Abstract
Precision medicines exert selective pressure on tumour cells that leads to the preferential growth of resistant subpopulations, necessitating the development of next-generation therapies to treat the evolving cancer. The PIK3CA–AKT–mTOR pathway is one of the most commonly activated pathways in human cancers1, which has led to the development of small-molecule inhibitors that target various nodes in the pathway. Among these agents, first-generation mTOR inhibitors (rapalogs) have caused responses in ‘N-of-1’ cases, and second-generation mTOR kinase inhibitors (TORKi) are currently in clinical trials2,3,4. Here we sought to delineate the likely resistance mechanisms to existing mTOR inhibitors in human cell lines, as a guide for next-generation therapies. The mechanism of resistance to the TORKi was unusual in that intrinsic kinase activity of mTOR was increased, rather than a direct active-site mutation interfering with drug binding. Indeed, identical drug-resistant mutations have been also identified in drug-naive patients, suggesting that tumours with activating MTOR mutations will be intrinsically resistant to second-generation mTOR inhibitors. We report the development of a new class of mTOR inhibitors that overcomes resistance to existing first- and second-generation inhibitors. The third-generation mTOR inhibitor exploits the unique juxtaposition of two drug-binding pockets to create a bivalent interaction that allows inhibition of these resistant mutants.
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References
Vivanco, I. & Sawyers, C. L. The phosphatidylinositol 3-kinase AKT pathway in human cancer. Nature Rev. Cancer 2, 489–501 (2002)
Basu, B. et al. First-in-human pharmacokinetic and pharmacodynamic study of the dual m-TORC 1/2 inhibitor AZD2014. Clin. Cancer Res. 21, 3412–3419 (2015)
Iyer, G. et al. Genome sequencing identifies a basis for everolimus sensitivity. Science 338, 221 (2012)
Wagle, N. et al. Activating mTOR mutations in a patient with an extraordinary response on a phase I trial of everolimus and pazopanib. Cancer Discov . 4, 546–553 (2014)
Wagle, N. et al. Response and acquired resistance to everolimus in anaplastic thyroid cancer. N. Engl. J. Med. 371, 1426–1433 (2014)
Feldman, M. E. et al. Active-site inhibitors of mTOR target rapamycin-resistant outputs of mTORC1 and mTORC2. PLoS Biol. 7, e38 (2009)
Thoreen, C. C. et al. An ATP-competitive mammalian target of rapamycin inhibitor reveals rapamycin-resistant functions of mTORC1. J. Biol. Chem. 284, 8023–8032 (2009)
Dowling, R. J. O. et al. mTORC1-mediated cell proliferation, but not cell growth, controlled by the 4E-BPs. Science 328, 1172–1176 (2010)
Brown, E. J. et al. Control of p70 s6 kinase by kinase activity of FRAP in vivo. Nature 377, 441–446 (1995)
Chen, J., Zheng, X. F., Brown, E. J. & Schreiber, S. L. Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue. Proc. Natl Acad. Sci. USA 92, 4947–4951 (1995)
Hara, K. et al. Regulation of eIF-4E BP1 phosphorylation by mTOR. J. Biol. Chem. 272, 26457���26463 (1997)
Lorenz, M. C. & Heitman, J. TOR mutations confer rapamycin resistance by preventing interaction with FKBP12-rapamycin. J. Biol. Chem. 270, 27531–27537 (1995)
Yang, H. et al. mTOR kinase structure, mechanism and regulation. Nature 497, 217–223 (2013)
Grabiner, B. C. et al. A diverse array of cancer-associated MTOR mutations are hyperactivating and can predict rapamycin sensitivity. Cancer Discov . 4, 554–563 (2014)
Cerami, E. et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov . 2, 401–404 (2012)
Mammen, M., Choi, S. K. & Whitesides, G. M. Polyvalent interactions in biological systems: Implications for design and use of multivalent ligands and inhibitors. Angew. Chem. Int. Ed. 37, 2754–2794 (1998)
Hsieh, A. C. et al. The translational landscape of mTOR signalling steers cancer initiation and metastasis. Nature 485, 55–61 (2012)
Molecular Operating Environment (Chemical Computing Group Inc., Montreal, Canada, 2016)
Szczepankiewicz, B. G. et al. Discovery of a potent, selective protein tyrosine phosphatase 1B inhibitor using a linked-fragment strategy. J. Am. Chem. Soc. 125, 4087–4096 (2003)
Marinec, P. S. et al. FK506-binding protein (FKBP) partitions a modified HIV protease inhibitor into blood cells and prolongs its lifetime in vivo. Proc. Natl Acad. Sci. USA 106, 1336–1341 (2009)
Patel, M. R. et al. A phase I study evaluating continuous and intermittent AZD2014 in combination with fulvestrant in patients with ER+ advanced metastatic breast cancer (abstract). Proc. AACR 106th Ann. Meeting CT233.25 (AACR, 2015)
Valant, C., Robert Lane, J., Sexton, P. M. & Christopoulos, A. The best of both worlds? Bitopic orthosteric/allosteric ligands of G protein-coupled receptors. Annu. Rev. Pharmacol. Toxicol. 52, 153–178 (2012)
Russo, A. A., Jeffrey, P. D., Patten, A. K., Massagué, J. & Pavletich, N. P. Crystal structure of the p27Kip1 cyclin-dependent-kinase inhibitor bound to the cyclin A–Cdk2 complex. Nature 382, 325–331 (1996)
Wei, L. et al. Design and synthesis of benzoazepin-2-one analogs as allosteric binders targeting the PIF pocket of PDK1. Bioorg. Med. Chem. Lett. 20, 3897–3902 (2010)
Brennan, D. F. et al. A Raf-induced allosteric transition of KSR stimulates phosphorylation of MEK. Nature 472, 366–369 (2011)
Juric, D. et al. Convergent loss of PTEN leads to clinical resistance to a PI(3)Kα inhibitor. Nature 518, 240–244 (2015)
Yao, Z. et al. BRAF mutants evade ERK-dependent feedback by different mechanisms that determine their sensitivity to pharmacologic inhibition. Cancer Cell 28, 370–383 (2015)
Cheng, A. C., Eksterowicz, J., Geuns-Meyer, S. & Sun, Y. Analysis of kinase inhibitor selectivity using a thermodynamics-based partition index. J. Med. Chem. 53, 4502–4510 (2010)
Rodrik-Outmezguine, V. S. et al. mTOR kinase inhibition causes feedback-dependent biphasic regulation of AKT signaling. Cancer Discov . 1, 248–259 (2011)
Acknowledgements
N.R. would like to thank the National Institutes of Health (NIH) (P01 CA094060) for funding, as well as the Breast Cancer Research Foundation grant and the National Cancer Institute Cancer Center Support grant P30 CA008748, W. H. Goodwin and A. Goodwin, the Commonwealth Foundation for Cancer Research, The Center for Experimental Therapeutics at Memorial Sloan Kettering Cancer Center, and the team up for a Cure Fund. K.M.S. would like to thank the NIH P50 AA017072, the Stand Up 2 Cancer Lung Cancer Dream Team, The Samuel Waxman Cancer Research Foundation and the Howard Hughes Medical Institute for funding. We would like to thank R. Mukherjee, S. Schwartz, J. Taunton and B. Roth for helpful comments.
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V.S.R.-O., M.O., Z.Y., C.J.N., N.R. and K.M.S. conceived the project, designed and analysed the experiments, and wrote the manuscript. V.S.R.-O., M.O., Z.Y., C.J.N., C.M., A.B., W.W., D.G.B., S.C. and T.K. performed and supervised the laboratory experiments. H.W. and M.B. performed and supervised the IMPACT sequencing and analysis. E.d.S. designed and supervised the in vivo experiments.
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K.M.S. is an inventor on patents related to MLN0128 held by the University of California San Francisco (UCSF), and sublicensed to Takeda Pharmaceuticals. N.R. and K.M.S. are consultants and M.O. is an employee at Takeda Pharmaceuticals Company Limited, which is conducting MLN0128 clinical trials. C.M., D.G.B., S.C. and T.K. are employees at AstraZeneca, which is conducting AZD2014 (mTOR kinase inhibitor) trials. K.M.S. and M.O. are inventors on a patent application related to RapaLink held by UCSF and licensed to Kura Oncology. K.M.S. is a shareholder in Kura Oncology, K.M.S. and N.R. are consultants to Kura Oncology.
Extended data figures and tables
Extended Data Figure 1 Acquired-mTOR mutations promote resistance to mTOR inhibitors in MCF-7 cells.
a, The RNA from MCF-7 parental, RR1, RR2 and TKi-R cells was isolated and the polymerase chain reaction with reverse transcription (RT–PCR) products were submitted to Sanger sequencing at Genewiz. b, MCF-7 parental, RR1, RR2 and TKi-R cells were treated with either dimethylsulfoxide (DMSO) or 50 nM of RAD001 for 4 h. Immunoblot analyses were performed on mTOR effectors. c, d, MCF-7 parental, RR1, RR2 and TKi-R cells were treated with either DMSO as a control or 500 nM of either KU006, WY354 or PP242 mTOR inhibitors (c), or with different doses of MLN0128 (d) for 4 h. Immunoblot analyses were performed on mTOR effectors. All cellular experiments were repeated at least three times.
Extended Data Figure 2 Acquired-mTOR mutations promote resistance to mTOR inhibitors in MDA-MB-468 cells
a, b, Dose-dependent cell growth inhibition of the MDA-MB-468 cells expressing green fluorescent protein (GFP), wild-type mTOR or different mTOR variants (A2034V, F2108L and M2327I) upon rapamycin (a) or AZD8055 treatment (b). Cells were pre-treated for 24 h with doxycycline (1 μg ml−1) to induce the expression of exogenous mTOR. The cell growth was determined as described in Fig. 1d. c–e, MDA-MB-468 cells expressing GFP, wild-type mTOR or different mTOR variants were treated with different concentrations of rapamycin (c), AZD8055 (d) or MLN0128 (e) for 4 h. Immunoblot analyses were performed on mTOR effectors. All cellular experiments were repeated at least three times.
Extended Data Figure 3 Synthesis of the mTOR bivalent inhibitor RapaLink-1.
a, Compound design of RapaLink-1, -2, and -3 possessing a polyethylene glycol unit of varying lengths. b, Calculated potential energy units (U) (kcal mol−1) of modelled compounds of varying methylene (CH2)n linker lengths for bivalent interactions with the catalytic site and the FKBP12 site. c, A convergent synthetic route for a bivalent mTOR inhibitor RapaLink-1.
Extended Data Figure 4 RapaLink-1 requires FKBP12 for binding to the mTOR FRB domain.
a, Dose-dependent cell growth inhibition curves of the MCF-7 parental cell line treated with rapamycin, MLN0128, a combination of rapamycin and MLN0128, or RapaLink-1. The cell growth was determined as described in Fig. 1d. b, mTOR–Flag wild type and variants were transfected into 293H cells. The mTORC1 complex was isolated, and an in vitro competition assay in the presence of FKBP12 was performed as described in Fig. 2b. c, MCF-7 cells were treated with either DMSO, RapaLink-1 (10 nM), FK506 (10 μM), or a combination of both for 24 h, at which time the cells were collected. Immunoblot analyses were performed on mTOR signalling. All experiments were repeated at least three times.
Extended Data Figure 5 RapaLink-1 is a potent mTOR inhibitor in wild-type and mutant mTOR cells.
a–d, MCF-7, RR1, RR2 and TKi-R cells were treated with different concentrations of rapamycin (a), MLN0128 (b), combination treatment (c) or RapaLink-1 (d) over 3 days. The cell growth was determined as described in Fig. 1d. Each dot and error bar on the curves represents mean ± s.d. (n = 8).
Extended Data Figure 6 RapaLink-1 has a prolonged intracellular half-life in wild-type mTOR cells.
a, MCF-7 F2039S cells were treated with different concentrations of rapamycin, MLN0128, combination treatment or RapaLink-1 for 4 h, at which time the cells were collected. Immunoblot analyses were performed on mTOR signalling. b, MCF-7 cells were treated for 4 h with either DMSO control, 30 nM of rapamycin, 30 nM of MLN0128, a combination of 30 nM of both or 30 nM of RapaLink-1 for 4 h, at which time the treatments were washed out three times with PBS and fresh media was re-added for the indicated times. Immunoblot analyses were performed on mTOR effectors. c, MCF-7 cells were treated with 10 nM of RapaLink-1 and collected at the indicated times. Immunoblot analyses were performed as described earlier. All experiments were repeated at least three times. d, Mice bearing MCF-7 xenograft tumours were treated with one single dose of vehicle or RapaLink-1 (1.5 mg kg−1), tumours were collected at different days after treatment as indicated. Immunoblot analyses were performed on mTOR effectors. e, The weight of the mice treated in the efficacy study shown in f is reported here. f, Mice bearing MCF-7 xenograft tumours were treated as described in Fig. 4c (n = 5 for each group). The results were reported as percentage tumour volume ± s.d.
Extended Data Figure 7 RapaLink-1 is a more potent mTOR inhibitor than rapamycin.
a, MCF-7 cells were treated for 4 h with either RapaLink-1 (10 nM) or rapamycin (10 nM) with simultaneous addition of increasing doses of either rapamycin (left) or RapaLink-1 (right). Immunoblot analyses were performed on mTOR effectors. b, c, Mice bearing RR1 (b) or TKi-R (c) xenograft tumours were treated for 24 h with a single dose of either vehicle, rapamycin (10 mg kg−1), AZD8055 (75 mg kg−1) or RapaLink-1 (1.5 mg kg−1) (n = 4 for each group). Immunoblot analyses were performed on mTOR effectors. d, MDA-MB-468 cells inducibly expressing mTOR wild type were treated with either rapamycin, MLN0128, a combination of rapamycin and MLN0128, or RapaLink-1 for 4 h. Immunoblot analyses were performed on mTOR effectors with the indicated antibodies. Rapamycin and MLN0128 panels are the same shown for wild type in Extended Data Fig. 2c and e, respectively.
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This file contains Supplementary Figures 1-6 (the uncropped blots), Supplementary Table 1 and Supplementary Methods. (PDF 4365 kb)
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Rodrik-Outmezguine, V., Okaniwa, M., Yao, Z. et al. Overcoming mTOR resistance mutations with a new-generation mTOR inhibitor. Nature 534, 272–276 (2016). https://doi.org/10.1038/nature17963
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DOI: https://doi.org/10.1038/nature17963
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