Abstract C66: Development of mechanistic assays to differentiate PI3K and mTOR inhibitors

Abstract The phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway plays a key role in the regulation of cellular growth, survival, and proliferation. Inappropriate activation of the pathway as a result of mutations and amplifications has been implicated in a variety of human cancers. Pathway dysregulation drives tumorigenesis through phosphorylation of proteins that directly regulate protein synthesis, cell cycle, and metabolism. The complexity of the mTOR and PI3K pathways, which include two functionally distinct mTOR complexes, feedback loops, and parallel regulatory pathways, clearly invokes a need for functional assays that can clarify the mechanism of action for drugs targeting mTOR and PI3K either as single agents or in combination. In this work, we used three different types of cell based assays to investigate the effects of PI3K versus mTORc1 inhibition on signaling pathways, cell cycle, and autophagy. The first set of assays differentiates an inhibitor of the mTorc1 complex versus the mTorc2 complex by utilizing two different cell lines and stimulation with either nutrient or growth factors. We show that the mTorc1 inhibitor, rapamycin, has potent activity in the nutrient stimulated mTorc1 assay, while PI3K inhibitors have very weak activity in such an assay. In the second type of assay, we used high content image analysis to produce cell cycle distribution profiles in PC3 cells, clearly showing G1 arrest for both mTOR and PI3K compounds as both single agents and in combination. However, the profiles showed no apoptotic population for either class of compounds in the cell lines tested. Therefore, as a means to investigate an alternative route of cell death to apoptosis, we also developed an autophagy assay using high content image analysis. Our results show that treatment of cells with a PI3K inhibitor or rapamycin as single agents or in combination can have different effects on autophagy and may potentially help differentiate the mechanism of action between the two classes of inhibitors. Taken together, we have generated a panel of mechanistic cellular assays which can help differentiate and characterize the effects of mTor and PI3K inhibition as both single agents and in combination. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C66.