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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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.

Author information

Author notes

    • Vanessa S. Rodrik-Outmezguine
    • , Masanori Okaniwa
    •  & Zhan Yao

    These authors contributed equally to this work.


  1. Program in Molecular Pharmacology, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA

    • Vanessa S. Rodrik-Outmezguine
    • , Zhan Yao
    • , Arpitha Banaji
    •  & Neal Rosen
  2. Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, California 94158, USA

    • Masanori Okaniwa
    • , Chris J. Novotny
    •  & Kevan M. Shokat
  3. AstraZeneca, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK

    • Claire McWhirter
    • , Derek G. Barratt
    • , Sabina Cosulich
    •  & Teresa Klinowska
  4. Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA

    • Helen Won
    •  & Mike Berger
  5. Anti-Tumor Assessment Core, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA

    • Wai Wong
    •  & Elisa de Stanchina
  6. Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA

    • Neal Rosen
  7. Department of Chemistry, University of California Berkeley, Berkeley, California 94720, USA

    • Kevan M. Shokat


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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.

Competing interests

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.

Corresponding authors

Correspondence to Neal Rosen or Kevan M. Shokat.

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    Supplementary Information

    This file contains Supplementary Figures 1-6 (the uncropped blots), Supplementary Table 1 and Supplementary Methods.

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