MDM2 antagonist nutlin plus proteasome inhibitor velcade ...

Research paper

Cancer Biology & Therapy 9:11, 936-944; June 1, 2010; © 2010 Landes Bioscience

MDM2 antagonist nutlin plus proteasome inhibitor velcade combination displays a synergistic anti-myeloma activity Manujendra N. Saha,1,2 Hua Jiang,1,2,3 Jennifer Jayakar,1,2 Donna Reece,4 Donald R. Branch1,2 and Hong Chang1,2,4,* Division of Molecular and Cellular Biology; Toronto General Hospital Research Institute; 2Department of Laboratory Medicine & Pathobiology; University of Toronto; 3 Department of Laboratory Hematology/Oncology, Shanghai Children's Medical Center, Shanghai Jiaotong University, Shanghai, China. 4 Department of Laboratory Hematology and Medical Oncology; University Health Network; Toronto, ON Canada

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Key words: multiple myeloma, p53, nutlin, velcade, apoptosis

Mutliple myeloma (MM) has one of the poorest prognosis of the hematological malignancies and novel therapeutic approaches are needed. Therapeutic induction of p53 might be important to evaluate the drugs either individually or in combination. Direct inhibition of MDM2 function by an MDM2 antagonist nutlin or blocking proteasomal degradation of p53 by a selective proteasome inhibitor velcade can stabilize p53 and activate the p53 apoptotic signaling pathway. We examined if inhibition of p53-MDM2 interaction by nutlin might potentiate the cytotoxic effects of velcade in MM cell lines and primary MM samples. Nutlin or velcade resulted in a reduction in cell proliferation or viability in MM cells harboring wild type p53. Nutlin plus velcade showed a synergistic anti-myeloma activity as evidenced by a significant increase of cytotoxicity with respect to each agonist alone. These effects were accompanied by accumulation of p53 and its two immediate downstream targets, p21 and MDM2, as well as caspase activation and induction of proapoptotic targets, PUMA, BAX and BAK. The induction of p53 target genes induced by nutlin and/or velcade was further validated by gene expression profiling and expression of some selective targets was quantified by qRT-PCR. These preclinical studies provide the framework for clinical trial of nutlin, alone and in combination with conventional and novel therapies such as velcade to increase efficacy and improve patient outcome in MM.

Introduction Multiple myeloma (MM) is a plasma-cell malignancy that remains incurable despite conventional treatment and recent advances in novel therapies.1 Defects in the Ubiquitin-Proteasome Signaling (UPS) pathway are linked to the pathogenesis of various human diseases.2 Since UPS regulates normal cellular processes via ubiquitin-dependent proteolysis of regulatory proteins such as p53, targeting proteasomes therefore offers great promise as a novel therapeutic strategy. Degradation of p53 is regulated by interaction with the MDM2 protein3,4 which functions as a ubiquitin ligase and shuttles from the nucleus to the cytoplasm, where degradation of p53 is thought to take place.5-7 The non-genotoxic small molecule MDM2 antagonist nutlin and the conventional chemotherapeutic agents such as velcade (also known as bortezomib) activate the p53 pathway by preventing at least in part, the proteasomemediated degradation of p53. Nutlin binds MDM2 in the p53 binding pocket with high selectivity, disrupts p53-MDM2 interaction and releases p53 from negative control. Therefore, nutlin protects p53 from ubiquitin-mediated proteasomal degradation leading to effective stabilization of p53 and activation

of the p53 pathway.8 Importantly, nutlin-mediated activation of the p53 pathway leads to apoptosis in various malignancies including MM.8-15 Velcade is a specific inhibitor of the chymotryptic activity of the proteasome that has cytotoxic activity in several malignant cell lines. It inhibits the degradation of multi-ubiquitinated target proteins such as cyclins and cyclin-dependent kinase inhibitors, regulates cell cycle progression16 and induces apoptosis of tumor cells, despite the accumulation of p21 and p27.17,18 Although the clinical trials of nutlin are ongoing, velcade has shown its efficacy in Phase II/III trials in patients with relapsed,refractory MM and malignant lymphoma with 20–50% response rates as a single agent.19-22 Even though velcade therapy is a major advance in the treatment of MM and lymphoma, it has been associated with possible off-target toxicities and the development of drug-resistance.19,23 In addition, a majority of patients do not respond to single agent therapy.24,25 Therefore, a combination of velcade with nongenotoxic agents such as nutlin may have a potential therapeutic value. The combination of nutlin and velcade has not been extensively studied. There have been only two very recent reports exploring the combined response of these drugs in mantle cell

*Correspondence to: Hong Chang; Email: [email protected] Submitted: 02/17/10; Revised: 03/26/10; Accepted: 03/27/10 Previously published online: www.landesbioscience.com/journals/cbt/article/11882 936

Cancer Biology & Therapy

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Research Paper

Research paper

Figure 1. Nutlin or velcade induces cytotoxicity and activates p53 in human MM cell lines harboring wt p53. (A) MM cells were treated with different doses of nutlin or velcade for 48 h and viability of the cells was quantified by MTT assay. Viability of MM1.S and H929 cells harboring wt p53 was decreased up to 60% by nutlin or velcade. No significant changes in cell viability was observed in nutlin-treated LP1 cells, however, viability of velcade treated LP1 cells was reduced up to 40% at similar doses. The percentage of cell viability (mean ± SD) is expressed as relative to DMSO control. Each experimental condition was repeated at least twice in triplicates. Data reported are mean ± SD of representative experiments. (B) Nutlin or velcade induces transcriptional activity of p53 in MM cell lines with wt p53. Western blot analysis of p53, p21 and MDM2 in MM cells after 24 h incubation with nutlin and 6 h incubation with velcade at different doses. Dose-dependent upregulation of p53, p21 and MDM2 was observed in MM1.S cells retaining wt p53 but not in mt p53 expressing LP1 cells.

lymphoma (MCL) cell lines15 and epithelial carcinoma and MM cell lines.26 Synergistic cytotoxic responses of nutlin and velcade have been observed in MCL and epithelial carcinoma, but only additive responses found in MM cells. In this study, we analyzed apoptotic pathways induced by nultlin and velcade, and demonstrated that nutlin in combination with velcade had synergistic cytotoxic responses in MM cells harboring wild type (wt) p53. Results Nutlin or velcade induces dose-dependent reduction in cell viability associated with activation of the p53 pathway. To investigate the effects of nutlin or velcade on cell viability, MM cell lines harboring either wt or mutant (mt) p53 were incubated with increasing concentrations of nutlin or velcade and assayed for cell viability by MTT assay at 48 h. The nutlin and velcade concentration required to induce cell death in 50% of MM cells (IC50) retaining wt p53 (MM1.S and H929) relative to control was 5 µM and 7 nM, respectively (Fig. 1A). In this respect, it should be noted that the specificity of nutlin in interrupting the MDM2-p53 interaction is lost at concentrations greater than 10 µM.8 Although the viability of LP1 cells retaining mt p53

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declined to up to 40% upon velcade treatment, viability of these cells showed only a modest decline of 20% upon nutlin treatment, suggesting a p53-dependent cytoxic effect of nutlin in MM cells (Fig. 1A). Next, we examined whether nutlin or velcade induces accumulation of p53 protein. Results showed p53-dependent upregulation of p53 and its two immediate downstream targets, p21 and MDM2 in nutlin or velcade treated MM cells (Fig. 1B). On the basis of these results, the concentration of 1.0 µM nutlin and 5 nM velcade was selected for further studies to explore the synergistic responses of these two drugs. Velcade interacts synergistically with nutlin to induce cell death and activate the p53 pathway in MM cells. Human MM cell lines (MM1.S and H929) were treated with nutlin and velcade at different concentrations for 48 h, followed by assessment for cell viability by MTT assays. For these studies, we utilized velcade and nutlin at concentrations lower than their maximal cytotoxic concentration for each cell line. A significant decrease in viability of both MM1.S and H929 cells was observed in response to treatment with combined low doses of velcade and nutlin than with either agent alone. Figure 2A shows representative results from minimally toxic and maximally additive concentrations of each agent. For example, treatment of


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Figure 2. Nutlin and velcade display synergistic cytotoxic responses in MM cells. (A) Combined treatment of MM1.S cells with a relatively low concentration (2–8 µM) of nutlin, with a fixed concentration of 6 nM velcade showed a substantial decrease of cell growth by 90% in MM1.S cells (left) and 75% in H929 cells (right) at 48 hrs treatment. By contrast, treatment with 4 µM nutlin or 6 nM velcade alone resulted in 98% of CD138 positive


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Table 2. Nucleotide sequences of the primers used for qRT-PCR Gene name P21

Primer (5' to 3') Forward: AGG CAC CGA GGC ACT CAG AG Reverse: AGT GGT AGA AAT CTG TCA TGC TG

FAS

Forward: GCT CTT TCA CTT CGG AGG ATT GC Reverse: GCC TTC CAA GTT CTG AGT CTC AAC

GADD45A

Forward: AAG GAT GGA TAA GGT GGG G

TNFRSF10B

Forward: AAG ACC CTT GTG CTC GTT GT

BAX

Forward: ACC AGG GTG GTT GGA CCG TGA

SPC25

Forward: GAG ATA CCT ACA AGG ATT CCA

Reverse: CTG GAT CAG GGT GAA GTG G Reverse: AGG TGG ACA CAA TCC CTC TG Reverse: GTC CAA GGC AGC TGG GGG C Reverse: GCT GAT CTG ATT TTG ATA TTC C UBE2L6

Forward: CCC GAG ATG ATG GCC AGC AAG Reverse: GAA GGC TTT GAG GCC ATA GGG

plasma cells. Primary MM cells were resuspended in IMDM medium and were incubated on plastic petridishes for 1 h at 37°C to remove the monocytes. Nonadherent cells were removed by gently washing the dishes and maintained in normal growth medium. Antibodies. Antibodies against the following proteins were used in this study: mouse monoclonal antibodies to p53 (DO-7), p21 and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); caspase-8 from BD Biosciences (San Diego, CA, USA) and MDM2 from Calbiochem (San Diego, CA, USA); Rabbit polyclonal antibodies to PUMA, BAX, and BAK and mouse monoclonal antibody to caspase-3 and cleaved PARP (Asp214) from Cell Signaling Technology (Cell Signaling, Danvers, MA, USA); caspase-9 from R&D Systems (Minneapolis, MN, USA); survivin from Abcam (Cambridge, MA, USA); Peroxidase-conjugated goat anti-mouse and antirabbit IgG were purchased from Cell Signaling and Santa Cruz Biotechnology, respectively. Drug treatment. Nutlin (nutlin-3) was purchased from Cayman Chemical (Ann Arbor, MI, USA) and dissolved in dimethyl sulfoxide (DMSO) to create a 1 mM stock solution and stored at -20°C. Velcade was obtained from Orthobiotech, Toronto, Ontario, Canada. Cell lines were harvested in log-Phase growth and exposed to nutlin (0–10 µM) for 24 h, with velcade (0–10 nM) added during the last 6 h of treatment. Primary MM samples (50 x 104 cells/ml) were cultured in six-well plates for 24 h and then treated with the drugs for another 24 h period, after which cells were harvested for further analysis. In each experiment, the final DMSO concentration was kept constant and did not exceed 0.1% (vol/vol). In some experiments, cells were exposed to only nutlin or velcade or simultaneously exposed to these agents. After drug treatment, cells were harvested and subjected to further analysis as described below. Cell viability and proliferation assay. Cell viability was assessed by MTT (3-[4,5-dimethilthiazol-2yl]-2,5-diphenyl tetrazolium bromide) colorimetric assay. For this, cells were cultured in 96-well micro-titer plates with different concentrations

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of drugs for a 72 h period. To assess the effect of the drugs on cell viability and proliferation of primary samples, 20 x 104 cells/ml were cultured in 96‑well plates for 24 h and then treated with the drugs for 48 h. After incubation, MTT (0.5 mg/ml) was added and the cells were further incubated for an additional 4 h. This was followed by the addition of acidified isopropanol to the wells and overnight incubation at 37°C. Following incubation, the optical density of the cells was read with a microplate reader set at a test wavelength of 570 nm and a reference wavelength of 630 nm. Each experiment was made in triplicate, and the mean value was calculated. Apoptosis assay. For quantitation of apoptotic cells by annexin-V staining, cells with or without drug treatment were washed with PBS, resuspended in annexin-V binding buffer and stained with FITC-annexin-V and PI, according to the manufacturer’s instructions (Abcam). Stained cells were analyzed using a FACScan (Becton Dickinson, NJ, USA) flow cytometer and apoptosis quantified as the percent annexin-V positive cells. The extent of apoptosis was quantified as percentage of annexin-V positive cells and the extent of drug-specific apoptosis was assessed by the formula: % specific apoptosis = (test - control) x 100/(100 - control). Protein extraction and Western blot (WB) analysis. Whole cell lysates were prepared by extraction of cell pellets which were lysed for 10 min on ice in a buffer composed of 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 5 mM EDTA, 1% (v/v) Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride (PMSF), 20 µg/ml aprotinin and 25 µg/ml leupeptin. Protein concentrations were measured by using a Nano Drop 1000 spectrophotometer (Thermo Fisher Scientific Inc., San Diego, CA, USA). Equal amounts of protein extracts were resolved using 12% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene diflouride (PVDF) membrane (Perkin Elmer Inc., Waltham, MA, USA). After blocking for 1 h at room temperature with PBS containing either 5% skim milk or 3% bovine serum albumin (BSA) depending on the antibodies used for probing the blots, the filter was incubated with specific antibodies for at least 2 h but not more than 24 h. The filter was washed, incubated with a horseradish peroxidase (HRP)-labeled secondary antibody for 1 h and the blots developed using a chemiluminescent detection system (ECL, Perkin Elmer). Gene expression analysis. Total RNA was isolated using TRIzol reagent (Invitrogen) and the gene expression profile was evaluated using Illumina Whole-Genome Expression Beadchips (Illumina Inc., San Diego, CA, USA) representing ∼25,000 (HumanRef-8) human genes. GeneChip arrays were scanned on a GeneArray Scanner (Illumina). Array normalization, expression value calculation and clustering analysis were performed using GenomeStudio Gene Expression Module (Illumina). Expression of key genes in nutlin-induced MM1.S cells involved in DNA repair, cell cycle arrest and apoptosis was analyzed. Quantitative Real Time PCR (qRT-PCR). To quantify and validate the expression of some of the p53 target genes at their mRNA level, qRT-PCR assays using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as reference gene were performed. Total RNA was isolated in the same method as described above


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for the gene expression analysis by microarray. cDNA was synthesized using the SuperScriptTM III first strand cDNA synthesis kit (Invitrogen). Samples for the qRT-PCR was prepared using Platinum SYBR Green qPCR SuperMix-UDG with Rox (Invitrogen) and run on the StepOnePlusTM Real-Time PCR System (Applied Biosystems, Foster, CA, USA) using a thermal profile of an initial 2 min UDG incubation step at 50°C and 2 min melting step at 95°C, followed by 40 cycles at 95°C for 20 s and 55°C for 40 s. The primers used for analysis by qRTPCR are listed in Table 2. To verify the presence of only one amplicon, a melting curve was processed after each run. After normalization with GAPDH expression, regulation was calculated between treated and untreated cells. All reactions were carried out at least twice in triplicate. Statistical analysis. Synergism, additive effects or antagonism were assessed using the Chou-Talalay method.27 The dose-effect References 1. Hideshima T, Richardson P, Anderson KC. Novel therapeutic approaches for multiple myeloma. Curr Treat Options Oncol. 2004; 5:227-38 2. Adams J. The proteasome: a suitable antineoplastic target. Nat Rev Cancer 2004; 4:349-60. 3. Haupt Y, Maya R, Kazaz A, Oren M. Mdm2 promotes the rapid degradation of p53. Nature 1997; 387:296-9. 4. Kubbutat MHG, Jones SN, Vousden KH. Regulation of p53 stability by Mdm2. Nature 1997; 387:299-303. 5. Roth J, Dobbelstein M, Freedman DA, Shenk T, Levine AJ. Nucleo-cytoplasmic shuttling of the hdm2 oncoprotein regulates the levels of the p53 protein via a pathway used by the human immunodeficiency virus rev protein. EMBO J 1998; 17:554-64. 6. Lain S, Midgley C, Sparks A, Lane EB, Lane DP. An inhibitor of nuclear export activates the p53 response and induces the localization of HDM2 and p53 to U1A-positive nuclear bodies associated with the PODS. Exp Cell Res 1999; 248:457-72. 7. Tao W, Levine AJ. Nucleocytoplasmic shuttling of oncoprotein Hdm2 is required for Hdm2-mediated degradation of p53. Proc Natl Acad Sci USA 1999; 96:3077-80. 8. Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z, et al. In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 2004; 303:844-8. 9. Stuhmer T, Chatterjee M, Hildebrandt M, Herrmann P, Gollasch H, Gerecke C, et al. Nongenotoxic activation of the p53 pathway as a therapeutic strategy for multiple myeloma. Blood 2005; 106:3609-17. 10. kojima K, konopleva M, Samudio IJ, Shikami M, Cabreira-Hansen M, McQueen T, et al. MDM2 antagonists induce p53-dependent apoptosis in AML: implications for leukemia therapy. Blood 2005; 106:3150-9. 11. Kojima K, Konopleva M, McQueen T, O’Brien S, Plunkett W, Andreeff M. Mdm2 inhibitor Nutlin3A induces p53-mediated apoptosis by transcriptiondependent and transcription-independent mechanisms and may overcome Atm-mediated resistance to fludarabine in chronic lymphocytic leukemia. Blood 2006; 108:993-1000. 12. Secchiero P, Barbarotto E, Tiribelli M, Zerbinati C, di Iasio MG, Gonelli A. Functional integrity of the p53-mediated apoptotic pathway induced by the nongenotoxic agent nutlin-3 in B-cell chronic lymphocytic leukemia (B-CLL). Blood 2006; 107:4122-9.

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curve for each drug alone was determined based on the experimental observations using the median-effect principle; the combination index (CI) for each experimental combination was then calculated according to the following equation: CI = (D)1/ (Dx)1 + (D)2 /(Dx)2 + (D)1(D)2 /(Dx)1(Dx)2, where (D)1 and (D)2 are the doses of drug 1 and drug 2 that have x effect when used in combination and (D)1 and (D)2 are the doses of drug 1 and drug 2 that have the same x effect when used alone. The combination is additive when CI = 1, synergistic when CI < 1.0, and antagonistic when CI > 1.0. Acknowledgements

This work was supported in part by research grants from Canadian Institute of Health Research (CIHR) and Leukemia & Lymphoma Society of Canada (LLSC). We thank P. Hu for his assistance with microarray data analysis.

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