The PI3K-mTOR pathway is one of the most commonly dysregulated pathways in human tumors. Rapalogs have been used extensively in human clinical trials but exhibit modest clinical benefit for the most part, possibly due to their lack of effect on 4E-BP1, a key target downstream of mTORC1. ATP-competitive mTOR inhibitors can fully inhibit mTORC1 but are poorly tolerated possibly due to their inhibition of mTORC2. A new class of selective mTORC1 inhibitors has been developed and termed ‘bi-steric', which comprise a rapamycin-like core moiety covalently linked to an mTOR active-site inhibitor. RMC-5552 is the first clinical candidate of this class and clinical testing is planned in 2021. RM-001 and RM-006 (also known as RMC-6272) are representative bi-steric tool compounds that exhibit potent and selective (> 10-fold) inhibition of mTORC1 over mTORC2. We report that bi-steric mTORC1-selective inhibitors have greater activity than rapalogs for tumors with mTORC1 hyperactivation secondary to TSC1/2 loss. RM-001 and RM-006 showed more effective growth inhibition in multiple TSC1 or TSC2 mutant tumor cell lines compared to rapamycin. RM-001 and RM-006 caused a more profound growth inhibition in the TSC1 or TSC2 mutant cells than the wild type cells. Two-week low dilution clonogenic assays in TSC1 or TSC2 mutant cells showed clone numbers were significantly reduced by rapamycin, RM-001, and RM-006, whereas in the wild type cells, Rapamycin and RM compounds had less reduction in clone numbers, indicating a differential sensitivity in TSC-null cells. Both RM-001 and RM-006 at ~1 nM showed near complete inhibition of p4E-BP1T37/46, which was not seen with rapamycin treatment, while inhibition of pS6S240/244 levels was similar for rapamycin and RM compounds. Further, the RM compounds had prolonged anti-proliferative activity in TSC-null cells, as shown by washout studies. Rapamycin, MLN0128 and both RM compounds markedly reduced kidney tumor burden in Tsc2+/- A/J mice after four weeks of treatment. Tumor regrowth was assessed two months after treatment cessation, tumor burden was significantly reduced in the RM-006 group as compared to the rapamycin and MLN0128 groups. Finally, TUNEL staining showed RM-006 treatment led to a greater induction of apoptosis in kidney tumor cells relative to rapamycin and MLN0128 after a single dose of each compound in Tsc2+/- A/J mice. This is the first time that clear evidence for cell death after mTORC1 inhibition has been seen in TSC rodent models. In summary, RM-001 and RM-006 demonstrate improved in vitro and in vivo inhibition of mTORC1 in comparison to rapamycin, and induced more cell death in TSC2 null tumors in vivo, indicating the potential of bi-steric mTORC1-selective inhibitors as a novel therapeutic strategy to treat tumors with mTORC1 dysregulation.

Citation Format: Heng Du, Yu Chi Yang, Mallika Singh, Heng-jia Liu, David Kwiatkowski. Bi-steric mTORC1-selective inhibitors demonstrate improved potency and efficacy in tumors with mTORC1 hyperactivation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1026.