Deregulation of the phosphatidylinositol 3-kinases (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway plays a central role in tumor formation and progression, providing validated targets for cancer therapy. S9, a hybrid of \#945;-methylene-\#947;-lactone and 2-phenyl indole compound, was found to abrogate EGF-activated PI3K-Akt-mTOR signaling cascade and Akt translocation to cellular membrane. S9 possesses inhibitory activity against both PI3K and mTOR with little effect on other tested 30 kinases. S9 also completely impedes hyper-phosphorylation of Akt as a negative feedback of inhibition of mTOR by rapamycin. S9 unexpectedly arrested cells in M phase other than G1 phase, which was distinct in compounds targeting PI3K-Akt-mTOR pathway. Further study revealed that S9 inhibited tubulin polymerization via binding to colchicines-binding site of tubulin and resulted in microtubule disturbance. Molecular modeling indicated that S9 could potentially bind to the kinase domains of PI3K p110\#945; subunit and mTOR, and shared similar hydrophobic interactions with colchicines in the complex with tubulin. Moreover, S9 induced rapid apoptosis in tumor cell, which might reflect a synergistic cooperation between blockade of both PI3-Akt-mTOR signaling and tubulin cytoskeleton. Finally, S9 displayed potent antiproliferative activity in a panel of tumor cells originated from different tissue types including drug-resistant cells and in nude mice bearing human tumor xenografts. Taken together, we herein show for the first time that S9 functions as a multi-inhibitor simultaneously targeting both PI3K-mTOR axis and microtubule cytoskeleton. The converged outcome of both cytostatic and cytotoxic effects of S9 in vitro and in vivo , together with its appreciable anti-MDR profile, help prove to be a promising alternative template for rational designing of anticancer drugs. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 3391.