Abstract B47: Blockade of MDM2 function synergizes with front-line chemotherapy to promote cell death in p53-wildtype and p53-mutant MYCN-amplified neuroblastoma cells

Abstract

Abstract Neuroblastoma (NB) arises in the sympathetic nervous system and is the most common extracranial solid tumor of childhood. While treatment outcome for NB has improved over the years, the survival of children with disseminated, high-risk NB tumors is less than 50%. To improve treatment outcome in these patients, inhibition of murine double minute-2 (MDM2) function in the context of front-line therapy used to treat NB (cisplatin and etoposide) is being explored. MDM2 is a multi-functional protein that plays a critical role in controlling cell growth, cell migration, and the DNA damage response. Moreover, MDM2 is a downstream target of MYCN which can be commonly amplified in high-risk NB. The objective of the present study was to evaluate the extent to which the MDM2 antagonist nutlin3a alone or in combination with front-line therapies, could augment cell death in NB cells with clinically relevant molecular profiles. Cisplatin and etoposide are currently used in the clinic as front-line therapy, and when administered alone produced dose-related increases in cell death in SK-N-SH (wtp53, MYCN-nonamplified), IMR32 (wtp53, MYCN-amplified), SK-N-FI (mtp53, MYCN-nonamplified) and SK-N-DZ (mtp53, MYCN-amplified) NB cell lines. Nutlin3a also produced dose-related increases in cell death irrespective of p53 status, suggesting that p53-independent mechanisms, may play a role in increasing cell death produced by blockade of MDM2-mediated signaling. However, IC50 values for nutlin3a administered alone in the absence of DNA damage were >10x higher in the mtp53 cell lines compared with the wtp53 cell lines, suggesting that mechanisms other than MDM2-blockade could also be involved in promoting death in mtp53 NB cells. Using isobolographic analysis and broad ranges of compound ratios, we found that nutlin3a and cisplatin were moderately synergistic in wtp53 MYCN-nonamplified SK-N-SH and markedly synergistic in wtp53 MYCN-amplified IMR32 cells. Moreover, nutlin3a and etoposide were also moderately synergistic in these wtp53 cell lines. In mtp53 NB cells, nutlin3a was also synergistic with both cisplatin and etoposide irrespective of MYCN status. Interestingly, cisplatin and etoposide, which are frequently used together clinically, were strictly additive in all four cell lines by isobolographic analysis. Pharmacodynamic analysis was conducted in IMR32 cells (wtp53, MYCN-amplified) since this molecular profile is commonly found in high-risk NB. IMR32 cells were treated with vehicle, nutlin3a, cisplatin, and the combination of cisplatin/nutlin3a, and activation of p53 and the status of signaling pathways that regulate cell growth were assessed by Western analyses. Optimal activation of the p53 pathway was observed by 24-48 hours post-treatment which correlated with a robust downregulation of MYCN and the anti-apoptotic protein survivin. These data suggest that a combination therapy that incorporates efficient modulation of the MDM2 pathway and builds upon front-line therapies enhanced cell death of neuroblastoma cells that are highly resistant to conventional therapies. This combinatorial approach that builds upon front-line therapy is now under evaluation in orthotopic NB xenograft models. Citation Format: Karen E. Pollok, Barbara J. Bailey, Kania Katarzyna, Lawrence M. Gelbert, Harlan E. Shannon. Blockade of MDM2 function synergizes with front-line chemotherapy to promote cell death in p53-wildtype and p53-mutant MYCN-amplified neuroblastoma cells. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B47.