Abstract 612: MDMX expression is linked with MYCN amplification or expression in neuroblastoma, and sensitizes cells to MDM2-p53 antagonists

Abstract Introduction: Neuroblastoma is the most common solid extracranial pediatric tumor. Despite intense treatment, survival rates for patients with high risk disease remain below 40%. MYCN amplification is a major biomarker of poor prognosis and is an important oncogene that plays roles in driving proliferation and sensitizing to apoptosis. MDM2 is the principal negative regulator of p53 and is often hyperactive in neuroblastoma through either co-amplification with MYCN or inactivation of its negative regulator p14ARF. Increased activity of MDM2 may be an important mechanism by which MYCN amplified neuroblastomas evade MYCN mediated p53 dependent apoptosis. Since most neuroblastomas have wildtype p53, reactivation of p53 by MDM2-p53 antagonists is being investigated as a potential therapeutic approach. Like MDM2, MDMX is a negative regulator of p53 but whereas MDM2 regulates p53 stability and activity, MDMX regulates activity only. We hypothesised that MDMX removal or inhibition is necessary to fully activate p53 in neuroblastoma in response to MDM2-p53 antagonists. Methods: MDMX expression in Tet21N MYCN regulatable cells and a panel of MYCN amplified and non-amplified neuroblastoma cell lines was determined by western blot. Knockdown of MDMX by siRNA was achieved in 3 MYCN amplified cell lines, followed by treatment with the MDM2-p53 antagonists Nutlin and MI-63. Induction of p53 responsive genes and apoptotic markers were observed by western blot and changes in cell cycle investigated using flow cytometry. Apoptosis was assessed by caspase activity. Results: Activation of p53 was observed following Nutlin or MI-63 treatment in all 3 cell lines, as shown by a G1 arrest and/or induction of apoptosis. Following MDMX knockdown alone, an increase in p21 expression and/or apoptosis was observed suggesting that MDMX removal may be necessary to activate p53. However, MDMX knockdown and MDM2-p53 antagonist treatment resulted in no further effect on the cell cycle compared to Nutlin or MI-63 alone, and unexpectedly, a decrease in levels of apoptotic markers and caspase 3/7 activity (p<0.001). We previously found that knockdown of MYCN resulted in decreased apoptosis after MDM2 inhibitor treatment (Gamble et al., submitted). Because a similar trend was observed with MDMX, we looked for a relationship between MYCN and MDMX. We found that MYCN+ Tet21N cells have increased MDMX expression compared to MYCN- cells, and MYCN amplified cell lines had increased MDMX expression compared to non-amplified cell lines. Conclusions: Neuroblastoma cell lines are more resistant to MDM2-p53 antagonist mediated apoptosis following MDMX knockdown, suggesting that retention of MDMX activity is beneficial for response to MDM2-p53 antagonists in neuroblastoma. The link seen between MYCN and MDMX protein expression may therefore contribute to the sensitivity of MYCN amplified cells to MDM2-p53 antagonists. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 612. doi:10.1158/1538-7445.AM2011-612