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News Article | October 26, 2016
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Lapatinib, PLX-4032, trametenib, tarceva, ABT-199 and ABT-263 were purchased from Selleck-chem; QVD-OPh from Sigma; MG132 from Calbiochem; idarubicin and araC from Pharmacia and Upjohn. A-1210477 was made according to published methods26. Synthesis and characterization of S63845 is provided in the Supplementary Methods. Owing to light sensitivity, S63845 was stored in the dark. Following the previously published structure of MCL1 (PDB ID4WGI)43, a construct was designed with residues 173–321 of human MCL1 as a C-terminal fusion with maltose binding protein (MBP). In addition to the surface entropy-reducing (SER) mutations in MCL1 (K194A, K197A and R201A (ref. 43)), we also introduced E198A, E199A and K265A mutations into MBP (ref. 44). The plasmid encoding the MBP–MCL1 fusion protein was transformed into BL21(DE3)pLysS bacteria. A single colony was used to inoculate 5 ml terrific broth (Fisher BioReagents, (BP2468-2)) containing kanamycin and chloramphenicol at 100 μg ml−1 and 34 μg ml−1, respectively. After 3 h growth at 37 °C, the 5 ml culture was used to inoculate 2 l terrific broth containing the same antibiotics. At an OD of 0.7, the temperature was reduced to 18 °C before induction of MBP–MCL1 protein expression by addition of IPTG to a final concentration of 1 mM. Cells were harvested by centrifugation. Harvested cells were resuspended in 3 volumes of 20 mM Tris–HCl pH 7.4, 200 mM NaCl, 2 mM EDTA, 1 mM DTT and lysed by passing three times through an emulsiflex-C5 (Avestin). The lysate was clarified by centrifugation at 40,000 g, at 4 °C, for 60 min and applied to a 5-ml MBPTrap column (GE Healthcare). The MBP–MCL1 fusion protein was eluted in 20 mM Tris-HCl pH 7.4, 200 mM NaCl, 2 mM EDTA, 1 mM DTT, 10 mM maltose and further purified by size exclusion chromatography in 20 mM HEPES, 100 mM NaCl and 1 mM DTT. Protein eluted as a monomer was concentrated and used in crystallization studies. Apo crystals were grown at a concentration of 34 mg ml−1 (20 mM HEPES pH 7.5, 150 mM NaCl and 2 mM DTT) by the sitting drop vapour diffusion. 2 μl of the protein solution was mixed with 2 μl of the crystallization reservoir (25% PEG 3350, 0.2 M magnesium formate, 1 mM maltose) in a sitting drop plate. The plate was incubated at 284 K and suitable rod-like crystals appeared overnight. Individual crystals were harvested from the crystallization drops and transferred to a drop containing 4.5 μl of the crystallization reservoir solution plus 0.5 μl of S63845 (20 mM in DMSO). The mixture was incubated for 72 h at 284 K. Crystals were flash frozen in liquid nitrogen after cryoprotection using crystallization reservoir plus 20% ethylene glycol. Diffraction data were collected at the Soleil Synchrotron (France) on a beamline Proxima1 and were processed and scaled using XDS (ref. 45). The structure was solved by molecular replacement using MOLREP (ref. 46), using another crystal structure of an MBP–MCL1 fusion protein43. The data were subsequently refined using REFMAC5 (ref. 47). Interactive graphical model building was carried out with COOT. The ligand was clearly defined by the initial electron density maps. The progress of the refinement was assessed using R and the conventional R factor. Once refinement was completed, the structures were validated using various programs from the CCP4i package, CCP4. Statistic parameters are detailed in Extended Table 1. Fluorescence polarization assays were carried out in black-walled, flat-bottomed, low-binding, 384-well plates (Corning) in buffer A (10 mM HEPES, 150 mM NaCl, 0.05% Tween 20 pH 7.4 and 5% DMSO) in the presence of 10 nM fluorescein-PUMA (3-(((3′,6′-dihydroxy-3-oxo-3H-spiro(2-benzofuran-1,9′-xanthene)-5-yl)carbamothioyl)amino)-N-(6-oxohexyl)propanamide-AREIGAQLRRMADDLNAQY, from the polypeptide group, France). Final concentrations of MCL1, BCL-2 and BCL-X proteins were 10, 10 and 20 nM, respectively. The assay plates were incubated for 2 h at room temperature and the fluorescence polarization was measured on a Synergy 2 reader (exitation, 528 nm; emission, 640 nm; cut-off, 510 nm). The binding of increasing doses of the compound was expressed as a percentage reduction in mP compared to the window established between the ‘DMSO only’ and ‘total inhibition’ control (30 μM PUMA). The inhibitory concentrations of the drugs that gave a 50% reduction in mP (IC ) were determined, from 11-point dose response curves, in XL-Fit using a 4-parameter logistic model (Sigmoidal dose–response model). The K was subsequently calculated as described in ref. 48. All SPR measurements were performed on a BIAcore T200 instrument (BIAcore GE Healthcare). Direct binding experiments were performed at 20 °C on Series S NTA chips. 10 mM HEPES pH 7.4, 175 mM NaCl, 25 μM EDTA, 1 mM TCEP, 0.01% P20 and 1% DMSO was used as a running buffer (buffer B). The ligand surface was generated using double His-tagged proteins essentially as described in refs 49, 50. Serial dilutions of the compound in buffer B were injected over the protein surface. All sample measurements were performed at a flow rate of 30 μlper min (injection time 120 s, dissociation time 360 s). The sensor surface was regenerated by consecutive injections of 0.35 M EDTA pH 8.0 with 0.1 mg/ml−1 trypsin, 0.5 M imidazole and 45% DMSO (60 s, 15 μl per min). Data processing was performed using BIAevaluation 2.1 (BIAcore GE Healthcare Bio-Sciences Corp) software. Sensorgrams were double referenced before global fitting of the concentration series to a 1:1 binding model. Affinity determination by competition in solution experiments were performed at 30 °C in 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 1 mM TCEP, 2% glycerol, 0.05% P20 and 1% DMSO (buffer C). An MCL1-specific compound was immobilized on Series S CM5 chips by amine coupling as advised in the BIAcore GE Healthcare protocol. Serial dilutions of compounds in buffer B supplemented with fixed concentrations of protein were injected over the generated surface at a flow rate of 15 μl per min for 90 s. Calibration curves were generated using the same procedure by injecting different concentrations of protein over the same sensor chip. Affinity evaluations were performed using the affinity in solution model of BIA evaluation 2.1 (BIAcore GE Healthcare Bio-Sciences Corp) software. Mice were kept in either the Servier Research Institute or the Walter and Eliza Hall Institute (WEHI) specified pathogen-free animal areas for mouse experimental purpose (for Servier, facility license number B78-100-2). The care and use of animals was in accordance with European and national regulations for the protection of vertebrate animals used for experimental and other scientific purposes (directives 86/609 and 2003/65) and the requirements of the Servier Research Institute and WEHI Animal Ethics Committees. Sample sizes were chosen to reach statistical significance, and tumour measurements and all data analysis were performed in a blinded fashion. The Eμ-Myc transgenic mice are kept on a C57BL/6–Ly5.2+ background and have been described previously51. 8–10 week old female SCID mice (for transplantation with human AMO1 and H929 tumour cells) or Swiss Nude mice (Crl:NU(Ico)-Foxn1nu) (for transplantation with human MV4-11 tumour cells) were inoculated with 0.1 ml containing 5 × 106 of the indicated tumour cells subcutaneously into the right flank. The H929 and MV4-11 cells were resuspended in 100% matrigel (BD Biosciences) and the AMO1 cells in a 50:50 mixture of growth medium and matrigel. The width and length of the tumours were measured 2–3 times a week using an electronic caliper. Tumour volume was calculated using the formula: (length × width2)/2. When the tumour volume reached approximately 200 mm3, mice were randomized into different groups; that is, treatment with drug (different concentrations) or vehicle (n = 8 for each group). S63845 was formulated extemporaneously in 25 mM HCl, 20% 2-hydroxy propyl β-cyclo dextrin 20% (Fisher Scientifics) and administrated at the doses and schedules described in the figure legends. Tumour growth inhibition (TGI ) was calculated at the greatest response using the following equation: where day x is the day maximum where the number of animals per group in the control group is sufficient to calculate the TGI (%). For the statistical analysis of differences in tumour volume between treatment groups, a two-way ANOVA with repeated measures on day factor was performed on log-transformed data followed by Dunnett adjustment in order to compare each dose of drug to the control group. A complete tumour regression response was considered for the population with tumours 25 mm3 for at least three consecutive measurements. For ethical reasons, mice carrying tumours exceeding 2,000 mm3 were euthanized. Data are represented as mean of tumour volume ± s.e.m. over time (days) until at least one mouse per cohort had to be killed. Single-cell suspensions of 106 Eμ-Myc lymphoma cell lines (AH15A, AF47A, BRE966, 2253, MRE 721, 560), resuspended in phosphate-buffered saline (PBS), were injected into the tail vein of 8–9 week old female C57BL/6–Ly5.1+ mice. Mice were treated with either vehicle (25 mM HCl, 20% 2-hydroxy propyl β-cyclo dextrin) or 25 mg kg−1 S63845 (reconstituted in vehicle) on days 5–9 after transplant, administered by tail vein injection or, in some incidences when the tails became damaged, by retro-orbital injection. To generate the survival curves of the mice bearing the Eμ-Myc lymphoma cells, mice were killed when deemed unwell by experienced animal technicians. For the toxicity experiments, female C57BL/6–Ly5.1+ mice bearing Eμ-Myc lymphomas or non-tumour bearing C57BL/6–Ly5.1+ mice were killed 4 days after the 5-day drug treatment regimen had been completed (this equated to 13 days after transplantation of the tumour cells in the mice bearing the Eμ-Myc lymphoma cells). For the three mice injected with the AH15A Eμ-Myc lymphoma cells, those treated with vehicle were analysed after only 4 days of treatment because they were deemed too unhealty from the lymphoma to complete their prescribed regimen. For the maximal tolerated dose experiments, 7–8 week old C57BL/6 mice (3 male and 3 female mice in each arm) were treated with a dose of vehicle or S63845 (25 mg per kg, 40 mg per kg, 50 mg per kg or 60 mg per kg body weight) for 5 consecutive days by i.v. tail vein injection or by retro-orbital injection if the tails became damaged. The mice were analysed as they were killed, or for the mice surviving the entire course of treatment, 3 days after the 5-day treatment had been completed. For the initial toxicity studies, sections of spleen, lymph nodes, thymus, ovaries, uterus, kidneys, liver, pancreas, intestines, colon, heart, lung, sternum, backbone and muscle were fixed in 10% formalin and stained with haematoxylin and eosin. The weights of the spleen, thymus, (axillary, brachial and inguinal) lymph nodes, liver and kidneys were recorded. Cells were flushed from the bone marrow (two femurs and one tibia) and single cell suspensions of the spleen, thymus and lymph nodes were generated. The red blood cells were lysed by treatment with 0.168 M ammonium chloride and the white blood cell count was determined using a CASY cell counter (Scharfe System GmbH). All bone marrow or peripheral blood samples from patients with AML were collected after informed consent in accordance with guidelines approved by the Alfred and Royal Melbourne Hospital human research ethics committees. Mononuclear cells were isolated by Ficoll-Paque (GE Healthcare) density-gradient centrifugation, followed by red cell depletion in ammonium chloride (NH Cl) lysis buffer at 37 °C for 10 min. Cells were then resuspended in PBS containing 2% fetal bovine serum (FBS, Sigma). Mononuclear cells were suspended in RPMI-1640 (Gibco) medium containing penicillin and streptomycin (Gibco) and 15% heat-inactivated FBS (Sigma). Normal CD34+ progenitor cells from healthy donors were collected from granulocyte colony stimulating factor (G-CSF)-mobilized blood harvests and purified after Ficoll separation by CD34 positive selection using Miltenyi Biotec micobeads (Miltenyi Biotec. Cat. No. 130-046-703). The research with primary human cells was approved by the Human Research Ethics Committee (HREC) of Alfred Health. AML cells from patients and cells from normal donors were obtained following informed consent processes approved by the HRECs of Alfred Health and Melbourne Health. Colony-forming assays were performed on freshly purified and frozen mononuclear fractions from AML patients or normal cells from G-CSF mobilized normal, healthy donors. Primary cells were cultured in duplicate in 35-mm dishes (Griener-bio) at 104 to 105 cells. Cells were plated in 0.6% agar (Difco): AIMDM 2× (IMDM powder, Invitrogen), supplemented with NaHCO , dextran, penicillin and streptomycin, β-mercaptoethanol and asparagine, FBS (Sigma) at a 2:1:1 ratio of agar:AIMDM:FBS. For optimal growth conditions, all plates contained granulocyte/macrophage colony stimulating factor (100 ng per plate, genzyme), IL-3 (100 ng per plate, R&D Systems), stem cell factor (100 ng per plate, R&D Systems) and erythropoietin (4U per plate, Roche). Cells were cultured for 2–3 weeks in the presence or absence of drugs at 37 °C at 5% CO in a high humidity incubator. After incubation, plates were fixed with 2.5% glutaraldehyde in saline and scored using GelCount (Oxford Optronix). NCI-H929, RS4;11, MV4-11, HCT-116, BT-474, SK-Mel-2, PC-9 and H146 cells were cultured in RPMI 1640 medium, A2058 cells were cultured in DMEM medium and SK-MEL-28 cells were cultured in EMEM medium. All media were supplemented with 10% heat-inactivated FBS, 2 mM l-glutamine, 100 U ml−1 penicillin, 100 μg ml−1 streptomycin, and 10 mM HEPES pH = 7.4, at 37 °C, in 5% CO . For RS4;11 cells the medium was additionally supplemented with 4.5 g l−1 glucose. AMO1 cells were cultured in RPMI 1640 medium supplemented with 20% heat-inactivated FBS, 2 mM l-glutamine, 100 U ml−1 penicillin, 100 μg ml−1 streptomycin, and 10mM HEPES pH = 7.4. HeLa cells were cultured in DMEM medium (containing 10% heat-inactivated FBS, 10 mM HEPES, 100 U ml−1 penicillin, 100 μg ml−1 streptomycin). Cells were grown at 37 °C in a humidified atmosphere with 5% CO . All of the cell lines were purchased from the ATCC or DSMZ. Human Burkitt lymphoma (BL)-derived cell lines (Rael-BL, Kem-BL, BL2, BL30, BL31, BL41, and Ramos-BL, a gift from A.B. Rickinson and M. Rowe, University of Birmingham, UK) were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 1 mM glutamine, 1 mM sodium pyruvate, 50 μM α-thiogycerol (Sigma), and 20 nM bathocuproine disulfonic acid (Sigma) in a humidified incubator at 37 °C, 5% CO . The mouse Eμ-Myc lymphoma cell lines (AH15A, AF47A, 2253, BRE966, MRE 721 and 560) were cultured in high-glucose DMEM supplemented with 10% heat-inactivated FBS, 50 μM β-mercaptoethanol (Sigma), 100 μM asparagine (Sigma), 100 U ml−1 penicillin and 100 mg ml−1 streptomycin in a humidified incubator at 37 °C, 10% CO . The myeloma-derived cell lines were purchased from the ATCC, DSMZ or JCRB or provided by the laboratory of A. Spencer (XG1, KMS-26, ANBL6, WL-2 and OCI-MY1) and cultured as recommended by the suppliers at 37 °C in the presence of 5% CO . Bax−/−,Bak−/− H929, KMS-12-PE and AMO1 cells were generated using CRISPR/Cas9 genome editing as described below. HEK293T cells were cultured in DMEM supplemented with 10% heat-inactivated FBS at 37 °C in the presence of 10% CO . Media and supplements were purchased from Life Technologies unless specified otherwise. To test the sensitivity of 152 cell lines derived from several types of solid tumours or haematological malignancies (AML, lymphoma, bladder, central nervous system, colorectal, gastric, head and neck, liver, lung, breast, melanoma, ovarian, pancreas, prostate, renal, sarcoma and uterine) to S63845, cells were grown at 37 °C in a humidified atmosphere with 5% CO in RPMI 1640 medium (25 mM HEPES, with l-glutamine, Biochrom) supplemented with 10% (v/v) FBS (Sigma) and 0.1 mg ml−1 gentamicin (Life Technologies). Different culture media were used for VCap (DMEM, 10% FCS, 1% gentamycin), CALU1 (McCoy, 10% FCS 1% gentamycin) and U87MG (EMEM, 10% FCS 1% gentamycin). These cell lines were provided by the Children’s Hospital Cologne, the University Hospital Freiburg or the NCI or were purchased from ATCC, DSMZ, JCRB, ECACC or KCBL. Two cell lines used in this study were present in the list of commonly misidentified cell lines maintained by the International Cell Line Authentication Committee (ICLAC): NCI-H929 and U-937. For our study, H929 cells were obtained from authentic stocks (ATCC CRL-9068 and DSMZ ACC-163) and U937 cells were authenticated by STR analysis. All cell lines used in this study were verified to be mycoplasma negative before undertaking any experiments with them. Cell viability was measured using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colourimetric assay. Cells were seeded in 96-well microplates at a density to maintain control (untreated) cells in an exponential phase of growth during the entire experiment. Cells were incubated with compounds for 48 h followed by incubation with 1 mg ml−1 MTT for 4 h at 37 °C. Lysis buffer (20% SDS) was added and absorbance was measured at 540 nm 18 h later. All experiments were repeated at least three times. The percentage of viable cells was calculated and averaged for each well: per cent growth = (OD treated cells/OD control cells) × 100, and the IC , the concentration where the optical density was reduced by 50%, was calculated by a linear regression performed on the linear zone of the dose–response curve. Cells were harvested from exponential phase cultures, counted and plated in 96-well flat-bottom microtitre plates at a cell density depending on the cell line’s growth rate (4,000 to 30,000 cells for solid-tumour-derived cell lines, 10,000 to 60,000 for haematological cancer-derived cell lines). After a 24-h recovery period to allow the cells to resume exponential growth, 10 μl of culture medium (four control wells per plate) or of culture medium with the test compound were added by a liquid-handling robotic system and treated or untreated cells were cultured for a further 3 days. Compounds were applied in half-log increments at 10 concentrations in duplicate. After treatment of cells, 20 μl per well CellTiter-Blue reagent (Promega) was added. After incubation for up to 4 h, fluorescence was measured by using the Enspire Multimode Reader (Perkin Elmer, excitation λ = 531 nm, emission λ = 615 nm). IC values were calculated by 4-parameter, nonlinear curve fit using Oncotest Warehouse Software. To test the activity of S63845 in combination with the kinase inhibitors trametenib, tarceva, PLX-4032 and lapatinib, SK-MEL-28, BT-474, A2058, SK-Mel-2 and PC-9 cells were seeded into 96-well plates. After 24 h, cells were treated with the indicated compounds for 72 h and assayed for viability using the CellTiter-Glo reagent (Promega). Luminescence was measured at 0, 24, 48 and 72 h on independent plates seeded and treated at the same time. Results were normalized to the samples without treatment at time 0 h. To test the sensitivity of the multiple myeloma cell lines to S63845 cells were seeded into 96-well plates at 5,000 cells per well and treated at 5-point 1:8 serial dilutions of compounds starting from 10 μM. Cell viability was assessed at 48 h using the CellTiter-Glo Assay (Promega) following the manufacturer’s instructions and the plates were read using an Envision luminescence plate reader (Perkin Elmer). Results were normalized to the viability of cells that had been treated with 0.1% DMSO (vehicle) in medium for 48 h. The IC values were calculated using nonlinear regression algorithms in Prism software (GraphPad). To test the dependence of H929 cells on BCL-2, BCL-X , BCL-W, MCL1 or A1/BFL1 for their sustained survival, pools of cells stably expressing Cas9 (mCherry+) and inducibly expressing the relevant single guide RNA (sgRNA) (GFP+) were purified by flow cytometry (BD Biosciences) and seeded into 96-well plates (5,000 cells per well) in triplicates and their viability was determined by using the CellTiter-Glo assay 72 h after the addition of doxocycline (1 μg ml−1) to induce expression of the sgRNA targeting the corresponding genes. The data were normalized to the viability of cells infected with the empty vector. In some experiments, the viability (determined by propidium iodide exclusion) of the cells with or without co-treatment with the pan-caspase inhibitor QVD-OPh (25 μM; MP Biomedicals) for 12 h was also determined. Freshly purified mononuclear cells from AML patient samples were adjusted to a concentration of 2.5 × 105 per ml1 and 100 μl of cell suspensions were aliquoted per well into 96-well plates (Sigma). Cells were then treated with S63845, cytarabine (Pfizer), ABT-199 (Active Biochem) or idarubicin (Sigma) over a 6 log concentration range from 1 nM to 10 μM for 48 h and incubated at 37 °C, 5% CO . Cells were then stained with the Sytox blue nucleic acid stain (Invitrogen) and fluorescence measured by flow cytometric analysis using a LSR-II Fortessa machine (Becton Dickinson). FACSDiva software was used for data collection, and FlowJo software for data analysis. Blast cells were gated using forward and side light scatter properties. Viable cells excluding Sytox blue were determined at six concentrations for each drug and the 50% lethal concentration (LC , in μM) was calculated using nonlinear regression algorithms in Prism software (GraphPad). NCI-H929 cells were treated with the indicated compounds for 4 h, centrifuged and washed with binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl ). Cells were incubated with 200 μl of binding buffer containing Annexin V–Alexa fluor 488 (Invitrogen) and propidium iodide (Sigma) for 15 min at 20 °C in the dark. 400 μl of binding buffer was added and samples were kept at 4 °C before flow cytometry analysis. For each sample, 104 cells were analysed by flow cytometry in an Epics XL/MCL flow cytometer (Beckman Coulter). Fluorescence was collected at 520 nm (Alexa fluor 488) and 630 nm ( propidium iodide). Human Burkitt lymphoma-derived cell lines and mouse Eμ-Myc lymphoma cell lines were plated at a density of 4 × 104 cells per well in flat-bottomed 96-well plates. These cells were treated with increasing doses of S63845 (typically 0.008, 0.025, 0.04, 0.2, 1, 5 μM) for 24 h. Cells were stained with Annexin V-FITC and propidium iodide, analysed on a FACS Calibur and live cells (Annexin V negative/propidium iodide negative) were recorded. Data are presented as per cent cell death induction relative to cells cultured in medium alone. Twenty-four hours after seeding, cells were treated with the indicated compounds for 6 h and harvested in lysis buffer (10 mM HEPES pH 7.4, 142.5 mM KCl, 5 mM MgCl , 1 mM EDTA, 1% NP40, protease and phosphatase inhibitors cocktails (Calbiochem)). Cleared lysates (5 μg protein) were prepared for immunodetection of cleaved PARP (a marker of apoptosis) by using the MSD apoptosis panel whole cell lysate kit (MSD) in 96-well plates according to manufacturer’s instructions, and were analysed on the Sector Image 2400. NCI-H929 cells were treated with S63845 for 4 h, washed with PBS and harvested in lysis buffer delivered with the cytochrome c release apoptosis assay kit (Qiagen). Cells were then homogenized using an ice-cold tissue grinder (40 passes). Homogenates were centrifuged at 700 g for 10 min at 4 °C. The supernatants were transferred into fresh tubes and centrifuged at 10 000 g for 30 min at 4 °C. The supernatants were collected as cytosolic fractions. Cytochrome c release was determined by western blotting using the cytochrome c antibody provided in the kit. Lysates were also analysed by immunoblotting using an anti-LDH antibody (Rockland 200-1173; used as protein loading control). Total protein extracts of myeloma cells were generated in lysis buffer (20 mM Tris-HCl pH 7.4, 135 mM NaCl, 1.5 mM MgCl , 1 mM EDTA, 10% glycerol) containing 1% Triton X-100 and complete protease inhibitors (Roche). Protein extracts of the other cell lines were generated in lysis buffer containing 10 mM HEPES pH 7.4, 142.5 mM KCl, 5 mM MgCl , 1 mM EDTA, 1% NP40, protease and phosphatase inhibitors cocktails (Calbiochem). Lysates were stored at −80 °C. Protein content was quantified using the Bradford assay (Bio-Rad). Lysates were diluted with LDS sample buffer (Invitrogen) at a 3:1 ratio and denatured at 95 °C for 7–10 min. 20–40 μg of protein extracts were separated by SDS–PAGE (NuPAGE 10% Bis Tris gels) and proteins transferred onto nitrocellulose membranes. The membranes were blocked in 5% skimmed milk in PBS and 0.1% Tween20 (blocking buffer) before incubation with antibodies. Rat monoclonal antibodies to BAX (21C10; WEHI) or BAK (7D10; WEHI) and mouse monoclonal antibody against HSP70 (N6; used as a loading control) were used. All antibodies were diluted in blocking buffer. Commercially available antibodies were also used: rabbit polyclonal antibodies against MCL1 (Santa Cruz, S-19, sc-819), PARP (Cell Signaling, 9542), BIM (Cell Signaling, C34C5 2933), Phospho-ERK (Cell Signaling, 9101), total ERK (Cell Signaling, 9102), BAK (BD 556396), BAX (Santa Cruz, sc-493) BCL-X (Transduction Laboratory, 610212) and mouse monoclonal antibodies against actin (Millipore, MAB1501R; used as a loading control), NOXA (Calbiochem, OP180), Flag-M2 (Sigma) and p53 (Santa Cruz, sc-126). HeLa cells were transiently transfected, using the Effecten reagent (Qiagen), with expression vectors encoding 3× Flag-tagged MCL1, BCL-X or BCL-2 (p3×Flag–CMV10, Sigma). After 24 h, cells were treated for 4 h with S63845 and then harvested in lysis buffer (10 mM HEPES pH 7.5, 150 mM KCl, 5 mM MgCl , 1 mM EDTA, 0.4% Triton X100, protease and phosphatase inhibitors cocktails (Calbiochem)). The cleared lysates were subjected to immunoprecipitation with anti-Flag M2 agarose beads (Sigma). The immunoprecipitates and inputs were analysed by immunoblotting using the antibodies listed above. Total RNA was extracted using RNeasy mini kit with DNase I treatment (Qiagen) and reverse transcripted using a high-capacity cDNA reverse transcription kit with RNAse inhibitor (Life Technologies). Conventional real-time PCR was performed on an ABI 7900HT system in 50 μl reaction volumes containing 2× TaqMan universal PCR master mix, 2.5 μl of 20× target/control assay mix and 5 μl of respective cDNA in an optical 96-well plate. NTCs (no template controls) using RNase-free water were included in the plate. Cycling conditions were 95 °C (10 min), followed by 40 PCR cycles at 95 °C (15 s) and 60 °C (1 min). TaqMan Gene Expression Assays (Life Technologies) for MCL1 evaluated the anti-apoptotic long (L) isoform NM_021960 (reference assay Hs01050896_m1). Two out of five reference genes including GAPDH, PPIA, 18S, UBC and SDHA, were selected on geNorm software as the optimal number of reference target genes (geNorm pairwise variation cut off V < 0.15). As such, the optimal normalization factor was calculated as the geometric mean (GM) of reference targets SDHA and PPIA (ref genes) and calculation of −ΔC was achieved as follows: Data are presented as fold change of relative quantification calculated as 2–ΔΔCt, with . Pair-wise comparisons were evaluated with a t-test. Aliquots of cells were stained in 24G2 (anti-FcγR, (Fcγ gamma receptor)) antibody containing hybridoma supernatant, containing fluorescently (FITC, R-PE or APC) labelled monoclonal antibodies against cell surface markers, and analysed on an LSR11C (Becton Dickinson) excluding propidium iodide + (dead) cells. The following antibodies were used: anti-CD25 (clone PC61), anti-CD4 (clones GK1.5 (Biolegend) and H129), anti-CD8 (clones 53-6.7 (Biolegend) and YTS 169), CD44 (clone IM7), anti-B220 (220 kDa form of CD45 expressed on B cells, clones RA3-6B2 (Biolegend)), anti-GR1 (granulocyte antigen 1, clone RB6-8C5), anti-MAC1 macrophage antigen 1, clone M1/70), anti-SCA1 (stem cell antigen 1, clone E13-161.7), anti-c-KIT (clone 2B8 (Biolegend)), anti-TCR (T cell receptor, Biolegend), anti-TER119 (clone TER-119), anti-IgM (clone 5.1), anti-IgD (clone 11-26C), anti-Ly5.1 (clone A20.1) and anti-Ly5.2 (clone S.450-15.2). Data were processed using FlowJo Version 9.9 (TreeStar). Blood cell counts and cell subset composition were determined using an ADVIA 2120 haematology analyser (Siemens). The vectors for the constitutive expression of Cas9 and the inducible expression of the sgRNAs have been previously described30. To target the BCL-2 family members, sgRNAs were designed ( http://crispr.mit.edu) and cloned into pFH1tUTG (ref. 30) with the exception of sgRNAs for MCL1, which have been previously described30. The sequences of the sgRNAs used in this study as well as the primers for amplifying the targeted regions for DNA sequence analysis are detailed in Supplementary Tables 1 and 2, respectively. The vectors to express the BIM variants have been previously described12, 13. To produce lentiviruses, the constructs of interest were co-transfected into HEK293T cells with the packaging viruses pMDLg/pRRE, pRSV RRE and pCMV VSV-G (all from Addgene) using the Effectene transfection reagent (Qiagen). The lentiviruses were harvested, filtered and used to infect target cells as previously described13, 30. Multiple-myeloma-derived cell lines were serially infected with lentiviruses that stably co-express Cas9 and the fluorescent marker, mCherry, and inducibly express the indicated sgRNAs plus stably express GFP. Double positive cells (mCherry+ GFP+) were purified using a BD FACSAria Fusion Sorter (BD Biosciences). Expression of the sgRNA was induced by the addition of doxycycline (1 μg ml−1; Sigma). The experiments targeting BCL-2, BCL-X , BCL-W, MCL1 and BFL1 were undertaken with pools of infected cells. To generate the BAX−/−,BAK−/− H929 clone, a BAX-deficient H929 clone was infected with a lentivirus expressing a sgRNA to target BAK, re-cloned and verified by DNA sequencing and western blotting (Fig. 2c). Sequences of sgRNAs and primers for targeted PCR used in this study are shown in Supplementary Tables 1 and 2. DNA sequence verification was carried out as previously described30. Briefly, genomic DNA was isolated using the DNeasy kit (Qiagen) and mutation of targeted DNA confirmed by the Illumina MiSeq30. The unique PCR primers with overhang sequences for each sgRNA are listed in Supplementary Table 2. Graphpad Prism software was used for generating Kaplan–Meier animal survival plots of vehicle and S63845 treated mice and performing statistical analysis (using a log-rank test (Mantel–Cox)). Graphpad Prism was also used to perform multiple unpaired two-tailed t-tests of vehicle-treated and S63845-treated mice to look for significant changes in the data generated from the ADVIA analysis of the blood and from the FACS analysis of the number of different cells present in the spleen, thymus, lymph nodes and bone marrow. Graphpad Prism was used to generate IC curves for cell lines treated with S63845 in vitro. GraphPad Software was used for statistical analysis. All data are expressed as mean ± s.d. P < 0.05 was considered to be significant. The PDB deposition code for the X-ray structure of the MBP-MCL1 complex with S63845 is 5LOF.


Li S.,New York Medical College | Labaj P.P.,University of Vienna | Zumbo P.,New York Medical College | Sykacek P.,University of Vienna | And 12 more authors.
Nature Biotechnology | Year: 2014

High-throughput RNA sequencing (RNA-seq) enables comprehensive scans of entire transcriptomes, but best practices for analyzing RNA-seq data have not been fully defined, particularly for data collected with multiple sequencing platforms or at multiple sites. Here we used standardized RNA samples with built-in controls to examine sources of error in large-scale RNA-seq studies and their impact on the detection of differentially expressed genes (DEGs). Analysis of variations in guanine-cytosine content, gene coverage, sequencing error rate and insert size allowed identification of decreased reproducibility across sites. Moreover, commonly used methods for normalization (cqn, EDASeq, RUV2, sva, PEER) varied in their ability to remove these systematic biases, depending on sample complexity and initial data quality. Normalization methods that combine data from genes across sites are strongly recommended to identify and remove site-specific effects and can substantially improve RNA-seq studies. © 2014 Nature America, Inc.


PubMed | University of Vienna, WEHI, Loma Linda University, Fudan University and 3 more.
Type: Journal Article | Journal: Nature biotechnology | Year: 2014

High-throughput RNA sequencing (RNA-seq) enables comprehensive scans of entire transcriptomes, but best practices for analyzing RNA-seq data have not been fully defined, particularly for data collected with multiple sequencing platforms or at multiple sites. Here we used standardized RNA samples with built-in controls to examine sources of error in large-scale RNA-seq studies and their impact on the detection of differentially expressed genes (DEGs). Analysis of variations in guanine-cytosine content, gene coverage, sequencing error rate and insert size allowed identification of decreased reproducibility across sites. Moreover, commonly used methods for normalization (cqn, EDASeq, RUV2, sva, PEER) varied in their ability to remove these systematic biases, depending on sample complexity and initial data quality. Normalization methods that combine data from genes across sites are strongly recommended to identify and remove site-specific effects and can substantially improve RNA-seq studies.


News Article | February 27, 2017
Site: www.eurekalert.org

New light on a key factor involved in diseases such as Parkinson's disease, gastric cancer and melanoma has been cast through latest University of Otago, New Zealand, research carried out in collaboration with Australian scientists. In new findings published in leading international journal PNAS, the team of researchers, led by Otago Department of Biochemistry's Dr Peter Mace, studied a protein called Apoptosis signal-regulating kinase 1 (ASK1). Along with other kinases, ASK1 acts as a signalling protein that controls many aspects of cellular behaviour. Kinases put tags onto other proteins that can turn them on, off, which in turn can make a cell divide, die, move or any number of other responses. Dr Mace says ASK1 plays an important role in controlling how a cell responds to cell damage, and can push the cell towards a process of programmed cell death for the good of the body, if damage to a cell is too great. This key role is reflected in ASK1's name - apoptosis is an Ancient Greek word meaning "falling off" - and is used to describe the process of programmed dying of cells, rather than their loss by injury. The research team determined ASK1's molecular structure through crystallography studies and also performed biochemical experiments to better understand the protein. They found that ASK1 has unexpected parts to its structure that help control how the protein is turned on, and that an entire family of ASK kinases share these features. "We now know a lot more about how ASK1 gets turned on and off - this is important because in diseases such as Parkinson's, stomach cancer and melanoma, there can be either too much or too little ASK1 activity". Dr Mace says that the new findings add to our understanding of how cells can trigger specific responses to different threats or damage encountered. Such threats can include oxidants, which damage the body's tissues by causing inflammation. He adds that kinases are excellent targets for developing new drugs because they have a "pocket" in their structure that such compounds can bind to, but to develop better drugs we need to understand far more about how they are controlled. This is the goal of several projects in his lab, he says. The study is a collaboration between Otago researchers and scientists at the Walter and Eliza Hall Institute (WEHI) in Melbourne, and at the Australian Synchrotron. Otago alumnus Tom Caradoc-Davies, who works at the MX Beamline, collected data that was critical to the project. Synchrotron access was enabled by the New Zealand Synchrotron Group, which is coordinated by the Royal Society of New Zealand and supported by all New Zealand universities in partnership with the Government. The synchrotron is crucial to many other research projects from Otago and throughout New Zealand.


News Article | February 27, 2017
Site: phys.org

In new findings published in leading international journal PNAS, the team of researchers, led by Otago Department of Biochemistry's Dr Peter Mace, studied a protein called Apoptosis signal-regulating kinase 1 (ASK1). Along with other kinases, ASK1 acts as a signalling protein that controls many aspects of cellular behaviour. Kinases put tags onto other proteins that can turn them on, off, which in turn can make a cell divide, die, move or any number of other responses. Dr Mace says ASK1 plays an important role in controlling how a cell responds to cell damage, and can push the cell towards a process of programmed cell death for the good of the body, if damage to a cell is too great. This key role is reflected in ASK1's name - apoptosis is an Ancient Greek word meaning "falling off" - and is used to describe the process of programmed dying of cells, rather than their loss by injury. The research team determined ASK1's molecular structure through crystallography studies and also performed biochemical experiments to better understand the protein. They found that ASK1 has unexpected parts to its structure that help control how the protein is turned on, and that an entire family of ASK kinases share these features. "We now know a lot more about how ASK1 gets turned on and off - this is important because in diseases such as Parkinson's, stomach cancer and melanoma, there can be either too much or too little ASK1 activity". Dr Mace says that the new findings add to our understanding of how cells can trigger specific responses to different threats or damage encountered. Such threats can include oxidants, which damage the body's tissues by causing inflammation. He adds that kinases are excellent targets for developing new drugs because they have a "pocket" in their structure that such compounds can bind to, but to develop better drugs we need to understand far more about how they are controlled. This is the goal of several projects in his lab, he says. The study is a collaboration between Otago researchers and scientists at the Walter and Eliza Hall Institute (WEHI) in Melbourne, and at the Australian Synchrotron. Otago alumnus Tom Caradoc-Davies, who works at the MX Beamline, collected data that was critical to the project. Synchrotron access was enabled by the New Zealand Synchrotron Group, which is coordinated by the Royal Society of New Zealand and supported by all New Zealand universities in partnership with the Government. The synchrotron is crucial to many other research projects from Otago and throughout New Zealand. More information: Structural basis of autoregulatory scaffolding by apoptosis signal-regulating kinase 1, PNAS, www.pnas.org/cgi/doi/10.1073/pnas.1620813114


News Article | December 16, 2015
Site: www.scientificamerican.com

It can be hard to visualize how biology works at the molecular level, but biomedical animators like Drew Berry at the Walter and Eliza Hall Institute of Medical Research make it easy, using a wealth of data to create vibrant motion pictures of cellular processes in vivid color, complete with sound effects. Cells act like small cities, with proteins and other molecules moving between different factories. But they can’t move themselves. They need motor proteins to pull them or walk around. At 3:10 in this animation, you can watch two motor proteins, dynein (pale green) and kinesin (orange), strut their stuff. Animators like these integrate studies about structure, energy, and more to know just where and when to place every molecule. The hues and the sound of footsteps give them personality and bring them to life. You can watch animations on more cellular processes on WEHI’s online platforms, without needing to walk with any motor proteins to get there.


Czabotar P.E.,WEHI | Lee E.F.,WEHI | Thompson G.V.,WEHI | Wardak A.Z.,WEHI | And 4 more authors.
Journal of Biological Chemistry | Year: 2011

Pro-survival members of the Bcl-2 family of proteins restrain the pro-apoptotic activity of Bax, either directly through interactions with Bax or indirectly by sequestration of activator BH3-only proteins, or both. Mutations in Bax that promote apoptosis can provide insight into how Bax is regulated. Here, we describe crystal structures of the pro-survival proteins Mcl-1 and Bcl-xL in complex with a 34-mer peptide from Bax that encompasses its BH3 domain. These structures reveal canonical interactions between four signature hydrophobic amino acids from the BaxBH3 domain and the BH3-binding groove of the pro-survival proteins. In both structures, Met-74 from the Bax peptide engages with the BH3-binding groove in a fifth hydrophobic interaction. Various Bax Met-74 mutants disrupt interactions between Bax and all pro-survival proteins, but these Bax mutants retain pro-apoptotic activity. Bax/Bak-deficient mouse embryonic fibroblast cells reconstituted with several Bax Met-74 mutants are more sensitive to the BH3 mimetic compound ABT-737 as compared with cells expressing wild-type Bax. Furthermore, the cells expressing Bax Met-74 mutants are less viable in colony assays even in the absence of an external apoptotic stimulus. These results support a model in which direct restraint of Bax by pro-survival Bcl-2 proteins is a barrier to apoptosis. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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