Torin1

TFEB (transcription factor EB) is a critical regulator of lysosomal biogenesis, macroautophagy/autophagy and energy homeostasis through controlling expression of genes belonging to the coordinated lysosomal expression and regulation network. AMP-activated protein kinase (AMPK) has been reported to phosphorylate TFEB at three conserved C-terminal serine residues (S466, S467, S469) and these phosphorylation events were reported to be essential for transcriptional activation of TFEB. In sharp contrast to this proposition, we demonstrate that AMPK activation leads to the dephosphorylation of the C-terminal sites. We show that a synthetic peptide encompassing the C-terminal serine residues of TFEB is a poor substrate of AMPK in vitro. Treatment of cells with an AMPK activator (MK-8722), glucose deprivation or MTOR inhibitor (torin1) robustly dephosphorylated TFEB not only at the MTORC1-targeted N-terminal serine sites, but also at the C-terminal sites. Loss of function of AMPK abrogated MK-8722- but not torin1-induced dephosphorylation and induction of the TFEB target genes.Abbreviations: AMPK: 5'-adenosine monophosphate-activated protein kinase; ACAC/ACC: acetyl-CoA carboxylase; AICAR: 5-aminoimidazole-4-carbox-amide ribonucleotide; CLEAR: coordinated lysosomal expression and regulation; DKO: double knockout; DMEM: Dulbecco's modified Eagle's medium; DMSO: dimethyl sulfoxide; DQ-BSA: self-quenched BODIPY® dye conjugates of bovine serum albumin; KI: knock-in; KO: knockout; MEFs: mouse embryonic fibroblasts; MTORC1: mechanistic target of rapamycin kinase complex 1; RRAGC: Ras related GTP binding C; RPTOR: regulatory associated protein of MTOR complex 1; RPS6KA/RSK: ribosomal protein S6 kinase A; RPS6KB1/S6K1: ribosomal protein S6 kinase B1; RT-qPCR: reverse transcription quantitative polymerase chain reaction; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcription factor EB; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type.

Pathological remodeling in cardiomyocytes during heart failure is driven by excessive activation of mTORC1. Preclinical studies in mice demonstrated that reducing mTORC1 activity protects against cardiac dysfunction and hypertrophic remodeling. However, clinical application of current mTOR inhibitors is limited by incomplete mTORC1 inhibition and off-target effects, including suppression of mTORC2-mediated pro-survival signaling. To address these challenges, the therapeutic effects of the third-generation bi-steric mTORC1 inhibitor RMC-6272 was tested in pressure overload induced heart failure models. The potency and selectivity of RMC-6272 were evaluated in isolated cardiomyocytes. Hypertrophy was induced by phenylephrine in vitro and aortic banding in vivo. Cardiomyocyte-specific mRNA translation changes were assessed in αMHC-Cre Ribo-tag mice. RMC-6272 demonstrated superior potency and specificity for mTORC1 compared to rapamycin and Torin1. RMC-6272 prevented pathological hypertrophic growth in vitro and suppressed mTORC1-dependent mRNA translation, particularly those encoding components of the translational machinery. Preventive RMC-6272 treatment preserved cardiac function under pressure overload, maintaining contractile dysfunction in mice. Therapeutic treatment improved function in established hypertrophy, demonstrating therapeutic potential even after disease onset.