Abstract 1726: Acquired resistance to rapamycin and mTOR kinase inhibitors is mediated by non-overlapping mutations in distinct sites in the mTOR protein
Authors: Rodrik-Outmezguine Vanessa S. , Yao Zhan , Mukherjee Radha , Cai Liqun , Barratt Derek , Ward Richard , Klinowska Teresa , Stanchina Elisa D. , Berger Michael , Baselga Jose , Rosen Neal 
Published: May 2015

Abstract
The prevalence of mTOR activation in cancer has led to the development of two classes of inhibitors of the protein as therapeutics: the natural product rapamycin and its analogs as well as direct inhibitors of mTOR kinase. mTOR exists in at least two multi-protein complexes. The mTORC1 complex phosphorylates S6K and 4EBP and stimulates protein translation, metabolism as well as other processes. The mTORC2 complex phosphorylates and activates AKT. Rapamycin binds to the immunophillin FKBP12. Drug-bound FKBP12 complex binds to mTOR FRB domain and selectively inhibits the activity of mTORC1. However, rapalogs preferentially inhibit S6K phosphorylation compared to 4EBP phosphorylation. Rapalogs have undergone extensive clinical testing and have significant antitumor activity in renal cell and pancreatic neuroendocrine tumors and, in combination with aromatase inhibitors, in resistant, ER positive breast cancers. In contrast, mTOR kinase inhibitors suppress both mTORC1 and mTORC2 functions and potently inhibit S6K, 4EBP and AKT S473 phosphorylation, these drugs are in early clinical testing.
In order to better understand the mechanism of action of these drugs and potential mechanisms of tumor escape from mTOR inhibition, we selected breast tumor cells for resistance to growth inhibition in cell culture by treatment with either rapamycin or an mTOR kinase inhibitor. In rapamycin resistant cells, phosphorylation of S6K and S6 were insensitive to the drug, but remained sensitive to mTOR kinase inhibitors. Conversely, in clones resistant to mTOR kinase inhibitors, mTORC1 and mTORC2 substrates were insensitive to the drugs, but S6K and S6 phosphorylation remained sensitive to rapamycin. Deep sequencing results explained these findings: rapamycin resistant clones harbored mutations in the FRB domain of mTOR, in the sites shown to interact with the FKBP12-rapamycin complex; mTOR kinase resistant clones harbored mutations in the mTOR catalytic domain. These mutations were not observed in the parental cells. It is likely that the mutations identified in each domain prevent binding of the drug.
Consistent with these data, growth of rapamycin resistant cells retain sensitivity to mTOR kinase inhibitors and mTOR kinase resistant cells retain sensitivity to rapalogs, both in tissue culture and in vivo. The results suggest that tumor cells with acquired resistance to mTOR inhibitors retain a requirement for mTOR signaling for proliferation. Furthermore, tumors resistant to either class of drug may not be cross-resistant to the other and combined therapy with both might delay the onset of resistance. Further studies on the genetics of human tumors with acquired resistance to these agents will determine the clinical importance of these findings.
Citation Format: Vanessa S. Rodrik-Outmezguine, Zhan Yao, Radha Mukherjee, Liqun Cai, Derek Barratt, Richard Ward, Teresa Klinowska, Elisa De Stanchina, Michael Berger, Jose Baselga, Neal Rosen. Acquired resistance to rapamycin and mTOR kinase inhibitors is mediated by non-overlapping mutations in distinct sites in the mTOR protein. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1726. doi:10.1158/1538-7445.AM2014-1726