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. 2015 Apr 21;11(3):446-59.
doi: 10.1016/j.celrep.2015.03.040. Epub 2015 Apr 9.

Identification of a Non-Gatekeeper Hot Spot for Drug-Resistant Mutations in mTOR Kinase

Tzung-Ju Wu  1 , Xiaowen Wang  1 , Yanjie Zhang  2 , Linghua Meng  2 , John E Kerrigan  2 , Stephen K Burley  3 , X F Steven Zheng  4
Affiliations

Identification of a Non-Gatekeeper Hot Spot for Drug-Resistant Mutations in mTOR Kinase

Tzung-Ju Wu et al. Cell Rep. .

Abstract

Protein kinases are therapeutic targets for human cancer. However, "gatekeeper" mutations in tyrosine kinases cause acquired clinical resistance, limiting long-term treatment benefits. mTOR is a key cancer driver and drug target. Numerous small-molecule mTOR kinase inhibitors have been developed, with some already in human clinical trials. Given our clinical experience with targeted therapeutics, acquired drug resistance in mTOR is thought likely, but not yet documented. Herein, we describe identification of a hot spot (L2185) for drug-resistant mutations, which is distinct from the gatekeeper site, and a chemical scaffold refractory to drug-resistant mutations. We also provide new insights into mTOR kinase structure and function. The hot spot mutations are potentially useful as surrogate biomarkers for acquired drug resistance in ongoing clinical trials and future treatments and for the design of the next generation of mTOR-targeted drugs. Our study provides a foundation for further research into mTOR kinase function and targeting.

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Figures

Figure 1
Figure 1. Developing a yeast system to assay for mTOR kinase inhibition
(A) Wild type (WT) yeast cells were spread onto YPD plates and tested for sensitivity to structurally diverse mTOR kinase inhibitors by disc halo assay. Rapamycin was used as a positive control. (B) The N-terminus of TOR2 (1-2080 aa) was fused in frame with mTOR kinase domain (2140-2549 aa). The TOR2-mTOR fusion is expressed under the control of TOR2 promoter in a centromeric plasmid. (C) Yeast strain expressing WT TOR2 or TOR2-mTOR fusion was analyzed for expression by immunoblot with an antibody specific for mTOR kinase domain. PGK1 was used as a loading control and extracts from MCF7 breast cells were used as a positive control for mTOR. (D) TOR2-mTOR fusion was expressed in tor2-dg and tested for its ability to complement TOR2 function by growth at permissive and restrictive temperatures. (E) tor2-dg cells expressing TOR2 or TOR2-mTOR were serially diluted by 10-fold and tested for drug sensitivity on plates containing BEZ235 and OSI-027.
Figure 2
Figure 2. Enhancement of yeast cell permeability to structurally diverse mTOR kinase inhibitors by amphotericin B
(A) tor2-dg cells expressing TOR2-mTOR were spread on synthetic complete (SC)-leucine plate and tested for drug sensitivity by disc halo assay using filter discs containing different mTOR kinase inhibitors supplemented with the drug carrier DMSO or amphotericin B (10 μM). (B) Similar to Figure 2A except the filter discs were supplemented with miconazole 50 μM). (C) Similar to Figure 2A except the filter discs were supplemented with caspofungin 50 μM).
Figure 3
Figure 3. Mutational analysis of the gatekeeper residue in mTOR kinase
(A) Sequence alignment of the gatekeeper site for PKA, c-Kit, EGFR, ABL, p110-PI3Kα, and mTOR. Arrowhead marks the gatekeeper residue. (B) Electrostatic model of the ATP-binding pocket of mTOR kinase (PDB ID code 4JSP). The I2237 position is as indicated. Atom is colored as follows: N, blue; O, red; P, orange; S, yellow; Mg+2, green. Surface representation is as follows: hydrophobic residue, red; neutral residue, white; hydrophilic residue, blue. (C) tor2-dg cells expressing WT or mutant TOR2-mTOR were serially diluted by 10-fold and assayed for drug sensitivity on SC-leucine plates containing BEZ235, OSI-027, or Torin2, or AZD8055, BEZ235, INK128, PF-04691502 or PKI-587 in the presence of amphotericin B. (D) tor2-dg cells expressing WT or mutant TOR2-mTOR were serially diluted by 10-fold and assayed for cell growth at different temperatures. Vector and TOR2 plasmids were used as a negative and positive control, respectively. (E) Summary of gatekeeper mutations and their effects on mTOR kinase function. “+”: normal function; “−”: minor defect; “−−”: moderate defect, and “−−−”: severe defect.
Figure 4
Figure 4. Identification of a hotspot for drug-resistant mutations in mTOR kinase domain
(A) Scheme of a yeast-based screen for drug-resistant mutations in mTOR kinase domain. mTOR kinase domain is amplified by error-prone PCR to generate randomized mutations, which is then recombined in frame into the TOR2-mTOR plasmid by gap-repair in tor2-dg cells, and is selected on SC-leucine minus plates. Replica plating is then made onto SC-leucine plates containing DMSO or mTOR kinase inhibitor for selection of drug resistant clones. (B) tor2-dg cells expressing WT or mutant TOR2-mTOR were serially diluted by 10-fold and assayed for sensitivity to different mTOR kinase inhibitors in the presence of amphotericin B. Vector and TOR2 were used as controls. Drug resistant assay was performed at 37°C in the presence of amphotericin B (except OSI-027). (C) Systematic mutational analysis of L2185 on drug resistance. tor2-dg cells expressing WT or mutant TOR2-mTOR carrying all possible mutations at L2185 were serially diluted by 10-fold and tested for sensitivity to different mTOR kinase inhibitors at 37°C. AZD8055, BEZ235, INK128, PF-04691502 and PKI-587 were supplemented with amphotericin B.
Figure 5
Figure 5. L2185A mutation confers resistance to mTOR kinase inhibitors in colorectal cancer models
(A) SW480 cells carrying homozygous WT mTOR or L2185A mutant alleles were treated with various concentrations of AZD8055, INK-128, OSI-027 and PP242 for 2 day. Growth of SW480 cells was measured by SRB assay. Data represent means ± SD in three independent experiments. (B) SW480 cells carrying homozygous WT and L2185A mutant mTOR allele were treated with a single dose of AZD8055 (100 nM), INK-128 (100nM), OSI-027 (6,000 nM), and PP242 (2,000 nM) for different times. Cell growth was measured by SRB assay. The drug carrier DMSO was used as a control. Data represent means ± SD in three independent experiments. (C) SW480 cells carrying homozygous WT and L2185A mutant mTOR were treated with various concentrations of INK-128, OSI-027, AZD8055 and PP242 for 1 hr. The effect on the level of P-S6K, S6K, P-4E-BP1, 4EB-P1, P-AKT and AKT was analyzed by immunoblot. (D) SW480 cells carrying homozygous WT and L2185A mutant mTOR alleles were treated with various concentrations of BEZ235, PF-0691502 and Torin2 for 2 day. The growth of SW480 cells was measured by SRB assay. Data represent means ± SD in three independent experiments.
Figure 6
Figure 6. L2185A mutation renders resistance of xenograft tumors to mTOR kinase inhibitors
(A) Mice bearing xenograft tumors derived from SW480 cells expressing WT mTOR were administered with INK128 at 1 mg/kg or 0.3 mg/kg, once daily via intraperitoneal injection. Shown are representative animals and excised tumors after drug treatment. (B) Same as Figure 6A except xenograft tumors were derived from SW480 carrying homozygous mTOR(L2185A) alleles. (C) Tumor volume measurement for SW480 xenograft tumors expressing mTOR (WT) (expressed as means ± SD; n = 8, *P < 0.01, vs. vehicle control). (D) Tumor volume measurement for SW480 xenograft tumors expressing mTOR(L2185A) mutant (expressed as means ± SD; n = 8). (E) Tissue extracts from xenograft tumors at the end of treatment with or without 1 mg/kg INK128 were analyzed for the level of P-S6K, S6K, P-S6, S6, P-4E-BP1, 4E-BP1, P-AKT and AKT was analyzed by immunoblot. Six tumor samples from each animal group were shown with each lane representing an individual tumor sample.
Figure 7
Figure 7. Saturation mutagenesis of highly conserved hydrophobic residues of mTOR kinase domain
(A) Shown is hydrophobic surface representation of the ATP-binding pocket of mTOR kinase bound with an ATP molecule (PDB ID code 4JSP). ATP atoms are colored as follows: N, blue; O, red; P, orange; S, yellow; Mg+2, green. Protein surface representation is as follows: hydrophobic residue, red; neutral residue, white; hydrophilic residue, blue. (B) Summary of the effect of mutations at different conserved hydrophobic residues in mTOR kinase domain. “+”: normal; “−”: minor defect; “−−”: moderate defect; “−−−”: severe defect. (C) Stacked bar graph summarizes each category of mutations in terms of function as a percentage of total mutations. (D) Stacked bar graph summarizing hydrophobic, neutral, and hydrophilic mutations as a percentage of total mutations with normal mTOR kinase function. (E) Shown is yeast growth-based assay for several representative mutations with severe loss-of-function in mTOR kinase. (F) WT and mutant Flag-mTOR were transiently expressed in HEK293T cells, immunoprecipitated, and assayed for mTOR kinase activity toward recombinant 4E-BP1 in vitro. Phosphorylation of 4E-BP1 was analyzed by immunoblot using a P-4E-BP1 specific antibody. Data represent means ± SD in three independent experiments.
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References

    1. Alarcon CM, Cardenas ME, Heitman J. Mammalian RAFT1 kinase domain provides rapamycin-sensitive TOR function in yeast. Genes & development. 1996;10:279–288. - PubMed
    1. Azam M, Latek RR, Daley GQ. Mechanisms of autoinhibition and STI-571/imatinib resistance revealed by mutagenesis of BCR-ABL. Cell. 2003;112:831–843. - PubMed
    1. Bell DW, Gore I, Okimoto RA, Godin-Heymann N, Sordella R, Mulloy R, Sharma SV, Brannigan BW, Mohapatra G, Settleman J, et al. Inherited susceptibility to lung cancer may be associated with the T790M drug resistance mutation in EGFR. Nat Genet. 2005;37:1315–1316. - PubMed
    1. Bjornsti M-A, Houghton PJ. The tor pathway: a target for cancer therapy. Nat Rev Cancer. 2004;4:335–348. - PubMed
    1. Chen J, Zheng XF, Brown EJ, Schreiber SL. Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue. PNAS. 1995;92:4947–4951. - PMC - PubMed

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