Novel HSP90 Inhibitor NVP-HSP990 Targets Cell-Cycle Regulators to Ablate Olig2-Positive Glioma Tumor–Initiating Cells

Published OnlineFirst March 14, 2013; DOI: 10.1158/0008-5472.CAN-12-2033

Cancer Therapeutics, Targets, and Chemical Biology Research

Jun Fu1,2, Dimpy Koul1,2, Jun Yao1, Shuzhen Wang1, Ying Yuan3, Howard Colman1, Erik. P. Sulman4, Frederick. F. Lang5, and W.K. Alfred Yung1

Abstract Genetic heterogeneity and signaling alterations diminish the effectiveness of single-agent therapies in glioblastoma multiforme (GBM). HSP90 is a molecular chaperone for several signaling proteins that are deregulated in glioma cells. Thus, HSP90 inhibition may offer an approach to coordinately correct multiple signaling pathways as a strategy for GBM therapy. In this study, we evaluated the effects of a novel HSP90 inhibitor, NVP-HSP990, in glioma tumor–initiating cell (GIC) populations, which are strongly implicated in the root

pathobiology of GBM. In GIC cultures, NVP-HSP990 elicited a dose-dependent growth inhibition with IC50 values in the low nanomolar range. Two GIC subgroups with different responses were observed with an Olig2-expressing subset relatively more sensitive to treatment. We also showed that Olig2 is a functional marker associated with cell proliferation and response to NVP-HSP990, as NVP-HSP990 attenuated cell proliferation in Olig2-high GIC lines. In addition, NVP-HSP990 disrupted cell-cycle control mechanism by decreasing CDK2 and CDK4 and elevating apoptosis-related molecules. Mechanistic investigations revealed molecular interactions between CDK2/CDK4 and Olig2. Inhibition of CDK2/CDK4 activity disrupted Olig2–CDK2/CDK4 interactions and attenuated Olig2 protein stability. In vivo evaluation showed a relative prolongation of median survival in an intracranial model of GIC growth. Our results suggest that GBM characterized by high-expressing Olig2 GIC may exhibit greater sensitivity to NVP-HSP990 treatment, establishing a foundation for further investigation of the role of HSP90 signaling in GBM. Cancer Res; 73(10); 1–13. 2013 AACR.

Introduction neously targeting multiple molecules that are deregulated is Glioblastoma multiforme (GBM), the most common adult critical to designing a successful therapeutic strategy for GBM. glioma, is associated with a dismal prognosis not only because HSPs are a highly conserved family of molecular chaperones, of the high degree of genetic heterogeneity among patients and which can be upregulated to protect cells from potentially even within individual tumors but also because of its dynamic lethal stress. Upregulated HSPs may partially account for genetic instability. The most frequently altered genes are glioma cell's ability to survive the otherwise fatal hypoxic CDKN2A, TP53, EGFR, PTEN, and RB (1). Signal transduction environment and tolerate genetic alterations (3). HSP90 is pathways are not linear; they are complex, overlapping, and induced in response to cellular stress and stabilizes client crosstalking, which may allow alternative pathways to com- proteins involved in cell-cycle control and proliferation/anti- pensate when one is disrupted, potentially leading to resis- apoptotic signaling (4). By binding and chaperoning proteins, tance to single agents that affect only one target (2). Simulta- HSP90 can buffer the genetic variation at the protein level (5). Many of HSP900s more than 100 client proteins, including P53, CDK4, ErbB2, PI3K, PTEN, AKT, Raf, c-MET, and EGFR, are reported to be involved in the major aberrant signal transduction pathways identified by The Cancer Genome Atlas (TCGA; Authors' Affiliations: 1Brain Tumor Center, and Departments of 2Neuro- Oncology, 3Biostatistics, 4Radiation Oncology and 5Neurosurgery, The refs. 6–9). An advantage of HSP90 inhibitors is their ability to University of Texas M. D. Anderson Cancer Center, Houston, Texas affect multiple oncoproteins simultaneously, including targets Note: Supplementary data for this article are available at Cancer Research considered "undruggable," and thus they may largely avoid Online (http://cancerres.aacrjournals.org/). generating resistant phenotypes arising from mutation, acti- J. Fu and D. Koul contributed equally to this work. vation of alternative signaling pathways, or feedback loops seen with therapeutics targeting a single oncogene or pathway (10). Corresponding Author: W. K. Alfred Yung, Department of Neuro-Oncol- fi ogy, Unit 431, The University of Texas M. D. Anderson Cancer Center, 1515 Tumor-initiating cells are functionally de ned through their Holcombe Blvd., Houston, TX 77030. Phone: 713-794-1285; Fax: 713-794- capacity for sustained self-renewal and tumorigenicity. Glioma 4999; E-mail: [email protected] tumor–initiating cells (GIC) retain relevant molecular features doi: 10.1158/0008-5472.CAN-12-2033 of GBMs and enable preclinical models for evaluation of both 2013 American Association for Cancer Research. tumor biology and therapeutics (11). GIC maintenance is

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regulated by an interconnected regulatory circuit consisting of Lentiviral vectors and virus production many HSP90 client proteins including AKT and STAT3 (12, 13). Lentiviral vectors encoding Olig2 or short hairpin RNA Because HSP90 is involved in redundant pathways for main- (shRNA) for Olig2 was purchased from Genecopoie Inc. taining cell viability, its inhibition has the potential to block Sequences of shRNA hairpin are listed in Supplementary Data. expression of multiple client proteins involved in tumorigen- The VSV-G pseudotyped lentiviral vectors were produced by esis. Therefore, there seems to be a compelling rationale to transient cotransfection of 3 plasmids into 293T cells and evaluate an HSP90 inhibitor in GIC models. The aim of the produced lentiviruses stock was titered and stored at 80C. current study therefore was to evaluate the effects and mechanism of an HSP90 inhibitor in GICs both in vitro and in vivo. Western blot analysis Cells were harvested in a lysis solution as previously described Materials and Methods (17) and subjectedto Western blotting. Membranes were probed Cell lines and reagents with the following primary antibodies: anti-HSP90, anti-HSP70, a The GIC lines were established by isolating neurosphere- anti-HSP27, anti-PDGFR , anti-AKT, anti-CDK4, anti-CDK2, forming cells from surgical specimens of human GBM using a anti-cleaved-caspase-7, anti-cleaved-caspase-8, anti-Bim, anti- method described previously (14, 15) The study was approved phosphop-Rb (Ser807/811), and anti-p27 (all from Cell Signal- b by the Institutional Review Board of M. D. Anderson Cancer ing), Olig2 antibody was purchased from Chemicon, and -actin Center, and informed consent was obtained from all subjects. antibody was from Sigma-Aldrich. These GIC lines were cultured as GBM neurospheres in DMEM/F12 medium containing B27 supplement (Invitrogen) Cell proliferation analysis m and bFGF and EGF (20 ng/mL each). 17-N-allylamino-17- Dissociated GICs were plated at 10 cells/ L in 6-well plates demethoxygeldanamycin (17-AAG) and retinoic acid (RA) were and incubated with various concentrations of NVP-HSP990 for obtained from Sigma. The HSP90 inhibitor NVP-HSP990 was 7 days. Formed tumorspheres were dissociated into single cells provided by Novartis. NVP-HSP990 was dissolved in dimethyl and counted with hemocytometer using 0.2% Trypan blue sulfoxide to a concentration of 10 mmol/L. Normal human exclusion. astrocyte (NHA) was maintained in Dulbecco's Modified Eagle fl Medium (DMEM) supplemented with 10% FBS. Human fetal Indirect immuno uorescence staining fl brain neural stem cell line HFB2050 was kindly provided by Dr. Immuno uorescence staining was conducted as described fl Evan Snyder (Burnham Institute, La Jolla, CA) and cultured as previously (18). Brie y, GICs were grown on chamber slides – fi described previously (15). precoated with poly-lysine coated coverslips, cells were xed with 4% paraformaldehyde, permeated with 0.25% Triton X- Drug cytotoxicity analysis 100, and blocked with 3% normal goat serum. Immunostaining Cells were seeded in 96-well plates (2 103 cells/well) and was conducted using the appropriate primary and secondary incubated at 37C for 24 hours before serial dilutions of antibodies, and images were acquired using a confocal laser NVP-HSP990 were added. Growth inhibition was determined scanning microscope (Carl Zeiss Microscopy). using the CellTiter-Blue cell viability assay (Promega). Cell viability in the vehicle control was considered as to be 100%. Immunohistochemistry analysis The IC50 value was calculated by using Calcusyn version 2.0 Immunohistochemistry was conducted as described previ- software (Biosoft) as the mean drug concentration required ously (19). Briefly, after dewaxing and antigen retrieval, tissue to inhibit cell proliferation by 50% compared with vehicle- sections were incubated with appropriate primary and sec- treated controls. ondary antibodies. Sections were stained 3,3-diaminobenzidine and hydrogen peroxide chromogen substrate (DAKO) and Cell-cycle analysis counterstained with hematoxylin and mounted. Images were GIC tumorspheres were dissociated using Accutase enzyme acquired using a Zeiss Microsystems fluorescence microscope cocktails (Invitrogen) and plated at a density of 2 105 cells per linked to a DFC340FX camera (Carl Zeiss Microscopy). well in 60 mm plates. The cells were treated with NVP-HSP990 for 24 hours, fixed in 70% ethanol, and stored at 20C. Cells TUNEL assay were stained with propidium iodide for cell-cycle analysis Apoptosis was determined using the terminal deoxynucleo- using a BD FACSCalibur flow cytometer and CellQuest soft- tidyltransferase–mediated deoxyuridine triphosphate nick end ware (BD Biosciences). labeling (TUNEL) method using in situ cell death detection reagent (Roche Applied Science). The percentage of TUNEL- Immunoprecipitation labeled cells in each section was determined at a magnification The immunoprecipitation procedure was conducted as of 400 by counting 500 cells in a randomly selected field. described previously (16). Briefly, after NVP-HSP990 (50 nmol/L) or 17-AAG (500 nmol/L) treatment for 2 hours, cells Reverse-phase protein arrays were lysed in immunoprecipitation lysis buffer, and precleared After NVP-HSP990 (50 nmol/L) treatment for 24 hours, cells by incubation with poly(G) immunoprecipitation beads for 1 were collected and lysed in a buffer consisting of a 2.5% hour at 4C. The pellet was discarded, and the supernatant (200 solution of 2-mercaptoethanol in loading buffer/T-PER mg of lysate protein) was subjected to immunoprecipitation. (Pierce) plus phosphatase and protease inhibitors. All samples

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were diluted to a final concentration of 1 mg/mL, and then 30 schedule of once per week for 5 weeks. After NVP-HSP990 mL of each sample, arrayed in a series of dilutions, was printed treatment for 2 or 4 weeks, 2 animals from each group were in duplicate on slides. The slides were then subjected to euthanized for biologic assessment of tumor response. immunostaining with a panel of 207 commercially available antibodies (20). Slides were stained on an automated slide Statistical analysis strainer (DAKO) using biotin-linked peroxidase catalyzed sig- Statistical analysis was conducted with using the Student nal amplification. unpaired t test. Results are presented as the mean SD of at least 3 independent experiments. Survival analysis was con- Animal studies ducted using the log-rank analysis module in SPSS 10.0 (SPSS fi < All animal studies were conducted in the veterinary facilities Inc). Differences were considered signi cant at P 0.05 for all of M.D Anderson Cancer Center (Houston, TX) in accordance the statistical analysis conducted in our study. with institutional rules. The antitumor efficacy of NVP-HSP990 was examined in intracranial xenografts derived from 2 GIC Results lines, GSC11 and GSC20. To create the intracranial disease NVP-HSP990 displays dose- and time-dependent effects model, we engrafted GICs (5 105) into the caudate nucleus of on HSP90 client proteins Nude (nu/nu) 6- to 8-week-old mice using a previously Examination of some known HSP90 client proteins, includ- described guide-screw system (21) and then randomly divided ing PDGFRa, CDK4, and AKT, revealed a dose- and time- the mice into 4 groups of 14 mice in each group. Starting on day dependent decrease in GIC line GSC11, which is consistent 4 after the tumor cells implantation, mice were treated by oral with the idea that the responsiveness of these cells to NVP- gavages with 10 mg/kg NVP-HSP990 in methylcellulose or with HSP990 is a results of the degradation of HSP90 client proteins an equal volume of methylcellulose alone (vehicle control) on a (Fig. 1A and B). GIC lines such as GSC13, GSC16, and GSC23

Figure 1. NVP-HSP990 suppresses HSP90 activity in a dose- or time-dependent manner. A and B, GSC11 cells were treated with the indicated doses of NVP- HSP990 for 24 hours or with 20 nmol/L NVP-HSP990 for the indicated time intervals. Western blotting was conducted to analyze the cellular protein levels of known client proteins (PDGFRa, AKT, CDK4) and negative indicators of HSP90 activities (HSP27, HSP70). b-Actin was used as loading control. C, a panel of GIC lines was treated with various concentrations of NVP-HSP990, and cell viability was measured by Cell-Titer Blue assay. The graph depicts cell viability at 72 hours. D, waterfall diagram of IC50 of 14 GIC lines. , P < 0.05 responder GICs versus nonresponder GICs.

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Figure 2. NVP-HSP990 suppresses GIC proliferation and induces neuronal differentiation. A, micrographs of GSC11 treated with NVP-HSP990 at 20 nmol/L for 7 days. Bars, 200 mm. B, NVP-HSP990 attenuated cell proliferation in responder GICs. GICs were treated with NVP-HSP990 at 20 or 50 nmol/L for 7 days and viable cells were counted. The number of viable cells in the vehicle control was considered as to be 100%. n ¼ 4; , P < 0.05, as compared with vehicle control. C, NVP-HSP990 attenuated Ki-67 index in responder GIC lines. n ¼ 4; , P < 0.05, as compared with vehicle control. D, NVP-HSP990 induces neuronal differentiation. GIC lines were treated with differentiation media (1 mmol/L RA þ 1% FBS) or NVP-HSP990 (50 nmol/L) for 5 days. Cells were stained with neural lineage markers: neuronal lineage (TuJ1, NeuN); astrocytic lineage (GFAP); oligodendrocytic lineage (CNPase). Micrographs showed the representative staining results of for GSC11 cells. Bars, 50 mm. E, NVP-HSP990 induces neuronal differentiation in responder GIC lines (GSC11, GSC13). n ¼ 4; , P < 0.05, as compared with marker staining positivity in vehicle control.

also showed similar changes after NVP-HSP990 treatment lines in responder group exhibited high sensitivity to NVP- (Supplementary Fig. S1A). We also observed increased HSP70 HSP990 with IC50 values less than 60 mol/L. Although the and HSP27 levels following NVP-HSP990 treatment. levels of HSP90 protein expression was similar in responder and nonresponder GICs, the IC50 values in responder GIC NVP-HSP990 targets a subset of GICs lines were signiﬁcantly lower than those in nonresponder We tested the effects of NVP-HSP990 against a panel of 14 group (Supplementary Fig. S1B and Fig. 1D; P ¼ 0.0002). GIC lines. NVP-HSP990 inhibited GIC proliferation in all GIC Treatment of responder GIC with NVP-HSP990 for an lines, with IC50 values ranging approximately between 10 extended time from 3 to 5 days did not seem to produce and 500 nmol/L (Fig. 1C). Furthermore, we observed a more cytotoxicity on responder GICs (Supplementary Fig. differential response in these GIC lines and divided them S1C). We also evaluated the effect of NVP-HSP990 on NHAs into responder and nonresponder groups (Fig. 1D). All GIC and human fetal brain neural stem cells (HFB2050). The IC50

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Figure 3. NVP-HSP990 targets a subset of GICs with high Olig2 expression. A, immunostaining positivity of thirteen neural lineage markers in responder and nonresponder GIC lines. B, Western blot analysis on Olig2 protein in GIC lines. C, NVP-HSP990 treatment reduces Olig2 levels in GICs. GSC11 cells were treated with 50 nmol/L NVP-HSP990 for the indicated time intervals from 24 to 72 hours. D, immunostaining on Olig2 and NeuN in GSC11 cells incubated with NVP-HSP990 for 5 days. Bars, 25 mm.

values were much higher than responder GICs (NHA, 225.2 NeuN after NVP-HSP990 treatment (Fig. 2E). Nevertheless, RA- 28.5 nmol/L; HFB2050, 132.4 20.3 nmol/L). differentiated GICs seemed less sensitive to NVP-HSP990 treatment, as the IC50 value of the RA-differentiated GICs were NVP-HSP990 attenuates GIC proliferation and induces higher than GICs grown in stem cell medium (Supplementary neuronal differentiation Fig. S2C). We selected 2 GIC lines in the responder group (GSC11 and GSC13) and 2 cell lines in the nonresponder group (GSC2 NVP-HSP990 targets GICs with high Olig2 expression and GSC20) to study NVP-HSP990 effect on cell proliferation To correlate the drug response with the lineage character- and differentiation. Tumorspheres that formed from NVP- istics of GICs, immunostaining on 13 lineage markers showed HSP990–treated GICs were smaller and fewer than those that all responder GIC lines exhibited high Olig2 expression, formed from control untreated GSC11 cells (Fig. 2A). Treat- also shown by Western blotting analysis (Fig. 3B), as compared ment with NVP-HSP990 markedly decreased the proliferation with nonresponder GIC lines (Fig. 3A). Gene expression anal- of responder GICs (Fig. 2B). In addition, NVP-HSP990 treat- ysis also conﬁrmed that Olig2 is the top marker associated with ment markedly impaired the proliferative potential of respond- response to NVP-HSP990 (Supplementary Fig. S3). Treatment er GICs, as shown by the decreased positivity of Ki-67 staining of responder GIC line, GSC11, with HSP990 (50 nmol/L) for in treated cells (Fig. 2C). Limiting dilution assay also showed different times points showed that Olig2 protein levels marked- that NVP-HSP990 attenuated the self-renewal potential in ly decreased after 24 hours of treatment and disappeared after GSC11 cells (Supplementary Fig. S2A). Thus, our data indicated 72 hours (Fig. 3C) accompanied with increased NeuN staining that inhibition of HSP90 by NVP-HSP990 is associated with (Fig. 3D). attenuation of GIC proliferation at the functional levels. To further determine the effect of inhibiting HSP90 on GIC Olig2 might be a functional marker associated with cell differentiation, exposure of GICs to either differentiation con- proliferation and response to NVP-HSP990 dition solution (1% FBS þ 1 mmol/L retinoic acid in DMEM/ Olig2 has a well-established function in regulating the F12) or to NVP-HSP990 (50 nmol/L) for 5 days differentiated proliferation of neural progenitors and glioma cells. In our GICs into 3 neural lineages, as GFAP, TuJ1, NeuN, and CNPase- study, we showed that Olig2-high glioma cells displayed high stainined cells increased following induction by 1% FBS þ 1 Ki-67 labeling in GSC11 cell line (Fig. 4A). To further evaluate mmol/L retinoic acid. Surprisingly, NVP-HSP990 treatment the role of Olig2 on GIC proliferation, Olig2 knockdown GICs only considerably increased neuronal marker TuJ1 and NeuN and cell lines ectopically expressing Olig2 in GSC23 were expression and did not alter GFAP and CNPase in responder generated. Olig2-high GSC11 and GSC13 cells showed slower GICs, suggesting that NVP-HSP990 mainly triggered neuronal cell proliferation rates following Olig2 knockdown (Fig. 4B and lineage-restricted differentiation (Fig. 2D). Consistently, the C) and GSC23-expressing ectopic Olig2 showed increased cell expression of stem cell related genes such as CD133, Nestin, proliferation (Fig. 4D and E) indicating Olig2 might regulate the Sox2, and Olig2 decreased following NVP-HSP990 treatment proliferation of GICs. (Supplementary Fig. S2B). However, the nonresponder cells The observation that NVP-HSP990 attenuates the cell pro- (GSC2 and GSC20) did not show much staining of TuJ1 and liferation of GICs with high Olig2 prompted us to hypothesize

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Figure 4. Olig2 is a functional marker associated with cell proliferation and response to NVP- HSP990. A, coimmunostaining on Olig2 and Ki-67 in GSC11 cells. Bars, 25 mm. B, Western blot analysis conﬁrmed the knockdown effect of Olig2 shRNA lentivirus on GSC11 and GSC13 cells. C, Olig2 knockdown attenuates GIC proliferation. The number of viable cells in shscr control was treated as 100%. n ¼ 4; , P < 0.05, as compared with shscr control. D and E, ectopic expression of Olig2 promotes GIC proliferation. Lentivirus containing GFP control or Olig2 was constitutively expressed in GSC23 cells. The number of viable cells in GFP control was considered as 100%. n ¼ 4; , P < 0.05, as compared with GFP control. F, Olig2 knockdown desensitizes GICs to NVP-HSP990 treatment. n ¼ 4; , P < 0.05, as compared with shscr control. G, NVP-HSP990 showed mild effect on Ki-67 index in Olig2 knockdown GICs. n ¼ 4; , P < 0.05, as compared with shscr control. , P < 0.05, as compared with vehicle control. H, ectopic Olig2 expression sensitizes nonresponder GICs to NVP- HSP990 treatment. n ¼ 4; , P < 0.05, as compared with GFP control.

that Olig2 might be a functional molecule associated with marker associated with cell proliferation and response to response to NVP-HSP990. To confirm this hypothesis, we NVP-HSP990. tested the effect of NVP-HSP990 on various cell models manip- ulating Olig2 expression. Our results showed that Olig2-knock- NVP-HSP990 disrupts cell-cycle regulation and induces down GIC lines (GSC11/shOlig2 and GSC13/shOlig2) were less apoptosis in GICs sensitive to NVP-HSP990 treatment as compared with GSC11/ We analyzed the alteration of various signaling components shscr and GSC13/shscr (P < 0.05; Fig. 4F). Consistent with this by hierarchical clustering analysis of the RPPA data from 10 finding, we showed that Ki-67 index in the Olig2 knockdown GIC lines (4 responder cell lines and 6 nonresponder cell lines) GICs is lower than shscr, whereas it decreased slightly, as treated with NVP-HSP990 by comparing patterns of protein compared with shscr control following NVP-HSP990 treatment expression in the responder group versus the nonresponder (Fig. 4G). In parallel, overexpression of Olig2 in GSC23-a low group to identify altered signaling events associated with Olig2-expressing cell line sensitized GSC23 to NVP-HSP990 response to NVP-HSP990. We identified a series of cell-cycle treatment, as compared with GFP control (Fig. 4H, P < 0.05). regulatory proteins in responder GIC lines that showed These results suggested that Olig2 might be a functional changes, specifically an increase in p27, and a decrease in

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Figure 5. NVP-HSP990 disrupts cell-cycle regulation and induces apoptosis in GICs. A, RPPA protein array analysis on of GICs treated with NVP-HSP990. Color bars indicate an increase (red) or decrease (green) of signaling proteins following NVP-HSP990 treatment (50 nmol/L, 24 hours). Differential changes of in signaling proteins between responder GICs versus and nonresponder GICs were selected, with P < 0.05. B, Western blotting validation of signaling proteins listed in A. C, flow cytometry analysis on of GIC lines treated with NVP-HSP990 at 50 nmol/L for 24 hours. D, TUNEL staining on of GIC lines treated with NVP-HSP990 at 50 nmol/L for 48 hours. n ¼ 4; , P < 0.05, as compared with vehicle control. phosphorylated Rb (Ser 807/811), and appreciable induction of labeling, implicating a potential combinational strategy to proapoptotic proteins, including cleaved caspase-7/8, and Bim. eliminate GICs (Supplementary Fig. S4). These results sug- Such changes were not observed in nonresponder GIC lines gested that the disruption of cell-cycle regulation and the (Fig. 5A). Western blot analysis validated the RPPA results as induction of apoptosis might be crucial for NVP-HSP9900s shown by reduced expression of phosphorylated Rb and effect on responder GICs. increased p27 levels in GSC11 cells and increased in the apoptosis-related molecules cleaved caspase-7/8 and Bim in NVP-HSP990 promotes degradation of HSP90 client GSC11 after NVP-HSP990 treatment (Fig. 5B). proteins CDK2 and CDK4 in GICs Flow cytometry analysis showed that NVP-HSP90 caused Because CDK2 and CDK4 were previously reported to be the a dramatic depletion of cells in the S-phase in 2 of the major kinases for Rb phosphorylation at S807/S811 and to be responder GIC lines (P < 0.001; Fig. 5C). We did not observe client proteins for HSP90 (22–24), we tested the possibility that significant change in the proportion of cells in S-phase in the alteration of cell-cycle–related molecules by NVP-HSP990 nonresponder GIC lines (P > 0.05). TUNEL staining con- might result from the attenuation of CDK2 and CDK4 expres- firmed that the NVP-HSP990 induced apoptosis in both sion. Coimmunoprecipitation assay showed that HSP90 was GSC11 and GSC13 cell lines, whereas minimal induction of associated with both CDK2 and CDK4 in GSC11 and GSC13 cell apoptosis was observed in the nonresponder GIC lines GSC2 lysate (Fig. 6A) and inhibition of HSP90 by either NVP-HSP990 and GSC20 (Fig. 5D). The combined treatment of NVP- (50 nmol/L, 2 hours) or the reference inhibitor 17-AAG (500 HSP990 with chemotherapeutic drugs etoposide and topo- nmol/L, 2 hours) disrupted the interactions between HSP90 tecan (known apoptosis inducer) showed enhanced TUNEL and CDK2/4 (Fig. 6B).

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Figure 6. NVP-HSP990 targets cell- cycle regulators CDK2 and CDK4 in responder GICs. A, coimmunoprecipitation assay on of endogenous HSP90 reveals its interaction with CDK2 and CDK4 in GSCs. Rabbit IgG was used as negative control for HSP90 antibody. B, treatment with NVP- HSP990 or 17-AAG disrupts the interaction between HSP90 and CDK2/4. C, CDK2/4 inhibition by NVP-HSP990 or FLP reduces phosphorylated Rb and increases p27 in GICs. GSC11 and GSC13 were treated with NVP-HSP990 (50 nmol/L) or FLP (100 nmol/L) for 24 hours. D, FLP targets Olig-high GICs. GIC lines were treated with various doses of FLP for 3 days, and cell viability was then assessed by CTB assay. n ¼ 4; , P < 0.05, as compared with nonresponder group. E, FLP treatment reduces Ki-67 index in Olig2-high GICs. GIC cells were treated with 100 nmol/L FLP for 48 hours. n ¼ 4; , P < 0.05, as compared with vehicle control. F, FLP treatment induces apoptosis in Olig2-high GICs. TUNEL staining was conducted on GIC lines treated with FLP at 100 nmol/L for 48 hours. n ¼ 4; , P < 0.05, as compared with vehicle control.

To further investigate the function of CDK2 and CDK4 in the showed that endogenous Olig2 physiologically interacts with stem cell maintenance, treatment with ﬂavopiridol (FLP: a CDK2/4 in GSC11 cells (Fig. 7A) and treatment with FLP selective inhibitor of CDK2 and CDK4) decreased phosphor- disrupted this interaction between CDK2/4 and Olig2 in ylated Rb levels and increased p27 expression (Fig. 6C). Similar Olig2-transfected 293T cells suggesting that CDK2/4 activities to NVP-HSP990, FLP treatment showed similar distribution of might be crucial to maintain this interaction between CDK2/4 responder and nonresponder GIC lines in cytotoxicity assay and Olig2 (Fig. 7B). (Fig. 6D), a marked reduction of Ki-67 index (Fig. 6E), and To show that interaction of CDK2/4 with Olig2 might induction of apoptosis in responder GIC lines GSC11 and regulate Olig2 expression and more importantly Olig2 pro- GSC13 (Fig. 6F). Therefore, NVP-HSP990 might promote the tein stability, we measured the protein stability of Olig2 protein degradation of CDK2 and CDK4 and consequently protein following CDK2/4 inhibition after protein synthesis impair GIC proliferation. was blocked by cycloheximide (CHX). Results show that Olig2 levels were decreased more rapidly in FLP-treated CDK2/4 interacts with Olig2 and regulates its protein GSC11 cells, as Olig2 protein almost disappeared after FLP stability treatment for 2 hours (Fig. 7C). Time course treatment with We showed that NVP-HSP990 affects both Olig2 and CDK2/4 FLP in GSC11 cells showed that Olig2 protein dramatically function in regulating GIC proliferation, and HSP90 does not decreased after 12-hour treatment and completely disap- directly interact with Olig2 protein. Therefore, we hypothe- peared after 24 hours. This decrease in Olig2 was also sized that HSP90 client protein CDK2/4 might interact with accompanied with downregulatedp-Rbandincreasedp27 Olig2 to regulate its function. Coimmunoprecipitation assays (Fig. 7D). Therefore, NVP-HSP990 might affect Olig2 protein

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Figure 7. CDK2/4 interacts with Olig2 and regulates its protein stability. A, coimmunoprecipitation assay of endogenous Olig2 reveals its interaction with CDK2 and CDK4 in GICs. Rabbit IgG was used as negative control for Olig2 antibody. B, inhibition on CDK2/4 activities disrupts the interaction between CDK2/4 and Olig2. 293T cells were transfected with ﬂag-tagged Olig2 and treated with FLP (100 nmol/L) for 2 hours. Cells were lysed by radioimmunoprecipitation analysis lysis buffer and 200 mg of total protein was used for coimmunoprecipitation analysis. Mouse IgG was used as negative control for ﬂag antibody. C, CDK2/4 activities are required to maintain Olig2 protein stability. GSC11 cells were pretreated with CHX (100 mg/mL) for 1 hour, then FLP was added and incubated for 0, 0.5, 1, 2, 4 and 6 hours, cells were harvested and lysed, and cell lysates were collected for Western blot analysis on endogenous Olig2 protein level. b-actin was used as loading control. D, CDK2/4 inhibition by FLP reduces Olig2 protein levels in GICs. GSC11 cells were treated with FLP (100 nmol/L) for 0, 12, 24, and 48 hours, cells were harvested and lysed, and cell lysates were collected for Western blot analysis. b-actin was used as loading control.

expression through attenuation of CDK2/4 activities in client proteins CDK2 and CDK4 were lower in NVP-HSP990– GICs. treated GSC11 tumors than in untreated tumors, suggesting NVP-HSP990 suppressed HSP90 activities in vivo. In addition, NVP-HSP990 suppresses tumor growth and improves the treated tumors showed decreased Ki-67 positivity and survival in an orthotopic mouse model of GBM increased cell TUNEL labeling (Fig. 9 and Supplementary Fig. Therapeutic efficacy of NVP-HSP990 (10 mg/kg) was shown S5) indicating that NVP-HSP990 suppressed proliferation and by increase in median survival times to 58 days (95% confi- induced apoptosis in the responder GIC tumors. Furthermore, dence interval: 52–64 days) in comparison with 46 (95% reduced Olig2 expression was accompanied by increased p27 confidence interval: 45–47 days) days in the vehicle control and TuJ1 positivity after NVP-HSP990 treatment. group for GSC11 (P < 0.01; Fig. 8B, Supplementary Table S1). However, NVP-HSP990 treatment had no effect on the median survival of GSC20 xenografted animals (P ¼ 0.4897). Histo- Discussion pathologic staining revealed that NVP-HSP990 considerably We have shown that NVP-HSP990, a HSP90 inhibitor, sup- reduced the tumor growth in GSC11 xenografts, as shown by presses the oncogenic properties of a subgroup of high Olig2 decreased tumor mass in animals treated for 4 weeks with expressing GICs with respect to cell proliferation, induces NVP-HSP990 in comparison with vehicle control. However, the neuronal differentiation, and affects survival in mice. Further- tumor mass in GSC20 xenograft showed mild reduction fol- more, our results indicate that NVP-HSP990 treatment in GICs lowing NVP-HSP990 treatment (Fig. 8A). disrupts cell-cycle control via the CDK2/CDK4 pathway by regulating the protein degradation of these cell-cycle NVP-HSP990 suppresses proliferation and induces regulators. apoptosis and differentiation in GIC xenografts NVP-HSP990 is highly potent and selective for HSP90 and The orthotopic tumors after NVP-HSP990 treatment for 4 represents one of the most potent oral HSP90 inhibitors weeks were analyzed for biologic effects. The levels of HSP90 reported. NVP-HSP990 also displayed significant HSP90-

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Figure 8. NVP-HSP990 suppressed tumor growth and prolonged animal survival in an orthotopic mouse xenograft model. GSC11 and GSC20 cells were used to generate orthotopic xenografts in mouse brains. Tumor-bearing mice were administered either vehicle or NVP-HSP990 at the indicated dose/schedules. Two mice were sacriﬁced at indicated time point (2 week, 4 week) after NVP- HSP990 treatment. A, representative hematoxylin and eosin (H&E)-stained whole brain sections at 4 weeks after treatment; arrows indicate central necrosis. B, Kaplan–Meier survival probability plots of tumor-bearing mice in vehicle or NVP-HSP990 treatment groups (n ¼ 10), using the log-rank method to test for a difference between groups.

dependent antitumor activity in glioma cell lines by inter- showed that acquisition of Olig2 in the Id1high GICs popu- acting with HSP90 and leading to the destabilization and lation is a critical factor in its tumorigenic potential (32). degradation of several proteins that depend on HSP90 for Consistently, we showed that Olig2 might regulate prolifer- their stability (25, 26). Furthermore, NVP-HSP990 exhibited ation of GICs, as Olig2-high GICs exhibit higher Ki-67 index. promising drug selectivity on responder GICs, as IC50 values Knockdown of Olig2 slowed the GIC proliferation, whereas of responder GIC lines is much less than those of astrocytes overexpression of Olig2 promotes cell proliferation in Olig2- or neural stem cells. NVP-HSP990 significantly impaired GIC low GICs. Therefore, Olig2 might be a functional marker in proliferation and triggered neuronal differentiation of the maintaining the proliferative/rapid cycling state of GICs, GICs, confirming that HSP90 plays a role in maintaining which might render them susceptible to drugs that disrupt GICs biologic properties. the cell-cycle progression. GICs cultured under neural stem cell conditions can Cell-cycle regulators are inserted in complex networks display heterogeneous biologic characteristics and lineage whose alteration might underlie tumorigenesis in the ner- profiles that reflect the heterogeneous features of GBMs (27). vous system (33). Accumulating evidence indicates that the Olig2, a bHLH transcription factor, shows both antineural regulatory mechanisms in the G1 phase in neural precursors functions and proneural functions in oligodendrocyte pro- play an important role in the precursors' decision to progenitors (28, 29, 30). The responder GIC lines highly liferate or differentiate and that factors modulating G1 might expressed Olig2, whereas NVP-HSP990 treatment reduced be used to influence this decision (34). CDK2 and CDK4 are its expression, suggesting NVP-HSP990 might preferentially the master regulators controlling G1–S progression, which target GICs that might originate from early neural progeni- they do by phosphorylating the Rb protein. CDK2 is also tors such as oligodendrocyte progenitors. More importantly, critical for the proliferation and self-renewal of neural Olig2 might play a crucial role in promoting the proliferation progenitor cells in the adult subventricular zone (35, 36). of neural stem/progenitor cells and malignant glioma. Ligon In this study, we showed that CDK2 and CDK4 are com- and colleaguesindicated that Olig2-regulated lineage- plexed with HSP90 in GICs; NVP-HSP990 attenuates CDK2 restricted pathway controls replication competence in neu- and CDK4 protein levels, consequently decrease cell popula- ral stem cells and malignant glioma (31). Recent study also tions in the S-phase, and impair cell proliferation.

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Figure 9. NVP-HSP990 treatment inhibits cell proliferation and induces apoptosis and neuronal differentiation in GIC xenografts. Immunostaining of the brain sections of animals treated with NVP-HSP990 for 4 weeks (n ¼ 2). The tissue section was incubated with antibodies against CDK2, CDK4, Ki-67, p27, Olig2, and TuJ1 (antibody speciﬁcfor human TuJ1). Diaminobenzidine was used as a chromogen, followed by counterstaining with hematoxylin. TUNEL staining was also conducted to evaluate the proapoptotic effects of NVP-HSP990 on GICs in xenograft sections. Bar, 50 mm.

Furthermore, our data indeed showed that similar to NVP- One challenge for molecular inhibitor development is the HSP990, Olig2-high GIC lines are more sensitive to CDK2/4 selection of cancer patients who would benefit from this inhibitor flavopiridol. Given the potential role of Olig2 as a treatment. TCGA described a robust gene expression-based functional marker in maintaining the rapid cycling state of molecular classification of GBMs, with distinct gene expres- GICs, disrupting cell-cycle regulation by targeting CDK2 and sion profiles in GBM subclasses (37). Establishing the associ- CDK4 (NVP-HSP990 or flavopiridol) might help to kill GICs ation between drug responses and GBM molecular subclasses with high Olig2 expression. may help to identify potential cohorts of patients for targeted The mechanism that regulates CDK2/4 and Olig2 function therapy. In this study, we identified proneural gene Olig2 as a is unknown. Our findings indicate that CDK2/4 can interact functional marker to predict the GICs' response to NVP- with Olig2 in GICs. Nevertheless, CDK2/4 activities are HSP990. These data suggest that NVP-HSP990 would be effec- crucial to maintain Olig2 stability, as CDK2/4 inhibition by tive in targeting as well as eventually minimizing recurrence by Flavopiridol disrupts the CDK2/4/Olig2 interaction and inhibiting the proliferation of GICs. Results of this study are destabilize Olig2 protein. The above findings might impli- therefore anticipated to help identify potential GBM patient cate a mechanism through which HSP90 regulate cell cohorts who would gain the most from HSP90 inhibition and proliferation in Olig2-high GICs. Although the detailed could also be potentially valuable in designing future combi- mechanisms still remains to be understood regarding how national strategy to target the subset of GICs with high Olig2 CDK2/4 interacts with Olig2. expression.

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Disclosure of Potential Conflicts of Interest Acknowledgments W.K.A. Yung has a commercial research grant from Novartis; has a honoraria The authors thank Verlene Henry and Lindsay Holmes for performing the from speakers' bureau from Novartis, Merck, and Actelion; and is a consultant/ animal studies and Kathryn Carnes (Department of Scientific Publications, advisory board member of Novartis. No potential conflicts of interest were The University of Texas M. D. Anderson Cancer Center) for editing the disclosed by the other authors. manuscript.

Authors' Contributions Conception and design: J. Fu, D. Koul, W.K.A. Yung Grant Support Development of methodology: J. Fu, H. Colman, W.K.A. Yung This study was supported by grants from the National Cancer Institute Acquisition of data (provided animals, acquired and managed patients, (CA56041 and CA127001 to W.K.A. Yung), Cancer Center Support Grant provided facilities, etc.): D. Koul, E.P. Sulman, F.F. Lang (CA16672) and a sponsored research grant from Novartis. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, The costs of publication of this article were defrayed in part by the payment of computational analysis): D. Koul, J. Yao, Y. Yuan, W.K.A. Yung page charges. This article must therefore be hereby marked advertisement in Writing, review, and/or revision of the manuscript: D. Koul, Y. Yuan, H. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Colman, W.K.A. Yung Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): S. Wang, W.K.A. Yung Received May 24, 2012; revised January 18, 2013; accepted February 21, 2013; Study supervision: D. Koul, W.K.A. Yung published OnlineFirst March 14, 2013.

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Novel HSP90 Inhibitor NVP-HSP990 Targets Cell-Cycle Regulators to Ablate Olig2-Positive Glioma Tumor−Initiating Cells

Jun Fu, Dimpy Koul, Jun Yao, et al.

Cancer Res Published OnlineFirst March 14, 2013.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-12-2033

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