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Targeting survivin and p53 in pediatric acute lymphoblastic leukemia

Targeting survivin and p53 in pediatric acute lymphoblastic leukemia Leukemia (2012) 26, 623–632 & 2012 Macmillan Publishers Limited All rights reserved 0887-6924/12 www.nature.com/leu ORIGINAL ARTICLE 1 2 3 1,4 2 JW Tyner , AM Jemal , M Thayer , BJ Druker and BH Chang 1 2 Division of Hematology and Medical Oncology, and OHSU Knight Cancer Institute, OHSU, Portland, OR, USA; Division of Hematology and Oncology, Department of Pediatrics and OHSU Knight Cancer Institute, OHSU, Portland, OR, USA; Department of Biochemistry and Molecular Biology and OHSU Knight Cancer Institute, OHSU, Portland, OR, USA and Howard Hughes Medical Institute Despite advances in treatment and outcomes for patients with malignancy with little to no expression in normal terminally pediatric acute lymphoblastic leukemia (ALL), there continue to differentiated tissue. Furthermore, survivin overexpression has be subsets of patients who are refractory to standard correlated with resistant and refractory disease in many different chemotherapy and hematopoietic stem cell transplant. There- malignancies including ALL. fore, novel gene targets for therapy are needed to further Survivin is a small 16 kD protein that belongs to the inhibitor advance treatment for this disease. RNA interference technol- of apoptosis (IAP) family and also functions as a member of the ogy has identified survivin as a potential therapeutic target. Survivin, a member of the inhibitor of apoptosis (IAP) proteins chromosome passenger complex. Survivin is a unique member and chromosome passenger complex, is expressed in hemato- of the IAPs in that it is both the smallest member and may not logic malignancies and overexpressed in relapsed pediatric directly interact with caspases. Instead, it may interact with ALL. Our studies show that survivin is uniformly expressed at another IAP, smac/Diablo, to regulate apoptosis within the high levels in multiple pediatric ALL cell lines. Furthermore, mitochondria. Transcription of survivin shows several splice silencing of survivin expression in pediatric ALL cell lines as variants with conserved N-terminal domains, with most of the well as primary leukemic blasts reduces viability of these cells. This includes cell lines derived from patients with relapsed divergence occurring within or after the baculovirus IAP repeat 5–7 disease featuring cytogenetic anomalies such as t(12;21), domain. Recent published reports would suggest that these Philadelphia chromosome t(9;22), t(1;19) as well as a cell line splice variants may have different subcellular localization carrying t(17;19) from a patient with de novo ALL. Furthermore, including the mitochondria and different apoptotic activities. inhibition of survivin increases p53-dependent apoptosis that The role of survivin as a member of the chromosome can be rescued by inhibition of p53. Finally, a screen of passenger complex, in which it plays a critical role during randomly selected primary patient samples confirms that survivin-specific small interfering RNA and survivin-targeted mitosis, is better defined. As such, survivin expression is cell- drug, YM155, effectively reduce viability of leukemic blasts. cycle dependent, with the highest expression during G2/M Leukemia (2012) 26, 623–632; doi:10.1038/leu.2011.249; through canonical cell cycle-dependent and cell-cycle homol- published online 30 September 2011 ogy regions within the proximal promoter. During mitosis, Keywords: RNAi; targeted therapy; IAP survivin specifically localizes with INCENP and Aurora B kinase within the mitotic apparatus from the centromeres in prophase, kinetochores in metaphase, the mid-plate during anaphase and Introduction the mid-body during cytokinesis. Furthermore, it is the direct interaction of survivin with both Aurora B kinase and INCENP 11–13 Over the past 50 years, the prognosis for pediatric acute that is essential for cell division. Survivin phosphorylation lymphoblastic leukemia (ALL) has changed from a terminal at Ser 20 by PLK1 (polo-like kinase) is required for the priming of diagnosis to a treatable disease. The success of current Aurora B kinase activity to undergo cell division. therapeutic regimens has given rise to the identification of a In this study we verified the cell-cycle dependence of survivin subset of patients who will have recurrent or refractory disease. expression within pediatric ALL cells. We further tested whether Unfortunately, patients within these subsets have highly resistant several pediatric leukemic cell lines as well as primary patient disease that may not be overcome even with myeloablative samples were sensitive to manipulation of survivin expression therapy and hematopoietic stem cell transplant. These diseases and activity. Pediatric ALL cell lines have similar expression may have approached the limit of treatment by standard levels of total survivin. In addition, the expression patterns of chemotherapeutic regimens. Therefore, new targets for therapy survivin among cell lines were similar with an increase in are imperative in the hope of improving outcomes. survivin expression during G /M. Virtually all of the ALL cell A report from the Children’s Oncology Group has shown a lines tested were sensitive to silencing of survivin including REH differential expression profile of relapsed ALL compared with (ETV6-RUNX1), SUPB15 (BCR-ABL) RCH-ACV (E2A-PBX1) and initial diagnosis. One of the genes showing a marked increase HAL01 (E2A-HLF). We also identified that targeting survivin in expression in patients with recurrent disease is survivin (aka either by small interfering RNA (siRNA) or by the survivin- BIRC5). Survivin is an attractive target for therapy because it is suppressor YM155 in these cell lines increased cell death expressed mainly during development and in the setting of through the p53-dependent apoptosis pathway. This increase in cell death could then be rescued by silencing p53. Finally, early Correspondence: Dr BH Chang, Division of Hematology and screening of patient samples with survivin siRNA or YM155 Oncology, Department of Pediatrics, OHSU Knight Cancer Institute, showed sample-specific variation of sensitivity to survivin Mailcode CDRCP, 3181 SW Sam Jackson Park Road, Portland, silencing. The heterogeneity of YM155 responses would suggest OR, USA. that other factors may have importance for primary lympho- E-mail: changb@ohsu.edu blasts to respond to survivin inhibition beyond p53. As such, Received 5 February 2011; revised 10 July 2011; accepted 11 August 2011; published online 30 September 2011 early disease selection through in vitro screening may become p53, apoptosis, survivin, acute lymphoblastic leukemia JW Tyner et al important for future clinical strategies that would employ 2–4 h at 37 1C. The cell media was then supplemented with 0.5 ml survivin as a therapeutic gene target. of RPMI containing FBS such that the final concentration of FBS was 10–20% (depending on cell line). A total of 50 000 cells per well were plated in triplicate and grown for 4 days for either MTS or for Materials and methods Gauva Nexin assay. The remaining cells were harvested 48 h after treatment for immunoblot. Reagents Fetal bovine serum (FBS) was obtained from Hyclone Laboratories Immunoblot analysis Inc. (South Logan, UT, USA). All other tissue culture reagents Cells were washed with PBS and lysed with 1 SDS loading buffer were obtained from Invitrogen Corporation (Carlsbad, CA, USA). (75 mM Tris,pH6.8,3%SDS,15% glycerol,8% b-mercaptoetha- The siRNAs (Supplementary Table 1) were from the siGenome nol, 0.1% bromophenol blue). For mitochondrial fractionation, cells SMARTpool designed by Dharmacon (ThermoFisher Scientific, were processed using ApoALert Cell Fractionation Kit (Clontech, Waltham, MA, USA). Viability assays were performed with Mountain View, CA, USA). All samples were separated by standard CellTiter 96 AQ One Solution Cell Proliferation Assay from ueous SDS-polyacrylamide gel electrophoresis and transferred onto poly- Promega Corporation (Madison, WI, USA). Apoptosis assays were vinylidene fluoride membrane (Immobilon-FL, Millipore). Mem- performed using the Guava Nexin Assay (Millipore, Billerica, MA, branes were blocked with Aquablock tm/EIA/WB (EastCoast Bio, USA). YM155 was purchased from Selleck (Houston, TX, USA) Inc., North Berwick, ME, USA) for 1 h, and then incubated with and solubilized in dimethylsulfoxide at 100 mM stock. Graphical primary antibodies to survivin (Cell Signaling Technology, Inc., and statistical data were generated using either Microsoft Excel or Danvers, MA, USA), (Ser 10) phospho-histone H3 (Upstate GraphPad Prism (GraphPad Software, Inc., La Jolla, CA, USA). Biotechnology, Lake Placid, NY, USA), tubulin (Sigma-Aldrich Corp., St Louis, MO, USA), (Ser 15) phospho-p53 (Cell Signaling), Cell lines and tissue culture p53 (Santa Cruz, Biotechnology, Inc., Santa Cruz, CA, USA) and RCH-ACV (RCH) (Deutsche Sammlung von Mikroorganismen ABL (BD Biosciences, San Jose, CA, USA) in Aquablock/0.1% und Zellkulturen GmbH (DSMZ), Braunschweig, Germany) is a Tween-20 overnight at 4 1C. Secondary fluorescent antibodies pediatric ALL cell line from a patient with recurrent disease (Molecular Probes) were used and detected and quantified with carrying the E2A-PBX1 t(1;19) chimeric protein. REH (ATCC) is a Odyssey (LI-COR, Corporate Offices, Lincoln, NE, USA). pediatric ALL cell line from a patient with recurrent disease carrying the ETV6-RUNX1 t(12;21) chimeric protein. SUPB15 Immunofluorescence (American Type Culture Collection (ATCC), Manassas, VA, USA) 5 ALL cells were grown in complete media and then B5 10 is a pediatric ALL cell line also from a patient with recurrent 4 cells were harvested and resuspended in 0.5 ml of PBS. 2 10 disease carrying the BCR-ABL t(9;22) translocation. HAL01 cells cells were spotted onto a cover slip for 10 min. Cells were fixed (DSMZ) are from a pediatric patient with de novo ALL with the with 4% formaldehyde at 37 1C for 10 min. Samples were then E2A-HLF t(17;19). RCH, REH and HAL01 cells were maintained permeabilized with 0.25% Triton X-100 and washed with PBS. in RPMI with 10% FBS, 4 mM glutamine and 1% penicillin and Cells were then stained overnight in 4 1C with primary streptomycin. SUPB15 cells were maintained in RPMI with 20% antibodies and subsequently stained for 1 h with secondary FBS, 4 mM glutamine, 50 nM 2-mercaptoethanol and 1% antibody at room temperature. All images were captured with penicillin and streptomycin. All patient samples were obtained an Olympus (San Jose, CA, USA) BX Fluorescent Microscope with informed consent approved by the institutional review using a  50–100 objective, automatic filter-wheel and Cytovi- board of Oregon Health and Science University. sion workstation. Small interfering RNA knockdown, proliferation and Cell sorting induction of apoptosis ALL cell lines were grown in RPMI/10% FBS to a concentration Standard electroporation was modified from a previously described of 5–10 10 cells/ml. Cells were then washed with PBS/1% 15 5 protocol. Briefly, 1.5 10 cells per condition were resuspended FBS and fixed in 70% ethanol and stored at 20 1C. The samples in 75 ml siPORT buffer (Applied Biosystems, Life Technologies were then washed with PBS and stained with buffer containing Corporation, Carlsbad, CA, USA). To the samples, 1–2mM of siRNA 3mM EDTA, pH 8.0, 0.05% NP-40, 50 mg/ml propidium iodide was added. Cells were electroporated at 200 V, 250 ms, 2 pulses, and 1 mg/ml RNAse A in PBS. Cells were sorted by DNA and 20 000 cells per well were plated in triplicate containing content with BD FACS/Aria (BD Biosciences) and harvested with 100 ml of culture media. The remaining 60 000 cells were plated SDS loading buffer. into a well containing 500 ml of culture media. For determination of cell viability, the triplicate plates containing 20 000 cells were YM155 dose response subjected to the CellTiter 96 AQ One Solution Cell Prolifera- ALL cells lines (5000 cells per well) and primary patient samples ueous tion Assay (MTS). For subsequent immunoblot analysis, the plate (50 000 cells per well) were incubated with graded concentra- containing 60 000 cells were harvested and lysed in 20 mlof 1 tions of YM155 (0–10 mM) in RPMI with 10% FBS. After 3 days, sodium dodecyl sulfate (SDS) loading buffer. Identification of cells were subjected to MTS for assessment of cell viability. induction of apoptosis was performed using the Guava Nexin assay All values were normalized to the no drug control from each (Millipore). Briefly, triplicate samples containing 20 000 cells were respective cell line. incubated with 60 ml of the Guava Nexin reagent and then analyzed through the microcapillary flow cytometer at varying time points up to 96 h. Results Cells were also treated with transductin (Integrated DNA Technologies (IDT), Inc., Coralville, IA, USA) for introduction of Pediatric ALL cell lines express high levels of survivin siRNA into the cells. A total of 500 nM of siRNA was incubated in mostly at G /M phosphate buffered saline (PBS) with 5mM transductin and added to Prior studies have suggested that there are varying amounts of 2.5 10 cells in 0.5 ml of RPMI with 1% bovine serum albumin for survivin expression in primary pediatric ALL samples and in Leukemia p53, apoptosis, survivin, acute lymphoblastic leukemia JW Tyner et al G1 S1 S2 G2 G1 S1 S2 G2 DNA Content G1 S1 S2 G2/M G1 S1 S2 G2/M Survivin pH3 Tubulin REH (ETV6-RUNX1) RCH (E2A-PBX1) Survivin pH3 Tubulin G1 S1 S2 G2/M G1 S1 S2 G2/M Survivin pH3 Tubulin HAL01 (E2A-HLF) SUPB15 (BCR-ABL) 0.2 0.04* REH (ETV6-RUNX1) 2 RCH (E2A-PBX1) HAL01 (E2A-HLF) SUPB15 (BCR-ABL) 1 2 pH3 tubulin late interphase prophase metaphase telophase REH RCH HAL01 SUPB15 C M C M C M C M survivin cytochrome c Figure 1 Relative expression of survivin in ALL cell lines and cell-cycle dependence. (a) Immunoblot of asynchronous populations ALL cell lines for survivin, (Ser 10) phospho-histone H3 (pH3) and tubulin. The cell lines tested include REH (ETV6-RUNX1), RCH-ACV (E2A-PBX1), HAL01 (E2A-HLF) and SUPB15 (BCR-ABL). The immunoblot shown is representative of three independent experiments. (b) Survivin level is dependent on the cell cycle in ALL cell lines. An asynchronous population of ALL cell lines was fixed in 70% ethanol and stained with propidium iodide. Cells were flow sorted by DNA content for G1, early S (S1), late S (S2) and G2/M. Top panels represent the histograms of the cells that were sorted. Bottom panels represent the immunoblots for survivin, pH3 and tubulin. (c) Quantification of survivin expression in relation to tubulin and pH3. (Left panel) Each band from immunoblots was quantified by Odyssey for fluorescence intensity and normalized either to tubulin for pH3. Ratios were then compared with REH as control. (Right panel) Graphical representation of the relative quantity of survivin normalized to tubulin from (b). Quantity is expressed in comparison with REH in G1. *Denotes Po0.05 by student’s t-test. (d) Subcellular localization of survivin in lymphoblasts. (Upper panel) Each cell line showed similar staining patterns. Representative images of later interphase, prophase, metaphase and telophase are shown from each cell line. 5 10 cells of an asynchronous population were dropped onto coverslips and fixed with formaldehyde, and processed for dual immunofluorescence labeling; survivin (green), pH3 (red), DAPI (blue). (Lower panel). Immunoblot of survivin and cytochrome c after fractionation with Apoalert Fractionation. C, cytosolic fraction; M, mitochondrial fraction. 2,17 17 pediatric ALL cell lines. To validate this finding, we assessed E2A-HLF found in t(17;19). Immunoblot of a population of survivin expression level in a variety of cell lines derived from asynchronous cells showed varying amounts of survivin divergent subsets of pediatric ALL patients, including HAL01 when normalized to tubulin expression, with SUPB15 cells cells that have been used to describe the dependence of survivin showing a statistically significant difference to REH (t-test 0.04; overexpression through the chimeric transcription factor Figures 1a and c). Leukemia REH (ETV6-RUNX1) Survivin expression normalized to REH RCH (E2A-PBX1) HAL01 (E2A-HLF) SUPB15 (BCR-ABL) Count Survivin expression normalized to REH G1 G1 S1 S2 G2/M G1 S1 S2 G2/M G1 S1 S2 G2/M G1 S1 S2 G2/M p53, apoptosis, survivin, acute lymphoblastic leukemia JW Tyner et al Survivin expression is regulated by the cell cycle and plays a be seen in a dose-dependent manner even at 24 h after exposure 11–13 role within the chromosome passenger complex. These to YM155 (Supplementary Figure 1). To test whether YM155 chromosome passenger complex proteins show relatively lower sensitivity was specific to inhibition of survivin expression, RCH expression in G /S phase with the highest expression in G /M. cells were transfected with pMIG-Survivin. Cells were treated 1 2 To determine whether the small variations of survivin expression with 1 mM YM155 for 48 h, and then assayed for apoptosis by observed in cell lines is dependent on the proportion of cells in Annexin V staining. Ectopic expression of survivin in RCH cells G /M phase at the time of cell lysis, we also examined cell partially rescues apoptosis when treated with 1 mM YM155, lysates for abundance of Ser 10 phosphorylation on histone H3 further validating the selective inhibition of survivin (Supple- (pH3), a marker for G /M phase. After normalization of survivin mentary Figure 1d). Interestingly, there was some variation of expression to pH3, there appeared to be no significant sensitivity to this compound with REH cells being the most difference in the amount of survivin expression between cell sensitive (half-maximal inhibitory concentration (IC )17nM) lines (Figures 1a and c). Furthermore, most of the expressed and HAL01 showing the least sensitivity (IC 560 nM). RCH and survivin appears to be in G2/M as shown by flow sorting cells SUPB15 cells had IC ’s that ranged between 10 and 50 nM. from different stages of the cell cycle followed by immunoblot These results would suggest a possible heterogeneity of response for survivin, pH3 and tubulin (Figures 1b and c). among ALL lymphoblasts to inhibition of survivin. The cell cycle-dependent expression of survivin would suggest that in ALL cells lines the major role of survivin would Survivin inhibits the p53-dependent apoptotic pathway be as a member of the chromosome passenger complex. in ALL cell lines However, survivin has been shown to exhibit divergent Previous studies have suggested that overexpression of survivin subcellular localizations with certain splice variants exhibiting inhibits the p53-dependent apoptosis pathway. Therefore, cytosolic and mitochondrial distribution, suggesting that survi- inhibition of survivin may allow for re-activation of this p53- vin may also play a role as an IAP. As such, we next wanted to mediated apoptotic program. Prior data suggest that most pediatric assess whether there are different subcellular pools of survivin ALL cell lines are wild type for TP53 by gene expression within ALL cell lines. We examined survivin expression patterns 21,22 patterns. To test whether the cells lines evaluated in this in RCH, REH, SUPB15 and HAL01 cells by immunofluores- study had an intact p53-dependent cell-cycle arrest, the cells were cence. In each cell line, the majority of survivin staining was treated with 0.1 mg/ml of doxorubicin. Doxorubicin is an evident in cells undergoing mitosis (Figure 1d and data not anthracycline that is known to inhibit resealing of DNA breaks, shown). There is an increase in survivin staining in early thereby activating a p53-dependent cell-cycle arrest and apoptosis prophase with evident colocalization with chromosomal mate- through the intrinsic pathway. RCH, REH, SUP B15 and HAL01 rial, and there is a subsequent increase in the intensity of cells all showed activation of p53 by phosphorylation at Ser 15 survivin staining through metaphase. As the chromosomes (pTP53) within 4 h of treatment (Figure 3a). Furthermore, these segregate, survivin stays at the mid-body through cytokinesis. cells showed cell-cycle arrest by decrease in pH3. These results are consistent with prior published data on the To test whether the increase in cell death observed after 8,11 subcellular localization of survivin in monolayer cells. silencing of survivin in ALL cell lines was due to p53 activity, Interestingly, we do observe some variation in punctate RCH and REH cells were treated with a combination of survivin cytosolic staining for survivin in interphase cells. The antibody and p53 siRNA. As previously shown, knockdown of survivin to survivin, which is generated against the N-terminus of the significantly decreased the viability and increased annexin V protein (Cell Signaling), is predicted to react to all splice staining of both RCH and REH cells. Interestingly, treatment variants. Furthermore, cell fractionation does show that there is with a combination of siRNA to p53 and survivin partially a small population of survivin in both the cytosolic and rescued cell viability and decreased annexin V staining mitochondrial fractions (Figure 1d). (Figure 3b). Immunoblots performed showed similar amounts of protein silencing (Figure 3c), whether alone or in combina- tion. Interestingly, treatment of cells with siRNA to survivin Inhibition of survivin expression reduces cell viability in increases the quantity of p53 within the cell (Figure 3c). This ALL cell lines increase in p53 is also abrogated by siRNA to p53, suggesting As ALL cell lines appear to express similar amounts of survivin, that this phenomenon is from increased transcription or stability we next tested whether survivin knockdown would have of the p53 transcript rather than enhanced stability of the p53 deleterious effects on viability of these cells. Leukemia cell protein. These results are consistent with prior findings that lines treated with survivin-specific siRNA consistently showed a inhibition of survivin expression increases p53-dependent significant reduction in viability, with RCH, REH, SUPB15 and apoptosis and may begin to shed new light as to the mechanism HAL01 cells each exhibiting at least 40% decrease in cell of this inhibition. Prior studies have shown conflicting data as viability (Figure 2a). Immunoblots performed 48 h after siRNA to whether survivin interacts with or is independent of treatment confirmed siRNA-mediated silencing of survivin of 17,24,25 19 caspases. For example, Nakahara et al. have shown B50% reduction in levels (Figure 2b and data not shown). that caspase 3 is activated by YM155 in HRPC cells whereas Recently, a novel small-molecule inhibitor, YM155, was Okuya et al. describe caspase-independent cell death in developed by Astellas Pharma US, Inc. (Deerfield, IL, USA) as a t(17;19) ALL. Our studies would further agree with Okuya potent inhibitor of survivin expression. This compound was et al. that neither caspase 3 nor caspase 9 is activated when developed using a screen that identified small molecules that treated with either siRNA or YM155, yet they still activate would only inhibit survivin expression at the promoter. As this apoptosis in ALL cells (Supplementary Figures 1 and 2). reagent offered a different mode of survivin suppression, we also tested the impact of this compound on viability of ALL cell lines. Consistent with siRNA results, each of the cell lines tested Phþ ALL cells are sensitive to knockdown of survivin showed a dose-dependent sensitivity to YM155 as measured by independent of imatinib cell viability 72 h after exposure (Figure 2c). Furthermore, As previously discussed, Phþ ALL (BCR-ABL) is a model of inhibition of survivin expression and increase in apoptosis can resistant disease in which the primary oncogene is well Leukemia p53, apoptosis, survivin, acute lymphoblastic leukemia JW Tyner et al Figure 2 Inhibition of survivin expression inhibits cell viability for REH, RCH, SUPB15 and HAL01. (a) Cell viability as measured by MTS 96 h after treatment with siRNA for survivin. All samples were treated with nonspecific (NS) siRNA as a control and normalized to 100%. s.e.m. bars are inserted. *Po0.05, **Po0.01. (b) Representative immunoblots for the expression level of survivin treated with NS siRNA or survivin siRNA. Tubulin expression was used as a control for loading. (c) Dose response of ALL cell lines to YM155. Asynchronous populations of cells were treated with increasing doses of YM155 for 72 h. Then, viability was measured by MTS and normalized to no drug control. S.e.m. bars are inserted. Dotted line represents 50% viability. characterized. Although the addition of imatinib as targeted siRNA for survivin and p53 in the presence of 500 nM imatinib therapy has improved outcome, there remain patients who (Figure 4d). In contrast to cells treated with ABL siRNA where no continue to relapse. To test whether Phþ ALL would also be additivity with survivin was observed, knockdown of survivin did sensitive to targeting of survivin, SUPB15 cells were treated with show an additive increase in cell death in the presence of siRNA to survivin, BCR-ABL (ABL),and p53 (Figure 4). Knock- imatinib. One possibility for this difference could involve minor down of survivin increases Annexin V staining that is then variations in signaling that occur after elimination of BCR-ABL rescued by siRNA to p53 (Figures 4a and b). Silencing of protein from cells (siRNA) versus inhibition of BCR-ABL activity survivin or BCR-ABL decreased cell viability by B50% without eliminating the protein (imatinib). Another possibility (Figure 4c). Furthermore, simultaneous knockdown of BCR- could include inhibition of other non-BCR-ABL pathways by ABL and p53 had a minimal effect on the cells, suggesting that imatinib that could synergize with silencing of survivin. Knock- cell death after silencing of BCR-ABL occurs mainly through a down of p53 did not rescue the effects on cell viability of either p53-independent mechanism. imatinib or ABL siRNA. In contrast, silencing of p53 did rescue Interestingly, the combination of survivin and BCR-ABL the effects of both survivin siRNA and YM155 (Figure 4d, lower silencing did not show an additive effect. It has previously been panel), and YM155 appears to activate p53 (Supplementary shown that in certain CML cell lines, the BCR-ABL/mitogen- Figure 1a). Cumulatively, these results support the role of survivin activated protein kinase pathway increases expression of survivin as an inhibitor of p53-dependent apoptosis in Phþ ALL, which is and that silencing of survivin enhances imatinib-induced cell a pathway independent of BCR-ABL function, suggesting that death. To test whether survivin knockdown could also enhance targeting of survivin could enhance therapy by inhibiting imatinib-induced cell death, SUPB15 cells were treated with independent pathways to imatinib. Leukemia p53, apoptosis, survivin, acute lymphoblastic leukemia JW Tyner et al Figure 3 Knockdown of p53 rescues cell death because of silencing of survivin. (a) Activation of p53 by treatment with doxorubicin. 1 10 cells of RCH, REH, SUPB15 and HAL01 were treated with 0.1 mg/ml of doxorubicin (Sigma) for 0, 4 and 8 h. The cells were then harvested, separated and immunoblotted for (Ser 15)-phospho-p53 (pp53), pH3 and tubulin. S15P p53 evaluated the phosphorylation state of p53 at Ser 15 by activation of the replication checkpoint. pH3 was again used as a marker for cell-cycle arrest reflecting a decrease in the number of cells entering mitosis. (b) Partial rescue of cell viability by knockdown of p53. RCH and REH cells were treated with siRNA to NS, survivin, p53 and a combination of p53/survivin. Viability was tested by MTS and normalized to NS (left panel). Apoptosis was then evaluated by Annexin V binding (right panel). (c) Immunoblots of representative duplicate experiments to quantify the expression of survivin, and p53 after treatment with siRNA. Lower panels show the relative expression normalized to tubulin quantified by fluorescence intensity. Primary patient samples are sensitive to inhibition of E2A-HLF. Immunoblots were also performed to identify the survivin expression levels of survivin as compared with tubulin and pH3 Our results would suggest that patients with pediatric ALL could (Figures 5c and d). There was a distribution of variability of benefit from inhibition of survivin. For initial, preclinical expression that does not appear to correlate with sensitivity to validation of this strategy, we isolated leukemic blasts from YM155. For example, patient 5 (relapsed with ETV6-RUNX1) fresh primary ALL samples and treated the cells with siRNA to was most sensitive to YM155, yet had one of the lowest levels of survivin. Treatment of two, randomly selected primary patient survivin expression when normalized to tubulin. However, this samples with survivin siRNA showed response with a 30–50% patient had relatively high expression when normalized to pH3. decrease in cell viability (Figure 5a). To further validate survivin This would suggest that the cell cycle-independent expression of as a bona fide therapeutic target, we treated fresh primary survivin is a more important correlate for YM155 sensitivity than patient samples with YM155. Treatment of four patient samples overall survivin expression. In contrast, samples with E2A-HLF revealed a range of sensitivity to this drug from IC values (patient 3, Figure 5, and HAL01, Figure 2) exhibited less p10 nM to IC ’s exceeding 1 mM consistent with the cell lines sensitivity to YM155 despite high expression of survivin, (Figure 5b). Interestingly, the samples that showed the highest suggesting that other factors may play an important role in IC ’s were the HAL01 cells and the patient sample with YM155 sensitivity. E2A-HLF cell lines have previously been Leukemia p53, apoptosis, survivin, acute lymphoblastic leukemia JW Tyner et al p53 NS 1 41% 1 30% 10 10 10 20 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 4 p53/survivin survivin NS SUR ABL ABL/SUR p53 p53/SUR 55% 35% 80 Control 10 60 500nM 0 1 2 3 4 10 10 10 10 10 0 1 2 3 4 10 10 10 10 10 40 imatinib Annexin V-PE NS SUR p53 p53/SUR NS SUR ABL ABL/SUR p53 p53/SUR p53/ABL BCR-Abl Survivin NS NS/100nM p53 p53/100nM p53 YM155 YM155 Tubulin Figure 4 Sensitivity of inhibition of survivin in SUPB15 cells is dependent on intact p53. (a) Flow cytometric representation of apoptosis by staining for Annexin V and 7-aminoactinomycin D (7-AAD). SUPB15 cells (BCR-ABL) were treated with siRNA to NS, SUR, p53 and p53/SUR. (b) A graphical representation of Annexin V staining in SUPB15 treated with NS, survivin, ABL, ABL/survivin, p53 and p53/survivin. (c) Graphical representation of the percent viability of SUPB15 cells treated with NS, survivin, p53 and p53/survivin. The lower panel represents the immunoblots performed on the samples treated with the siRNA and stained for BCR-ABL, survivin, p53 and tubulin. Viability was tested by MTS and normalized to nonspecific siRNA (NS). (d) Treatment of SUPB15 cells with imatinib or YM155. Top panel shows cells treated with siRNA in the presence of 500 nM imatinib. Cells were treated with siRNA, and then subsequently incubated in 500 nM imatinib for 96 h. Bottom panel shows the partial rescue of SUPB15 cells treated with 100 nM YM155 for 96 h. S.e.m. bars are inserted into each graph. shown to overexpress the drug efflux protein ABCB1, which shown that overexpression of survivin has the ability to inhibit may reduce the amount of YM155 within the cell, thereby degradation of Mdm2 (murine double minute 2), thereby increasing the IC . These studies would suggest that preselec- promoting degradation of p53. Other studies have shown that tion of patients by in vitro screening for sensitivity to YM155 p53 has the ability to decrease survivin expression. In human would be important in future studies using this compound for lung and ovarian cancer cell lines, activation of p53 decreases clinical trials. expression of survivin by changing acetylation of the survivin promoter rather than direct binding. In our studies, silencing of survivin did show a qualitative increase in p53 expression. Discussion These findings would suggest that there is a critical relationship with survivin and p53 whereby higher levels of survivin inhibit In contrast to prior reports, our data have shown that ALL cell p53 either by degradation or inhibition of expression, whereas inhibition of survivin activates p53 and increases p53 expres- lines express similar amounts of survivin and that most of the protein is expressed during G /M. Meanwhile, primary patient sion. Taken together, our studies support the critical role for the interaction of survivin with the p53-dependent apoptotic path- samples do show significant variability in survivin expression. This study also demonstrates that knockdown of survivin in ALL way as an important regulator of growth in pediatric lympho- blasts. increases p53-dependent apoptosis. Furthermore, in the BCR- ABL line SUPB15, reduction of BCR-ABL activity or expression Molecular targeting of survivin continues to be an intriguing concept for therapy. It is a protein whose expression is almost does not increase the p53-dependent apoptotic pathway. exclusively confined to dividing cells such as malignant tissue Finally, we show that screening of primary samples with either siRNA to survivin or the selective suppressor YM155 has the and normal hematopoietic stem cells and colonic epithe- 29,30 lium. Although survivin is expressed differentially within potential to identify samples that are more sensitive to survivin inhibition. the cell cycle with the highest expression at G /M, some studies have suggested a baseline increase in expression in malignant There is evidence to support the interaction of survivin with the p53-dependent apoptotic pathway. In BaF3 cells, it has been cells. Also, higher expression of survivin has been shown in Leukemia 7-AAD Percent viable Percent viability Percent viability Percent annexin V staining p53, apoptosis, survivin, acute lymphoblastic leukemia JW Tyner et al Figure 5 Primary patient (Pat.) samples show sensitivity to inhibition of survivin. See Supplementary Table 2 for patient details. (a) Two random fresh primary patient samples were treated with siRNA to NS and survivin. Viability was then assayed 4 days later by MTS, and normalized to viability to NS. (b) Dose response of patient samples to YM155. Fresh mononuclear cells were separated by Ficoll and B50 000 cells were plated into each well containing increasing concentrations of YM155 from 1 nM to 1 mM. Viability was assayed 3 days later by MTS and normalized to no drug control. Viability was then assayed 3 days later by MTS. All plots contain s.e.m. bars. (c) Immunoblots of patient samples for survivin pH3 and tubulin. Approximately 1 10 cells were lysed in 100 ml of SDS loading buffer and 20 ml was run on polyacrylamide gel electrophoresis (PAGE). (d) Graphical representation of survivin expression normalized to either tubulin or pH3 intensity. These ratios were then compared with REH ratios as a control. 3 29 recurrent and relapsed disease. Furthermore, in cell lines there mitosis (including normal hematopoietic cells), one could does appear to be a weak but direct correlation with the quantity argue that complete inhibition of survivin as a therapy may have of survivin expression and sensitivity to YM155. Our studies increased side effects because of its essential role within the cell. would suggest that there is a distribution of expression of Instead, perhaps selective inhibition of survivin as an IAP may survivin in primary patient samples and the expression level may provide a better therapy. Our data, which were largely collected not correlate with sensitivity to targeting of survivin. using siRNA in which only partial silencing of survivin was There are many ways of targeting survivin such as antisense achieved, may suggest that a therapeutic strategy that also oligonucleotides, ribozymes, siRNA, dominant-negative mu- achieved only partial inhibition of survivin could increase tants, small-molecule antagonists and immunotherapy. Many p53-dependent apoptosis in a select group of pediatric ALL of these compounds are in early clinical phase I and II trials. including Phþ ALL. Therefore, partial inhibition of survivin may Interestingly, most of these compounds are well tolerated with have a selective role in adjuvant therapy for these diseases. In minimal toxicities and appear to have modest effects on heavily contrast, cell line studies using YM155 have not shown a 19,32 pretreated patients when administered as monotherapy. Our correlation with sensitivity to survivin inhibition and p53 status. studies would suggest that pediatric ALL patients will also These observations would suggest that other mechanisms must be benefit from inhibition of survivin. Future studies are currently involved regarding inhibition of survivin and cell death. under development for combination of these survivin-targeted Future studies must be performed to address several unan- agents with other therapeutics. swered questions. The first is the mechanism by which survivin In our studies we chose to utilize YM155 because this drug inhibits the p53-dependent apoptotic pathway in leukemic cells. has the advantage over siRNA or antisense oligos in that it does Previous data in t(17;19) cell lines would suggest that survivin not require a specific delivery system. When the drug is given as sensitivity is mediated through a mechanism independent of a continuous intravenous infusion for 7 days, it was well caspases 3 and 9. Yet, we have observed that both the cell line tolerated and also had modest effects on the diseases, including HAL01 and the primary t(17;19) patient sample were relatively diffuse large B-cell lymphomas. Our studies would suggest insensitive to YM155. This may suggest that there are other that specific patient samples or disease subtypes may respond mechanisms involved in this disease as opposed to the other differently to these drugs. Therefore, selection of patients based samples such as drug efflux. Another concern would be the on in vitro sensitivity screens may improve response rates to this relative utility of targeting survivin in pediatric patients with and other survivin-targeted compounds. ALL. Our data would suggest that there may be a selective Clearly, there are a variety of potential mechanisms that may population that is more sensitive to inhibition of survivin. explain the heterogeneous responses observed to inhibition of Currently, there are clinical trials using antisense oligonucleo- survivin. As knockout studies have confirmed survivin as an tides (EZN-3042) to survivin either under development or in essential protein and it is also expressed in all cells undergoing phase I trials for relapsed leukemias. Our prediction would be a Leukemia p53, apoptosis, survivin, acute lymphoblastic leukemia JW Tyner et al heterogeneous response in these trials as only a subset of 7 Ryan BM, O’Donovan N, Duffy MJ. Survivin: a new target for anti- cancer therapy. Cancer Treat Rev 2009; 35: 553–562. patients within each disease subtype would be predicted to 8 Caldas H, Jiang Y, Holloway MP, Fangusaro J, Mahotka C, exhibit survivin sensitivity, based on our findings. Furthermore, Conway EM et al. Survivin splice variants regulate the our studies show the importance of preselection of subjects by balance between proliferation and cell death. Oncogene 2005; in vitro inhibitory screens to identify subjects more likely to 24: 1994–2007. respond to survivin inhibition. Therefore, we would propose to 9 Mita AC, Mita MM, Nawrocki ST, Giles FJ. Survivin: key regulator perform in vitro screening to assist with subject selection. of mitosis and apoptosis and novel target for cancer therapeutics. Clin Cancer Res 2008; 14: 5000–5005. In conclusion, our data support the concept that targeting 10 Li F, Altieri DC. Transcriptional analysis of human survivin gene survivin may be of clinical benefit in pediatric ALL. Further- expression. Biochem J 1999; 344 (Pt 2): 305–311. more, this targeting would be selective to the role of survivin as 11 Wheatley SP, Carvalho A, Vagnarelli P, Earnshaw WC. INCENP is an IAP as opposed to its fundamental role in the chromosome required for proper targeting of Survivin to the centromeres and the passenger complex. Future studies will be needed to verify these anaphase spindle during mitosis. Curr Biol 2001; 11: 886–890. results in primary patient samples and to identify patients who 12 Uren AG, Wong L, Pakusch M, Fowler KJ, Burrows FJ, Vaux DL et al. Survivin and the inner centromere protein INCENP show will respond to therapies targeting survivin. similar cell-cycle localization and gene knockout phenotype. Curr Biol 2000; 10: 1319–1328. 13 Bolton MA, Lan W, Powers SE, McCleland ML, Kuang J, Conflict of Interest Stukenberg PT. Aurora B kinase exists in a complex with survivin and INCENP and its kinase activity is stimulated by survivin BHC, JWT and MT have no competing financial interests. BJD binding and phosphorylation. Mol Biol Cell 2002; 13: 3064–3077. has financial interest in MolecularMD; BJD receives clinical trial 14 Chu Y, Yao PY, Wang W, Wang D, Wang Z, Zhang L et al. Aurora funding from Novartis and BMS. B kinase activation requires survivin priming phosphorylation by PLK1. J Mol Cell Biol 2011; 3: 260–267. 15 Tyner JW, Deininger MW, Loriaux MM, Chang BH, Gotlib JR, Acknowledgements Willis SG et al. RNAi screen for rapid therapeutic target identification in leukemia patients. Proc Natl Acad Sci USA 2009; 106: 8695–8700. We thank Leslie Smith for her expertise in microscopy. We also 16 Azuara V. Profiling of DNA replication timing in unsynchronized thank Dr Peter Rotwein for access to Odyssey. This work was cell populations. Nat Protoc 2006; 1: 2171–2177. supported in part by the Leukemia & Lymphoma Society. BHC is 17 Okuya M, Kurosawa H, Kikuchi J, Furukawa Y, Matsui H, Aki D supported in part by the Oregon Child Health Research Center et al. Up-regulation of survivin by the E2A-HLF chimera is (National Institute of Child Health and Development K12) and the indispensable for the survival of t(17;19)-positive leukemia cells. St Baldrick’s Foundation. JWT is supported by grants from the J Biol Chem Jan 15; 285: 1850–1860. 18 Dohi T, Beltrami E, Wall NR, Plescia J, Altieri DC. Mitochondrial National Cancer Institute (NCI) as well as grants from the William survivin inhibits apoptosis and promotes tumorigenesis. J Clin Lawrence and Blanche Hughes Foundation and the Oregon Invest 2004; 114: 1117–1127. Clinical and Translational Research Institute (OCTRI) Grant 19 Nakahara T, Takeuchi M, Kinoyama I, Minematsu T, Shirasuna K, number UL1 RR024140 from the National Center for Research Matsuhisa A et al. YM155, a novel small-molecule Resources (NRCC), a component of the NIH, and NIH Roadmap survivin suppressant, induces regression of established human for Medical Research. hormone-refractory prostate tumor xenografts. Cancer Res 2007; 67: 8014–8021. 20 Mirza A, McGuirk M, Hockenberry TN, Wu Q, Ashar H, Black S Author contributions et al. Human survivin is negatively regulated by wild-type p53 and participates in p53-dependent apoptotic pathway. Oncogene BHC and JWT designed the experiments; BHC and AMJ 2002; 21: 2613–2622. performed the experiments; and BHC, JWT, AMJ, MT and BJD 21 Fenaux P, Jonveaux P, Quiquandon I, Preudhomme C, Lai JL, wrote the manuscript. Vanrumbeke M et al. Mutations of the p53 gene in B-cell lymphoblastic acute leukemia: a report on 60 cases. Leukemia 1992; 6: 42–46. References 22 Zhou M, Gu L, Yeager AM, Findley HW. Sensitivity to Fas- mediated apoptosis in pediatric acute lymphoblastic leukemia is associated with a mutant p53 phenotype and absence of Bcl-2 1 Kersey JH. Fifty years of studies of the biology and therapy of expression. Leukemia 1998; 12: 1756–1763. childhood leukemia. Blood 1997; 90: 4243–4251. 23 Zhou M, Gu L, Li F, Zhu Y, Woods WG, Findley HW. DNA 2 Bhojwani D, Kang H, Moskowitz N, Min D, Lee H, Potter J et al. damage induces a novel p53-survivin signaling pathway regulating Biologic pathways associated with relapse in childhood acute cell cycle and apoptosis in acute lymphoblastic leukemia cells. lymphoblastic leukemia: a Children’s Oncology Group study. J Pharmacol Exp Ther 2002; 303: 124–131. Blood 2006; 108: 711–717. 24 Conway EM, Pollefeyt S, Cornelissen J, DeBaere I, Steiner-Mosonyi 3 Troeger A, Siepermann M, Escherich G, Meisel R, Willers R, M, Ong K et al. Three differentially expressed survivin cDNA Gudowius S et al. Survivin and its prognostic significance variants encode proteins with distinct antiapoptotic functions. in pediatric acute B-cell precursor lymphoblastic leukemia. Blood 2000; 95: 1435–1442. Haematologica 2007; 92: 1043–1050. 25 Liu T, Brouha B, Grossman D. Rapid induction of mitochondrial 4 Song Z, Yao X, Wu M. Direct interaction between survivin events and caspase-independent apoptosis in Survivin-targeted and Smac/DIABLO is essential for the anti-apoptotic activity of melanoma cells. Oncogene 2004; 23: 39–48. survivin during taxol-induced apoptosis. J Biol Chem 2003; 278: 26 Carter BZ, Mak DH, Schober WD, Cabreira-Hansen M, Beran M, 23130–23140. McQueen T et al. Regulation of survivin expression through Bcr- 5 Mahotka C, Wenzel M, Springer E, Gabbert HE, Gerharz CD. Abl/MAPK cascade: targeting survivin overcomes imatinib resis- Survivin-deltaEx3 and survivin-2B: two novel splice variants of the apoptosis inhibitor survivin with different antiapoptotic properties. tance and increases imatinib sensitivity in imatinib-responsive Cancer Res 1999; 59: 6097–6102. CML cells. Blood 2006; 107: 1555–1563. 6 Badran A, Yoshida A, Ishikawa K, Goi T, Yamaguchi A, Ueda T 27 Baudis M, Prima V, Tung YH, Hunger SP. ABCB1 over-expression et al. Identification of a novel splice variant of the human and drug-efflux in acute lymphoblastic leukemia cell lines with anti-apoptosis gene survivin. Biochem Biophys Res Commun t(17;19) and E2A-HLF expression. Pediatr Blood Cancer 2006; 47: 2004; 314: 902–907. 757–764. Leukemia p53, apoptosis, survivin, acute lymphoblastic leukemia JW Tyner et al 28 Wang Z, Fukuda S, Pelus L. Survivin regulates the P53 tumor 32 Nakahara T, Kita A, Yamanaka K, Mori M, Amino N, Takeuchi M suppressor gene family. Oncogene 2004; 23: 8146–8153. et al. Broad spectrum and potent antitumor activities of YM155, a 29 Fukuda S, Foster RG, Porter SB, Pelus LM. The antiapoptosis novel small-molecule survivin suppressant, in a wide variety of protein survivin is associated with cell cycle entry of normal human cancer cell lines and xenograft models. Cancer Sci 2011; cord blood CD34(+) cells and modulates cell cycle and prolifera- 102: 614–621. tion of mouse hematopoietic progenitor cells. Blood 2002; 100: 33 Ryan B, O’Donovan N, Duffy M. Survivin: a new target for 2463–2471. anti-cancer therapy. Cancer Treat Rev 2009; 35: 553–562. 30 Gianani R, Jarboe E, Orlicky D, Frost M, Bobak J, Lehner R et al. Expression of survivin in normal, hyperplastic, and neoplastic This work is licensed under the Creative Commons colonic mucosa. Hum Pathol 2001; 32: 119–125. Attribution-NonCommercial-No Derivative Works 31 Papapetropoulos A, Fulton D, Mahboubi K, Kalb RG, O’Connor 3.0 Unported License. To view a copy of this license, visit http:// DS, Li F et al. Angiopoietin-1 inhibits endothelial cell apoptosis via the Akt/survivin pathway. J Biol Chem 2000; 275: 9102–9105. creativecommons.org/licenses/by-nc-nd/3.0/ Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu) Leukemia http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Leukemia Springer Journals

Targeting survivin and p53 in pediatric acute lymphoblastic leukemia

Leukemia , Volume 26 (4) – Sep 30, 2011

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Springer Journals
Copyright
Copyright © 2012 by The Author(s)
Subject
Medicine & Public Health; Medicine/Public Health, general; Internal Medicine; Intensive / Critical Care Medicine; Cancer Research; Oncology; Hematology
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0887-6924
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1476-5551
DOI
10.1038/leu.2011.249
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Abstract

Leukemia (2012) 26, 623–632 & 2012 Macmillan Publishers Limited All rights reserved 0887-6924/12 www.nature.com/leu ORIGINAL ARTICLE 1 2 3 1,4 2 JW Tyner , AM Jemal , M Thayer , BJ Druker and BH Chang 1 2 Division of Hematology and Medical Oncology, and OHSU Knight Cancer Institute, OHSU, Portland, OR, USA; Division of Hematology and Oncology, Department of Pediatrics and OHSU Knight Cancer Institute, OHSU, Portland, OR, USA; Department of Biochemistry and Molecular Biology and OHSU Knight Cancer Institute, OHSU, Portland, OR, USA and Howard Hughes Medical Institute Despite advances in treatment and outcomes for patients with malignancy with little to no expression in normal terminally pediatric acute lymphoblastic leukemia (ALL), there continue to differentiated tissue. Furthermore, survivin overexpression has be subsets of patients who are refractory to standard correlated with resistant and refractory disease in many different chemotherapy and hematopoietic stem cell transplant. There- malignancies including ALL. fore, novel gene targets for therapy are needed to further Survivin is a small 16 kD protein that belongs to the inhibitor advance treatment for this disease. RNA interference technol- of apoptosis (IAP) family and also functions as a member of the ogy has identified survivin as a potential therapeutic target. Survivin, a member of the inhibitor of apoptosis (IAP) proteins chromosome passenger complex. Survivin is a unique member and chromosome passenger complex, is expressed in hemato- of the IAPs in that it is both the smallest member and may not logic malignancies and overexpressed in relapsed pediatric directly interact with caspases. Instead, it may interact with ALL. Our studies show that survivin is uniformly expressed at another IAP, smac/Diablo, to regulate apoptosis within the high levels in multiple pediatric ALL cell lines. Furthermore, mitochondria. Transcription of survivin shows several splice silencing of survivin expression in pediatric ALL cell lines as variants with conserved N-terminal domains, with most of the well as primary leukemic blasts reduces viability of these cells. This includes cell lines derived from patients with relapsed divergence occurring within or after the baculovirus IAP repeat 5–7 disease featuring cytogenetic anomalies such as t(12;21), domain. Recent published reports would suggest that these Philadelphia chromosome t(9;22), t(1;19) as well as a cell line splice variants may have different subcellular localization carrying t(17;19) from a patient with de novo ALL. Furthermore, including the mitochondria and different apoptotic activities. inhibition of survivin increases p53-dependent apoptosis that The role of survivin as a member of the chromosome can be rescued by inhibition of p53. Finally, a screen of passenger complex, in which it plays a critical role during randomly selected primary patient samples confirms that survivin-specific small interfering RNA and survivin-targeted mitosis, is better defined. As such, survivin expression is cell- drug, YM155, effectively reduce viability of leukemic blasts. cycle dependent, with the highest expression during G2/M Leukemia (2012) 26, 623–632; doi:10.1038/leu.2011.249; through canonical cell cycle-dependent and cell-cycle homol- published online 30 September 2011 ogy regions within the proximal promoter. During mitosis, Keywords: RNAi; targeted therapy; IAP survivin specifically localizes with INCENP and Aurora B kinase within the mitotic apparatus from the centromeres in prophase, kinetochores in metaphase, the mid-plate during anaphase and Introduction the mid-body during cytokinesis. Furthermore, it is the direct interaction of survivin with both Aurora B kinase and INCENP 11–13 Over the past 50 years, the prognosis for pediatric acute that is essential for cell division. Survivin phosphorylation lymphoblastic leukemia (ALL) has changed from a terminal at Ser 20 by PLK1 (polo-like kinase) is required for the priming of diagnosis to a treatable disease. The success of current Aurora B kinase activity to undergo cell division. therapeutic regimens has given rise to the identification of a In this study we verified the cell-cycle dependence of survivin subset of patients who will have recurrent or refractory disease. expression within pediatric ALL cells. We further tested whether Unfortunately, patients within these subsets have highly resistant several pediatric leukemic cell lines as well as primary patient disease that may not be overcome even with myeloablative samples were sensitive to manipulation of survivin expression therapy and hematopoietic stem cell transplant. These diseases and activity. Pediatric ALL cell lines have similar expression may have approached the limit of treatment by standard levels of total survivin. In addition, the expression patterns of chemotherapeutic regimens. Therefore, new targets for therapy survivin among cell lines were similar with an increase in are imperative in the hope of improving outcomes. survivin expression during G /M. Virtually all of the ALL cell A report from the Children’s Oncology Group has shown a lines tested were sensitive to silencing of survivin including REH differential expression profile of relapsed ALL compared with (ETV6-RUNX1), SUPB15 (BCR-ABL) RCH-ACV (E2A-PBX1) and initial diagnosis. One of the genes showing a marked increase HAL01 (E2A-HLF). We also identified that targeting survivin in expression in patients with recurrent disease is survivin (aka either by small interfering RNA (siRNA) or by the survivin- BIRC5). Survivin is an attractive target for therapy because it is suppressor YM155 in these cell lines increased cell death expressed mainly during development and in the setting of through the p53-dependent apoptosis pathway. This increase in cell death could then be rescued by silencing p53. Finally, early Correspondence: Dr BH Chang, Division of Hematology and screening of patient samples with survivin siRNA or YM155 Oncology, Department of Pediatrics, OHSU Knight Cancer Institute, showed sample-specific variation of sensitivity to survivin Mailcode CDRCP, 3181 SW Sam Jackson Park Road, Portland, silencing. The heterogeneity of YM155 responses would suggest OR, USA. that other factors may have importance for primary lympho- E-mail: changb@ohsu.edu blasts to respond to survivin inhibition beyond p53. As such, Received 5 February 2011; revised 10 July 2011; accepted 11 August 2011; published online 30 September 2011 early disease selection through in vitro screening may become p53, apoptosis, survivin, acute lymphoblastic leukemia JW Tyner et al important for future clinical strategies that would employ 2–4 h at 37 1C. The cell media was then supplemented with 0.5 ml survivin as a therapeutic gene target. of RPMI containing FBS such that the final concentration of FBS was 10–20% (depending on cell line). A total of 50 000 cells per well were plated in triplicate and grown for 4 days for either MTS or for Materials and methods Gauva Nexin assay. The remaining cells were harvested 48 h after treatment for immunoblot. Reagents Fetal bovine serum (FBS) was obtained from Hyclone Laboratories Immunoblot analysis Inc. (South Logan, UT, USA). All other tissue culture reagents Cells were washed with PBS and lysed with 1 SDS loading buffer were obtained from Invitrogen Corporation (Carlsbad, CA, USA). (75 mM Tris,pH6.8,3%SDS,15% glycerol,8% b-mercaptoetha- The siRNAs (Supplementary Table 1) were from the siGenome nol, 0.1% bromophenol blue). For mitochondrial fractionation, cells SMARTpool designed by Dharmacon (ThermoFisher Scientific, were processed using ApoALert Cell Fractionation Kit (Clontech, Waltham, MA, USA). Viability assays were performed with Mountain View, CA, USA). All samples were separated by standard CellTiter 96 AQ One Solution Cell Proliferation Assay from ueous SDS-polyacrylamide gel electrophoresis and transferred onto poly- Promega Corporation (Madison, WI, USA). Apoptosis assays were vinylidene fluoride membrane (Immobilon-FL, Millipore). Mem- performed using the Guava Nexin Assay (Millipore, Billerica, MA, branes were blocked with Aquablock tm/EIA/WB (EastCoast Bio, USA). YM155 was purchased from Selleck (Houston, TX, USA) Inc., North Berwick, ME, USA) for 1 h, and then incubated with and solubilized in dimethylsulfoxide at 100 mM stock. Graphical primary antibodies to survivin (Cell Signaling Technology, Inc., and statistical data were generated using either Microsoft Excel or Danvers, MA, USA), (Ser 10) phospho-histone H3 (Upstate GraphPad Prism (GraphPad Software, Inc., La Jolla, CA, USA). Biotechnology, Lake Placid, NY, USA), tubulin (Sigma-Aldrich Corp., St Louis, MO, USA), (Ser 15) phospho-p53 (Cell Signaling), Cell lines and tissue culture p53 (Santa Cruz, Biotechnology, Inc., Santa Cruz, CA, USA) and RCH-ACV (RCH) (Deutsche Sammlung von Mikroorganismen ABL (BD Biosciences, San Jose, CA, USA) in Aquablock/0.1% und Zellkulturen GmbH (DSMZ), Braunschweig, Germany) is a Tween-20 overnight at 4 1C. Secondary fluorescent antibodies pediatric ALL cell line from a patient with recurrent disease (Molecular Probes) were used and detected and quantified with carrying the E2A-PBX1 t(1;19) chimeric protein. REH (ATCC) is a Odyssey (LI-COR, Corporate Offices, Lincoln, NE, USA). pediatric ALL cell line from a patient with recurrent disease carrying the ETV6-RUNX1 t(12;21) chimeric protein. SUPB15 Immunofluorescence (American Type Culture Collection (ATCC), Manassas, VA, USA) 5 ALL cells were grown in complete media and then B5 10 is a pediatric ALL cell line also from a patient with recurrent 4 cells were harvested and resuspended in 0.5 ml of PBS. 2 10 disease carrying the BCR-ABL t(9;22) translocation. HAL01 cells cells were spotted onto a cover slip for 10 min. Cells were fixed (DSMZ) are from a pediatric patient with de novo ALL with the with 4% formaldehyde at 37 1C for 10 min. Samples were then E2A-HLF t(17;19). RCH, REH and HAL01 cells were maintained permeabilized with 0.25% Triton X-100 and washed with PBS. in RPMI with 10% FBS, 4 mM glutamine and 1% penicillin and Cells were then stained overnight in 4 1C with primary streptomycin. SUPB15 cells were maintained in RPMI with 20% antibodies and subsequently stained for 1 h with secondary FBS, 4 mM glutamine, 50 nM 2-mercaptoethanol and 1% antibody at room temperature. All images were captured with penicillin and streptomycin. All patient samples were obtained an Olympus (San Jose, CA, USA) BX Fluorescent Microscope with informed consent approved by the institutional review using a  50–100 objective, automatic filter-wheel and Cytovi- board of Oregon Health and Science University. sion workstation. Small interfering RNA knockdown, proliferation and Cell sorting induction of apoptosis ALL cell lines were grown in RPMI/10% FBS to a concentration Standard electroporation was modified from a previously described of 5–10 10 cells/ml. Cells were then washed with PBS/1% 15 5 protocol. Briefly, 1.5 10 cells per condition were resuspended FBS and fixed in 70% ethanol and stored at 20 1C. The samples in 75 ml siPORT buffer (Applied Biosystems, Life Technologies were then washed with PBS and stained with buffer containing Corporation, Carlsbad, CA, USA). To the samples, 1–2mM of siRNA 3mM EDTA, pH 8.0, 0.05% NP-40, 50 mg/ml propidium iodide was added. Cells were electroporated at 200 V, 250 ms, 2 pulses, and 1 mg/ml RNAse A in PBS. Cells were sorted by DNA and 20 000 cells per well were plated in triplicate containing content with BD FACS/Aria (BD Biosciences) and harvested with 100 ml of culture media. The remaining 60 000 cells were plated SDS loading buffer. into a well containing 500 ml of culture media. For determination of cell viability, the triplicate plates containing 20 000 cells were YM155 dose response subjected to the CellTiter 96 AQ One Solution Cell Prolifera- ALL cells lines (5000 cells per well) and primary patient samples ueous tion Assay (MTS). For subsequent immunoblot analysis, the plate (50 000 cells per well) were incubated with graded concentra- containing 60 000 cells were harvested and lysed in 20 mlof 1 tions of YM155 (0–10 mM) in RPMI with 10% FBS. After 3 days, sodium dodecyl sulfate (SDS) loading buffer. Identification of cells were subjected to MTS for assessment of cell viability. induction of apoptosis was performed using the Guava Nexin assay All values were normalized to the no drug control from each (Millipore). Briefly, triplicate samples containing 20 000 cells were respective cell line. incubated with 60 ml of the Guava Nexin reagent and then analyzed through the microcapillary flow cytometer at varying time points up to 96 h. Results Cells were also treated with transductin (Integrated DNA Technologies (IDT), Inc., Coralville, IA, USA) for introduction of Pediatric ALL cell lines express high levels of survivin siRNA into the cells. A total of 500 nM of siRNA was incubated in mostly at G /M phosphate buffered saline (PBS) with 5mM transductin and added to Prior studies have suggested that there are varying amounts of 2.5 10 cells in 0.5 ml of RPMI with 1% bovine serum albumin for survivin expression in primary pediatric ALL samples and in Leukemia p53, apoptosis, survivin, acute lymphoblastic leukemia JW Tyner et al G1 S1 S2 G2 G1 S1 S2 G2 DNA Content G1 S1 S2 G2/M G1 S1 S2 G2/M Survivin pH3 Tubulin REH (ETV6-RUNX1) RCH (E2A-PBX1) Survivin pH3 Tubulin G1 S1 S2 G2/M G1 S1 S2 G2/M Survivin pH3 Tubulin HAL01 (E2A-HLF) SUPB15 (BCR-ABL) 0.2 0.04* REH (ETV6-RUNX1) 2 RCH (E2A-PBX1) HAL01 (E2A-HLF) SUPB15 (BCR-ABL) 1 2 pH3 tubulin late interphase prophase metaphase telophase REH RCH HAL01 SUPB15 C M C M C M C M survivin cytochrome c Figure 1 Relative expression of survivin in ALL cell lines and cell-cycle dependence. (a) Immunoblot of asynchronous populations ALL cell lines for survivin, (Ser 10) phospho-histone H3 (pH3) and tubulin. The cell lines tested include REH (ETV6-RUNX1), RCH-ACV (E2A-PBX1), HAL01 (E2A-HLF) and SUPB15 (BCR-ABL). The immunoblot shown is representative of three independent experiments. (b) Survivin level is dependent on the cell cycle in ALL cell lines. An asynchronous population of ALL cell lines was fixed in 70% ethanol and stained with propidium iodide. Cells were flow sorted by DNA content for G1, early S (S1), late S (S2) and G2/M. Top panels represent the histograms of the cells that were sorted. Bottom panels represent the immunoblots for survivin, pH3 and tubulin. (c) Quantification of survivin expression in relation to tubulin and pH3. (Left panel) Each band from immunoblots was quantified by Odyssey for fluorescence intensity and normalized either to tubulin for pH3. Ratios were then compared with REH as control. (Right panel) Graphical representation of the relative quantity of survivin normalized to tubulin from (b). Quantity is expressed in comparison with REH in G1. *Denotes Po0.05 by student’s t-test. (d) Subcellular localization of survivin in lymphoblasts. (Upper panel) Each cell line showed similar staining patterns. Representative images of later interphase, prophase, metaphase and telophase are shown from each cell line. 5 10 cells of an asynchronous population were dropped onto coverslips and fixed with formaldehyde, and processed for dual immunofluorescence labeling; survivin (green), pH3 (red), DAPI (blue). (Lower panel). Immunoblot of survivin and cytochrome c after fractionation with Apoalert Fractionation. C, cytosolic fraction; M, mitochondrial fraction. 2,17 17 pediatric ALL cell lines. To validate this finding, we assessed E2A-HLF found in t(17;19). Immunoblot of a population of survivin expression level in a variety of cell lines derived from asynchronous cells showed varying amounts of survivin divergent subsets of pediatric ALL patients, including HAL01 when normalized to tubulin expression, with SUPB15 cells cells that have been used to describe the dependence of survivin showing a statistically significant difference to REH (t-test 0.04; overexpression through the chimeric transcription factor Figures 1a and c). Leukemia REH (ETV6-RUNX1) Survivin expression normalized to REH RCH (E2A-PBX1) HAL01 (E2A-HLF) SUPB15 (BCR-ABL) Count Survivin expression normalized to REH G1 G1 S1 S2 G2/M G1 S1 S2 G2/M G1 S1 S2 G2/M G1 S1 S2 G2/M p53, apoptosis, survivin, acute lymphoblastic leukemia JW Tyner et al Survivin expression is regulated by the cell cycle and plays a be seen in a dose-dependent manner even at 24 h after exposure 11–13 role within the chromosome passenger complex. These to YM155 (Supplementary Figure 1). To test whether YM155 chromosome passenger complex proteins show relatively lower sensitivity was specific to inhibition of survivin expression, RCH expression in G /S phase with the highest expression in G /M. cells were transfected with pMIG-Survivin. Cells were treated 1 2 To determine whether the small variations of survivin expression with 1 mM YM155 for 48 h, and then assayed for apoptosis by observed in cell lines is dependent on the proportion of cells in Annexin V staining. Ectopic expression of survivin in RCH cells G /M phase at the time of cell lysis, we also examined cell partially rescues apoptosis when treated with 1 mM YM155, lysates for abundance of Ser 10 phosphorylation on histone H3 further validating the selective inhibition of survivin (Supple- (pH3), a marker for G /M phase. After normalization of survivin mentary Figure 1d). Interestingly, there was some variation of expression to pH3, there appeared to be no significant sensitivity to this compound with REH cells being the most difference in the amount of survivin expression between cell sensitive (half-maximal inhibitory concentration (IC )17nM) lines (Figures 1a and c). Furthermore, most of the expressed and HAL01 showing the least sensitivity (IC 560 nM). RCH and survivin appears to be in G2/M as shown by flow sorting cells SUPB15 cells had IC ’s that ranged between 10 and 50 nM. from different stages of the cell cycle followed by immunoblot These results would suggest a possible heterogeneity of response for survivin, pH3 and tubulin (Figures 1b and c). among ALL lymphoblasts to inhibition of survivin. The cell cycle-dependent expression of survivin would suggest that in ALL cells lines the major role of survivin would Survivin inhibits the p53-dependent apoptotic pathway be as a member of the chromosome passenger complex. in ALL cell lines However, survivin has been shown to exhibit divergent Previous studies have suggested that overexpression of survivin subcellular localizations with certain splice variants exhibiting inhibits the p53-dependent apoptosis pathway. Therefore, cytosolic and mitochondrial distribution, suggesting that survi- inhibition of survivin may allow for re-activation of this p53- vin may also play a role as an IAP. As such, we next wanted to mediated apoptotic program. Prior data suggest that most pediatric assess whether there are different subcellular pools of survivin ALL cell lines are wild type for TP53 by gene expression within ALL cell lines. We examined survivin expression patterns 21,22 patterns. To test whether the cells lines evaluated in this in RCH, REH, SUPB15 and HAL01 cells by immunofluores- study had an intact p53-dependent cell-cycle arrest, the cells were cence. In each cell line, the majority of survivin staining was treated with 0.1 mg/ml of doxorubicin. Doxorubicin is an evident in cells undergoing mitosis (Figure 1d and data not anthracycline that is known to inhibit resealing of DNA breaks, shown). There is an increase in survivin staining in early thereby activating a p53-dependent cell-cycle arrest and apoptosis prophase with evident colocalization with chromosomal mate- through the intrinsic pathway. RCH, REH, SUP B15 and HAL01 rial, and there is a subsequent increase in the intensity of cells all showed activation of p53 by phosphorylation at Ser 15 survivin staining through metaphase. As the chromosomes (pTP53) within 4 h of treatment (Figure 3a). Furthermore, these segregate, survivin stays at the mid-body through cytokinesis. cells showed cell-cycle arrest by decrease in pH3. These results are consistent with prior published data on the To test whether the increase in cell death observed after 8,11 subcellular localization of survivin in monolayer cells. silencing of survivin in ALL cell lines was due to p53 activity, Interestingly, we do observe some variation in punctate RCH and REH cells were treated with a combination of survivin cytosolic staining for survivin in interphase cells. The antibody and p53 siRNA. As previously shown, knockdown of survivin to survivin, which is generated against the N-terminus of the significantly decreased the viability and increased annexin V protein (Cell Signaling), is predicted to react to all splice staining of both RCH and REH cells. Interestingly, treatment variants. Furthermore, cell fractionation does show that there is with a combination of siRNA to p53 and survivin partially a small population of survivin in both the cytosolic and rescued cell viability and decreased annexin V staining mitochondrial fractions (Figure 1d). (Figure 3b). Immunoblots performed showed similar amounts of protein silencing (Figure 3c), whether alone or in combina- tion. Interestingly, treatment of cells with siRNA to survivin Inhibition of survivin expression reduces cell viability in increases the quantity of p53 within the cell (Figure 3c). This ALL cell lines increase in p53 is also abrogated by siRNA to p53, suggesting As ALL cell lines appear to express similar amounts of survivin, that this phenomenon is from increased transcription or stability we next tested whether survivin knockdown would have of the p53 transcript rather than enhanced stability of the p53 deleterious effects on viability of these cells. Leukemia cell protein. These results are consistent with prior findings that lines treated with survivin-specific siRNA consistently showed a inhibition of survivin expression increases p53-dependent significant reduction in viability, with RCH, REH, SUPB15 and apoptosis and may begin to shed new light as to the mechanism HAL01 cells each exhibiting at least 40% decrease in cell of this inhibition. Prior studies have shown conflicting data as viability (Figure 2a). Immunoblots performed 48 h after siRNA to whether survivin interacts with or is independent of treatment confirmed siRNA-mediated silencing of survivin of 17,24,25 19 caspases. For example, Nakahara et al. have shown B50% reduction in levels (Figure 2b and data not shown). that caspase 3 is activated by YM155 in HRPC cells whereas Recently, a novel small-molecule inhibitor, YM155, was Okuya et al. describe caspase-independent cell death in developed by Astellas Pharma US, Inc. (Deerfield, IL, USA) as a t(17;19) ALL. Our studies would further agree with Okuya potent inhibitor of survivin expression. This compound was et al. that neither caspase 3 nor caspase 9 is activated when developed using a screen that identified small molecules that treated with either siRNA or YM155, yet they still activate would only inhibit survivin expression at the promoter. As this apoptosis in ALL cells (Supplementary Figures 1 and 2). reagent offered a different mode of survivin suppression, we also tested the impact of this compound on viability of ALL cell lines. Consistent with siRNA results, each of the cell lines tested Phþ ALL cells are sensitive to knockdown of survivin showed a dose-dependent sensitivity to YM155 as measured by independent of imatinib cell viability 72 h after exposure (Figure 2c). Furthermore, As previously discussed, Phþ ALL (BCR-ABL) is a model of inhibition of survivin expression and increase in apoptosis can resistant disease in which the primary oncogene is well Leukemia p53, apoptosis, survivin, acute lymphoblastic leukemia JW Tyner et al Figure 2 Inhibition of survivin expression inhibits cell viability for REH, RCH, SUPB15 and HAL01. (a) Cell viability as measured by MTS 96 h after treatment with siRNA for survivin. All samples were treated with nonspecific (NS) siRNA as a control and normalized to 100%. s.e.m. bars are inserted. *Po0.05, **Po0.01. (b) Representative immunoblots for the expression level of survivin treated with NS siRNA or survivin siRNA. Tubulin expression was used as a control for loading. (c) Dose response of ALL cell lines to YM155. Asynchronous populations of cells were treated with increasing doses of YM155 for 72 h. Then, viability was measured by MTS and normalized to no drug control. S.e.m. bars are inserted. Dotted line represents 50% viability. characterized. Although the addition of imatinib as targeted siRNA for survivin and p53 in the presence of 500 nM imatinib therapy has improved outcome, there remain patients who (Figure 4d). In contrast to cells treated with ABL siRNA where no continue to relapse. To test whether Phþ ALL would also be additivity with survivin was observed, knockdown of survivin did sensitive to targeting of survivin, SUPB15 cells were treated with show an additive increase in cell death in the presence of siRNA to survivin, BCR-ABL (ABL),and p53 (Figure 4). Knock- imatinib. One possibility for this difference could involve minor down of survivin increases Annexin V staining that is then variations in signaling that occur after elimination of BCR-ABL rescued by siRNA to p53 (Figures 4a and b). Silencing of protein from cells (siRNA) versus inhibition of BCR-ABL activity survivin or BCR-ABL decreased cell viability by B50% without eliminating the protein (imatinib). Another possibility (Figure 4c). Furthermore, simultaneous knockdown of BCR- could include inhibition of other non-BCR-ABL pathways by ABL and p53 had a minimal effect on the cells, suggesting that imatinib that could synergize with silencing of survivin. Knock- cell death after silencing of BCR-ABL occurs mainly through a down of p53 did not rescue the effects on cell viability of either p53-independent mechanism. imatinib or ABL siRNA. In contrast, silencing of p53 did rescue Interestingly, the combination of survivin and BCR-ABL the effects of both survivin siRNA and YM155 (Figure 4d, lower silencing did not show an additive effect. It has previously been panel), and YM155 appears to activate p53 (Supplementary shown that in certain CML cell lines, the BCR-ABL/mitogen- Figure 1a). Cumulatively, these results support the role of survivin activated protein kinase pathway increases expression of survivin as an inhibitor of p53-dependent apoptosis in Phþ ALL, which is and that silencing of survivin enhances imatinib-induced cell a pathway independent of BCR-ABL function, suggesting that death. To test whether survivin knockdown could also enhance targeting of survivin could enhance therapy by inhibiting imatinib-induced cell death, SUPB15 cells were treated with independent pathways to imatinib. Leukemia p53, apoptosis, survivin, acute lymphoblastic leukemia JW Tyner et al Figure 3 Knockdown of p53 rescues cell death because of silencing of survivin. (a) Activation of p53 by treatment with doxorubicin. 1 10 cells of RCH, REH, SUPB15 and HAL01 were treated with 0.1 mg/ml of doxorubicin (Sigma) for 0, 4 and 8 h. The cells were then harvested, separated and immunoblotted for (Ser 15)-phospho-p53 (pp53), pH3 and tubulin. S15P p53 evaluated the phosphorylation state of p53 at Ser 15 by activation of the replication checkpoint. pH3 was again used as a marker for cell-cycle arrest reflecting a decrease in the number of cells entering mitosis. (b) Partial rescue of cell viability by knockdown of p53. RCH and REH cells were treated with siRNA to NS, survivin, p53 and a combination of p53/survivin. Viability was tested by MTS and normalized to NS (left panel). Apoptosis was then evaluated by Annexin V binding (right panel). (c) Immunoblots of representative duplicate experiments to quantify the expression of survivin, and p53 after treatment with siRNA. Lower panels show the relative expression normalized to tubulin quantified by fluorescence intensity. Primary patient samples are sensitive to inhibition of E2A-HLF. Immunoblots were also performed to identify the survivin expression levels of survivin as compared with tubulin and pH3 Our results would suggest that patients with pediatric ALL could (Figures 5c and d). There was a distribution of variability of benefit from inhibition of survivin. For initial, preclinical expression that does not appear to correlate with sensitivity to validation of this strategy, we isolated leukemic blasts from YM155. For example, patient 5 (relapsed with ETV6-RUNX1) fresh primary ALL samples and treated the cells with siRNA to was most sensitive to YM155, yet had one of the lowest levels of survivin. Treatment of two, randomly selected primary patient survivin expression when normalized to tubulin. However, this samples with survivin siRNA showed response with a 30–50% patient had relatively high expression when normalized to pH3. decrease in cell viability (Figure 5a). To further validate survivin This would suggest that the cell cycle-independent expression of as a bona fide therapeutic target, we treated fresh primary survivin is a more important correlate for YM155 sensitivity than patient samples with YM155. Treatment of four patient samples overall survivin expression. In contrast, samples with E2A-HLF revealed a range of sensitivity to this drug from IC values (patient 3, Figure 5, and HAL01, Figure 2) exhibited less p10 nM to IC ’s exceeding 1 mM consistent with the cell lines sensitivity to YM155 despite high expression of survivin, (Figure 5b). Interestingly, the samples that showed the highest suggesting that other factors may play an important role in IC ’s were the HAL01 cells and the patient sample with YM155 sensitivity. E2A-HLF cell lines have previously been Leukemia p53, apoptosis, survivin, acute lymphoblastic leukemia JW Tyner et al p53 NS 1 41% 1 30% 10 10 10 20 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 4 p53/survivin survivin NS SUR ABL ABL/SUR p53 p53/SUR 55% 35% 80 Control 10 60 500nM 0 1 2 3 4 10 10 10 10 10 0 1 2 3 4 10 10 10 10 10 40 imatinib Annexin V-PE NS SUR p53 p53/SUR NS SUR ABL ABL/SUR p53 p53/SUR p53/ABL BCR-Abl Survivin NS NS/100nM p53 p53/100nM p53 YM155 YM155 Tubulin Figure 4 Sensitivity of inhibition of survivin in SUPB15 cells is dependent on intact p53. (a) Flow cytometric representation of apoptosis by staining for Annexin V and 7-aminoactinomycin D (7-AAD). SUPB15 cells (BCR-ABL) were treated with siRNA to NS, SUR, p53 and p53/SUR. (b) A graphical representation of Annexin V staining in SUPB15 treated with NS, survivin, ABL, ABL/survivin, p53 and p53/survivin. (c) Graphical representation of the percent viability of SUPB15 cells treated with NS, survivin, p53 and p53/survivin. The lower panel represents the immunoblots performed on the samples treated with the siRNA and stained for BCR-ABL, survivin, p53 and tubulin. Viability was tested by MTS and normalized to nonspecific siRNA (NS). (d) Treatment of SUPB15 cells with imatinib or YM155. Top panel shows cells treated with siRNA in the presence of 500 nM imatinib. Cells were treated with siRNA, and then subsequently incubated in 500 nM imatinib for 96 h. Bottom panel shows the partial rescue of SUPB15 cells treated with 100 nM YM155 for 96 h. S.e.m. bars are inserted into each graph. shown to overexpress the drug efflux protein ABCB1, which shown that overexpression of survivin has the ability to inhibit may reduce the amount of YM155 within the cell, thereby degradation of Mdm2 (murine double minute 2), thereby increasing the IC . These studies would suggest that preselec- promoting degradation of p53. Other studies have shown that tion of patients by in vitro screening for sensitivity to YM155 p53 has the ability to decrease survivin expression. In human would be important in future studies using this compound for lung and ovarian cancer cell lines, activation of p53 decreases clinical trials. expression of survivin by changing acetylation of the survivin promoter rather than direct binding. In our studies, silencing of survivin did show a qualitative increase in p53 expression. Discussion These findings would suggest that there is a critical relationship with survivin and p53 whereby higher levels of survivin inhibit In contrast to prior reports, our data have shown that ALL cell p53 either by degradation or inhibition of expression, whereas inhibition of survivin activates p53 and increases p53 expres- lines express similar amounts of survivin and that most of the protein is expressed during G /M. Meanwhile, primary patient sion. Taken together, our studies support the critical role for the interaction of survivin with the p53-dependent apoptotic path- samples do show significant variability in survivin expression. This study also demonstrates that knockdown of survivin in ALL way as an important regulator of growth in pediatric lympho- blasts. increases p53-dependent apoptosis. Furthermore, in the BCR- ABL line SUPB15, reduction of BCR-ABL activity or expression Molecular targeting of survivin continues to be an intriguing concept for therapy. It is a protein whose expression is almost does not increase the p53-dependent apoptotic pathway. exclusively confined to dividing cells such as malignant tissue Finally, we show that screening of primary samples with either siRNA to survivin or the selective suppressor YM155 has the and normal hematopoietic stem cells and colonic epithe- 29,30 lium. Although survivin is expressed differentially within potential to identify samples that are more sensitive to survivin inhibition. the cell cycle with the highest expression at G /M, some studies have suggested a baseline increase in expression in malignant There is evidence to support the interaction of survivin with the p53-dependent apoptotic pathway. In BaF3 cells, it has been cells. Also, higher expression of survivin has been shown in Leukemia 7-AAD Percent viable Percent viability Percent viability Percent annexin V staining p53, apoptosis, survivin, acute lymphoblastic leukemia JW Tyner et al Figure 5 Primary patient (Pat.) samples show sensitivity to inhibition of survivin. See Supplementary Table 2 for patient details. (a) Two random fresh primary patient samples were treated with siRNA to NS and survivin. Viability was then assayed 4 days later by MTS, and normalized to viability to NS. (b) Dose response of patient samples to YM155. Fresh mononuclear cells were separated by Ficoll and B50 000 cells were plated into each well containing increasing concentrations of YM155 from 1 nM to 1 mM. Viability was assayed 3 days later by MTS and normalized to no drug control. Viability was then assayed 3 days later by MTS. All plots contain s.e.m. bars. (c) Immunoblots of patient samples for survivin pH3 and tubulin. Approximately 1 10 cells were lysed in 100 ml of SDS loading buffer and 20 ml was run on polyacrylamide gel electrophoresis (PAGE). (d) Graphical representation of survivin expression normalized to either tubulin or pH3 intensity. These ratios were then compared with REH ratios as a control. 3 29 recurrent and relapsed disease. Furthermore, in cell lines there mitosis (including normal hematopoietic cells), one could does appear to be a weak but direct correlation with the quantity argue that complete inhibition of survivin as a therapy may have of survivin expression and sensitivity to YM155. Our studies increased side effects because of its essential role within the cell. would suggest that there is a distribution of expression of Instead, perhaps selective inhibition of survivin as an IAP may survivin in primary patient samples and the expression level may provide a better therapy. Our data, which were largely collected not correlate with sensitivity to targeting of survivin. using siRNA in which only partial silencing of survivin was There are many ways of targeting survivin such as antisense achieved, may suggest that a therapeutic strategy that also oligonucleotides, ribozymes, siRNA, dominant-negative mu- achieved only partial inhibition of survivin could increase tants, small-molecule antagonists and immunotherapy. Many p53-dependent apoptosis in a select group of pediatric ALL of these compounds are in early clinical phase I and II trials. including Phþ ALL. Therefore, partial inhibition of survivin may Interestingly, most of these compounds are well tolerated with have a selective role in adjuvant therapy for these diseases. In minimal toxicities and appear to have modest effects on heavily contrast, cell line studies using YM155 have not shown a 19,32 pretreated patients when administered as monotherapy. Our correlation with sensitivity to survivin inhibition and p53 status. studies would suggest that pediatric ALL patients will also These observations would suggest that other mechanisms must be benefit from inhibition of survivin. Future studies are currently involved regarding inhibition of survivin and cell death. under development for combination of these survivin-targeted Future studies must be performed to address several unan- agents with other therapeutics. swered questions. The first is the mechanism by which survivin In our studies we chose to utilize YM155 because this drug inhibits the p53-dependent apoptotic pathway in leukemic cells. has the advantage over siRNA or antisense oligos in that it does Previous data in t(17;19) cell lines would suggest that survivin not require a specific delivery system. When the drug is given as sensitivity is mediated through a mechanism independent of a continuous intravenous infusion for 7 days, it was well caspases 3 and 9. Yet, we have observed that both the cell line tolerated and also had modest effects on the diseases, including HAL01 and the primary t(17;19) patient sample were relatively diffuse large B-cell lymphomas. Our studies would suggest insensitive to YM155. This may suggest that there are other that specific patient samples or disease subtypes may respond mechanisms involved in this disease as opposed to the other differently to these drugs. Therefore, selection of patients based samples such as drug efflux. Another concern would be the on in vitro sensitivity screens may improve response rates to this relative utility of targeting survivin in pediatric patients with and other survivin-targeted compounds. ALL. Our data would suggest that there may be a selective Clearly, there are a variety of potential mechanisms that may population that is more sensitive to inhibition of survivin. explain the heterogeneous responses observed to inhibition of Currently, there are clinical trials using antisense oligonucleo- survivin. As knockout studies have confirmed survivin as an tides (EZN-3042) to survivin either under development or in essential protein and it is also expressed in all cells undergoing phase I trials for relapsed leukemias. Our prediction would be a Leukemia p53, apoptosis, survivin, acute lymphoblastic leukemia JW Tyner et al heterogeneous response in these trials as only a subset of 7 Ryan BM, O’Donovan N, Duffy MJ. Survivin: a new target for anti- cancer therapy. Cancer Treat Rev 2009; 35: 553–562. patients within each disease subtype would be predicted to 8 Caldas H, Jiang Y, Holloway MP, Fangusaro J, Mahotka C, exhibit survivin sensitivity, based on our findings. Furthermore, Conway EM et al. Survivin splice variants regulate the our studies show the importance of preselection of subjects by balance between proliferation and cell death. Oncogene 2005; in vitro inhibitory screens to identify subjects more likely to 24: 1994–2007. respond to survivin inhibition. Therefore, we would propose to 9 Mita AC, Mita MM, Nawrocki ST, Giles FJ. Survivin: key regulator perform in vitro screening to assist with subject selection. of mitosis and apoptosis and novel target for cancer therapeutics. Clin Cancer Res 2008; 14: 5000–5005. In conclusion, our data support the concept that targeting 10 Li F, Altieri DC. Transcriptional analysis of human survivin gene survivin may be of clinical benefit in pediatric ALL. Further- expression. Biochem J 1999; 344 (Pt 2): 305–311. more, this targeting would be selective to the role of survivin as 11 Wheatley SP, Carvalho A, Vagnarelli P, Earnshaw WC. INCENP is an IAP as opposed to its fundamental role in the chromosome required for proper targeting of Survivin to the centromeres and the passenger complex. Future studies will be needed to verify these anaphase spindle during mitosis. Curr Biol 2001; 11: 886–890. results in primary patient samples and to identify patients who 12 Uren AG, Wong L, Pakusch M, Fowler KJ, Burrows FJ, Vaux DL et al. Survivin and the inner centromere protein INCENP show will respond to therapies targeting survivin. similar cell-cycle localization and gene knockout phenotype. Curr Biol 2000; 10: 1319–1328. 13 Bolton MA, Lan W, Powers SE, McCleland ML, Kuang J, Conflict of Interest Stukenberg PT. Aurora B kinase exists in a complex with survivin and INCENP and its kinase activity is stimulated by survivin BHC, JWT and MT have no competing financial interests. BJD binding and phosphorylation. Mol Biol Cell 2002; 13: 3064–3077. has financial interest in MolecularMD; BJD receives clinical trial 14 Chu Y, Yao PY, Wang W, Wang D, Wang Z, Zhang L et al. Aurora funding from Novartis and BMS. B kinase activation requires survivin priming phosphorylation by PLK1. J Mol Cell Biol 2011; 3: 260–267. 15 Tyner JW, Deininger MW, Loriaux MM, Chang BH, Gotlib JR, Acknowledgements Willis SG et al. RNAi screen for rapid therapeutic target identification in leukemia patients. Proc Natl Acad Sci USA 2009; 106: 8695–8700. We thank Leslie Smith for her expertise in microscopy. We also 16 Azuara V. Profiling of DNA replication timing in unsynchronized thank Dr Peter Rotwein for access to Odyssey. This work was cell populations. Nat Protoc 2006; 1: 2171–2177. supported in part by the Leukemia & Lymphoma Society. BHC is 17 Okuya M, Kurosawa H, Kikuchi J, Furukawa Y, Matsui H, Aki D supported in part by the Oregon Child Health Research Center et al. Up-regulation of survivin by the E2A-HLF chimera is (National Institute of Child Health and Development K12) and the indispensable for the survival of t(17;19)-positive leukemia cells. St Baldrick’s Foundation. JWT is supported by grants from the J Biol Chem Jan 15; 285: 1850–1860. 18 Dohi T, Beltrami E, Wall NR, Plescia J, Altieri DC. Mitochondrial National Cancer Institute (NCI) as well as grants from the William survivin inhibits apoptosis and promotes tumorigenesis. J Clin Lawrence and Blanche Hughes Foundation and the Oregon Invest 2004; 114: 1117–1127. Clinical and Translational Research Institute (OCTRI) Grant 19 Nakahara T, Takeuchi M, Kinoyama I, Minematsu T, Shirasuna K, number UL1 RR024140 from the National Center for Research Matsuhisa A et al. YM155, a novel small-molecule Resources (NRCC), a component of the NIH, and NIH Roadmap survivin suppressant, induces regression of established human for Medical Research. hormone-refractory prostate tumor xenografts. Cancer Res 2007; 67: 8014–8021. 20 Mirza A, McGuirk M, Hockenberry TN, Wu Q, Ashar H, Black S Author contributions et al. Human survivin is negatively regulated by wild-type p53 and participates in p53-dependent apoptotic pathway. Oncogene BHC and JWT designed the experiments; BHC and AMJ 2002; 21: 2613–2622. performed the experiments; and BHC, JWT, AMJ, MT and BJD 21 Fenaux P, Jonveaux P, Quiquandon I, Preudhomme C, Lai JL, wrote the manuscript. Vanrumbeke M et al. Mutations of the p53 gene in B-cell lymphoblastic acute leukemia: a report on 60 cases. 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To view a copy of this license, visit http:// DS, Li F et al. Angiopoietin-1 inhibits endothelial cell apoptosis via the Akt/survivin pathway. J Biol Chem 2000; 275: 9102–9105. creativecommons.org/licenses/by-nc-nd/3.0/ Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu) Leukemia

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Published: Sep 30, 2011

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