ET-25THERAPEUTIC MODULATION OF MDM2 IN NEUROBLASTOMA

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. In high-risk NB, the MYCN gene is amplified in approximately 25% of all cases, and is considered to be a negative prognostic factor. MYCN can transcriptionally activate the murine double minute-2 (MDM2) gene, leading to increased expression of MDM2, downregulation of the tumor suppressor p53, and increased tumorigenesis. To improve treatment outcome in NB, inhibition of MDM2 function with front-line chemotherapy is being explored. The E3 ubiquitin ligase, MDM2, which ubiquitinates and targets p53 for proteosomal degradation, is a multifunctional protein that regulates the p53 signaling network and the DNA damage response. Cell growth assays indicate that inhibition of MDM2 function by the small molecule antagonist nutlin3a is synergistic to additive in combination with frontline chemotherapeutics in IMR32 NB cells (wt p53, MYCN amplified). To determine the effects of the combination of nutlin3a and cisplatin on a cell line without MYCN amplification, cell growth assays using SK-N-SH NB cells (wt p53, single MYCN copy) were performed. Results demonstrated that combined treatment with nutlin3a and cisplatin is synergistic in SK-N-SH NB cells. To delineate the mechanism of action in IMR32 cells exposed to cisplatin and nutlin3a, the impact of the two drugs, alone and in combination, was investigated in the context of MDM2 expression, modulation of the cell cycle, and DNA damage response. Western blot analyses were performed to monitor expression of cell cycle proteins and DNA damage/repair responses that we hypothesized are regulated by nutlin3a-mediated MDM2 blockade. Results demonstrated that combination treatment in IMR32 NB cells enhanced the DNA damage response and inhibited cell growth. The data provide further support for the hypothesis that MDM2 is a target that can be therapeutically exploited in combination with front-line chemotherapy to improve treatment outcome in MYCN non-amplified and MYCN amplified neuroblastoma.