Overcoming mTOR resistance mutations with a new-generation mTOR inhibitor (original) (raw)

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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.

Author information

Author notes

  1. Vanessa S. Rodrik-Outmezguine, Masanori Okaniwa and Zhan Yao: These authors contributed equally to this work.

Authors and Affiliations

  1. Program in Molecular Pharmacology, Memorial Sloan-Kettering Cancer Center, New York, 10065, New York, 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, 94158, California, USA
    Masanori Okaniwa, Chris J. Novotny & Kevan M. Shokat
  3. AstraZeneca, Alderley Park, Macclesfield, SK10 4TG, Cheshire, UK
    Claire McWhirter, Derek G. Barratt, Sabina Cosulich & Teresa Klinowska
  4. Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, 10065, New York, USA
    Helen Won & Mike Berger
  5. Anti-Tumor Assessment Core, Memorial Sloan Kettering Cancer Center, New York, 10065, New York, USA
    Wai Wong & Elisa de Stanchina
  6. Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, 10065, New York, USA
    Neal Rosen
  7. Department of Chemistry, University of California Berkeley, Berkeley, 94720, California, USA
    Kevan M. Shokat

Authors

  1. Vanessa S. Rodrik-Outmezguine
  2. Masanori Okaniwa
  3. Zhan Yao
  4. Chris J. Novotny
  5. Claire McWhirter
  6. Arpitha Banaji
  7. Helen Won
  8. Wai Wong
  9. Mike Berger
  10. Elisa de Stanchina
  11. Derek G. Barratt
  12. Sabina Cosulich
  13. Teresa Klinowska
  14. Neal Rosen
  15. Kevan M. Shokat

Contributions

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.

Corresponding authors

Correspondence toNeal Rosen or Kevan M. Shokat.

Ethics declarations

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.

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.

Source data

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.

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.

Source data

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).

Source data

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.

Source data

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.

Extended Data Table 1 mTOR mutations found in human patient samples

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Extended Data Table 2 List of FRB domain mutations found in human patient samples

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Extended Data Table 3 List of mTOR kinase domain mutations found in human patient samples

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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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