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Cited by 49Glioma Diagnosis and TreatmentCancer-related Molecular PathwaysCancer Mechanisms and Therapy

Combination therapy in a xenograft model of glioblastoma: Enhancement of the antitumor activity of temozolomide by an MDM2 antagonist

Open access
Wang, Haiyan, Cai, Shanbao, Bailey, Barbara J., Reza Saadatzadeh, M., Ding, Jixin, Tonsing-Carter, Eva, Georgiadis, Taxiarchis M., Zachary Gunter, T. +17 more·2017·Journal of Neurosurgery
DOI 10.3171/2016.1.jns152513PMC PMC5280578
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Abstract

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

AI snapshot

Written from the paper’s full text.

TL;DR

Combining temozolomide (TMZ) with the MDM2 protein–protein interaction inhibitor nutlin3a enhanced DNA damage, downregulated DNA repair proteins, and increased survival in a TMZ-resistant intracranial GBM10 xenograft model. The authors report this as the first humanized intracranial patient-derived xenograft study showing efficacy of front-line TMZ plus an MDM2 inhibitor.

Key findings

  • In wild-type p53 GBM cells, TMZ/nutlin3a was additive to synergistic in inhibiting proliferation; GBM10 and MHBT91 were TMZ resistant, with TMZ IC50 values 11- to 47-fold higher than U87-MG. Combination treatment increased γH2AX foci, caused persistence of DNA strand breaks, activated p53 signaling, and decreased invasion.
  • In U87-MG flank xenografts, TMZ/nutlin3a further decreased tumor volume by approximately 50% compared with TMZ alone, and tumor growth was significantly inhibited for at least approximately 4 weeks beyond dosing.
  • In the intracranial GBM10 model, three 5-day cycles of TMZ/nutlin3a significantly increased survival compared with TMZ or nutlin3a alone (p < 0.05). TMZ alone produced an approximately 13% survival increase versus vehicle, while combination T/N produced an approximately 29% increase, with minimal bone marrow toxicity.
  • Nutlin3a was detected in intracranial tumor xenografts at 1–20 μM 4 hours after administration, higher than in normal brain; however, single-agent nutlin3a did not increase survival in mice with intracranial GBM tumors.

Methods

  • In vitro studies used human wild-type p53 GBM lines U87-MG, GBM10, and MHBT91, including patient-derived GBM10 and GBM43 cells. Assays included proliferation, Western blot, γH2AX foci, cell cycle, alkaline comet, apoptosis, wound healing, and Matrigel invasion.
  • In vivo studies used NSG mice with U87-MG or GBM10 flank xenografts and orthotopic intracranial GBM10 xenografts. Dosing included TMZ 40 or 66 mg/kg once daily and nutlin3a 100 or 200 mg/kg twice daily in 5-day cycles; 125 mice received intracranial implants, and 7 (6%) were censored because they did not die of tumor growth by 105 days.
  • Analyses included pharmacokinetic measurement of nutlin3a, IC50/isobologram analysis by the Chou-Talalay method, ANOVA with Holm-Šídák post hoc tests, and Kaplan-Meier survival analysis by logrank test, with significance at p < 0.05.

Limitations

  • The paper states that toxicity data in mice should be interpreted with caution because differential sensitivity to oncolytics between murine and human bone marrow has been reported; it also notes that clinical utility of MDM2 PPI inhibitors is still in early phases and that dosing regimens will need refinement to avoid normal tissue toxicity.
  • Evident from the text: this is a preclinical xenograft study, not a human trial, and the intracranial efficacy testing was conducted in one patient-derived model, GBM10, with wild-type p53; mutant p53 GBM was not tested in this model.
  • Evident from the text: single-agent nutlin3a did not improve survival in the intracranial GBM10 model despite in vitro activity, and the authors note that prolonged treatment may be required to block tumor growth.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.3171/2016.1.jns152513

Cites

52 of 52 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 52 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    After TMZ/nutlin3a exposure, reduced cell growth correlated with downregulation of DNA repair proteins.

    “2) downregulation of DNA repair proteins”
    Quote in the paperSubstantial claim
  • Findingown data

    Five days after treatment, nutlin3a or T/N caused significant rises in early and late apoptotic/necrotic cells relative to control or TMZ alone.

    “At Day 5 posttreatment, there were significant increases in early and late apoptotic/necrotic cells treated with nutlin3a or T/N compared with control or TMZ alone.”
    Quote in the paperSubstantial claim
  • Findingown data

    After TMZ/nutlin3a exposure, reduced cell growth correlated with activation of the p53 pathway.

    “Pharmacodynamic studies demonstrated that inhibition of cell growth following exposure to TMZ/nutlin3a correlated with: 1) activation of the p53 pathway”
    Quote in the paperSubstantial claim
  • Findingown data

    Nutlin3a was detected in human intracranial tumor xenografts.

    “Pharmacokinetic studies indicated that nutlin3a was detected in human intracranial tumor xenografts.”
    Quote in the paperSubstantial claim
  • Findingown data

    Exposure to nutlin3a or T/N caused G0/G1 arrest in both U87-MG and GBM10 cells.

    “indicated that both the U87-MG and GBM10 cells arrested in G0/G1 following exposure to nutlin3a or T/N”
    Quote in the paperNot a substantial claim
  • May combine two claims. 35
  • Classified as probably not a substantial claim. 22

  • May combine two claims.

    Findingown data

    Three 5-day cycles of TMZ plus nutlin3a significantly improved survival in mice with intracranial GBM10 tumors compared with single-agent therapy, with little bone marrow toxicity.

    “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.”
    • Also stated in: § CONCLUSIONS · plain
      “A combination regimen consisting of three 5-day cycles resulted in a significant increase in survival compared with mice treated with TMZ alone, with minimal bone marrow toxicity.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: APE1 and MGMT are needed to fix TMZ-induced DNA damage, and higher amounts of them can drive resistance in GBM.

  • May combine two claims.

    Findingown data

    This increase in apoptotic/necrotic cells correlated with more G0/G1 arrest and less TMZ-induced G2/M arrest.

    “This correlated with an increase in the percentage of cells arrested in G0/G1 and a decrease in TMZ-induced G2/M arrest”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Twenty-four hours after treatment, TMZ, nutlin3a, and T/N raised total p53, activated p53 (S15), and MDM2; nutlin3a or T/N also raised p21.

    “At 24 hours posttreatment (Fig. 3D), Western analyses indicated that levels of total p53, activated p53 (phosphorylation on serine 15-p53 [S15]), and MDM2 were increased by TMZ, nutlin3a, and T/N; p21 levels were increased following nutlin3a or T/N treatment.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The patient-derived GBM10 and MHBT91 cell lines were TMZ resistant, with TMZ IC50 values 11- to 47-fold higher than in U87-MG cells.

    “The primary patient cell lines GBM10 and MHBT91 were resistant to TMZ, i.e., the IC50 concentrations for TMZ were 11- to 47-fold higher than in U87-MG cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the present study, nutlin3a promoted apoptosis as a single agent in vitro or modestly blocked U87-MG flank tumor growth in vivo, but this did not translate to the intracranial xenograft model.

    “In the present study, although nutlin3a could promote apoptosis as a single agent in vitro or modestly block growth of U87-MG flank tumors in vivo, this did not translate to the in vivo intracranial xenograft model.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Combination T/N treatment produced about a 29% increase in survival with minimal bone marrow toxicity.

    “Combination T/N treatment further improved survival, resulting in an approximately 29% increase in survival with minimal toxicity to the bone marrow.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Body weights did not change and no apparent treatment-related toxicities were observed.

    “Body weights were unchanged (data not shown), and no apparent treatment-related toxicities were noted.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In the present study, nutlin3a potentiated TMZ-mediated GBM cell death and significantly increased survival of mice with orthotopic intracranial human GBM tumors that were highly resistant to TMZ.

    “In the present study, the MDM2 PPI inhibitor nutlin3a potentiated TMZ-mediated GBM cell death and significantly increased survival of mice with orthotopic intracranial human GBM tumors that were highly resistant to TMZ.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: GBM10 cells are relevant to the clinical challenges of GBM therapy.
    • Rests on an assumption: GBM10 cells are highly resistant to TMZ because of high levels of DNA repair proteins MGMT and APE1 and deletion of the CDKN2A/p16 gene.
    • Rests on an assumption: The GBM10 intracranial xenograft model is clinically relevant to human GBM.

  • May combine two claims.

    Findingown data

    T/N suppressed tumor growth relative to TMZ alone for roughly 4 weeks after dosing ended, then tumors started to regrow.

    “Tumor growth for T/N versus TMZ alone was significantly inhibited for at least approximately 4 weeks beyond the dosing window, but tumor volume began to rebound thereafter.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Mice at the predeath end point harbored intracranial tumors.

    “mice at the predeath end point harbored intracranial tumors (Fig. 7E).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Dose-response curves confirmed that nutlin3a was the more potent enantiomer.

    “Dose-response curves confirmed that nutlin3a was the more potent enantiomer (data not shown).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    MHBT91 has wild-type p53, ATM, Rb1, and CDKN2A/p16.

    “The focused molecular profile of the MHBT91 cell line, determined via the qBiomarker Somatic Mutation PCR array (SABiosciences, Qiagen) for the p53/RB1 pathway, is wild type for p53, ataxia-telangiectasia mutated (ATM), Rb1, and CDKN2A/p16.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Nutlin3a and T/N raised total p53 more than TMZ alone, and T/N raised total p53 and p53(S15) more than nutlin3a.

    “Moreover, total p53 levels were significantly increased by nutlin3a and T/N compared with TMZ alone, and T/N significantly increased total p53 and p53 (S15) compared with nutlin3a.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In initial studies, T/N did not significantly increase survival compared with other treatments, although body weights were maintained.

    “Although body weights were maintained, increases in survival of animals treated with T/N were not significant compared with other treatments (data not shown).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingcitation

    Pharmacokinetic analysis of racemic nutlin3 in NSG mice agreed with previously reported values in C57BL/6 mice.

    “results of pharmacokinetic (PK) analysis of racemic nutlin3 in NSG mice was in agreement with values previously reported in C57BL/6 mice (Table 1).51”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [51] Zhang F Tagen M Throm S Mallari J Miller L Guy RK Whole-body physiologically based pharmacokinetic model for nutlin-3a in mice after intravenous and oral administration Drug Metab Dispos 39 15 21 2011 4 hours 20947617 PMC3014266 source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Both GBM10 migration and invasion were significantly inhibited by nutlin3a and the T/N combination.

    “Both GBM10 migration (Fig. 6B) and invasion (Fig. 6C) were significantly inhibited by nutlin3a and the T/N combination.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingown data

    GBM10 cells express the mismatch repair proteins MSH6, PMS2, and MSH2, which is consistent with mismatch repair proficiency.

    “The mismatch repair proteins MSH6, PMS2, and MSH2 are expressed in GBM10 cells, which is consistent with mismatch repair proficiency (data not shown).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    There was a significant increase in survival of mice treated with T/N compared with TMZ alone, achievable with three 5-day treatment cycles.

    “There was a significant increase in survival of mice treated with TMZ/nutlin3a in combination compared with TMZ alone, which could be achieved with three 5-day treatment cycles.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    APE1 and MGMT were both downregulated to a greater extent in GBM cells treated with TMZ/nutlin3a compared with either agent alone.

    “APE1 and MGMT were both downregulated to a greater extent in GBM cells treated with TMZ/nutlin3a compared with either agent alone.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    After two treatment cycles, nutlin3 alone significantly slowed tumor growth and TMZ alone significantly reduced tumor volume compared with vehicle.

    “Following 2 treatment cycles, nutlin3 alone significantly slowed tumor growth, and TMZ alone produced a significant decrease in tumor volume compared with vehicle.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Changing MDM2 activity is associated with DNA damage and may interfere with DNA repair mechanisms.

    “These data indicate that modulation of MDM2 function is linked to DNA damage and may potentially block DNA repair mechanisms.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Nutlin3a's modulation of MDM2 function impairs DNA repair and is associated with enhanced TMZ-mediated cell death in vitro and in vivo.

    “We demonstrated that nutlin3a-mediated modulation of MDM2 function leads to an impairment in DNA repair that correlates with potentiation of TMZ-mediated cell death both in vitro and in vivo.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    MDM2 acts as an E3 ubiquitin ligase that sends p53 for proteasomal degradation, blocks p53's transactivation domain, and exports p53 from the nucleus.

    “MDM2 is an E3 ubiquitin ligase that targets p53 for proteasomal-mediated degradation, blocks the transactivation domain of p53, and is responsible for the export of p53 from the nucleus.26”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [26] Nag S Qin J Srivenugopal KS Wang M Zhang R The MDM2-p53 pathway revisited J Biomed Res 27 254 271 2013 23885265 PMC3721034

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Nutlin3a binds the N-terminal hydrophobic pocket of MDM2 and prevents MDM2 from interacting with p53 and other signal transducers such as p73α, E2F1, Rb, and HIF-1α.

    “The MDM2 protein–protein interaction (PPI) inhibitor nutlin3a binds to the N-terminal hydrophobic pocket of MDM2 and blocks interaction of MDM2 with not only p53 but also a number of other key signal transducers, including p73α, E2F1, Rb, and HIF-1α”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [2] Ambrosini G Sambol EB Carvajal D Vassilev LT Singer S Schwartz GK Mouse double minute antagonist Nutlin-3a enhances chemotherapy-induced apoptosis in cancer cells with mutant p53 by activating E2F1 Oncogene 26 3473 3481 2007 17146434 source

      Not checked in the free run.

    • cites [21] LaRusch GA Jackson MW Dunbar JD Warren RS Donner DB Mayo LD Nutlin3 blocks vascular endothelial growth factor induction by preventing the interaction between hypoxia inducible factor 1α and Hdm2 Cancer Res 67 450 454 2007 17234751 source

      Not checked in the free run.

    • cites [22] Lau LM Nugent JK Zhao X Irwin MS HDM2 antagonist Nutlin-3 disrupts p73-HDM2 binding and enhances p73 function Oncogene 27 997 1003 2008 17700533 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The chosen human wtp53 GBM lines-U87-MG, GBM10, and MHBT91-reflect molecular profiles often found in clinical GBM.

    “A panel of human wtp53 GBM cell lines (U87-MG, GBM10, and MHBT91) was selected that represents molecular profiles commonly found clinically in GBM (Fig. 1).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Mice with U87-MG tumors were randomized into cohorts of 6-8 animals.

    “When U87-MG tumors reached approximately 150 mm3, mice were randomized into cohorts of 6–8 animals.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    APE1 and MGMT can be expressed at high levels in GBM and are major predictors of TMZ resistance.

    “The DNA repair proteins APE1 and MGMT can be expressed at high levels in GBM and are major predictors of TMZ resistance.49”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [49] Yoshimoto K Mizoguchi M Hata N Murata H Hatae R Amano T Complex DNA repair pathways as possible therapeutic targets to overcome temozolomide resistance in glioblastoma Front Oncol 2 186 2012 23227453 PMC3514620 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    GBM10 cells came from a recurrent glioblastoma, are strongly resistant to temozolomide and radiation, and show high APE1 and MGMT levels in vivo.

    “The GBM10 cells were originally derived from a recurrent GBM, are highly resistant to TMZ and ionizing radiation, and express high levels of APE1 and MGMT in vivo.31”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [31] Sarkaria JN Carlson BL Schroeder MA Grogan P Brown PD Giannini C Use of an orthotopic xenograft model for assessing the effect of epidermal growth factor receptor amplification on glioblastoma radiation response Clin Cancer Res 12 2264 2271 2006 16609043 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    The 2A9 MDM2 antibody was used to avoid possible epitope masking due to MDM2 phosphorylation.

    “The 2A9 MDM2 antibody was used to avoid possible epitope masking due to phosphorylation of MDM2.11”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [11] Cheng Q Chen J The phenotype of MDM2 auto-degradation after DNA damage is due to epitope masking by phosphorylation Cell Cycle 10 1162 1166 2011 21386656 PMC3100889 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Nutlin3a levels achieved in the brain were sufficient to increase TMZ-mediated inhibition of tumor growth and to enhance survival of mice with GBM10 intracranial tumors.

    “Our data do suggest that nutlin3a levels obtained in the brain were sufficient to increase TMZ-mediated inhibition of tumor growth and to enhance survival of mice with GBM10 intracranial tumors.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: GBM10 cells are relevant to the clinical challenges of GBM therapy.
    • Rests on an assumption: The GBM10 intracranial xenograft model is clinically relevant to human GBM.

  • May combine two claims.

    Premisecitation

    After DNA damage, MDM2 is phosphorylated at serine 395 by ATM, serine 407 by ATR, and tyrosine 394 by c-Abl, which together with other modifications neutralizes MDM2/MDMX inhibition of p53 and sustains p53 signaling.

    “Following DNA damage, MDM2 is phosphorylated on serine 395 by ATM,24 on serine 407 by ATR,36 and on tyrosine 394 by c-abl,37,47 which along with other posttranslational modifications of MDMX and p53 neutralizes the inhibitory effect of MDM2/MDMX on p53 and results in sustained p53-mediated signaling.10”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [24] Maya R Balass M Kim ST Shkedy D Leal JF Shifman O ATM-dependent phosphorylation of Mdm2 on serine 395: role in p53 activation by DNA damage Genes Dev 15 1067 1077 2001 11331603 PMC312683 source

      Not checked in the free run.

    • cites [36] Shinozaki T Nota A Taya Y Okamoto K Functional role of Mdm2 phosphorylation by ATR in attenuation of p53 nuclear export Oncogene 22 8870 8880 2003 14654783 source

      Not checked in the free run.

    • cites [10] Chen J The roles of MDM2 and MDMX phosphorylation in stress signaling to p53 Genes Cancer 3 274 282 2012 23150760 PMC3494364 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Seven of 125 mice with intracranial implants (6%) survived past 105 days without tumor death and were censored.

    “Among the 125 mice used in this study for intracranial implants, 7 (6%) did not die of tumor growth by 105 days postimplantation and were censored from the study.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Nutlin3 can enhance TMZ's antitumor effect in GBM xenografts, and longer treatment is needed to stop tumor growth.

    “These data indicate that nutlin3 can potentiate the antitumor activity of TMZ in GBM xenografts and that prolonged treatment is required to block tumor growth.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    ATM, ATR, and c-Abl provide a well-documented link between chemotherapy-induced DNA damage and regulation of the MDM2/MDMX/p53 complex.

    “The DNA damage sensors ATM, ATM- and RAD3-related (ATR), and the stress-activated tyrosine kinase c-Abl provide a well-documented link between chemotherapy-mediated DNA damage and regulation of the MDM2/mouse double-minute X (MDMX)/p53 complex.10”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [10] Chen J The roles of MDM2 and MDMX phosphorylation in stress signaling to p53 Genes Cancer 3 274 282 2012 23150760 PMC3494364 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Nutlin3a can inhibit migration and invasion via cytoskeletal changes in other cell types.

    “others have demonstrated that nutlin3a can inhibit migration and invasion via changes in the cytoskeleton in other cell types.25”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [25] Moran DM Maki CG Nutlin-3a induces cytoskeletal rearrangement and inhibits the migration and invasion capacity of p53 wild-type cancer cells Mol Cancer Ther 9 895 905 2010 20371712 PMC3075611 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    A 20% increase in survival was considered biologically significant when comparing TMZ with T/N-treated mice.

    “An effect size of 20% increase in survival was considered to be biologically significant when comparing survival in TMZ versus combination T/N-treated mice.39”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [39] Stupp R Mason WP van den Bent MJ Weller M Fisher B Taphoorn MJ Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma N Engl J Med 352 987 996 2005 15758009 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    Investigators created a primary GBM cell line from fresh tumor tissue from a patient with pathologically confirmed GBM, with IRB approval.

    “A primary GBM cell line was established using a fresh tumor specimen obtained in a patient with pathologically diagnosed GBM (Aaron Cohen-Gadol IRB#0909–05, Dept. of Neurosurgery, Indiana University School of Medicine [IUSM]).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    GBM10 cells originated from a recurrent wild-type p53 GBM tumor with high APE1 and MGMT expression.

    “We developed a TMZ/nutlin3a dosing regimen in an intracranial xenograft model using GBM10 cells, which were derived from a recurrent wild-type p53 GBM tumor that expresses high levels of the base excision repair (BER) protein apurinic/apyrimidinic endonuclease I (APE1) and MGMT.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [9] Carlson BL Pokorny JL Schroeder MA Sarkaria JN 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 Chapter 14 Unit 14.16 2011 PMC3129784 21743824 source

      Not checked in the free run.

    • cites [31] Sarkaria JN Carlson BL Schroeder MA Grogan P Brown PD Giannini C Use of an orthotopic xenograft model for assessing the effect of epidermal growth factor receptor amplification on glioblastoma radiation response Clin Cancer Res 12 2264 2271 2006 16609043 source

      Not checked in the free run.

    • Rests on an assumption: APE1 and MGMT are needed to fix TMZ-induced DNA damage, and higher amounts of them can drive resistance in GBM.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    Before efficacy testing, pilot work set cell dose, checked tumor take rates, and followed survival.

    “Prior to initiation of efficacy studies, pilot studies were first performed in NSG mice to optimize cell dose, assess tumor take frequency, and track survival.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Stupp et al. reported that TMZ plus radiation in GBM patients gave about a 20% increase in progression-free survival compared with radiation alone.

    “Stupp et al. originally reported that treatment of patients with GBM with TMZ and radiation resulted in an approximately 20% increase in progression-free survival compared with radiation therapy alone.39”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [39] Stupp R Mason WP van den Bent MJ Weller M Fisher B Taphoorn MJ Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma N Engl J Med 352 987 996 2005 15758009 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Phosphorylation of p53 at serine 15 by ATM or DNA-dependent protein kinase indicates DNA-strand breaks.

    “Phosphorylation of p53 on serine 15 by ATM or DNA-dependent protein kinase is an indicator of DNA-strand breaks.26”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [26] Nag S Qin J Srivenugopal KS Wang M Zhang R The MDM2-p53 pathway revisited J Biomed Res 27 254 271 2013 23885265 PMC3721034

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All GBM cells were routinely tested for mycoplasma and were negative.

    “All GBM cells were routinely checked for mycoplasma and were negative.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductown data

    Cell line identity was confirmed by DNA fingerprinting, all cell lines were 100% human, and a 9-marker STR analysis is on file.

    “Cell line identity was confirmed by DNA fingerprint analysis (IDEXX BioResearch) for species and baseline short-tandem repeat analysis testing. All cell lines were 100% human, and a 9-marker short-tandem repeat analysis is on file.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    Animal procedures were approved by the IACUC and the animal facility is AAALAC accredited.

    “All procedures and predeath monitoring criteria were approved by the Institutional Animal Care and Use Committee. The IUSM-LARC is accredited by the American Association for the Accreditation of Laboratory Animal Care.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Higgins et al. proposed a modeling approach for strategic scheduling of MDM2 PPI inhibitors.

    “To this end, Higgins et al. have proposed a modeling approach for strategic scheduling of MDM2 PPI inhibitors.16”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [16] Higgins B Glenn K Walz A Tovar C Filipovic Z Hussain S Preclinical optimization of MDM2 antagonist scheduling for cancer treatment by using a model-based approach Clin Cancer Res 20 3742 3752 2014 24812409 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    p53 reactivation by nutlin3a leads to persistent DNA damage and downregulation of DNA repair proteins in GBM cells treated with TMZ plus nutlin3a.

    “We also showed that p53 reactivation by nutlin3a leads to a persistence of DNA damage and downregulation of DNA repair proteins in GBM cells treated with TMZ in combination with nutlin3a.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Sustained p53 pathway activation after TMZ plus an MDM2 PPI inhibitor correlated with downregulation of DNA repair proteins critical for repairing TMZ-mediated DNA damage, leading to increased GBM cell apoptosis and increased survival of mice with intracranial GBM.

    “We found that sustained p53 pathway activation following exposure to the combination of TMZ and an MDM2 PPI inhibitor correlated with downregulation of DNA repair proteins critical for the repair of TMZ-mediated DNA damage, leading to increased apoptosis of GBM cells and increased survival of mice with intracranial GBM.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: GBM10 cells are relevant to the clinical challenges of GBM therapy.
    • Rests on an assumption: GBM10 cells are highly resistant to TMZ because of high levels of DNA repair proteins MGMT and APE1 and deletion of the CDKN2A/p16 gene.
    • Rests on an assumption: The GBM10 intracranial xenograft model is clinically relevant to human GBM.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Iancu-Rubin et al. evaluated the impact of the nutlin3-derivative RG7112 on thrombopoiesis because this has been a dose-limiting toxicity in clinical trials.

    “Iancu-Rubin et al. evaluated the impact of the nutlin3-derivative RG7112 on thrombopoiesis because this has been a dose-limiting toxicity in clinical trials.18”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [18] Iancu-Rubin C Mosoyan G Glenn K Gordon RE Nichols GL Hoffman R Activation of p53 by the MDM2 inhibitor RG7112 impairs thrombopoiesis Exp Hematol 42 137 145 145.e1 145.e5 2014 24309210 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Administration of RG7112 for 10 days decreased platelet production in rats and monkeys, but the effects were reversible.

    “Administration of RG7112 for 10 days decreased platelet production in rats and monkeys, but the effects were reversible.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [18] Iancu-Rubin C Mosoyan G Glenn K Gordon RE Nichols GL Hoffman R Activation of p53 by the MDM2 inhibitor RG7112 impairs thrombopoiesis Exp Hematol 42 137 145 145.e1 145.e5 2014 24309210 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Response to combination therapy was in part dependent on p73α levels.

    “In addition, response to combination therapy was in part dependent on p73α levels.43”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [43] Tonsing-Carter E Bailey BJ Saadatzadeh MR Ding J Wang H Sinn AL Potentiation of carboplatin-mediated DNA damage by the Mdm2 modulator Nutlin-3a in a humanized orthotopic breast-to-lung metastatic model Mol Cancer Ther 14 2850 2863 2015 26494859 PMC4674357 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Initial clinical trials showed therapeutic responses in relapsed leukemia and liposarcoma, but also indicated that molecular profiling could be essential for identifying responders versus nonresponders.

    “Initial clinical trials have yielded evidence of therapeutic responses in relapsed leukemia3 and liposarcoma, but indicate that molecular profiling could be essential in identifying responders versus nonresponders. 28”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [3] Andreeff M Kelly KR Yee K Assouline S Strair R Popplewell L Results of the Phase I trial of RG7112, a small-molecule MDM2 antagonist in leukemia Clin Cancer Res 22 868 876 2016 26459177 PMC4809642 source

      Not checked in the free run.

    • cites [28] Ray-Coquard I Blay JY Italiano A Le Cesne A Penel N Zhi J 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 Oncol 13 1133 1140 2012 23084521 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Inhibition of MDM2 binding to p53 or p73α appears more effective at promoting p53- and p73α-mediated apoptosis in cancer cells than in normal cells.

    “Moreover, others have shown that inhibition of MDM2 binding to p53 or p73α appears to be more effective at promoting p53- and p73α-mediated apoptosis in cancer cells compared with normal cells.19,45”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [19] Jiang M Pabla N Murphy RF Yang T Yin XM Degenhardt K Nutlin-3 protects kidney cells during cisplatin therapy by suppressing Bax/Bak activation J Biol Chem 282 2636 2645 2007 17130128 PMC2859169 source

      Not checked in the free run.

    • cites [45] Vassilev LT Vu BT Graves B Carvajal D Podlaski F Filipovic Z In vivo activation of the p53 pathway by smallmolecule antagonists of MDM2 Science 303 844 848 2004 14704432 source

      Not checked in the free run.

52 of 52

  • The paper relies on this without support.

    APE1 and MGMT are needed to fix TMZ-induced DNA damage, and higher amounts of them can drive resistance in GBM.

    “Both of these DNA repair proteins play an integral role in the repair of TMZ-induced DNA lesions, and increased levels can lead to resistance mechanisms in GBM.”

  • The paper relies on this without support.

    Better GBM outcomes need a multipronged strategy because many signaling pathways are abnormal.

    “Improvement in treatment outcome for patients with glioblastoma multiforme (GBM) requires a multifaceted approach due to dysregulation of numerous signaling pathways.”

  • The paper relies on this without support.

    p53 and MDM2 regulate each other, with MDM2 being a transcriptional target downstream of p53.

    “Additionally, p53 and MDM2 form an autoregulatory network in which MDM2 is a downstream transcriptional target of p53.”

  • The paper relies on this without support.

    GBM10 cells are relevant to the clinical challenges of GBM therapy.

    “The GBM10 cells were selected for further in vivo efficacy studies, because they are relevant to the clinical challenges of GBM therapy.”

  • The paper relies on this without support.

    GBM10 cells are highly resistant to TMZ because of high levels of DNA repair proteins MGMT and APE1 and deletion of the CDKN2A/p16 gene.

    “GBM10 cells represent a particularly challenging tumor type to treat because they are highly resistant to TMZ due to high levels of DNA repair proteins (MGMT and APE1) and deletion of the CDKN2A/p16 gene.”

  • The paper relies on this without support.

    The GBM10 intracranial xenograft model is clinically relevant to human GBM.

    “in a clinically relevant humanized intracranial GBM xenograft model (GBM10)”
6 of 6

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Nutlin3a-mediated inhibition of MDM2/p53 interaction led to an impairment in DNA repair that correlated with potentiation of temozolomide-mediated cell death both in vitro and in vivo in glioblastoma.

    “Another study found that nutlin3a-mediated inhibition of MDM2/p53 interaction and led to an impairment in DNA repair that correlated with potentiation of temozolomide-mediated cell death both in vitro and in vivo in GBM [45].”

    From The p53 Pathway in Glioblastoma (2018) · cites this paper as [45]

    The Results (In Vitro and In Vivo Evaluation of TMZ- and Nutlin3a-Mediated Target Modulation in GBM10 Cells; page not provided) report increased apoptosis and persistent DNA damage with the combination in cultured GBM cells, as well as reduced DNA-repair protein levels in mouse tumors. The Discussion links these findings to enhanced temozolomide sensitivity. In vivo, however, the paper reports improved tumor-growth inhibition and mouse survival, not a direct measurement of potentiated cell death; it also presents blocked DNA repair as an interpretation rather than an established mechanism. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • When combined with temozolomide, use of the MDM2 inhibitor nutlin3a correlated with activation of the p53 pathway, downregulation of DNA repair proteins, persistence of DNA damage, and decreased cellular invasion.

    “When combined with temozolomide, use of the MDM2 inhibitor, nutlin3a, correlated with activation of the p53 pathway, downregulation of DNA repair proteins, persistence of DNA damage, and decreased cellular invasion [45].”

    From The p53 Pathway in Glioblastoma (2018) · cites this paper as [45]

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Related

Authors
Wang, H., Cai, S., Bailey, B. J., Reza Saadatzadeh, M., Ding, J., Tonsing-Carter, E., Georgiadis, T. M., Zachary Gunter, T., Long, E. C., Minto, R. E., Gordon, K. R., Sen, S. E., Cai, W., Eitel, J. A., Waning, D. L., Bringman, L. R., Wells, C. D., Murray, M. E., Sarkaria, J. N., Gelbert, L. M., Jones, D. R., Cohen-Gadol, A. A., Mayo, L. D., Shannon, H. E., & Pollok, K. E.
Affiliations
  • Indiana University Health (US)
  • Herman B. Wells Center for Pediatric Research, Department of Pediatrics, Section of Pediatric Hematology/Oncology, Riley Hospital for Children at Indiana University Health;
  • Indiana University – Purdue University Indianapolis (US)
  • Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis;
  • Indiana University Indianapolis (US)
  • Indiana University Melvin and Bren Simon Comprehensive Cancer Center (US)
  • Indiana University School of Medicine (US)
  • Mayo Clinic (US)
  • Division of Clinical Pharmacology;
  • Indiana University Simon Cancer Center;
ORCID
Venue
Journal of Neurosurgery
Publisher
Journal of Neurosurgery Publishing Group (JNSPG)
Published
2017-02
Volume
126
Issue
2
Pages
446-459
ISSN
  • 0022-3085 (ISSN-L)
  • 1933-0693
DOI
10.3171/2016.1.jns152513
PMC
PMC5280578
OpenAlex
W2381784874
Semantic Scholar
84611d7f2941d6cfa86cfb9ad31f53b99b728440
Cited by
49
References
52
Citations by year
2026: 1 · 2025: 5 · 2024: 6 · 2023: 6 · 2022: 6 · 2021: 4 · 2020: 9 · 2019: 5 · 2018: 5 · 2017: 2
FWCI
2.90
Type
journal article
Language
en
Open access
Locations
  • Journal of neurosurgery (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • IUScholarWorks (Indiana University) (submittedVersion)
  • Purdue University Indianapolis (Indiana University) (submittedVersion)
Funders
National Cancer Institute (R01CA138798, R01CA151765, P30CA082709)
Topics
  • Health Sciences › Medicine › Genetics › Glioma Diagnosis and Treatment
  • Health Sciences › Medicine › Oncology › Cancer-related Molecular Pathways
  • Health Sciences › Medicine › Pathology and Forensic Medicine › Cancer Mechanisms and Therapy
Keywords
glioblastoma, temozolomide, MDM2 inhibitors, combination therapy, p53 pathway, DNA repair proteins, tumor invasion, temozolomide resistance, pharmacokinetics, pharmacodynamics
MeSH terms
Temozolomide, Animals, Brain Neoplasms, Combined Modality Therapy, Disease Models, Animal, Glioblastoma, Humans, Imidazoles, Piperazines, Antineoplastic Agents, Alkylating, Xenograft Model Antitumor Assays, Proto-Oncogene Proteins c-mdm2

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-08.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-08.

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