Mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that acts in two distinct complexes, mTORC1 and mTORC2, and is dysregulated in many diseases including cancer. mLST8 is a shared component of both mTORC1 and mTORC2, yet little is known regarding how mLST8 contributes to assembly and activity of the mTOR complexes. We assessed mLST8 loss in a panel of normal and cancer cells, finding little to no impact on mTORC1 activity, nor on assembly of mTOR with the mTORC1 co-factor Raptor. However, mLST8 loss blocked mTOR association with the mTORC2 co-factors Rictor and Sin1, and impaired mTORC2 kinase activity, including its phosphorylation of AKT at S473. We identified two sites within mLST8 governing its interaction with mTOR, finding that simultaneous mutation of these sites together, but not individually, disrupted not only mTOR-mLST8 interactions, but also blocked mTOR assembly with Rictor and Sin1, thus disrupting mTORC2 activity. Similary, a single mutation on mLST8 with a corresponding mutation on mTOR interfered with mTORC2 assembly, without affecting mTORC1 assembly or activity. We further discovered a direct interaction between mLST8 and the NH2-terminal domain of the mTORC2 cofactor Sin1. Using PTEN-null prostate cancer xenografts, we found that mLST8 mutations disrupting the mTOR interaction motif inhibited AKT S473 phosphorylation, decreased tumor cell proliferation, and increased tumor cell death in vivo. Together, these data suggest that the scaffolding function of mLST8 is critical for assembly and activity of mTORC2, but not mTORC1, an observation which could enable therapeutic mTORC2-selective inhibition, while sparing the activity of mTORC1.

Citation Format: Laura C. Kim, Yoonha Hwang, Wenqiang Song, Rebecca S. Cook, Jin Chen. Selective inhibition of mTORC2 by disruption of mLST8 scaffolding function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3434.