Abstract
The clinical benefits of pan-mTOR active-site inhibitors are limited by toxicity and relief of feedback inhibition of receptor expression. To address these limitations, we designed a series of compounds that selectively inhibit mTORC1 and not mTORC2. These ‘bi-steric inhibitors’ comprise a rapamycin-like core moiety covalently linked to an mTOR active-site inhibitor. Structural modification of these components modulated their affinities for their binding sites on mTOR and the selectivity of the bi-steric compound. mTORC1-selective compounds potently inhibited 4EBP1 phosphorylation and caused regressions of breast cancer xenografts. Inhibition of 4EBP1 phosphorylation was sufficient to block cancer cell growth and was necessary for maximal antitumor activity. At mTORC1-selective doses, these compounds do not alter glucose tolerance, nor do they relieve AKT-dependent feedback inhibition of HER3. Thus, in preclinical models, selective inhibitors of mTORC1 potently inhibit tumor growth while causing less toxicity and receptor reactivation as compared to pan-mTOR inhibitors.
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Data availability
Source data for Extended Data Fig. 7 have been provided as Supplementary Dataset 3. Source data for Extended Data Fig. 7 have been deposited at the Gene Expression Omnibus with the accession number GSE138417. All other data supporting the findings of this study are available from the corresponding authors on reasonable request.
Material availability
Requests for materials should be addressed to N.R. and J.A.M.S. Source data are provided with this paper.
Code availability
Computational code used in this study is available from the corresponding authors on reasonable request.
Change history
29 June 2021
A Correction to this paper has been published: https://doi.org/10.1038/s41589-021-00843-1
06 October 2021
A Correction to this paper has been published: https://doi.org/10.1038/s41589-021-00905-4
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Acknowledgements
We thank S. Kelsey and M.A. Goldsmith for scientific guidance and critical review of the manuscript, and A.I. Bassan for invaluable discussions leading to this work. We gratefully acknowledge the contributions of M.J. Gliedt, J. Pitzen, C. Semko, G. Wang and W. Won for the syntheses of the bi-steric mTORC1 inhibitors. N.R. is supported by an NCI Outstanding investigator grant, R35 CA210085 and a Breast Cancer Research Foundation Grant, BCRF17-139. I.T. is supported by Senior Scholar Award from Le Fonds de recherche du Québec–Santé (FRQS) and acknowledges support from Cancer Research Society grant no. 102585. L.H. is supported by Junior 1 Scholar Award from Le Fonds de recherche du Québec–Santé (FRQS). O.L. is a Wallenberg Academy Fellow. We also thank the researchers at WuXi Apptec who supported our in vivo pharmacology efforts. This study was funded in part through the NIH/NCI Cancer Center Support Grant P30 CA008748.
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Contributions
B.J.L., J.A.B., N.T., S.L.W., T.H., A.M., L.H., G.K., S.M., I.T. and D.W. designed, conducted and interpreted in vitro cellular experiments that include cell proliferation, apoptosis, phosphorylation and cap-binding affinity assays, and gene overexpression, knockout and knockdown. H.L. and O.L. designed, conducted and interpreted the polysome profiling study. B.J.L. and N.T. designed, oversaw and interpreted the KiNativ studies. D.W. designed, oversaw and interpreted the TR–FRET competitive binding and ternary complex assays. B.J.L., J.A.B., E.G.L., J.W.E., D.L., Zhengping W., Zhican W., Y.Z., D.W. and M.S. contributed experimental design, conduct and interpretation of in vivo efficacy, PK/PD and GTT experiments. G.L.B., A.P.T., G.K., J.B.A. and A.L.G. contributed the design and synthesis of RMC-4287, RMC-4627, RMC-4529 and RMC-4745. J.A.M.S., N.R., I.T. and O.L. supervised and contributed to the design and interpretation of all experiments from their respective collaborating groups. B.J.L., J.A.B., D.W., J.A.M.S. and N.R. wrote the manuscript with input from all coauthors.
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N.R. reports grants from Revolutionary Medicines and Boehringer Ingelheim; grants and consulting fees from AstraZeneca and Pfizer Array; consulting fees (scientific advisory board) from Ribon Therapeutics, Tarveda Therapeutics, and Chugai Pharmaceuticals; scientific advisory board fees and equity in Beigene and Zai Laboratories; and equity in Kura Oncology and Fortress. I.T. and O.L. have consulted at and are recipients of research grants from Revolution Medicines. B.J.L., G.L.B., A.P.T., N.T., S.L.W., T.H., A.M., E.G.L., J.W.E., G.K., D.L., Zhengping W., Zhican W., Y.Z., D.W., J.B.A., M.S., A.L.G. and J.A.M.S. are current or former employees of Revolution Medicines, Inc. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Bi-steric inhibitors are selective for mTORC1 in multiple cell lines.
a, Immunoblot analysis of whole cell lysates from MDA-MB-468 (ER-, PR-, HER2-/PTEN null, EGFR amplified), MCF-7 (ER + /PIK3CA E545K), ZR-75-1 (ER + /PTEN PL108R), and HCC1954 (HER2 + /PIK3CA H1047R) breast cancer cell lines incubated with indicated compounds for 4 hours. Data are representative of at least n = 2 independent experiments. b, Immunoblot analysis of whole cell lysates from A375 (melanoma, BRAF V600E), MCF-7, and PC3 (prostate cancer, TP53 deletion) cells incubated with indicated compounds for 4 and 24 hours. Data are representative of at least n = 2 independent experiments.
Extended Data Fig. 2 Selectivity of bi-steric inhibitors for mTORC1 is independent of feedback activation of AKT.
Immunoblot analysis of whole cell lysates from MCF-7 cells transfected with HA-Myr-AKT or vector control for 24 hours followed by exposure to indicated compounds for 4 hours. Data are representative of at least n = 2 independent experiments.
Extended Data Fig. 3 Cellular activity of mTOR bi-steric inhibitors is dependent on formation of an FKBP12-mTOR inhibitor-FRB ternary complex.
a, Concentration-dependent formation of a ternary complex with emGFP-FKBP12, GST-mTOR (residues 1360-2549), and inhibitor detected by TR-FRET signal from LanthaScreen TB-anti-GST antibody. 100% ternary complex formation is defined by signal with 1 µM rapamycin. Data are the mean of technical duplicates from n = 1 experiment. b, Immunoblot analysis for FKBP12 protein in parental and FKBP12 knockout NCI-H358 cells (analysis performed in n = 1 experiment to verify FKBP12 knockout). c, Concentration responses of p4EBP1 T37/T46 determined from whole cell lysates of parental (filled circles) and FKBP12 knockout (open circles) NCI-H358 cells treated with increasing concentrations of indicated compounds for four hours. Data are the mean of n = 2 experiments each done in technical duplicates, with error bars representing SD. d, Concentration responses of cellular proliferation determined for parental (filled circles) and FKBP12 knockout (open circles) NCI-H358 cells treated with increasing concentrations of indicated compounds for 72 hours. Data are the mean of n = 2 experiments each done in technical duplicates, with error bars representing SD. e, Concentration responses of p4EBP1 T37/T46 determined from whole cell lysates of MDA-MB-468 cells treated with increasing concentrations of indicated compounds in the presence, or absence, of FK506 (10 μM) for four hours. Data are the mean of n = 3 experiments each done in technical duplicates, with error bars representing SD. f, Immunoblot analysis of whole cell lysates from parental MCF-7 cells and MCF-7 cells harboring mTOR F2108L treated with increasing concentrations of indicated compounds for four hours. Data are representative of at least n = 2 independent experiments.
Extended Data Fig. 4 mTORC1 bi-steric inhibitors cause reduced induction of HER3 in vitro and in vivo.
a, Immunoblot analysis of whole cell lysates from T47D cells treated with indicated compounds for 16 hours. Data are representative of at least n = 2 independent experiments. b, Immunoblot analysis of lysates from T47D xenografts over a period of 24 hours following in vivo administration of a single dose of the indicated compounds. Data are representative of at least n = 2 independent experiments.
Extended Data Fig. 5 Inhibition of 4EBP1 phosphorylation is important for maximal activity of mTOR bi-steric inhibitors.
a, Immunoblot analysis of whole cell lysates from MDA-MB-468 and MCF-7 sgGFP and sg4EBP1 cells incubated with increasing concentrations of RapaLink-1 for 4 hours. Data are representative of at least n = 2 independent experiments. b, In vitro cap-binding affinity assay and immunoblot analysis of whole cell lysates from MDA-MB-468 sgGFP and sg4EBP1 cells. Data are representative of at least n = 2 independent experiments. c, Cell viability of MDA-MB-468 sgGFP and sg4EBP1 cells incubated with increasing concentrations of RapaLink-1 for up to 5 days. Data are a mean of technical duplicates, and representative of at least n = 2 independent experiments. d, In vitro cap-binding affinity assay and immunoblot analysis of MCF-7 shScr and sh4EBP1 KD cells serum-starved (0% FBS) for 18 hours, then serum-stimulated (10% FBS) and incubated for two hours with compounds at EC80 concentrations: BiS-13x (3 nM), BiS-35x (35 nM), and MLN0128 (40 nM). Serum-starved cells served as a control. Data are representative of two independent experiments with similar results. e, Cell viability of MCF-7 shScr and sh4EBP1 cells (knockdown by scrambled shRNA and shRNA against 4EBP1, respectively) exposed to the indicated compounds for 72 hours. Viability was measured as viable cell counts normalized to DMSO-treated cells, with center bars representing the mean of n = 2 or 3 technical replicates and error bars representing SD of n = 3 technical replicates. Data are a representative experiment of at least n = 2 independent experiments. f, Cell viability of 4EBP1/2 WT and 4EBP1/2 DKO MEFs exposed to indicated compounds for 72 hours. Viability was measured as viable cell counts normalized to DMSO-treated cells, with center bars representing mean of n = 2 or 3 experiments and error bars representing SD of n = 3 experiments.
Extended Data Fig. 6 Inhibition of global protein synthesis by mTOR bi-steric inhibitors is partially dependent on 4EBP1.
a, Immunoblot analysis of whole cell lysates from MCF-7 cells incubated with indicated inhibitors for 4 hours and pulsed with 1 µM puromycin (Puro) for the last 30 minutes. Data are representative of at least n = 2 independent experiments. b, Immunoblot analysis of whole cell lysates from MCF-7 control (siScr) and 4EBP1-depleted (si4EBP1) cells incubated with increasing concentrations of RapaLink-1 for 4 hours and pulsed with 1 µM puromycin for the last 30 minutes. Data are representative of at least n = 2 independent experiments.
Extended Data Fig. 7 Translational reprogramming by bi-steric and active-site inhibitors of mTOR is modulated by inhibition of mTORC1.
a, Scatter plots of polysome-associated RNA vs. cytosolic RNA log2 fold changes for the four comparisons: DMSO (complete media) vs. starvation [top left], MLN0128 vs. DMSO [top right], BiS-13x vs. MLN0128 [bottom left] and BiS-35x vs. MLN0128 [bottom right]. For each comparison, genes are colored according to their mode for regulation of gene expression derived from analysis using anota2seq50 and the number of such regulated transcripts is indicated: ‘translation’ denotes transcripts whose change in polysome-association (up or down) could not be explained by a corresponding change in total mRNA level (that is changes in translational efficiency leading to altered protein level); ‘buffering’ denotes transcripts whose change in the total mRNA pool (up or down) was offset at the level of translation such that the association with polysomes remained largely unaltered (that is a change in total mRNA level opposed by a change in translational efficiency, which is expected to lead to unaltered protein levels despite changes in mRNA levels [as recently described]55,56; ‘abundance’ denotes transcript which show a similar change in the total mRNA pool and association with polysomes (that is a change in transcription or mRNA stability leading to altered protein level). b, Samples from the polysome-profiling experiment (normalized data) were projected on the 2 first components of a centered principal component analysis (the proportion of the variance explained by each component is indicated). Data points are colored according to their RNA source (total mRNA or polysome-associated mRNA), shapes denote treatments (DMSO, MLN0128, BiS-13, BiS-35x, or starvation) and the numbers indicate biological replicates (1-4). The library preparation was performed twice on biological replicate 1 providing 2 technical replicates labeled 1 A and 1B. Replicate 1 A was excluded from the downstream analysis.
Extended Data Fig. 8 mTORC1-selective bi-steric inhibitors suppress S6 phosphorylation in tumors and inhibit tumor growth in MCF-7 xenografts.
a, Levels of pS6 S240/S244 as percent of control determined for lysates from MCF-7 tumors at end of study over a period of 72 hours following the final dose of a repeat dosing schedule. Data are the mean signal of each group (n = 3) and error bars represent SD. b, Mean tumor volume of MCF-7 xenografts following daily oral administration (po qd) of MLN0128, and once weekly intraperitoneal (ip qw) administration of bi-steric inhibitors (n = 12 animals per group), with error bars representing SEM. c, Percent tumor volume change of individual MCF-7 xenografts with daily oral administration (po qd) of MLN0128, and once weekly intraperitoneal (ip qw) administration of bi-steric inhibitors. Gray zone represents 10% margin of error for tumor growth and shrinkage. Similar results for each agent were observed in at least n = 2 independent experiments.
Extended Data Fig. 9 mTORC1-selective bi-steric inhibitors suppress 4EBP1 phosphorylation in tumors and inhibit tumor growth in HCC1954 xenografts.
a, Level of p4EBP1 T37/T46 as percent of control determined for lysates from HCC1954 tumors at end of study over a period of 72 hours following the final dose of a repeat dosing schedule (left), and unbound plasma concentration of inhibitors over time (right). Data are the mean signal of each group (n = 3) and error bars represent SD. b, Level of p4EBP1 T37/T46 as percent of control determined for lysates from HCC1954 tumors after a single dose of inhibitors (left), and unbound plasma concentration of inhibitors over time (right). Data are the mean signal of each group (n = 3) and error bars represent SD. c, Waterfall plot of individual end of study tumor responses, with tumor volume expressed as a percentage of initial tumor volume at time of study. Each animal is represented as a separate bar. d, Mean tumor volume of HCC1954 xenografts following once weekly intraperitoneal (ip qw) administration of bi-steric inhibitors (n = 12 animals per group), with error bars representing SEM. e, Percent tumor volume change of individual HCC1954 xenografts with once weekly intraperitoneal (ip qw) administration of bi-steric inhibitors. Gray zone represents 10% margin of error for tumor growth and shrinkage. f, Mean percent body weight change of HCC1954 xenografts (n = 12 animals per group), with error bars representing SEM. Similar results for each agent were observed in at least n = 2 independent experiments.
Supplementary information
Supplementary Information
Supplementary Tables 1–3 and Note 1 (contains Supplementary Tables 4–11).
Supplementary Dataset 1
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Supplementary Dataset 2
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Lee, B.J., Boyer, J.A., Burnett, G.L. et al. Selective inhibitors of mTORC1 activate 4EBP1 and suppress tumor growth. Nat Chem Biol 17, 1065–1074 (2021). https://doi.org/10.1038/s41589-021-00813-7
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DOI: https://doi.org/10.1038/s41589-021-00813-7
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