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Aberrant activation of the PI3K/AKT/mTOR pathway is implicated in human cancer. The protein kinase mechanistic target of rapamycin (mTOR) is a key node in this pathway and participates in two complexes: mTORC1 and mTORC2. mTORC1 regulates cap-dependent protein translation, both by activating downstream effectors, including p70S6 kinase 1 (S6K1), and by deactivating the translation-initiation repressor eIF4E binding protein 1 (4EBP1). mTORC2 regulates cellular proliferation via AKT phosphorylation. mTOR inhibitors have diverse pharmacologic effects on the activity of these complexes. Rapamycin and its analogs (rapalogs) cause selective but incomplete inhibition of mTORC1, reducing phosphorylation of some substrates, including S6K1, but not 4EBP1. In contrast, ATP-competitive inhibitors, such as MLN0128, inhibit mTORC1, mTORC2, and various lipid kinases. The suboptimal antitumor activity of rapalogs has been attributed, at least in part, to their limited spectrum of inhibitory activity against mTORC1. Conversely, the broad inhibitory profile of the ATP competitive inhibitors may be the basis for observed toxicities in the clinic and lack of optimal inhibition of tumor mTORC1 at tolerated doses. Recently, a new class of mTOR inhibitor that exploits a bivalent interaction with both the ATP- and FRB-binding sites of mTOR has been reported, which we have termed “bi-steric” to reflect the interaction with both allosteric and orthosteric sites. The prototype bi-steric inhibitor, RapaLink-1, blocks phosphorylation of many mTORC1 substrates, including 4EBP1, but exhibits only modest differentiation between inhibition of mTORC1 and mTORC2 in vitro. Using our Revblocks® “modular synthesis” platform, we have generated novel bi-steric mTOR inhibitors that exhibit potent and selective inhibition of mTORC1 in vitro. By independently tuning the affinities of the rapamycin-derived and ATP-competitive moieties, we have designed bi-steric inhibitors that exhibit distinct mTORC1/2 selectivity profiles as well as different durations of action. Following intraperitoneal administration, mTORC1-selective bi-steric inhibitors produced sustained inhibition of tumor 4EBP1 phosphorylation in vivo in MCF7, an estrogen receptor-positive and PI3KCA mutant xenograft model of mammary carcinoma in nude mice, at doses that induced rapid and sustained tumor regression. In contrast, daily oral administration of everolimus at a clinically relevant dose did not alter tumor 4EBP1 phosphorylation and was less efficacious in comparison with bi-steric mTOR inhibitors. Naïve mice treated with mTORC1-selective inhibitors showed a reduced spike in blood glucose in a glucose tolerance test compared to nonselective mTORC1 inhibitors, indicative of reduced insulin resistance driven by mTORC2 inhibition. We will compare and contrast the biologic profiles of different levels of mTORC1/2 selectivity that can be achieved with selected examples from our collection of diverse bi-steric mTOR inhibitors. This abstract is also being presented as Poster A19. Citation Format: Nidhi Tibrewal, James B. Aggen, Abraham I. Bassan, G. Leslie Burnett, Jim Evans, Micah J. Gliedt, Daphne Hsieh, Gert Kiss, Bianca J. Lee, Dong Lee, Ed Lorenzana, Abby Marquez, Arun Thottumkara, Zhengping Wang, Stacy Wilson, Frances Zhao, Mark Goldsmith, Mallika Singh, Pete Wildes, Adrian L. Gill, Jacqueline A.M. Smith. 4EBP1 reactivation by potent and selective bi-steric inhibitors of mTORC1 [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr PR04.