Bivalent mTOR inhibitors overcome resistance to 1st and 2nd generation mTOR inhibitors.
Major finding: Bivalent mTOR inhibitors overcome resistance to 1st and 2nd generation mTOR inhibitors.
Concept: Third-generation mTOR inhibitors target two drug-binding pockets, an allosteric and kinase site.
Impact: RapaLink may effectively inhibit mTOR in tumors with mTOR activation or drug-resistance mutations.
The PIK3CA–AKT–mTOR pathway is commonly activated in cancer; thus, mTOR inhibitors have potential in precision medicine. First-generation allosteric mTOR inhibitors (including rapamycin) have had limited success in cancer, and second-generation mTOR kinase inhibitors (including AZD8055) are in clinical trials. Rodrik-Outmezguine, Okaniwa, Yao, and colleagues explored the resistance mechanisms of available mTOR inhibitors in order to develop third-generation mTOR inhibitors that may overcome the resistance mechanisms. Deep sequencing of breast cancer cells treated with rapamycin or AZD8055 to generate resistant colonies revealed two mutations (MTORA2034V and MTORF2108L) in the mTOR FKBP12–rapamycin-binding domain (FRB domain) of rapamycin-resistant clones, and a mutation (MTORM2327I) in the kinase domain of MTOR of the AZD8055-resistant clones. The FRB domain mutations targeted the rapamycin-binding pocket; however, the MTORM2327I mutation was more than 15 Å from the inhibitor site. Further, wild-type and M2327I mTOR had similar affinities for AZD8055, suggesting a resistance mechanism independent of reduced drug binding. In contrast to rapamycin-resistance mutations, M2327I increased the kinase activity of mTOR, suggesting the mutation confers a growth advantage by hyperactivating mTOR. Moreover, M2327I and other hyperactivating mutations were identified in drug-naïve patients with multiple tumor types. A molecular model revealed the juxtaposition of the rapamycin and AZD8055 binding sites, prompting development of a bivalent mTOR inhibitor with the rapamycin–FRB binding element linked to a second-generation inhibitor. Binding of the proposed inhibitor to one site would bring it in close proximity to the second site, potentially overcoming mutations that reduce drug binding or hyperactivate the kinase. The resulting inhibitor, RapaLink-1, inhibited phosphorylation of mTORC1 and mTORC2 targets and suppressed breast cancer cell growth. Further, it maintained activity in both rapamycin-resistant and AZD8055-resistant xenografts. Altogether, this study generated third-generation mTOR inhibitors that may overcome drug-resistance mutations associated with first- and second-generation inhibitors, guide future bivalent kinase inhibitor design, and warrant further clinical development.