Wang, Haiyan

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

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.