Abstract B108: 4EBP3 mRNA as a biomarker of therapeutic response to treatment with mTORC1 inhibitors

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