Shannon, Harlan E.

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 There is a significant unmet medical need to develop more effective treatments for cancers of the central and peripheral nervous systems. Glioblastoma multiforme (GBM) is the most common form of brain cancer, and successful long-term treatment has not been realized. Neuroblastomas (NB) arise in the sympathetic nervous system and are the most common extracranial solid tumor of childhood. Survival of children with disseminated, high-risk NB tumors is less than 50%, even with intensive multimodal treatment. To improve treatment outcome, inhibition of murine double minute 2 (MDM2) function in the context of front-line therapy is being explored. MDM2 is a multi-functional protein that plays a critical role in controlling cell growth and migration. MDM2 is a negative regulator of p53 and p73 and, under low stress conditions, sequesters them in the cytoplasm. However, inhibition of MDM2 interactions with p53 and p73 by the MDM2 antagonist nutlin3a can lead to activation of p53/p73-mediated cell-death in tumor cells. The objective of the present study was to evaluate the extent to which blockade of MDM2-p53/p73 interactions by nutlin3a, alone or in combination with front-line therapies, could augment cell death in GBM and NB cells with a variety of molecular profiles. Using the median effect model, we found that temozolomide (TMZ) and nutlin3a were synergistic in increasing cell death in wtp53 U87 and U373 but were predominantly additive in mtp53 U118 cells. Primary patient GBM cells were typically resistant to TMZ relative to established cell lines. However, resistance to TMZ was decreased in the presence of nutlin3a. Further, TMZ and nutlin3a were synergistic in primary GBM10 and GBM43 but not in recurrent primary IU-GBM16, IU-GBM23, IU-GBM27, and IU-GBM32 cells. Moreover, in NB cells with wtp53, nutlin3a and cisplatin were synergistic in MYCN nonamplified SK-N-SH and additive in MYCN amplified IMR5. In mtp53 NB cells, nutlin3a and cisplatin were synergistic in MYCN nonamplified SK-N-FI as well as in MYCN amplified SK-N-DZ. Nutlin3a alone produced dose-related increases in cell death of all GBM and NB cell lines irrespective of p53 status, suggesting that p53-independent mechanisms play a role in inhibition of cell growth produced by blockade of MDM2-mediated signaling. Consistent with this hypothesis, nutlin3a increased p73 protein levels in mtp53 GBM43 cells that were exposed to TMZ. Inhibition of MDM2 function also inhibited repair of TMZ-mediated DNA damage in GBM. In comet assays, nutlin3a+TMZ decreased DNA repair relative to TMZ alone in wtp53 U87 and GBM10 and in mtp53 GBM43 cells. The present results demonstrate that MDM2 is a valid therapeutic target in GBM and NB cells with varying genetic profiles. In conclusion, inhibition of MDM2 signaling by nutlin3a is generally synergistic to additive in combination with front-line chemotherapeutics used to treat cancers of the central and peripheral nervous systems. Citation Format: Harlan E. Shannon, Haiyan Wang, Shanbao Cai, Wei Michael Liu, Lawrence M. Gelbert, Aaron A. Cohen-Gadol, Jann N. Sarkaria, Lindsey D. Mayo, Karen E. Pollok. MDM2 as a therapeutic target: improving upon front-line chemotherapy for nervous system tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3435. doi:10.1158/1538-7445.AM2013-3435