Georgiadis, Taxiarchis M.

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.

Abstract Treatment of glioblastoma multiforme (GBM) continues to be a challenge due to its infiltrative nature, tumor heterogeneity, and lack of therapeutic agents that penetrate the blood-brain barrier (BBB). While the primary tumor is permeable to some degree, the ability of GBM cells to invade areas of parenchyma with an intact BBB indicates development of BBB-penetrable compounds is a necessity. The multifunctional protein MDM2 holds promise as a therapeutic target in a variety of cancers and plays a critical role in controlling cell survival, invasion, and DNA repair. MDM2 antagonists such as nutlin3a and RG7112 are being used to interrogate the impact of modulating MDM2 function in combination with front-line therapy. Our objective was to investigate whether MDM2 antagonists, alone or in combination with temozolomide (TMZ), can augment cell death in orthotopic GBM xenograft models. In vitro data indicate that TMZ and nutlin3a are synergistic in decreasing cell viability in wild type (wt) p53 U87-MG, primary human wt p53 GBM10 and mutant (mt) p53 GBM43 cells. Pharmacodynamic studies demonstrated that the mechanism of action for promoting cell death following exposure to TMZ/nutlin3a was multifactorial. Comet assays indicated that repair of TMZ-mediated DNA damage was significantly delayed in wt and mt p53 GBM cells treated with TMZ/nutlin3a compared to TMZ alone and the base excision repair protein Ape1 was downregulated in cells treated with TMZ/nutlin3a. Pharmacokinetic studies guided development of rational dosing regimens in which 2-3 five-day cycles of TMZ followed by nutlin3a 4 hours later were investigated. In ectopic U87-MG xenografts, nutlin3a sensitized xenografts to TMZ-mediated cell death. Orthotopic studies employing U87-MG, GBM10, and GBM43 tumors are being utilized to determine if nutlin3a levels detectable in the brain via HPLC-MS/MS(API 4000) are sufficient to modulate MDM2 function in the context of TMZ and increase survival. In the TMZ-resistant GBM10 (wtp53, MGMTpos, and PTENnull) orthotopic model, there was a modest increase in median survival from 63 days with TMZ to 73 days with TMZ/nutlin3a. Higher and more sustained brain levels of MDM2 antagonists will likely be necessary to improve survival. The MDM2 antagonist RG7112 has an improved PK profile and structural analysis of RG7112 and nutlin3a via QikProp 3.0 (www.schrodinger.com) indicates that RG7112 also has an improved predicted brain/blood partition coefficient (plog BB) compared to nutlin3a (RG7112 = -0.17; nutlin3a = -0.415). Studies are in progress to assess the brain penetration of RG7112 and its effect on GBM growth in vivo. Taken together, our data suggest that modulation of MDM2 function in the context of cytotoxic therapy has the potential to alter mechanisms involved in DNA repair that can promote cell death and improve survival. Citation Format: Haiyan Wang, Shanbao Cai, Barbara J. Bailey, Lawrence M. Gelbert, M. Reza Saadatzadeh, Aaron A. Cohen-Gadol, Jann N. Sarkaria, Paul Territo, Taxiarchis M. Georgiadis, T. Zachary Gunter, Samy Meroueh, Eric C. Long, David R. Jones, Lindsey D. Mayo, Shannon Harlan, Karen E. Pollok. Sensitization of temozolomide-mediated glioblastoma cell death by targeting MDM2: Assessment of PD biomarkers, brain penetration, and efficacy in humanized orthotopic xenograft models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2741. doi:10.1158/1538-7445.AM2014-2741

Abstract Development of efficacious strategies for eradication of glioblastoma multiforme (GBM) remains a significant challenge. While treatment of GBM with radiation and temozolomide (TMZ) has improved clinical outcome, lack of long-term efficacy can be due to the ability of GBM to acquire resistance to therapy by modulation of signaling pathways that control DNA repair and cell survival. The HDM2 antagonist, Nutlin3, blocks interactions of HDM2 with key signaling molecules such as p53, p73α, and HIF1α, and modulates their downstream effector function. A panel of human GBM cell lines is currently being used to monitor the extent to which Nutlin3 can sensitize cells to TMZ. In vitro survival assays indicate that Nutlin3 potentiates TMZ-mediated cell death; analysis of cell survival data using Dose Effect CalcuSyn software indicates that the combination index value was < 1 and indicative of a synergistic effect between Nutlin3 and TMZ. Western analyses of p53 wild-type GBM indicated increased p53 stabilization and activation of down-stream targets in cells treated with the combination of Nutlin3/TMZ compared to vehicle, TMZ, or Nutlin3. In addition, time-course studies indicated that U87-MG cells treated with Nutlin3/TMZ in comparison to single agent resulted in increased γ-H2AX and DNA-strand breaks. Efficacy studies using ectopic U87-MG xenografts indicated that 2 cycles of 5-consecutive days of Nutlin3/TMZ led to a significant decrease in tumor growth compared to vehicle or single agent (p < 0.001) with no signs of off-target toxicity. Regulation of DNA repair was further analyzed using the primary GBM.10 line that when grown as an ectopic xenograft, expresses the direct-reversal repair protein, O6-methylguanine DNA methyltransferase (MGMT) and proteins involved in base-excision repair (BER). In vivo target-validation studies using GBM.10 xenografts confirmed that repair of TMZ-mediated DNA damage is modulated in the presence of Nutlin3. GBM.10 xenografts were treated with vehicle, TMZ, nutlin3, or TMZ/Nutlin3 and modulation of critical targets determined by Western analyses. Increases in total p53, p53 phosphorylated at residue serine16, p73α, and substantial decreases in MGMT and the BER protein, APE-1, were observed following treatment with Nutlin3/TMZ compared to vehicle or single-agent exposure. Busso et al (Oncogene 2009) recently reported that HDM2 can monoubiquitinate APE-1 leading to proteosomal-mediated degradation. In addition, recombinant activity assays in our laboratory indicate that HDM2 directly monoubiquitinates recombinant MGMT and may play a role in its degradation. These data suggest that DNA repair proteins required for repair of TMZ-mediated DNA damage can be downregulated by altering HDM2-mediated signaling. Combination therapy that targets the p53-HDM2 E3 ligase network and DNA repair represents a novel approach towards improving treatment of GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5373.