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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.

Aberrantly activated PI3K/mTOR signaling is frequently implicated as an oncogenic driver in human cancer. In response to nutrient and growth factor stimuli, mTOR complex 1 (mTORC1) regulates cell growth and cap-dependent protein translation through phosphorylation and activation of S6K, and through phosphorylation and inactivation of the translational repressor 4EBP1. While suppression of mTORC1 activity causes rapid dephosphorylation of its substrates S6K and 4EBP1, it has also been reported that the translational repressor 4EBP3 is transcriptionally induced during prolonged mTORC1 inhibition.1 We have designed a series of complete and selective bi-steric inhibitors of mTORC1 by covalently linking rapamycin, a selective yet incomplete mTORC1 inhibitor, with mTOR kinase active-site inhibitors. These compounds potently and durably suppress phosphorylation of S6K and 4EBP1, induce growth suppression and apoptosis in multiple cancer cell lines, and cause tumor growth inhibition in xenograft models. In the present study we evaluated the utility of 4EBP3 mRNA expression as a pharmacodynamic (PD) biomarker of mTORC1 inhibition by our bi-steric inhibitors. We further interrogated whether tumor mTORC1 inhibition and anti-tumor efficacy could be predicted by monitoring this PD marker in surrogate tissue. First, we demonstrated that 4EBP3 mRNA and protein levels increased in response to mTORC1 inhibition in a concentration- and time-dependent manner in MDA-MB-468, MCF-7, and HCC1954 breast carcinoma cells. Kinetic studies in which we compared the response of bi-steric inhibitors to that of short duration-of-action active-site inhibitors revealed that 4EBP3 induction requires sustained mTORC1 inhibition, as loss of mTORC1 inhibition correlated with a rapid return to baseline 4EBP3 expression. Treatment with inhibitors of varying potency against mTORC1 also demonstrated a strong correlation between magnitude of cell growth inhibition and 4EBP3 mRNA induction. Additionally, suppression of tumor 4EBP1 phosphorylation and induction of 4EBP3 mRNA was observed in MCF-7 and HCC9154 tumor xenografts following in vivo administration of bi-steric inhibitors with a concomitant induction of murine 4ebp3 in peripheral blood mononuclear cells (PBMCs). Initial studies indicate that ex vivo exposure of human PBMCs to a mTORC1 inhibitor also promotes 4EBP3 mRNA expression. Together, these data suggest that 4EBP3 could provide a sensitive mRNA-based in vivo biomarker of prolonged mTORC1 inhibition, as is the case following administration of a single dose of a bi-steric mTORC1 inhibitor. The concordance between the responses in tumor and PBMC suggests it may be possible to monitor tumor mTORC1 inhibition by monitoring 4EBP3 in surrogate tissue. In summary, 4EBP3 mRNA represents a potential PD biomarker of mTORC1 inhibitor response as an alternative to measurement of the phosphorylation status of mTORC1 substrates. 1. Tsukumo, T., et al. Translation control during prolonged mTORC1 inhibition mediated by 4E-BP3. Nat Commun 7:11776. doi: 10.1038/ncomms11776 (2016). Citation Format: Bianca J. Lee, Nuntana Dinglasan, Tram Nguyen, Ed Lorenzana, Stacy Wilson, G. Leslie Burnett, James B. Aggen, Robert J. Nichols, Mallika Singh, David Wildes, Jaqueline A.M. Smith. 4EBP3 mRNA as a biomarker of therapeutic response to treatment with mTORC1 inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B108. doi:10.1158/1535-7163.TARG-19-B108