Improvement in treatment outcome for patients with glioblastoma multiforme (GBM) requires a multifaceted approach due to dysregulation of numerous signaling pathways. The murine double minute 2 (MDM2) protein may fulfill this requirement because it is involved in the regulation of growth, survival, and invasion. The objective of this study was to investigate the impact of modulating MDM2 function in combination with front-line temozolomide (TMZ) therapy in GBM.
The combination of TMZ with the MDM2 protein–protein interaction inhibitor nutlin3a was evaluated for effects on cell growth, p53 pathway activation, expression of DNA repair proteins, and invasive properties. In vivo efficacy was assessed in xenograft models of human GBM.
In combination, TMZ/nutlin3a was additive to synergistic in decreasing growth of wild-type p53 GBM cells. Pharmacodynamic studies demonstrated that inhibition of cell growth following exposure to TMZ/nutlin3a correlated with: 1) activation of the p53 pathway, 2) downregulation of DNA repair proteins, 3) persistence of DNA damage, and 4) decreased invasion. Pharmacokinetic studies indicated that nutlin3a was detected in human intracranial tumor xenografts. To assess therapeutic potential, efficacy studies were conducted in a xenograft model of intracranial GBM by using GBM cells derived from a recurrent wild-type p53 GBM that is highly TMZ resistant (GBM10). Three 5-day cycles of TMZ/nutlin3a resulted in a significant increase in the survival of mice with GBM10 intracranial tumors compared with single-agent therapy.
Modulation of MDM2/p53-associated signaling pathways is a novel approach for decreasing TMZ resistance in GBM. To the authors' knowledge, this is the first study in a humanized intracranial patient-derived xenograft model to demonstrate the efficacy of combining front-line TMZ therapy and an inhibitor of MDM2 protein–protein interactions.
ABBREVIATIONSAPE1 = apurinic/apyrimidinic endonuclease I; ATM = ataxia-telangiectasia mutated; BER = base excision repair; BSA = bovine serum albumin; FBS = fetal bovine serum; GAPDH = glyceraldehyde-3-phosphate-dehydrogenase; GBM = glioblastoma; IUSCC, IUSM = Indiana University Simon Cancer Center, Indiana University School of Medicine; MDM2 = murine double minute 2; MGMT = O6-methylguanine DNA methyltransferase; MS = mass spectroscopy; NSG = NOD.Cg-PrkdcscidIL2rgtm1Wjl/Sz; PBS = phosphate-buffered saline; PK = pharmacokinetic; PPI = protein–protein interaction; S15 = phosphorylation on serine 15-p53; TBS = Tris-buffered saline; TBST = TBS and Tween 20; TMZ = temozolomide; T/N = combination of TMZ and nutlin3a.
INCLUDE WHEN CITING Published online May 13, 2016; DOI: 10.3171/2016.1.JNS152513.
Drs. Wang and S. Cai contributed equally to this work.
Correspondence Karen E. Pollok, Departments of Pediatrics and Pharmacology and Toxicology, Herman B. Wells Center for Pediatric Research, 1044 W Walnut St., R4-302, Indianapolis, IN 46202-5525. email: email@example.com.
AmbrosiniGSambolEBCarvajalDVassilevLTSingerSSchwartzGK: Mouse double minute antagonist Nutlin-3a enhances chemotherapy-induced apoptosis in cancer cells with mutant p53 by activating E2F1. Oncogene26:3473–34812007
AndreeffM, KellyKR, YeeK, AssoulineS, StrairR, PopplewellL, : Results of the Phase I trial of RG7112, a small-molecule MDM2 antagonist in leukemia. 22:868–876, 201610.1158/1078-0432.CCR-15-048126459177)| false
Bijangi-VishehsaraeiKSaadatzadehMRHuangSMurphyMPSafaAR: 4-(4-Chloro-2-methylphenoxy)-N-hydroxybutanamide (CMH) targets mRNA of the c-FLIP variants and induces apoptosis in MCF-7 human breast cancer cells. Mol Cell Biochem342:133–1422010
Bijangi-VishehsaraeiK, SaadatzadehMR, HuangS, MurphyMP, SafaAR: 4-(4-Chloro-2-methylphenoxy)-N-hydroxybutanamide (CMH) targets mRNA of the c-FLIP variants and induces apoptosis in MCF-7 human breast cancer cells. 342:133–142, 20102044601910.1007/s11010-010-0477-7)| false
BocangelDSenguptaSMitraSBhakatKK: p53-Mediated down-regulation of the human DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT) via interaction with Sp1 transcription factor. Anticancer Res29:3741–37502009
BocangelD, SenguptaS, MitraS, BhakatKK: p53-Mediated down-regulation of the human DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT) via interaction with Sp1 transcription factor. 29:3741–3750, 200919846904)| false
CaiS, WangH, BaileyB, ErnstbergerA, JuliarBE, SinnAL, : Humanized bone marrow mouse model as a preclinical tool to assess therapy-mediated hematotoxicity. 17:2195–2206, 20112148706510.1158/1078-0432.CCR-10-1959)| false
CarlsonBLPokornyJLSchroederMASarkariaJN: Establishment, maintenance and in vitro and in vivo applications of primary human glioblastoma multiforme (GBM) xenograft models for translational biology studies and drug discovery. Curr Protoc Pharmacol Chapter14:Unit 14.162011
CarlsonBL, PokornyJL, SchroederMA, SarkariaJN: Establishment, maintenance and in vitro and in vivo applications of primary human glioblastoma multiforme (GBM) xenograft models for translational biology studies and drug discovery. 14:Unit 14.16201110.1002/0471141755.ph1416s52)| false
Erickson-MillerCLMayRDTomaszewskiJOsbornBMurphyMJPageJG: Differential toxicity of camptothecin, topotecan and 9-aminocamptothecin to human, canine, and murine myeloid progenitors (CFU-GM) in vitro. Cancer Chemother Pharmacol39:467–4721997
HigginsB, GlennK, WalzA, TovarC, FilipovicZ, HussainS, : Preclinical optimization of MDM2 antagonist scheduling for cancer treatment by using a model-based approach. 20:3742–3752, 201410.1158/1078-0432.CCR-14-046024812409)| false
LaRuschGAJacksonMWDunbarJDWarrenRSDonnerDBMayoLD: Nutlin3 blocks vascular endothelial growth factor induction by preventing the interaction between hypoxia inducible factor 1α and Hdm2. Cancer Res67:450–4542007
MoranDM, MakiCG: Nutlin-3a induces cytoskeletal rearrangement and inhibits the migration and invasion capacity of p53 wild-type cancer cells. 9:895–905, 20102037171210.1158/1535-7163.MCT-09-1220)| false
Ray-CoquardIBlayJYItalianoALe CesneAPenelNZhiJ: Effect of the MDM2 antagonist RG7112 on the P53 pathway in patients with MDM2-amplified, well-differentiated or dedifferentiated liposarcoma: an exploratory proof-of-mechanism study. Lancet Oncol13:1133–11402012
Ray-CoquardI, BlayJY, ItalianoA, Le CesneA, PenelN, ZhiJ, : Effect of the MDM2 antagonist RG7112 on the P53 pathway in patients with MDM2-amplified, well-differentiated or dedifferentiated liposarcoma: an exploratory proof-of-mechanism study. 13:1133–1140, 20122308452110.1016/S1470-2045(12)70474-6)| false
RudekMA, DonehowerRC, StatkevichP, BatraVK, CutlerDL, BakerSD: Temozolomide in patients with advanced cancer: phase I and pharmacokinetic study. 24:16–25, 200410.1592/phco.18.104.22.16880014740784)| false
SarkariaJNCarlsonBLSchroederMAGroganPBrownPDGianniniC: Use of an orthotopic xenograft model for assessing the effect of epidermal growth factor receptor amplification on glioblastoma radiation response. Clin Cancer Res12:2264–22712006
SarkariaJN, CarlsonBL, SchroederMA, GroganP, BrownPD, GianniniC, : Use of an orthotopic xenograft model for assessing the effect of epidermal growth factor receptor amplification on glioblastoma radiation response. 12:2264–2271, 200610.1158/1078-0432.CCR-05-251016609043)| false
SchneeklothAR, PucheaultM, TaeHS, CrewsCM: Targeted intracellular protein degradation induced by a small molecule: En route to chemical proteomics. 18:5904–5908, 200810.1016/j.bmcl.2008.07.11418752944)| false
ShangarySQinDMcEachernDLiuMMillerRSQiuS: Temporal activation of p53 by a specific MDM2 inhibitor is selectively toxic to tumors and leads to complete tumor growth inhibition. Proc Natl Acad Sci U S A105:3933–39382008
ShangaryS, QinD, McEachernD, LiuM, MillerRS, QiuS, : Temporal activation of p53 by a specific MDM2 inhibitor is selectively toxic to tumors and leads to complete tumor growth inhibition. 105:3933–3938, 20081831673910.1073/pnas.0708917105)| false
SionovRV, CoenS, GoldbergZ, BergerM, BercovichB, Ben-NeriahY, : c-Abl regulates p53 levels under normal and stress conditions by preventing its nuclear export and ubiquitination. 21:5869–5878, 20011148602610.1128/MCB.21.17.5869-5878.2001)| false
Tonsing-CarterEBaileyBJSaadatzadehMRDingJWangHSinnAL: Potentiation of carboplatin-mediated DNA damage by the Mdm2 modulator Nutlin-3a in a humanized orthotopic breast-to-lung metastatic model. Mol Cancer Ther14:2850–28632015
Tonsing-CarterE, BaileyBJ, SaadatzadehMR, DingJ, WangH, SinnAL, : Potentiation of carboplatin-mediated DNA damage by the Mdm2 modulator Nutlin-3a in a humanized orthotopic breast-to-lung metastatic model. 14:2850–2863, 201510.1158/1535-7163.MCT-15-0237)| false
YoshimotoK, MizoguchiM, HataN, MurataH, HataeR, AmanoT, : Complex DNA repair pathways as possible therapeutic targets to overcome temozolomide resistance in glioblastoma. 2:186, 20122322745310.3389/fonc.2012.00186)| false
ZakyABussoCIzumiTChattopadhyayRBassiounyAMitraS: Regulation of the human AP-endonuclease (APE1/Ref-1) expression by the tumor suppressor p53 in response to DNA damage. Nucleic Acids Res36:1555–15662008
ZakyA, BussoC, IzumiT, ChattopadhyayR, BassiounyA, MitraS, : Regulation of the human AP-endonuclease (APE1/Ref-1) expression by the tumor suppressor p53 in response to DNA damage. 36:1555–1566, 200810.1093/nar/gkm117318208837)| false
ZhangF, TagenM, ThromS, MallariJ, MillerL, GuyRK, : Whole-body physiologically based pharmacokinetic model for nutlin-3a in mice after intravenous and oral administration. 39:15–21, 2011. 4 hours2094761710.1124/dmd.110.035915)| false