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Malignant gliomas are characterized by heightened activity of the oncogenic kinase AKT, activated by growth factor signaling and deletion of inactivating phosphatases such as PTEN. In contrast, medulloblastoma (MB) malignancy often involves many of the same deregulated growth factor signaling pathways, but PTEN deletion is infrequent. The mechanisms by which AKT activity is increased in MB have not been studied. We have studied AKT signaling in MB by analyzing the expression and activity of AKT isotypes and downstream signaling in vitro and in vivo. Furthermore, using novel AKT inhibitors, we show that survival of medulloblastoma cells in vitro depends on maintenance of active AKT. We have used quantitative PCR, western blotting and in vitro kinase assays to assess the activity and expression levels of AKT1, 2, and 3. In medulloblastoma tissue samples (n=9), AKT1 levels were similar to control brain levels (n=3). In contrast, a subset of medulloblastomas (4/9, 44.4%) had 6.3 fold mean increase (ranging 2 to 15.4 fold) in AKT2 levels, and smaller subset (2/9, 18%) showed 3 fold greater expression of AKT3 expression, compared to normal brain or other PNET/MB samples. Further analysis of AKT isotypes in MB demonstrated that increased protein expression correlates with increased overall levels of activated AKT and activation of downstream signaling proteins such as GSK3β. AKT2 over-expression, but not AKT1 or 3, correlates with poor overall survival (Pearson’s, r=0.63) in this small sample set. Treatment of short term MB biopsy cultures or established cell lines with the AKT-selective inhibitor SH-6 (0.3-30 μM) demonstrated efficient cell kill using luminescence-ATP viability assays, similar to conventional chemotherapy agents such as Cisplatin and BCNU, with 67.2-82.2% cell kill achieved with doses of 10 μM SH-6. This study provides the first detailed characterization of AKT signaling and relation to survival mechanisms in medulloblastoma, and reports for the first time a role for AKT2 as a survival factor in the malignancy of primitive neuroectodermal tumors. Elevated AKT signaling was found to be mediated by both increased levels of activated AKT2 protein and increased overall expression levels of AKT2 in vivo. AKT-targeted therapeutics may therefore be useful in treating medulloblastoma.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]