Abstract 902: Effective and selective targeting of glioblastoma multiforme using an active-site inhibitor of mTORC1/mTORC2 kinase
Authors: Neil Jayson , Shannon Craig , Tobias Michael , Braun Alex , Murali Raj , Jhanwar-Uniyal Meena 
Published: August 2012

Abstract
The PI3K-AKT-mTOR signaling axis is central to the transformed phenotype of Glioblastoma multiforme (GBM) cells, due to frequent loss of tumor suppressor PTEN. The mammalian target of rapamycin (mTOR) kinase is present in two cellular multiprotein complexes, mTORC1 and mTORC2, which have distinct subunit composition, substrates and mechanisms of activation. Targeting the mTOR protein is a promising strategy for cancer therapy. However, neither of these complexes are fully inhibited by the allosteric inhibitor rapamycin or its analogs. The aim of this study is to provide evidence that a combined inhibition of mTORC1/2 using an ATP-competitive binding inhibitor, PP242, would effectively suppress GBM growth and dissemination as compared to an allosteric binding inhibitor of mTOR. In addition, we utilized another inhibitor, PI-103, a less selective TORC1/2 inhibitor that also targets PI3K, which is more immunosuppressive than PP242, to deter mTOR pathway. We observed a significant number of GBM tumors showed increased expression of pAKTSer473 and pmTORSer2448, as assessed by immunohistochemistry. GBM cells treated with PP242 significantly reduced the activation of mTORC1 and mTORC2, as shown by reduction in phosphorylation of their substrate levels, p-S6KSer235/236 /4E binding protein-1 (4E-BP1) and Akt, respectively, in a dose dependent manner. Furthermore, insulin induced activation of these kinases was abrogated by pre-treatment with PP242 as compared with rapamycin. The cell cycle analysis using the incorporation of 5-ethynyl-2-deoxyuridine (EdU) into the DNA demonstrated that PP242, but not rapamycin, completely blocked the EdU inclusion. To assess TORC2 function, we studied the phosphorylation forkhead box O (FOXO) transcription factors, which are substrates of both Akt and serum downstream of TORC2. PP242, but not rapamycin, reduced FOXO phosphorylation on Akt consensus sites of FOXO1 correlating with greater inhibition of cell cycling. A combined inhibitor of PI3K and mTOR, p103, an ATP binding inhibitor, caused no change in another substrate of mTORC1, suppressed p-S6KSer235/236 levels. In addition, GBM cell proliferation was significantly suppressed by PP242 which was more pronounced compared to rapamycin. Lastly, migration of GBM cells after treatment with PP242 was significantly suppressed, while analysis of cytoskeleton showed that this migration was associated with cellular behavior rather than cytoskeleton loss. In conclusion, these results suggest a novel combined active-site inhibitor of mTORC1/2 kinase; PP242 suppresses both GBM growth and dissemination, which underscores its potential therapeutic use in treatment of GBM.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 902. doi:1538-7445.AM2012-902