The primary objective of Project 4 is to determine if the efficacy of the recently approved mTOR inhibitor temsirolimus can be reproduced or even surpassed in RCC by blocking signaling events upstream of mTOR in the P13-K pathway. PI3-K inhibitors have numerous theoretical advantages over conventional mTOR inhibitors such as temsirolimus, not the least of which is the tendency of the latter to activate PI3-K through a feedback loop involving the mTOR substrate p70'^'*. We have recently begun studies ofthe effects ofthe dual PI3-K/mT0R inhibitor BEZ235 on intracellular signaling and tumor growth and have already observed single agent antitumor activity in 786-0 xenografts. We are now proposing to extend these studies by comparing the antitumor activities of BEZ235 with those ofthe mTOR inhibitor everolimus in xenografts generated from RCC short term cultures (STCs) that have not been propagated as monolayer cultures and therefore may be more representative of RCC in situ. These studies will also compare the effects of treatment with BEZ235 in paired tumor cell lines that differ only with respect to VHL status or constitutive Akt activity, both of which have been shown to affect the response to mTOR inhibitors. One of our proposed Aims involves a Phase 11 trial with BEZ235 in RCC patients. This trial will include a search for predictive biomarkers and a comprehensive pharmacodynamic analysis of the drug's effect on Akt, F0X03a, p53 and mTOR signaling in tumor tissue. BEZ235 induces growth arrest in RCC cell lines and its antiproliferative effects can be enhanced by the concurrent inhibition of other kinases associated with cell survival (MEK, GSK-3P). One of the objectives of this application is to determine if the in v/fro synergy between BEZ235 and inhibitors of MEK or GSK-3(3 can be duplicated in vivo in RCC xenograft models. Our proposed studies will determine if BEZ235 has pro-angiogenic effects similar to that reported for the PI3-K inhibitor Ly294002 and whether this effect can be blocked with the concurrent administration ofthe VEGF receptor antagonist sunitinib. Finally, in the final year ofthis grant, we propose to initiate a clinical trial of BEZ235 in combination with the drug that showed the greatest potential as an adjunct to BEZ235 in the aforementioned xenograft studies. Collectively, these studies will assess the antitumor activity of BEZ235 both as a single agent and in combination with other drugs as well as define the patient population most likely to respond this novel agent.