The mammalian target of rapamycin (mTOR) has been identified as a key target for the treatment of cancer. Rapamycin and its analogues (rapalogues) have been demonstrated to be effective against a number of tumour models preclinically, with CCI-779 approved for renal cell carcinoma. However, they only target the TORC1 complex, not TORC2. It has been postulated that, in part, the rapalogues may not achieve their full clinical potential due to the complexity of a feedback loop that activates Akt, a substrate of TORC2, in the signalling cascade. It has further been suggested that inhibition of multiple targets in the cascade may be best to overcome such feedback and be more efficacious. We hypothesised that by targeting the kinase activity of mTOR and inhibiting signalling through both TORC1 and TORC2, we would alleviate feedback signalling which may prove more effective against tumours driven through the mTOR pathway. KU-0063794 is a highly potent inhibitor of mTOR kinase and is highly selective (i.e. > 100 fold) to mTOR compared with other PIKK family members (e.g. DNA-PK, ATM, ATR, PI3Kα). KU-0063794 inhibits phosphorylation of 4EBP1 (T37/T46) or S6 (S235/236) in vitro in SF295 cells (PTEN-) and a number of other cell lines, indicating TORC1 inhibition. Analysis of TORC2 function by AKT (S473) phosphorylation in these cells indicated that this was inhibited with equal potency to TORC1, demonstrating the mTOR kinase inhibitory function of KU-0063794. We also demonstrated inhibition of TORC1 (S6 S235/236) and TORC2 (AKT S473) by immunocytochemistry. We will further describe our work to differentiate inhibition of mTOR by rapamycin and by KU-0063794 and demonstrate the effectiveness of KU-0063794 across a number of tumour cell types. The growth inhibitory effect of KU-0063794 was demonstrated via SRB assay. Cell cycle analysis showed that KU-0063794 inhibits progression through S phase with substantial G1 accumulation, similar to data obtained for rapalogues. A sub-G1 population was not evident in the cells and analysis of apoptosis has failed to identify evidence of cell death by this mechanism. We studied autophagy as an alternative death process and demonstrate that KU-0063794 initiates an autophagic response in cells. We conclude that the specific targeting of mTOR via TORC1/TORC2 inhibition with a small molecule inhibitor may prove efficacious in the treatment of tumours driven through PI3K/AKT/mTOR.
99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA