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. 2016 Jun 9;534(7606):272-6.
doi: 10.1038/nature17963. Epub 2016 May 18.

Overcoming mTOR resistance mutations with a new-generation mTOR inhibitor

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

Overcoming mTOR resistance mutations with a new-generation mTOR inhibitor

Vanessa S Rodrik-Outmezguine et al. Nature. .

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 cancers, 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 trials. 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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Figures

Extended Data Figure 1
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 RT-PCR products were submitted to Sanger sequencing at Genewiz, Inc. b, MCF-7 parental, RR1, RR2 and TKi-R cells were treated with either DMSO or 50 nM of RAD001 for 4 hours. Immunoblot analyses were performed on mTOR effectors. c, MCF-7 parental, RR1, RR2 and TKi-R cells were treated with either DMSO as a control or 500 nM of either KU006, WY354, PP242 mTOR inhibitors or with different doses of MLN0128 (d) for 4 hours. Immunoblot analyses were performed on mTOR effectors. All cellular experiments were repeated at least three times.
Extended Data Figure 2
Extended Data Figure 2. Acquired-mTOR mutations promote resistance to mTOR inhibitors in MDA-MB-468 cells
a, Dose-dependent cell growth inhibition of the MDA-MB-468 cells expressing GFP, WT mTOR or different mTOR variants (A2034V, F2108L and M2327I) upon rapamycin or AZD8055 treatment (b). Cells were pretreated for 24 hours with doxycycline (1 µg/mL) to induce the expression of exogenous mTOR. The cell growth was determined as described in Figure 1d. c, MDA-MB-468 cells expressing GFP, WT mTOR or different mTOR variants were treated with different concentration of rapamycin, AZD8055 (d) or with MLN0128 (e) for 4 hours. Immunoblot analyses were performed on mTOR effectors. All cellular experiments were repeated at least three times.
Extended Data Figure 3
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 energies U (kcal/mol) of modeled 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
Extended Data Figure 4. RapaLink-1 requires FKBP12 for binding to mTOR FRB domain
a, Dose-dependent cell growth inhibition curves of the MCF-7 parental cell line treated with rapamycin, MLN0128, combination of rapamycin and MLN0128 or RapaLink-1. The cell growth was determined as described in Figure 1d. b, mTOR-FLAG WT 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 Figure 2b. c, MCF-7 cells were treated with either DMSO, RapaLink-1 (10 nM), FK506 (10 µM) or combination of both for 24 hours at which time the cells were collected. Immunoblot analyses were performed on mTOR signaling. All experiments were repeated at least three times.
Extended Data Figure 5
Extended Data Figure 5. RapaLink-1 is a potent mTOR inhibitor in WT and mutant mTOR cells
a, MCF-7, (b) RR1, (c) RR2, (d) TKi-R cells were treated with different concentrations of rapamycin, MLN0128, combination treatment or RapaLink-1 over 3 days. The cell growth was determined as described in Figure 1d. Each dot and error bar on the curves represents mean ± standard deviation (n=8).
Extended Data Figure 6
Extended Data Figure 6. RapaLink-1 has a prolonged intracellular half-life in WT mTOR cells
a, MCF-7 F2039S cells were treated with different concentrations of rapamycin, MLN0128, combination treatment or RapaLink-1 for 4 hours at which time the cells were collected. Immunoblot analyses were performed on mTOR signaling. b, MCF-7 cells were treated for 4 hours with either DMSO control, 30 nM of rapamycin, 30 nM of MLN0128, combination of 30 nM of both or 30 nM of RapaLink-1 for 4 hours at which time the treatments were washed out 3 times with PBS and fresh media was readded 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 above. All experiments were repeated at least three times. d, Mice bearing MCF-7 xenograft tumors were treated with one single dose of Vehicle or RapaLink-1 (1.5 mg/kg), tumors 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 f is reported here. f, Mice bearing MCF-7 xenograft tumors were treated as described in Figure 4 c (n=5 for each group). The results were reported as % tumor volume ± standard deviation.
Extended Data Figure 7
Extended Data Figure 7. RapaLink-1 is a more potent mTOR inhibitor than rapamycin
a, MCF-7 cells were treated for 4 hours with either RapaLink-1 (10 nM) or rapamycin (10 nM) with simultaneous addition of increasing doses of either rapamycin or RapaLink-1 (left and right panels respectively). Immunoblot analyses were performed on mTOR effectors. b, Mice bearing RR1 or TKi-R (c) xenograft tumors were treated for 24 hours with a single dose of either vehicle, rapamycin (10 mg/kg), AZD8055 (75 mg/kg) or RapaLink-1 (1.5 mg/kg) (n=4 for each group). Immunoblot analyses were performed on mTOR effectors. d, MDA-MB-468 cells inducibly expressing mTOR WT were treated with either rapamycin, MLN0128, combination of rapamycin and MLN0128 or RapaLink-1 for 4 hours. Immunoblot analyses were performed on mTOR effectors with the indicated antibodies. Rapamycin and MLN0128 panels are the same shown for WT in Extended Data Figure 2c and e respectively.
Figure 1
Figure 1. Single amino acid mutation accounts for acquired resistance to mTOR inhibitors
a, Graphic representation of mTOR domains and site mutagenesis isolated in rapamycin- and AZD8055-resistant cells. b, The effects of rapamycin or AZD8055 (c) on mTOR signaling was assessed in MCF-7, RR1 and RR2 cells (or in TKi-R cells (c)) by immunoblotting 4 hours after treatment. For gel source data, see Supplemental Figure 1. d, Dose-dependent cell growth inhibition curves of MCF-7 and rapamycin-resistant MCF-7 A2034V (RR1) and MCF-7 F2108L (RR2) cells treated with rapamycin at day 3 or e, MCF-7 and AZD8055-resistant MCF-7 M2327I (TKi-R) cells treated with AZD8055. Each dot and error bar on the curves represents mean ± SD (n=8). All experiments were repeated at least three times.
Figure 2
Figure 2. Non-overlapping mechanisms of resistance mediated by mTOR mutations
a, mTOR-FLAG Wild-Type (WT) and variants were transfected into 293H cells. Cells were treated with rapamycin and lysates were immunoprecipitated (IP) with an anti-FLAG antibody. mTORC1 complex formation was assessed by immunoblotting. b, 293H cells were transfected and complex isolated as described in a, and an in vitro competition assay was performed followed by immunoblotting. For gel source data, see Supplemental Figure 2. c, Varying concentrations of AZD8055 were tested in vitro on WT and M2327I mTOR followed by a kinase reaction (see Methods). The IC50s were determined by fitting to a standard 4-parameter logistic using GraphPad Prism V.5. The diagram shows the mean of n=3 data. The error bars represent the standard deviation between experiments. d, 293H cells were transfected and the complex was isolated as described in a. An in vitro kinase assay was performed and the level of P-AKT (S473) was determined by immunoblotting. Dots represent on each curve the relative P-AKT at different time points. The kinase activity curves were generated using Pad Prism v.6 after densitometry analysis was performed. All experiments were repeated at least three times.
Figure 3
Figure 3. RapaLink-1 is a potent mTOR inhibitor
a, Molecular model constructed by two available co-crystal structures, mTOR catalytic domain bearing TORKi PP242 (4JT5) and mTOR FRB domain/rapamycin/FKBP12 (1FAP). Dotted line represents a guide line for the linker design of bivalent mTOR inhibitors. b, RapaLink-1 structure is displayed. c, MCF-7 cells were treated with RapaLink-1, -2, and -3 or (d) with rapamycin, MLN0128, combination of rapamycin and MLN0128 or RapaLink-1 for 4 hours followed by immunoblotting. The rapamycin panel is the same shown in Figure 1b and the RapaLink-1 panel is the same shown in Figure 3c. For gel source data, see Supplemental Figure 3.
Figure 4
Figure 4. RapaLink-1 reverses resistance due to mTOR FRB and kinase domain mutations
a, MDA-MB-468 cells inducibly expressing mTOR F2108L or M2327I (b) or F2108L/M2327I mTOR double mutant (e) were treated as in Figure 3d and followed by immunoblotting. For gel source data, see Supplemental Figures 4, 5 and 6. All experiments were repeated at least three times. c, Mice bearing RR1 or TKi-R (d) xenograft tumors (n=5 for each group) were randomized to 4 different groups; (1) Vehicle (M, W, F); (2) rapamycin (10 mg/kg; M, W, F); (3) AZD8055 (75 mg/kg; M, W, F); (4) RapaLink-1 (1.5 mg/kg; weekly). Tumor size was measured by caliper two times per week. The results were reported as tumor volume (mm^3) ± standard deviation.
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