nutlin-3a and Medulloblastoma

Ependymomas in children can arise throughout all compartments of the central nervous system (CNS). Highly malignant paediatric ependymoma subtypes are Group A tumours of the posterior fossa (PF-EPN-A) and RELA-fusion positive (ST-EPN-RELA) tumours in the supratentorial compartment. It was repeatedly reported in smaller series that accumulation of p53 is frequently observed in ependymomas and that immunohistochemical staining correlates with poor clinical outcome, while TP53 mutations are rare. Our TP53 mutation analysis of 130 primary ependymomas identified a mutation rate of only 3%. Immunohistochemical analysis of 398 ependymomas confirmed previous results correlating the accumulation of p53 with inferior outcome. Among the p53-positive ependymomas, the vast majority exhibited a RELA fusion leading to the hypothesis that p53 inactivation might be linked to RELA positivity.In order to assess the potential of p53 reactivation through MDM2 inhibition in ependymoma, we evaluated the effects of Actinomycin-D and Nutlin-3 treatment in two preclinical ependymoma models representing the high-risk subtypes PF-EPN-A and ST-EPN-RELA. The IC-50 of the agent as determined by metabolic activity assays was in the lower nano-molar range (0.2-0.7 nM). Transcriptome analyses of high-dose (100 nM), low-dose (5 nM) and non-treated cells revealed re-expression of p53 dependent genes including p53 upregulated modulator of apoptosis (PUMA) after low-dose treatment. At the protein level, we validated the Actinomycin-D induced upregulation of PUMA, and of p53 interaction partners MDM2 and p21. Proapoptotic effects of low-dose application of the agent were confirmed by flow cytometry. Thus, Actinomycin-D could constitute a promising therapeutic option for ST-EPN-RELA ependymoma patients, whose tumours frequently exhibit p53 inactivation.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Dactinomycin; DNA Mutational Analysis; Ependymoma; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Immunohistochemistry; Medulloblastoma; Mice; Neurons; Piperazines; Protein Synthesis Inhibitors; Proto-Oncogene Proteins c-mdm2; Stem Cells; Transcription Factor RelA; Treatment Outcome; Tumor Suppressor Protein p53

A critical component of the cellular stress response, the p53 tumor suppressor protein must be functional for many cancer therapies to be effective. Adjuvant therapies that augment p53 function are predicted to sensitize tumor cells to cancer therapies that rely upon p53 for their efficacy. Of those strategies currently being explored to enhance p53 function, inhibition of the ubiquitin ligase, MDM2, a negative regulator of p53, has shown promise. Here, we investigated whether MDM2 antagonism might be effective in inducing cell death in human medulloblastoma (MB) cells. Nutlin-3, a small-molecule inhibitor of MDM2, potently induced apoptosis in MB cells with wild-type TP53. Moreover, nutlin-3 potentiated p53 activation and growth impairment of MB cells in combination with the classic DNA-damaging agent doxorubicin. Together, these results support the concept that MDM2 antagonists may be therapeutically beneficial for patients with MB tumors.

Topics: Antibiotics, Antineoplastic; Apoptosis; Blotting, Western; Brain Neoplasms; Cell Cycle; Cell Death; Cell Line, Tumor; Coloring Agents; Doxorubicin; Humans; Imidazoles; Medulloblastoma; Piperazines; Proto-Oncogene Proteins c-mdm2; Tetrazolium Salts; Thiazoles; Tumor Suppressor Protein p53

Medulloblastomas account for 20% of pediatric brain tumors. With an overall survival of 40%-70%, their treatment is still a challenge. The majority of medulloblastomas lack p53 mutations, but even in cancers retaining wild-type p53, the tumor surveillance function of p53 is inhibited by the oncoprotein MDM2. Deregulation of the MDM2/p53 balance leads to malignant transformation. Here, we analyzed MDM2 mRNA and protein expression in primary medulloblastomas and normal cerebellum and assessed the mutational status of p53 and MDM2 expression in 6 medulloblastoma cell lines. MDM2 expression was elevated in medulloblastomas, compared with cerebellum. Four of 6 medulloblastoma cell lines expressed wild-type p53 and high levels of MDM2. The tumor-promoting p53-MDM2 interaction can be inhibited by the small molecule, nutlin-3, restoring p53 function. Targeting the p53-MDM2 axis using nutlin-3 significantly reduced cell viability and induced either cell cycle arrest or apoptosis and expression of the p53 target gene p21 in these 4 cell lines. In contrast, DAOY and UW-228 cells harboring TP53 mutations were almost unaffected by nutlin-3 treatment. MDM2 knockdown in medulloblastoma cells by siRNA mimicked nutlin-3 treatment, whereas expression of dominant negative p53 abrogated nutlin-3 effects. Oral nutlin-3 treatment of mice with established medulloblastoma xenografts inhibited tumor growth and significantly increased survival. Thus, nutlin-3 reduced medulloblastoma cell viability in vitro and in vivo by re-activating p53 function. We suggest that inhibition of the MDM2-p53 interaction with nutlin-3 is a promising therapeutic option for medulloblastomas with functional p53 that should be further evaluated in clinical trials.

Topics: Animals; Apoptosis; Blotting, Western; Case-Control Studies; Cell Cycle; Cell Proliferation; Cell Survival; Cerebellar Neoplasms; Cerebellum; Female; Humans; Imidazoles; Immunoenzyme Techniques; Medulloblastoma; Mice; Mice, Nude; Mutation; Piperazines; Proto-Oncogene Proteins c-mdm2; Signal Transduction; Tissue Array Analysis; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays