To understand the basis for cell sensitivity to temsirolimus as a single agent, we turned to an analysis of Akt activation, both at baseline and in response to treatment. We found that the baseline constitutive activation of Akt was predictive of cell sensitivity with the most resistant cells having very low basal S473 and T308 phosphorylation. After treatment with temsirolimus, a compensatory increase in Akt phosphorylation at both sites was detected in the most sensitive endometrial cancer cell lines tested, but the primarily resistant cellsdemonstrated no Akt phosphorylation at either site, and another resistant line, Hec50co, showed reduced phosphorylation. Thus, in contrast to sensitive cells, primarily resistant cells have low basal Akt phosphorylation and do not respond with compensatory hyper-phosphorylation after temsirolimus treatment. Of note, phospho-PDK1, the kinase responsible for Akt phosphorylation at T308, is low in several resistant cell lines such as Hec1A and KLE. This indicates primary cell resistance and a general lack of dependence on the Akt BMS-354825 signaling pathway for proliferation. On the other hand, the finding of compensatory activation of Akt in responsive cells is consistent with previous reports in the literature of rapalog-induced Akt phosphorylation in many cancer cell lines, human xenograft models, and patient tumors. Compensatory hyper-Akt phosphorylation suggests one potential mechanism whereby cells that are initially sensitive escape the growth inhibitory effects of the drug and become secondarily resistant. From these data, we distinguish between primary resistance with a lack of baseline Akt dependence versus acquired resistance demonstrated by hyper-Akt activation in response to temsirolimus. Temsirolimus is currently in phase II trials for advanced endometrial cancer and has shown some promise. However, lack of initial response to therapy as well as the development of acquired drug resistance continues to be problematic. To more fully understand the therapeutic potential of mTOR inhibition in endometrial cancer, we first examined the effect of temsirolimus alone on the viability of a panel of endometrial cancer cell lines. We sought to distinguish between cellular events which predict for primary resistance as well as those events which are linked to the MLN4924 905579-51-3 eventual development of acquired resistance. Consistent with other types of cancer, primary resistance to temsirolimus is found in a subset of these cell lines. Our data suggest that primarily resistant cells lack robust Akt signaling, are unable to phosphorylate Akt at baseline, and express PTEN. In contrast, the most sensitive cell lines have lost PTEN expression and have high baseline phosphorylation of Akt. Our data demonstrate that in these cells, temsirolimus treatment promotes a further increase in Akt phosphorylation, indicating that signaling through the prosurvival PI3K/Akt pathway is likely how these endometrial cancer cell lines eventually circumvent mTOR inhibition. These results are consistent with previous reports in other types of cancers documenting compensatory Akt phosphorylation in response to other rapalogs. This has been observed in xenograft models of lung canceras well as in advanced colon and breast cancer tissues following rapalog therapy. The elevated Akt phosphorylation is thought to be a predominant driving force in resistance to temsirolimus treatment in these cancers. To overcome 
resistance, we adopted a combination strategy. Dual treatment with temsirolimus and the PI3K inhibitor ZSTK474 or the PI3K/mTOR inhibitor BEZ235 overcame the temsirolimus-induced Akt hyper-phosphorylation, which is a marker for developing acquired resistance.