Abstract 5373: Modulation of temozolomide-mediated DNA damage in glioblastoma multiforme by the HDM2 antagonist, nutlin3 (2010)

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 &lt; 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 &lt; 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.