 0.11) 8.5 2.4 (P¼ 0.0079) 5-Aza in most AML cases. Consequently, dual or multiple ± ± OCI-AML3 2.4 1.0 (P¼ 0.067) 6.9 0.9 (P¼ 0.0084) simultaneous antiapoptotic BCL-2 family protein targeting may have greater antileukemic activity alone or with 5-Aza. Also BCL-XL and MCL-1 siRNA knockdown combined with ABT-199 drug-dose- supporting this concept, we show that knockdown of BCL-X or response in myeloid cells in vitro. Cell lines are listed in the left-most L column. The BCL-2 family member silenced by siRNA before assessing ABT- BCL-2 alone with siRNA did not significantly sensitize THP-1 or 199 drug-dose-response is shown as the heading for each column. ABT-199 ML-2 to 5-Aza; however, simultaneous inhibition of BCL-X , BCL-2 EC fold-shifts, determined relative to non-silencing siRNA, are listed with and BCL-w by ABT-737 resulted in potent 5-Aza sensitization in P-values associated with EC fold-shift measurements averaged for four these cells. In addition, siRNA knockdown of BCL-X or MCL-1 different siRNA sequences against each BCL-2 family member. strongly sensitized AML cell lines to BCL-2 inhibition with ABT-199, Figure 3. BCL-XL, MCL-1 and BCL-2 protein expression in primary specimens determined by Reverse Phase Protein Array. 577 primary AML patient samples are shown grouped by AML FAB classification. & 2014 Macmillan Publishers Limited Leukemia (2014) 1657 – 1665 5-Azacytidine sensitizers and response predictor JM Bogenberger et al De novo AML (FAB M4) MDS → AML_1 (FAB Unavailable) MDS → AML_2 (FAB M1) MDS → AML_3 (FAB M6) 5-Aza + ABT-737 - Algebraic estimate 5-Aza + ABT-737 - Algebraic estimate 5-Aza + ABT-737 - Algebraic estimate 5-Aza + ABT-737 - Algebraic estimate 1.0 1.0 2.0 1.0 0.8 0.8 0.8 1.5 0.6 0.6 0.6 1.0 0.4 0.4 0.4 0.5 0.2 0.2 0.2 0 0 0 0 0.2 0.4 0.6 0.8 1.0 00.2 0.4 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 1.0 Fractional Effect Fractional Effect Fractional Effect Fractional Effect PMF → AML (FAB M4) PV → MDS/MPN overlap RCMD MDS Low-grade MDS 5-Aza + ABT-737 - Algebraic estimate 5-Aza + ABT-737 - Algebraic estimate 5-Aza + ABT-737 - Algebraic estimate 5-Aza + ABT-737 - Algebraic estimate 1.0 1.0 1.0 2.0 0.8 0.8 0.8 1.5 0.6 0.6 0.6 1.0 0.4 0.4 0.4 0.5 0.2 0.2 0.2 0 0 0 0 00.2 0.4 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 1.0 Fractional Effect Fractional Effect Fractional Effect Fractional Effect ET PV_1 PV_2 5-Aza + ABT-737 - Algebraic estimate 5-Aza + ABT-737 - Algebraic estimate 5-Aza + ABT-737 - Algebraic estimate 1.0 1.0 1.0 0.8 0.8 0.8 0.6 0.6 0.6 0.4 0.4 0.4 0.2 0.2 0.2 0 0 0 0 0.2 0.4 0.6 0.8 1.0 00.2 0.4 0.6 0.8 1.0 00.2 0.4 0.6 0.8 1.0 Fractional Effect Fractional Effect Fractional Effect MDS→AML_1, ET, PV1 & 2: PV→MDS/MPN overlap: 5-Aza [uM] 5-Aza [uM] 7.4 22.2 66.7 1.0 3.1 9.3 27.8 83.3 78 48 12 78 48 12 16 20 156 3 3 7 7 11 156 37 7 11 15 19 313 2 2 6 10 313 2 6 10 14 18 625 1 5 9 625 1 5 9 13 17 PMF→AML, de novo AML, MDS→AML_3(M6), RCMD MDS, MDS→AML_2: Low-grade MDS: 5-Aza [uM] 5-Aza [uM] 2.5 7.4 22.2 66.7 0.8 2.5 7.4 22.2 66.7 78 4 8 12 16 78 4 8 12 16 20 1563 71115 156 3 71115 19 313 2 6 10 14 313 2 61014 18 625 1 5 9 13 625 1 5 9 13 17 Figure 4. 5-Azacytidine and ABT-737 synergistically combine in primary myeloid malignancy specimens (N¼ 11). Combination Index (CI) versus Fractional Effect (FE) plots were calculated using CalcuSyn Software Version 2.1. The numbers in the tables (Figure 4 key) correspond to the dose combinations shown on the CI versus FE plots for the indicated primary specimens. CI values o0.8 indicate synergy, whereas CI values 41.1 indicate antagonism. demonstrating that BCL-X and MCL-1 can cooperate with BCL-2 200-fold lower ABT-737 doses in five of seven AML cell lines). in governing apoptotic response. Furthermore, ABT-737 generally Single-agent activity of BCL-2 family inhibitors in AML cell lines resulted in broader and more potent sensitization than ABT-199, confirms this trend, as the median ABT-737 EC dose was found which occurred at lower doses of ABT-737 than ABT-199 (7- to to be 430-fold lower than that of ABT-199. Leukemia (2014) 1657 – 1665 & 2014 Macmillan Publishers Limited CI +/- 1.96 s.d. CI +/- 1.96 s.d. CI +/- 1.96 s.d. ABT-737 [nM] ABT-737 [nM] CI +/- 1.96 s.d. CI +/- 1.96 s.d. CI +/- 1.96 s.d. ABT-737 [nM] ABT-737 [nM] CI +/- 1.96 s.d. CI +/- 1.96 s.d. CI +/- 1.96 s.d. CI +/- 1.96 s.d. CI +/- 1.96 s.d. 5-Azacytidine sensitizers and response predictor JM Bogenberger et al Table 3. Ex vivo 5-Azacytidine EC fold-shift enhancement by ABT-737 Primary sample type Maximal 5-Aza EC fold-shift; (ABT-737 dose) Cytogenetics, mutations De novo AML 3.3 (1.3mM) Inv 16, FLT3 neg., NPM1 neg. MDS - AML_1 2.7 (630 nM) Restricted patient info. MDS - AML_2  6.8 (2.5mM) þ 8 (1 of 20), FISH normal, FLT3 neg., NPM1 pos. MDS - AML_3 1.1 (80 nM)  7 (20 of 20, 90% by FISH), NPM1 neg. PMF - AML 2.1 (630 nM) 46XY, JAK2 neg. PV - MDS/MPN 2.0 (1.3mM) 46XY, JAK2V617F pos. RCMD MDS 1.1 (160 nM) Del 20 (8 of 20, 41% by FISH), JAK2 neg. Low-grade MDS 1.5 (1.3mM) 46XX, MDS FISH neg. ET 2.3 (1.3mM) 46XY, JAK2 neg., MPL neg. PV_1 1.4 (310 nM) 46XY, JAK2 pos. PV_2 1.8 (1.3mM) JAK2 pos. Ex vivo 5-Azacytidine fold-shift enhancement by ABT-737. 5-Aza enhancement as determined by maximal 5-Aza EC shifts for the same 11 primary specimens shown in Figure 5 are shown with the dose of ABT-737 at which maximal EC enhancement occurred (shown in brackets), aside clinical cytogenetics and mutations. Although only doses corresponding to maximal enhancement are shown, significant enhancement often occurred at lower doses of ABT-737. Similarly, greater synergy by CI was frequently observed with increasing doses of 5-Aza beyond the EC dose. For example, see the MDS-to-AML_2 sample in Table 3 in comparison with this same sample in Figure 4, noting the antagonistic CI values near the lower, 2.5mM 5-Aza EC dose (points 1–4), yet strong synergistic CI and FE at the higher 7.4mM concentration of 5-Aza (points 5–8). ‘ ’ denotes antagonistic fold-shift. Figure 5. BH3-profiling metrics correlate with clinical 5-Azacytidine-based response. Clinical response to 5-Aza-based therapy (N¼ 22) is plotted against % priming by BH3 profiling for the indicated BH3 peptide(s). Clinicopathologic variables are shown in Supplementary Table 1. & 2014 Macmillan Publishers Limited Leukemia (2014) 1657 – 1665 5-Azacytidine sensitizers and response predictor JM Bogenberger et al Although the aforementioned collection of data suggests ACKNOWLEDGEMENTS partial functional redundancy of BCL-X , BCL-2 and MCL-1 in This work was supported in part by American Cancer Society Postdoctoral Fellowship myeloid malignancies, other interesting distinctions were Grant 119364-PF-10-123-01 awarded to JMB, in part by a grant from the IBIS Foundation for Individualized Medicine awarded to RT, in part by a Mayo Clinic observed in the proteomic data set. Median levels of BCL-2 Career Development award to RT, and in part by NCI-SBIR #HHSN261201200039C and were greater in normal CD34 progenitor cells as compared #HHSN261201299985C contracts to Eutropics Pharmaceuticals. Institutional support with most FAB groups. In contrast, median BCL-X and MCL-1 þ was provided by TGen and Mayo Clinic. levels were lower in normal CD34 progenitor cells. This raises speculation that compounds more selectively targeting BCL-X and MCL-1, without inhibiting BCL-2, could widen a therapeutic AUTHOR CONTRIBUTIONS index in some cases of AML. These preclinical observations can now be validated clinically with the availability of an JMB performed research, designed and executed experiments, analyzed and increasing number of BCL-2 family member-targeting drugs. interpreted data, and wrote the manuscript; SK performed research, RPPA In fact, the first clinical trials combining 5-Aza with BCL-2- experiments and analysis; WEP, RL, and MC performed BH3-profiling assays and targeting compounds are in development (Tibes, personal analysis; DC assisted RNAi assay development and experiments; C-XS assisted communication). with experiments; JM and GA isolated primary patient samples; IG assisted RNAi 41,42 As we previously reported, and recently confirmed by screening experiments; AC and RT performed RNAi screen analysis; RV, JC, VF, another group, we demonstrate formal synergy of ABT-737 and RAM provided primary patient samples and clinical information; YQ and and 5-Aza in AML. Herein, we also show for the first time that KRC performed RPPA experiments and analysis; EB performed research and ABT-737 and 5-Aza synergize in secondary AML arising from analysis; HHY directed assay development and provided essential infrastructure; MDS and MPNs, and in primary MDS, PV and ET samples ex vivo, DOA designed and analyzed experiments, and provided essential infrastructure; providing a preclinical rationale to include MDS and MPN AKS provided essential infrastructure; and RT conceived and directed the patients in future trials of 5-Aza and BCL-2 family-targeting project, performed research, designed experiments, analyzed and interpreted agents. data, and wrote the manuscript. Expression of BCL-2 alone is not sufficient for predicting 23,44 clinical response to navitoclax, although expression signatures of combined antiapoptotic BCL-2 family members REFERENCES may predict response in CLL. However, analyzing expression 1 Mesa RA, Tibes R. 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This work is licensed under a Creative Commons Attribution- The BH3 mimetic ABT-737 targets selective Bcl-2 proteins and efficiently induces NonCommercial-NoDerivs 3.0 Unported License. To view a copy of apoptosis via Bak/Bax if Mcl-1 is neutralized. Cancer Cell 2006; 10: 389–399. this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/ Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu) & 2014 Macmillan Publishers Limited Leukemia (2014) 1657 – 1665 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Leukemia Springer Journals

BCL-2 family proteins as 5-Azacytidine-sensitizing targets and determinants of response in myeloid malignancies

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Springer Journals
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Copyright © 2014 by The Author(s)
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Medicine & Public Health; Medicine/Public Health, general; Internal Medicine; Intensive / Critical Care Medicine; Cancer Research; Oncology; Hematology
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1476-5551
DOI
10.1038/leu.2014.44
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Abstract

Leukemia (2014) 28, 1657–1665 OPEN & 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14 www.nature.com/leu ORIGINAL ARTICLE BCL-2 family proteins as 5-Azacytidine-sensitizing targets and determinants of response in myeloid malignancies 1 2 3 3 4 1 1 1 4 4,6 JM Bogenberger , SM Kornblau , WE Pierceall , R Lena , D Chow , C-X Shi , J Mantei , G Ahmann , IM Gonzales , A Choudhary , 5 1 1 1 2 2 3 1 4 4 1 R Valdez , J Camoriano , V Fauble , RE Tiedemann , YH Qiu , KR Coombes , M Cardone , E Braggio , H Yin , DO Azorsa , RA Mesa , 1 1 AK Stewart and R Tibes Synergistic molecular vulnerabilities enhancing hypomethylating agents in myeloid malignancies have remained elusive. RNA-interference drug modifier screens identified antiapoptotic BCL-2 family members as potent 5-Azacytidine-sensitizing targets. In further dissecting BCL-X , BCL-2 and MCL-1 contribution to 5-Azacytidine activity, siRNA silencing of BCL-X and MCL-1, but not L L BCL-2, exhibited variable synergy with 5-Azacytidine in vitro. The BCL-X , BCL-2 and BCL-w inhibitor ABT-737 sensitized most cell lines more potently compared with the selective BCL-2 inhibitor ABT-199, which synergized with 5-Azacytidine mostly at higher doses. Ex vivo, ABT-737 enhanced 5-Azacytidine activity across primary AML, MDS and MPN specimens. Protein levels of BCL-X , BCL-2 and MCL-1 in 577 AML patient samples showed overlapping expression across AML FAB subtypes and heterogeneous expression within subtypes, further supporting a concept of dual/multiple BCL-2 family member targeting consistent with RNAi and pharmacologic results. Consequently, silencing of MCL-1 and BCL-X increased the activity of ABT-199. Functional interrogation of BCL-2 family proteins by BH3 profiling performed on patient samples significantly discriminated clinical response versus resistance to 5-Azacytidine-based therapies. On the basis of these results, we propose a clinical trial of navitoclax (clinical-grade ABT-737) combined with 5-Azacytidine in myeloid malignancies, as well as to prospectively validate BH3 profiling in predicting 5-Azacytidine response. Leukemia (2014) 28, 1657–1665; doi:10.1038/leu.2014.44 Keywords: 5-Azacytidine; BH3 profiling; myeloid malignancies INTRODUCTION secondary RNAi screen results indicated that inhibition of antiapoptotic BCL-2 family proteins constitutes an important Conventional therapies for poor-risk, elderly and secondary acute concept for modulating 5-Aza antileukemic activity. Antiapoptotic myeloid leukemia (AML), relapsed/refractory high-risk myelodys- BCL-2 family members have been proposed as targets in myeloid plastic syndrome (MDS) and accelerated and transformed malignancies, particularly MCL-1, BCL-2 and BCL-X , and (leukemic blast phase) myeloproliferative neoplasm (MPN) have therapeutic agents for these targets are currently in preclinical limited benefit. Response and long-term outcome with cytotoxic or clinical development. However, there is no direct comparison of chemotherapies are poor. Hypomethylating agents, 5-Azacytidine these antiapoptotic BCL-2 family members as targets in myeloid (5-Aza) and decitabine, demonstrate substantial single-agent 3,4 5 malignancies. Therefore, we focused on further studies to dissect activity in B25–50% of MDS and MPN, and are active in 6–8 how inhibition of BCL-X , BCL-2 and MCL-1, with siRNA and B15–25% of AML. Although these single-agent activities are 9,10 pharmacological inhibitors, modulates the antileukemic activity of encouraging, substantial room for improvement remains. The 5-Aza. These functional studies are complemented by proteomic development of rational combinations with hypomethylating data analyzing BCL-X , BCL-2 and MCL-1 in 577 primary AML agents has been hampered by a lack of precise mechanistic specimens, demonstrating that expression of antiapoptotic family understanding. Furthermore, although promising predictive 11–13 members is heterogeneous within AML FAB subtypes and biomarkers for 5-Aza have recently been reported, there is overlapping across subtypes, suggesting functional redundancy. no clinically validated assay or biomarker predicting response to We present additional evidence that the overall balance of BCL-2 hypomethylating agents. Therefore, we sought to identify family proteins, assessed with a functional BH3-profiling assay, therapeutically exploitable molecular vulnerabilities to design rational combinations with 5-Aza, and, in parallel, to develop predicts clinical response to 5-Aza. The results presented herein biomarkers of response to 5-Aza. have important implications for the design of clinical trials with As a mechanistically unbiased starting point to identify targets BCL-2 family-targeting agents in myeloid malignancies, the future that modulate the antileukemic activity of 5-Aza, we performed development of novel BCL-2 family-targeted agents with distinct replicate RNA-interference (RNAi) sensitizer screens of 861 kinase, inhibitory profiles and the clinical application of 5-Aza in myeloid cell cycle, apoptosis and other cancer-associated genes in myeloid malignancies through the potential development of a predictive cell lines in combination with 5-Aza treatment. Primary and biomarker of response to 5-Aza. 1 2 Department of Hematology and Oncology, Mayo Clinic, Scottsdale, AZ, USA; Section of Molecular Hematology and Therapy, Department of Leukemia, The University of Texas 3 4 M.D. Anderson Cancer Center, Houston, TX, USA; Eutropics Pharmaceuticals, Cambridge, MA, USA; Translational Genomics Research Institute, Phoenix, AZ, USA and Department of Lab Medicine and Pathology, Mayo Clinic, Scottsdale, AZ, USA. Correspondence: Dr R Tibes, Department of Hematology and Oncology, Mayo Clinic, 13400 East Shea Boulevard, Scottsdale, AZ 85259, USA. E-mail: tibes.raoul@mayo.edu Current address: Ashish Choudhary, Asuragen Inc., Austin, TX, USA. Roger Tiedemann, Ontario Cancer Institute, University of Toronto, Toronto, ON, Canada. Received 25 September 2013; revised 21 December 2013; accepted 2 January 2014; accepted article preview online 23 January 2014; advance online publication, 14 February 2014 5-Azacytidine sensitizers and response predictor JM Bogenberger et al MATERIALS AND METHODS AML specimens suspended in 1% FBS, 2 mM EDTA-PBS were stained with primary antibodies CD45-V450 (BD Biosciences, Franklin Lakes, NJ, USA), Cells, culture conditions and reagents CD3-Biotin (BD Biosciences) and CD20-Biotin (eBiosciences, San Diego, CA, Cell lines were obtained from ATCC or DSMZ, and MDS-L was kindly USA), and secondary antibody Streptavidin-APC (BD Biosciences). Speci- provided by Professor Kaoru Tohyama (Kawasaki Medical School, Kurashiki, 14 mens were then permeabilized with digitonin (Sigma-Aldrich) and Japan). Primary specimens were obtained in accordance with incubated with JC-1 mitochondrial dye (Enzo Life Sciences, Farmingdale, Institutional Review Board-approved protocols, separated by Ficoll NY, USA) and peptides (BIM 100mM, BIM 0.1mM, PUMA 100mM, PUMA 10mM, gradient centrifugation and/or treated with ACK lysis buffer before short- NOXA 100mM, BAD 100mM, BMF 100mM, HRK 100mM or PUMA2A 100mM)or term ex vivo culture. All cells were cultured in RPMI-1640 with 10% FBS, with dimethyl sulfoxide (DMSO (1%) or carbonyl cyanide m-chlorophenyl 2mML-glutamine, 100 IU/ml penicillin and 100mg/ml streptomycin hydrazone (CCCP (10mM)) at room temperature. Samples were run in (Invitrogen, Carlsbad, CA, USA) at 37 1C/5% CO . 5-Azacytidine was duplicate except in cases where insufficient viable cells were available. obtained from Sigma-Aldrich (St Louis, MO, USA), ABT-737 from Samples were analyzed on a FACS CantoII (BD Biosciences) using the BD ChemieTek (Indianapolis, IN, USA), and ABT-199 from SeleckChem FACS Diva software (BD Biosciences). The blast population was identified as (Houston, TX, USA). CD45 dim, CD3 and CD20 negative. Intensely stained CD45 cells (mature 20,21 lymphocytes) were excluded from analyses as described previously. siRNA drug-dose-response assays The quantifiable propensity of a pro-apoptotic peptide to induce mitochondrial depolarization relative to an uncoupling reagent control is Selected candidates from 5-Aza RNAi sensitizer screens were validated with siRNA drug-dose-response (siDDR) assays as described previously for referred to as percent priming. For the blast population, this was calculated using the median signal intensity of the PE channel normalized for DMSO primary RNAi screens. All siRNA were obtained from Qiagen, except for as background (negative control), and CCCP served as 100% priming two additional validation BCL-2 siRNA sequences, IDs: s224526 and (positive control). For calculation of % priming, the following formula is s194310 (Silencer Select, Ambion, Carlsbad, CA, USA). Four different utilized. siRNA sequences for each selected target, nonsilencing negative control siRNA, universal lethal positive control siRNA and buffer-transfection Peptide CCCP reagent were included on each 384-well siDDR assay plate. % priming ¼ 1 100 DMSO CCCP Drug-dose-response experiments and CalcuSyn analysis For ABT-737 and ABT-199 combination experiments with 5-Aza, com- Statistical Analysis pounds were added simultaneously and relative cell number was BH3-profiling biomarkers were analyzed by testing the association determined at 96 h with CTG. Prism Version 5.03 software (Prism Software between the biomarker status (% priming) and responder or nonresponder Corporation, Irvine, CA, USA) was used to calculate 5-Aza EC values at classification. Univariate comparisons were made using the Mann–Whitney various concentrations of ABT-737 and ABT-199. Synergy was assessed by test; all reported P-values are two sided. The predictive ability of markers calculating combination index values with CalcuSyn Version 2.1 software was assessed using the area under the curve (AUC) statistic. Analyses were (Biosoft, Cambridge, UK) according to the Chow and Talalay model. performed using SAS software, version 9.2 (SAS Institute Inc., Cary, NC, USA), R version 2.14.2 (R Core Team) and/or Graphpad Prism version 5.04 Cleaved caspase-3 analysis (Prism Software Corporation). Cells were processed according to the Cell Signaling Technology protocol. TF-1 cells were treated for 24 h with 625 nM ABT-737 before addition of RESULTS 1.0mM 5-Aza and fixed at 72 h total. HL-60 was dosed with 500 nM ABT-737 simultaneously with 1.0mM 5-Aza, before fixation at 8, 24 and 48 h. Cells Knockdown of antiapoptotic BCL-2 family proteins results in were incubated for 1 h with cleaved caspase-3 (Asp175)-Alexa Fluor 488 differential effects on viability and 5-Azacytidine sensitization antibody conjugate (Cell Signaling Technology, Danvers, MA, USA) at 1:50 Preliminary RNAi drug modifier screens targeting genes capable of dilution. Fluorescence intensity was measured on a CyAn flow cytometer sensitizing AML-derived cell lines TF-1 and ML-2 to 5-Aza (Beckman Coulter, Pasadena, CA, USA) and data analyzed with Summit identified BCL-2 family members as potential targets. Validation Version 4.3 software (DAKO, Carpinteria, CA, USA). studies confirmed that RNAi-mediated knockdown of BCL-X resulted in three- to fourfold 5-Aza sensitization in TF-1 (data not Protein expression/reverse phase protein array (RPPA) shown). To further explore the role of BCL-2 family members as Proteomic profiling was performed on primary AML specimens using therapeutic targets in myeloid malignancies, we examined the 17,18 validated methods described previously. Primary specimens were role of three major antiapoptotic BCL-2 family proteins, BCL-2, printed in five serial dilutions onto slides with normalization and MCL-1 and BCL-X , and tested their impact on cell proliferation expression controls. Slides were probed with validated primary and sensitivity to 5-Aza. siRNA silencing of BCL-X or MCL-1 antibodies (Cell Signaling Technology; Epitomics, Burlingame, CA, USA) L effectively reduced viability in most AML cell lines examined, at 1:500 dilution and secondary antibody to amplify the signal at 1:15 000 dilution. Stained slides were analyzed using Vigene Tech Microvigene whereas BCL-2 siRNA had less of an effect (Table 1a). In regards to Version 3.4 software (Carlisle, MA, USA) to produce quantified data as 5-Aza sensitization, silencing of BCL-X potently sensitized previously described. erythroid cells TF-1 and HEL (Supplementary Figure 1A), while MCL-1 sensitized more broadly across the AML cells examined mRNA expression from public data sets (Table 1b). Growth inhibitory effects of siRNA silencing alone generally paralleled the effects of siRNA sensitization to 5-Aza. Data from public data sets GEO accession numbers GSE19429, GSE6891, GSE12417 were MAS5 transformed using Expression Console Software Effective siRNA silencing was demonstrated for BCL-X by (Affymetrix, Santa Clara, CA, USA) and subsequently median normalized. reduction in protein levels in cells that were sensitized (TF-1) or The number of cases were as follows: CD34þ (17), MDS (50), M0 (16), M1 were not sensitized (THP-1) (Supplementary Figures 1B and C), (95), M2 (104), M3 (23), M4 (23), M5 (104), M6 (6). The ANOVA test statistical showing that differential 5-Aza sensitization was not due to analysis was performed across all groups, thus the P-value corresponds to differences in knockdown. simultaneous analysis of all French-American-British (FAB) classifications. The Valk and Metzeler2 data sets were obtained from Oncomine. ABT-737 synergizes with 5-Azacytidine more potently than ABT-199 in myeloid cell lines BH3-profiling assay Two therapeutic agents, ABT-263 and ABT-199, directly targeting Viably frozen patient specimens (N¼ 22) consisted of 11 bone marrow and antiapoptotic BCL-2 family members by acting as BH3-domain 11 peripheral blood mononuclear cells. For in vitro experiments, identical mimetics are currently undergoing clinical testing. Thus far, cell line passages were used for BH3-profiling assays and 5-Aza drug-dose- ABT-263 and ABT-199 have been tested primarily for the response experiments, performed simultaneously with the same lot of freshly prepared 5-Aza. treatment of solid tumors and lymphoid malignancies, and their Leukemia (2014) 1657 – 1665 & 2014 Macmillan Publishers Limited 5-Azacytidine sensitizers and response predictor JM Bogenberger et al 22–24 efficacy in myeloid malignancies remains to be determined. sensitization to 5-Aza was mediated by enhancing apoptosis ABT-263 (navitoclax), an orally available analog and the clinical (Supplementary Figures 2C and D). We conclude that, although grade compound of the experimental tool compound ABT-737 BCL-2 inhibition with ABT-199 is effective in myeloid malignancies, with a nearly identical binding profile, inhibits BCL-X , BCL-2 and in most cell lines simultaneous targeting of BCL-X , BCL-2 and Bcl- L L 25,26 BCL-w with K values o1nM. Because of the on-target effects w with ABT-737 is more potent than targeting BCL-2 alone, and in of ABT-263 on BCL-X , a megakaryocytic lineage gene, ABT-263 combination with 5-Aza. induces thrombocytopenia. Recently ABT-199, a more selective inhibitor of BCL-2 that does not inhibit BCL-X at low-to-moderate RNAi silencing of antiapoptotic BCL-2 family proteins in concentrations, has shown promising clinical responses in combination with ABT-199 or ABT-737 lymphoid malignancies, without some of the clinical toxicities of Next we aimed to explore the individual contribution of inhibiting 28,29 ABT-263, particularly thrombocytopenia. BCL-X or MCL-1 in combination with BCL-2. Thus, BCL-X or MCL-1 L L To determine which agent is more potent in myeloid was silenced by siRNA and ABT-199 activity subsequently malignancies, we assessed single-agent activity and 5-Aza assessed. BCL-X or MCL-1 silencing strongly potentiated ABT- sensitization with ABT-737 (the tool compound of ABT-263) versus 199 as shown by average ABT-199 EC fold-shifts (Table 2). MCL-1 ABT-199 across a spectrum of genomically heterogeneous AML has been shown to confer resistance to ABT-737. Conversely, cell lines. ABT-737 exhibited lower single-agent EC values inhibition of MCL-1 enhances ABT-737 sensitivity of several tumor (median 0.14mM for ABT-737 versus 4.3mM for ABT-199) 31 types; however, this has not been shown for myeloid cells. (Figure 1), and resulted in greater 5-Aza sensitization, as Indeed, siRNA silencing of MCL-1 strongly sensitized to ABT-737 determined by EC fold-enhancement and Combination Index (Supplementary Figure 3). These data confirm BCL-X and MCL-1 synergy with CalcuSyn (Figures 2a–c and Supplementary as critical targets in myeloid malignancies, and inhibiting either Figures 2A and B). Generally, higher concentrations of ABT-199, enhances effects of BCL-2 inhibition. than ABT-737, were required to enhance 5-Aza activity (Figure 2b). Moreover, ABT-737 exhibited dose-dependent sensitization in all Protein and mRNA expression of antiapoptotic BCL-2 family AML cells, whereas ABT-199 sensitization was not dose-dependent members in primary AML specimens except in MDS-L (Supplementary Figure 2A). ABT-737 also resulted To assess putative correlations between BCL-2 family expression in greater synergy than ABT-199 in most cell lines, as determined levels and pharmacological/functional siRNA data, we measured by lower combination index values and/or greater Fractional Effect the expression of BCL-X , BCL-2 and MCL-1 in 577 primary AML by CalcuSyn analysis, which again were seen at lower doses of samples using a reverse phase protein array (RPPA) proteomic ABT-737 than ABT-199 (low nM versus low mM concentrations, approach. There was no strong association between BCL-X , BCL-2 respectively) (Supplementary Figure 2B). As expected, ABT-737 or MCL-1 expression and cytogenetic-molecular features (data not shown), consistent with our previous report. However, when analyzed by AML FAB classification, BCL-X was highest in M6, Table 1a. In vitro % viability reduction by siRNA alone whereas BCL-2 was highest in M1 and M0 (Figure 3). Median BCL-2 levels were higher in normal CD34 -selected hematopoietic Cell line siRNA progenitor cells as compared with all FAB classifications except M0, M1 and M4 EOS. In contrast, median BCL-X and MCL-1 levels BCL-2 BCL-X MCL-1 þ were lower in normal CD34 progenitor cells as compared with the majority of FAB classifications. MCL-1 expression was more SET-2 0–2 39–52 22–65 uniformly distributed across FAB classifications than BCL-X or TF-1 0–4 11–71 0–17 BCL-2. There were often strong differences in expression levels of HEL 0–1 21–67 14–39 BCL-X , BCL-2 and MCL-1 within a given FAB subgroup, indicating THP-1 0–21 11–32 33–87 OCI-AML3 0–20 66–71 49–99 significant patient-to-patient variability. Similar trends were ML-2 0–4 11–38 18–35 confirmed by mRNA expression in AML data sets reanalyzed and in public databases (Supplementary Figures 4A–C). Antiapoptotic BCL-2 family siRNA drug-dose-response assays with 5-Azacytidine in myeloid cells in vitro. Cell lines are listed in the left-most column. The BCL-2 family member silenced by siRNA is shown as the 5-Azacytidine and ABT-737 exhibit synergy ex vivo across a heading for each column. The range of % viability reduction due to the spectrum of myeloid malignancies: AML, MDS and MPNs given siRNA alone (without 5-Aza) is shown (0¼ no affect and 100¼ To demonstrate that inhibition of antiapoptotic BCL-2 family maximal reduction in viability). proteins is a potent 5-Aza sensitization strategy for AML and Table 1b. In vitro 5-Azacytidine EC fold-shift enhancement by siRNA Cell line siRNA BCL-2 BCL-X MCL-1 ± ± ± SET-2 1.0 0.0 (P¼ 0.20) 1.4 0.2 (P¼ 0.052) 1.1 0.1 (P¼ 0.14) ± ± ± TF-1 1.2 0.1 (P¼ 0.011) 3.4 0.7 (P¼ 3.5e–5) 1.6 0.1 (P¼ 0.061) ± ± ± HEL 1.2 0.1 (P¼ 0.38) 4.4 1.8 (P¼ 0.031) 3.0 0.4 (P¼ 0.086) ± ± ± THP-1 1.0 0.1 (P¼ 0.82) 1.1 0.2 (P¼ 0.079) 1.2 0.0 (P¼ 0.076) ± ± ± OCI-AML3 1.2 0.5 (P¼ 0.61)  1.1 0.2 (P¼ 0.016) 2.2 0.8 (P¼ 0.056) ± ± ± ML-2 1.0 0.0 (P¼ 0.35) 1.1 0.1 (P¼ 0.026) 1.4 0.0 (P¼ 0.024) Anti-apoptotic BCL-2 family siRNA drug-dose-response assays with 5-Azacytidine in myeloid cells in vitro. Cell lines are listed in the left-most column. The BCL-2 family member silenced by siRNA is shown as the heading for each column. 5-Aza EC fold-shifts are listed with P values associated with EC fold-shift 50 50 measurements averaged for the different siRNA sequences against each BCL-2 family member. ‘ ’ denotes antagonistic fold-shift. & 2014 Macmillan Publishers Limited Leukemia (2014) 1657 – 1665 5-Azacytidine sensitizers and response predictor JM Bogenberger et al myeloid malignancies including MDS and MPNs, we evaluated the secondary AML (prior MDS or PMF) (Figure 4 and Table 3). We combination of ABT-737 with 5-Aza in short-term ex vivo cultures. reasoned that the combination would be particularly effective in ABT-737 sensitizes primary specimens from de novo AML and MPNs, given evidence for BCL-X as a lineage-specific proto- 32,33 oncogene, and its importance in erythroid progenitors. Indeed, primary samples from MDS and MPN patients were sensitive to the combination, exhibiting strong synergy (Figure 4 and Table 3). BH3 profiling discriminates clinical response to 5-Azacytdine- based therapies Although there are protein expression differences for BCL-X ,BCL-2 and MCL-1 by RPPA, within and between AML FAB subtypes, we did not find a strong correlation with clinical response to 5-Aza-based therapies (data not shown). This may be expected considering possible mechanistic distinctions and functional redundancies amongst the different antiapoptotic BCL-2 family members exhibiting overlapping expression patterns, as well as the distinct, yet overlapping binding affinities between anti- and proapoptotic 36,37 proteins. Thus, we aimed to functionally interrogate the overall balance of pro- and antiapoptotic BCL-2 family proteins, a cellular- Figure 1. Single-agent BCL-2 family inhibitor activity in AML cell molecular characteristic described as ‘primedness’, by BH3 lines. Seven AML-derived cells lines (TF-1, HEL, THP-1, U937, ML-2, profiling. This assay uses peptides derived from proapoptotic HL-60 and MDS-L) are plotted for ABT-737, whereas 11 are plotted BH3-only proteins and determines the degree to which these for ABT-199 activity (UKE-1, SET-2, M07e, TF-1, HEL, THP-1, ML-2, OCI- peptides induce apoptosis. As proof-of-concept in vitro,several AML3, OCI-AML2, HL-60 and MDS-L). Six cell lines were tested with both compounds (TF-1, HEL, THP-1, ML-2, HL-60 and MDS-L). BH3-profiling metrics significantly correlated with 5-Aza sensitivity ABT-737 ABT-199 12.0 10.1 10.0 8.0 8.0 6.0 4.8 3.8 3.4 3.3 4.0 4 30 2.9 2.1 2.0 1.8 1.6 2.0 0.0 -2.0 SET-2 M07e TF-1 THP-1 ML-2 HL-60 MDS-L -1.1 -1.1 Cell Lines ABT-737 ABT-199 2000 2000 2000 261 261 78 80 SET-2 M07e TF-1 THP-1 ML-2 HL-60 MDS-L Cell Lines Cell Line: SET-2 M07e TF-1 THP-1 ML-2 HL-60 MDS-L ABT Compound: 737 199 737 199 737 199 737 199 737 199 737 199 737 199 CI Value* 0.81 1.25 0.18 0.17 0.50 1.10 0.05 0.09 0.09 0.90 0.30 0.44 0.51 0.48 Figure 2. ABT-737 compared with ABT-199 in vitro synergy with 5-Azacytidine. (a) Maximal 5-Aza EC fold-shifts for ABT-737 and ABT-199 are shown side-by-side for each cell line. These EC fold-shifts are a ‘one-sided’ measurement of 5-Aza enhancement. (b) Corresponding ABT-737 or ABT-199 doses at which maximal 5-Aza EC fold-shifts occurred are shown side-by-side. (c) *CalcuSyn Combination Index (CI) values corresponding to the greatest synergy (thus doses shown in b) nearest to the 5-Aza EC dose are listed. It is important to note that CI values are a ‘two-sided’ measurement of drug synergy for two specific doses, thus a single CI value is not a universal characteristic of the interaction between two drugs because interactions can be dose dependent. See Supplementary Figure 2B for an extensive data set of CI values across multiple dose combinations plotted against the corresponding Fractional Effect, where 0¼ no effect and 1.0¼ maximal effect. CI values o0.8 indicate synergy, whereas CI values 41.1 indicate antagonism. Leukemia (2014) 1657 – 1665 & 2014 Macmillan Publishers Limited nM Dose of ABT Compounds At Maximal 5-Aza EC Fold- Enhancement Maximal 5-Aza EC Fold-Shift 5-Azacytidine sensitizers and response predictor JM Bogenberger et al (Supplementary Figure 5). To correlate clinical responses to 5-Aza underlying mechanisms of many therapeutic agents, including the 39,40 with BH3 profiling, specimens from 22 AML, MDS and MDS/MPN hypomethylating agent 5-Aza, are still poorly understood. This overlap patients treated with 5-Aza-based regimens, and for whom lack of mechanistic understanding has impeded development of clinical outcome was available, were assayed (Supplementary novel rational combinations. We aimed to circumvent these Table 1). Clinically, the best BH3 metric comprised combined values challenges by employing a previously successful RNAi sensitizer from NOXA and BIM peptides, which discriminated clinical screening approach as a mechanistically unbiased strategy to responses, defined as achieving a response (sensitive) versus a discover genes that modulate sensitivity to 5-Aza. Primary and patient being resistance/refractory, with statistical significance secondary RNAi screens indicated that inhibition of antiapoptotic (Mann–Whitney two-tailed P¼ 0.001) and an area under the BCL-2 family proteins constitutes an important concept for receiver operating characteristic curve of 0.950 (Figure 5). sensitizing myeloid cells to 5-Aza. Direct comparison of BCL-X , MCL-1 and BCL-2 inhibition by siRNA and pharmacologically suggests that, although inhibition of individual antiapoptotic BCL-2 proteins (that is, BCL-2 with DISCUSSION ABT-199) can be effective alone or in combination with 5-Aza, Therapeutic progress in the treatment of myeloid malignancies inhibiting more than one BCL-2 member simultaneously (that is, has been slow. Despite increased molecular knowledge, the BCL-X , BCL-2 and BCL-w with ABT-737, or siRNA against MCL-1 or BCL-X combined with ABT-199) has more potent antileukemic activity. Importantly, dual or multiple BCL-2 family targeting sensitizes to 5-Aza across a broader range of AML cell lines. Table 2. In vitro ABT-199 EC fold-shift enhancement by siRNA Proteomic data from 577 primary AML samples showed over- lapping expression of BCL-X , MCL-1 and BCL-2 across samples for Cell line siRNA the FAB subtypes. This overlap of expression, together with siRNA and pharmacological data, suggests functional redundancy BCL-X MCL-1 between BCL-X , BCL-2 and MCL-1 in myeloid malignancies. Thus, ± ± SET-2 1.2 0.1 (P¼ 0.032) 1.5 0.3 (P¼ 0.049) we conclude that two or more antiapoptotic BCL-2 family ± ± TF-1 1.6 0.2 (P¼ 0.0073) 2.3 1.0 (P¼ 0.076) members determine the apoptotic threshold and response to ± ± THP-1 4.2 2.8 (P¼ 0.11) 8.5 2.4 (P¼ 0.0079) 5-Aza in most AML cases. Consequently, dual or multiple ± ± OCI-AML3 2.4 1.0 (P¼ 0.067) 6.9 0.9 (P¼ 0.0084) simultaneous antiapoptotic BCL-2 family protein targeting may have greater antileukemic activity alone or with 5-Aza. Also BCL-XL and MCL-1 siRNA knockdown combined with ABT-199 drug-dose- supporting this concept, we show that knockdown of BCL-X or response in myeloid cells in vitro. Cell lines are listed in the left-most L column. The BCL-2 family member silenced by siRNA before assessing ABT- BCL-2 alone with siRNA did not significantly sensitize THP-1 or 199 drug-dose-response is shown as the heading for each column. ABT-199 ML-2 to 5-Aza; however, simultaneous inhibition of BCL-X , BCL-2 EC fold-shifts, determined relative to non-silencing siRNA, are listed with and BCL-w by ABT-737 resulted in potent 5-Aza sensitization in P-values associated with EC fold-shift measurements averaged for four these cells. In addition, siRNA knockdown of BCL-X or MCL-1 different siRNA sequences against each BCL-2 family member. strongly sensitized AML cell lines to BCL-2 inhibition with ABT-199, Figure 3. BCL-XL, MCL-1 and BCL-2 protein expression in primary specimens determined by Reverse Phase Protein Array. 577 primary AML patient samples are shown grouped by AML FAB classification. & 2014 Macmillan Publishers Limited Leukemia (2014) 1657 – 1665 5-Azacytidine sensitizers and response predictor JM Bogenberger et al De novo AML (FAB M4) MDS → AML_1 (FAB Unavailable) MDS → AML_2 (FAB M1) MDS → AML_3 (FAB M6) 5-Aza + ABT-737 - Algebraic estimate 5-Aza + ABT-737 - Algebraic estimate 5-Aza + ABT-737 - Algebraic estimate 5-Aza + ABT-737 - Algebraic estimate 1.0 1.0 2.0 1.0 0.8 0.8 0.8 1.5 0.6 0.6 0.6 1.0 0.4 0.4 0.4 0.5 0.2 0.2 0.2 0 0 0 0 0.2 0.4 0.6 0.8 1.0 00.2 0.4 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 1.0 Fractional Effect Fractional Effect Fractional Effect Fractional Effect PMF → AML (FAB M4) PV → MDS/MPN overlap RCMD MDS Low-grade MDS 5-Aza + ABT-737 - Algebraic estimate 5-Aza + ABT-737 - Algebraic estimate 5-Aza + ABT-737 - Algebraic estimate 5-Aza + ABT-737 - Algebraic estimate 1.0 1.0 1.0 2.0 0.8 0.8 0.8 1.5 0.6 0.6 0.6 1.0 0.4 0.4 0.4 0.5 0.2 0.2 0.2 0 0 0 0 00.2 0.4 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 1.0 Fractional Effect Fractional Effect Fractional Effect Fractional Effect ET PV_1 PV_2 5-Aza + ABT-737 - Algebraic estimate 5-Aza + ABT-737 - Algebraic estimate 5-Aza + ABT-737 - Algebraic estimate 1.0 1.0 1.0 0.8 0.8 0.8 0.6 0.6 0.6 0.4 0.4 0.4 0.2 0.2 0.2 0 0 0 0 0.2 0.4 0.6 0.8 1.0 00.2 0.4 0.6 0.8 1.0 00.2 0.4 0.6 0.8 1.0 Fractional Effect Fractional Effect Fractional Effect MDS→AML_1, ET, PV1 & 2: PV→MDS/MPN overlap: 5-Aza [uM] 5-Aza [uM] 7.4 22.2 66.7 1.0 3.1 9.3 27.8 83.3 78 48 12 78 48 12 16 20 156 3 3 7 7 11 156 37 7 11 15 19 313 2 2 6 10 313 2 6 10 14 18 625 1 5 9 625 1 5 9 13 17 PMF→AML, de novo AML, MDS→AML_3(M6), RCMD MDS, MDS→AML_2: Low-grade MDS: 5-Aza [uM] 5-Aza [uM] 2.5 7.4 22.2 66.7 0.8 2.5 7.4 22.2 66.7 78 4 8 12 16 78 4 8 12 16 20 1563 71115 156 3 71115 19 313 2 6 10 14 313 2 61014 18 625 1 5 9 13 625 1 5 9 13 17 Figure 4. 5-Azacytidine and ABT-737 synergistically combine in primary myeloid malignancy specimens (N¼ 11). Combination Index (CI) versus Fractional Effect (FE) plots were calculated using CalcuSyn Software Version 2.1. The numbers in the tables (Figure 4 key) correspond to the dose combinations shown on the CI versus FE plots for the indicated primary specimens. CI values o0.8 indicate synergy, whereas CI values 41.1 indicate antagonism. demonstrating that BCL-X and MCL-1 can cooperate with BCL-2 200-fold lower ABT-737 doses in five of seven AML cell lines). in governing apoptotic response. Furthermore, ABT-737 generally Single-agent activity of BCL-2 family inhibitors in AML cell lines resulted in broader and more potent sensitization than ABT-199, confirms this trend, as the median ABT-737 EC dose was found which occurred at lower doses of ABT-737 than ABT-199 (7- to to be 430-fold lower than that of ABT-199. Leukemia (2014) 1657 – 1665 & 2014 Macmillan Publishers Limited CI +/- 1.96 s.d. CI +/- 1.96 s.d. CI +/- 1.96 s.d. ABT-737 [nM] ABT-737 [nM] CI +/- 1.96 s.d. CI +/- 1.96 s.d. CI +/- 1.96 s.d. ABT-737 [nM] ABT-737 [nM] CI +/- 1.96 s.d. CI +/- 1.96 s.d. CI +/- 1.96 s.d. CI +/- 1.96 s.d. CI +/- 1.96 s.d. 5-Azacytidine sensitizers and response predictor JM Bogenberger et al Table 3. Ex vivo 5-Azacytidine EC fold-shift enhancement by ABT-737 Primary sample type Maximal 5-Aza EC fold-shift; (ABT-737 dose) Cytogenetics, mutations De novo AML 3.3 (1.3mM) Inv 16, FLT3 neg., NPM1 neg. MDS - AML_1 2.7 (630 nM) Restricted patient info. MDS - AML_2  6.8 (2.5mM) þ 8 (1 of 20), FISH normal, FLT3 neg., NPM1 pos. MDS - AML_3 1.1 (80 nM)  7 (20 of 20, 90% by FISH), NPM1 neg. PMF - AML 2.1 (630 nM) 46XY, JAK2 neg. PV - MDS/MPN 2.0 (1.3mM) 46XY, JAK2V617F pos. RCMD MDS 1.1 (160 nM) Del 20 (8 of 20, 41% by FISH), JAK2 neg. Low-grade MDS 1.5 (1.3mM) 46XX, MDS FISH neg. ET 2.3 (1.3mM) 46XY, JAK2 neg., MPL neg. PV_1 1.4 (310 nM) 46XY, JAK2 pos. PV_2 1.8 (1.3mM) JAK2 pos. Ex vivo 5-Azacytidine fold-shift enhancement by ABT-737. 5-Aza enhancement as determined by maximal 5-Aza EC shifts for the same 11 primary specimens shown in Figure 5 are shown with the dose of ABT-737 at which maximal EC enhancement occurred (shown in brackets), aside clinical cytogenetics and mutations. Although only doses corresponding to maximal enhancement are shown, significant enhancement often occurred at lower doses of ABT-737. Similarly, greater synergy by CI was frequently observed with increasing doses of 5-Aza beyond the EC dose. For example, see the MDS-to-AML_2 sample in Table 3 in comparison with this same sample in Figure 4, noting the antagonistic CI values near the lower, 2.5mM 5-Aza EC dose (points 1–4), yet strong synergistic CI and FE at the higher 7.4mM concentration of 5-Aza (points 5–8). ‘ ’ denotes antagonistic fold-shift. Figure 5. BH3-profiling metrics correlate with clinical 5-Azacytidine-based response. Clinical response to 5-Aza-based therapy (N¼ 22) is plotted against % priming by BH3 profiling for the indicated BH3 peptide(s). Clinicopathologic variables are shown in Supplementary Table 1. & 2014 Macmillan Publishers Limited Leukemia (2014) 1657 – 1665 5-Azacytidine sensitizers and response predictor JM Bogenberger et al Although the aforementioned collection of data suggests ACKNOWLEDGEMENTS partial functional redundancy of BCL-X , BCL-2 and MCL-1 in This work was supported in part by American Cancer Society Postdoctoral Fellowship myeloid malignancies, other interesting distinctions were Grant 119364-PF-10-123-01 awarded to JMB, in part by a grant from the IBIS Foundation for Individualized Medicine awarded to RT, in part by a Mayo Clinic observed in the proteomic data set. Median levels of BCL-2 Career Development award to RT, and in part by NCI-SBIR #HHSN261201200039C and were greater in normal CD34 progenitor cells as compared #HHSN261201299985C contracts to Eutropics Pharmaceuticals. Institutional support with most FAB groups. In contrast, median BCL-X and MCL-1 þ was provided by TGen and Mayo Clinic. levels were lower in normal CD34 progenitor cells. This raises speculation that compounds more selectively targeting BCL-X and MCL-1, without inhibiting BCL-2, could widen a therapeutic AUTHOR CONTRIBUTIONS index in some cases of AML. These preclinical observations can now be validated clinically with the availability of an JMB performed research, designed and executed experiments, analyzed and increasing number of BCL-2 family member-targeting drugs. interpreted data, and wrote the manuscript; SK performed research, RPPA In fact, the first clinical trials combining 5-Aza with BCL-2- experiments and analysis; WEP, RL, and MC performed BH3-profiling assays and targeting compounds are in development (Tibes, personal analysis; DC assisted RNAi assay development and experiments; C-XS assisted communication). with experiments; JM and GA isolated primary patient samples; IG assisted RNAi 41,42 As we previously reported, and recently confirmed by screening experiments; AC and RT performed RNAi screen analysis; RV, JC, VF, another group, we demonstrate formal synergy of ABT-737 and RAM provided primary patient samples and clinical information; YQ and and 5-Aza in AML. Herein, we also show for the first time that KRC performed RPPA experiments and analysis; EB performed research and ABT-737 and 5-Aza synergize in secondary AML arising from analysis; HHY directed assay development and provided essential infrastructure; MDS and MPNs, and in primary MDS, PV and ET samples ex vivo, DOA designed and analyzed experiments, and provided essential infrastructure; providing a preclinical rationale to include MDS and MPN AKS provided essential infrastructure; and RT conceived and directed the patients in future trials of 5-Aza and BCL-2 family-targeting project, performed research, designed experiments, analyzed and interpreted agents. data, and wrote the manuscript. Expression of BCL-2 alone is not sufficient for predicting 23,44 clinical response to navitoclax, although expression signatures of combined antiapoptotic BCL-2 family members REFERENCES may predict response in CLL. However, analyzing expression 1 Mesa RA, Tibes R. 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This work is licensed under a Creative Commons Attribution- The BH3 mimetic ABT-737 targets selective Bcl-2 proteins and efficiently induces NonCommercial-NoDerivs 3.0 Unported License. To view a copy of apoptosis via Bak/Bax if Mcl-1 is neutralized. Cancer Cell 2006; 10: 389–399. this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/ Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu) & 2014 Macmillan Publishers Limited Leukemia (2014) 1657 – 1665

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LeukemiaSpringer Journals

Published: Jan 23, 2014

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