The MYCN oncogene is the major negative prognostic marker in neuroblastoma with important roles in both the pathogenesis and clinical behavior of this aggressive malignancy. MYC oncogenes activate both proliferative and apoptotic cellular pathways and, accordingly, inhibition of p53-mediated apoptosis is a prerequisite for MYC-driven tumorigenesis. To identify novel transcriptional targets mediating the MYCN-dependent phenotype, we screened a MYCN-amplified neuroblastoma cell line by using chromatin immunoprecipitation (ChIP) cloning. We identified the essential p53 inhibitor and protooncogene MDM2 as a putative target. (1), and improvement in outcome clearly requires a better understanding of the pathogenesis and pathophysiology of this disease. The MYCN oncogene is amplified in 25% of neuroblastomas and is the most powerful clinical prognostic marker for poor survival (2). Tissue-targeted expression of MYCN is sufficient to induce neuroblastoma in transgenic mice (3), suggesting this oncogene plays an important role in the pathogenesis of neuroblastoma. In vitro studies demonstrate that MYCN overexpression induces an aggressive phenotype with decreased contact inhibition, decreased growth factor dependence, and increased metastatic potential (4, 5). These findings correlate with the malignant clinical behavior of MYCN-amplified tumors in children.MYCN is a transcription factor with well defined mechanisms of both transcriptional activation, when bound to promoter E-boxes as a MYCN͞Max heterodimer, and transcriptional repression, when bound as a heterodimer with Mnt, Mxi, Mad, or other negative cofactors (6, 7). MYCN is involved in many aspects of normal and oncogenic cellular physiology, including proliferation, cell cycle regulation, apoptosis, and genomic instability (8, 9). Clinically and therapeutically relevant insight into neuroblastoma biology thus can be achieved through identification of the downstream transcriptional targets of MYCN involved in these pathways.In vitro studies of neuroblastoma cell lines demonstrate that overexpression of MYCN induces the conflicting cellular processes of rapid proliferation and apoptotic cell death (10). MYCN shortens the G 1 -S phase transition, increases cell proliferation rates, and decreases cell dependence on paracrine growth factors (5,11,12). Concurrently, MYCN suppresses Bcl-2, activates Bax, and sensitizes cells to genotoxicity-mediated apoptosis through intrinsic apoptosis pathways (13). MYCC has been shown to activate the ARF tumor suppressor, leading to p53 activation and apoptosis through Bcl-x and Bcl-2-dependent and -independent pathways (14). Less than 2% of neuroblastomas have mutated p53, and the p53 pathways are functionally active in the majority of de novo tumors (15, 16). Therefore, for MYCN-expressing neuroblastoma precursor cells to escape p53-mediated cell death, proliferate, and progress to invasive malignancy, a balance must be struck between MYCN-driven proliferation and MYCN-driven apoptosis.In this study we use chromatin immunoprecipita...

Novel therapeutic approaches are urgently needed for high-stage neuroblastoma, a major therapeutic challenge in pediatric oncology. The majority of neuroblastoma tumors are p53 wild type with intact downstream p53 signaling pathways. We hypothesize that stabilization of p53 would sensitize this aggressive tumor to genotoxic chemotherapy via inhibition of MDM2, the primary negative upstream regulator of p53. We used pharmacologic inhibition of the MDM2-p53 interaction with the small-molecule inhibitor Nutlin and studied the subsequent response to chemotherapy in neuroblastoma cell lines. We did 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and terminal deoxynucleotidyl transferase assays to measure proliferation and apoptosis in several cell lines (IMR32, MYCN3, and JF) treated with combinations of cisplatin, etoposide, and Nutlin. We found consistent and robust decreases in proliferation and increases in apoptosis with the addition of Nutlin 3a to etoposide or cisplatin in all cell lines tested and no response to the inactive Nutlin 3b enantiomer. We also show a rapid and robust accumulation of p53 protein by Western blot in these cells within 1 to 2 hours of treatment. We conclude that MDM2 inhibition dramatically enhances the activity of genotoxic drugs in neuroblastoma and should be considered as an adjuvant to chemotherapy for this aggressive pediatric cancer and for possibly other p53 wild-type solid tumors. [Mol Cancer Ther 2006;5(9):2358 -65]