Rapalink-1 reveals an mTOR- and p53-regulated transcriptional program in Neuro-2a cells

The mammalian target of rapamycin (mTOR) is the catalytic subunit of two exclusive complexes referred to as mTORC1 and mTORC2. The mTORCs phosphorylate different substrates and control different cellular processes. Inhibitors of mTORC1 include rapamycin analogs (rapalogs) that are useful in many clinical settings. However, they have failed in cancer trials potentially due to resistance mechanisms and incomplete inhibition of mTORC1 phosphorylation of select substrates. The rapamycin sensitivity of mTORC1 substrates is likely encoded by the substrate's primary amino acid sequence determinants and the ability of substrates to fit into the catalytic region of the kinase super-complex. Thus, some mTORC1 substrates are unaffected by rapalogs. Second-generation ATP-binding pocket inhibitors are mTOR catalytic inhibitors that bind in an ATP-competitive manner. Torin1 is an exemplary second-generation mTOR inhibitor with superior inhibition of mTORC1 substrates, including 4EBP. However, these inhibitors fail to demonstrate selectivity between mTORC1 and mTORC2. Third-generation bisteric mTORC1 inhibitors are exemplified by Rapalink-1, which links a rapamycin moiety to the ATP-competitive inhibitor MLN-0128. Here, we report that Rapalink-1 inhibits three mTORC1 substrates, including 4EBP and Ulk1, which are rapamycin insensitive. To provide additional mechanistic insight into the Rapalink-1 mechanism of action, RNA sequencing of Neuro-2a cells treated with Rapalink-1 was performed. Rapalink-1 caused transcriptomic changes indicative of reductions in the mTOR pathway and brain development. Many transcripts contained a p53 binding site, and consistent with this finding, we also observed reduced levels of p53. These results confirm and expand upon the Rapalink-1 mechanism of action.