Research ArticleBiochemistry

Covalent inhibitors of EGFR family protein kinases induce degradation of human Tribbles 2 (TRIB2) pseudokinase in cancer cells

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Science Signaling  25 Sep 2018:
Vol. 11, Issue 549, eaat7951
DOI: 10.1126/scisignal.aat7951
  • Fig. 1 Full-length TRIB2 is a target for protein kinase inhibitors in vitro.

    (A) Top: Sequence alignment of human TRIB2, TRIB1, TRIB3, and STK40/SgK495, highlighting Cys-rich residues (numbered in red) in the TRIB2 pseudokinase domain. Bottom: Blot of the recombinant proteins used for in vitro analysis. The indicated purified proteins (5 μg each) were resolved by SDS–polyacrylamide gel electrophoresis (SDS-PAGE). WT, wild-type; BSA, bovine serum albumin. (B) Thermal denaturation profiles of recombinant proteins. A representative unfolding profile is shown. Tm values (±SD) were obtained from three separate fluorescence profiling experiments, each point assayed in duplicate. (C) The ability of GST-TRIB2 to interact with active [3-phosphoinositide-dependent protein kinase 1 (PDK1)–phosphorylated] or inactive (non–PDK1-phosphorylated) S473D AKT1 was assessed by glutathione-Sepharose pulldown followed by immunoblotting. Left: “Master-mix” input. (D) Transient transfection of TET-inducible FLAG-TRIB2 leads to increased AKT phosphorylation on Ser473. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (E) TRIB2 DSF screen using PKIS. His-TRIB2 (5 μM) was used for all DSF analysis. ΔTm values were calculated for each compound (N = 2). Scattergraph of data highlights a wide variety of compounds that either stabilize or destabilize TRIB2 in vitro. Cutoff values of >+3.5°C and <−2°C were used to designate hits. (F) Comparative DSF analysis of clinical and preclinical kinase inhibitors as potential TRIB2-binding compounds. LAP, lapatinib; TAK, TAK-285; AFA, afatinib; NER, neratinib; OSI, osimertinib; IBR, ibrutinib; ERL, erlotinib; GEF, gefitinib. (G) Dose-dependent analysis of thermal shifts induced by clinical TRIB2-binding compounds. Compounds were tested at 5, 10, 20, 40, 80, and 160 μM. (H) Profiling of TRIB2 and C104Y with selected inhibitors by DSF.

  • Fig. 2 TRIB2 thermal stability is modulated through Cys binding to covalent inhibitors.

    (A) Top: Schematic cartoon of TRIB2 with domain boundaries numbered and cysteine residues highlighted (red). Bottom: SDS-PAGE of 5-μg recombinant TRIB2 proteins. (B) Thermal denaturation profiles of 5 μM WT-TRIB2 (amino acids 1 to 343), three truncated variants, and an AQLAA triple-point mutant. Representative curves for each protein and average Tm values (±SD) are shown, calculated from N = 3 experiments. (C) Thermal shift analysis of TRIB2 deletion and AQLAA proteins measured in the presence of a panel of compounds (20 μM). The change in Tm value (ΔTm) is reported from N = 3 experiments, each performed in triplicate. (D) Thermal denaturation profiles for purified TRIB2 and C96S, C104S, and C96/104S proteins. (E) Thermal shift analysis of TRIB2 Cys-mutated proteins measured in the presence of a panel of compounds (20 μM). The change in Tm value (ΔTm) is reported from N = 3 experiments.

  • Fig. 3 Afatinib promotes rapid degradation of FLAG-TRIB2 in an inducible HeLa model.

    (A) Uninduced (−TET) or TET-induced (+TET) HeLa cells containing a stably integrated FLAG-TRIB2 transgene were serum-starved for 16 hours before the addition of serum and lysed at the indicated times. Whole-cell extracts were blotted with FLAG antibody to detect FLAG-TRIB2 and pSer473 AKT. Total AKT and total GAPDH served as loading controls. (B) A selection of clinically approved kinase inhibitors, including dual EGFR/HER2- and EGFR-specific compounds, were added to TET-induced cells at a final concentration of 10 μM. Stable cells were induced to express FLAG-tagged TRIB2 with TET for 16 hours before inhibitor treatment for 4 hours. Whole-cell extracts were immunoblotted with FLAG, phosphorylated ERK (pERK 1/2), total ERK, or GAPDH antibodies. (C) Stable HeLa cells were incubated with TET for 16 hours and then incubated with DMSO (top panels) or 10 μM afatinib (bottom panels) before lysis at the indicated time. One-hour and 16-hour samples were pretreated with λ protein phosphatase (λPP) before SDS-PAGE. Whole-cell extracts were immunoblotted with FLAG or GAPDH antibodies. Blots are representative of three independent experiments.

  • Fig. 4 “On-target” degradation of TRIB2 by afatinib: C96/104S TRIB2 double-point mutant is resistant to degradation.

    (A) The indicated concentration of afatinib, lapatinib, or TAK-285 was incubated for 4 hours with isogenic stable HeLa cells expressing FLAG-tagged WT-TRIB2, C96S, or C96/104S TRIB2 (induced by TET exposure for 16 hours). After lysis, whole-cell extracts were immunoblotted with the indicated antibodies. Right: FLAG-TRIB2 abundance was quantified after exposure to 10, 15, and 20 μM afatinib relative to DMSO controls using ImageJ densitometry software. Data are means ± SD from N = 3 independent biological replicates. (B) WT and C96/104S stable HeLa cell lines were subjected to serum block-and-release protocol in the presence (+TET) or absence (−TET) of TET. Subsequently, the indicated compounds (10 μM) were added for 4 hours before cell lysis and immunoblotting with the indicated antibodies. (C) FLAG-tagged TRIB2-expressing HeLa cells were incubated with 0.1% (v/v) DMSO or afatinib (10 μM), in the presence or absence of MG132 (10 μM for 4 hours, left) or at the indicated time points (right) before lysis and processing for immunoblotting. (D) Left: FLAG-TRIB2–expressing stable cells were incubated with the indicated concentration of MG132 in the presence or absence of 10 μM afatinib for 4 hours before cell lysis and immunoblotting. Right: FLAG-TRIB2–expressing stable cells were incubated for 1 hour with MG132 (10 μM), bortezomib (BOR; 10 μM), AICAR (AIC; 1 mM), or chloroquine (CLQ; 50 μM) before the addition of afatinib (10 μM) for an additional 4 hours followed by lysis and immunoblotting with the indicated antibodies. Blots are representative of three independent experiments. *P ≤ 0.05 , **P ≤ 0.01.

  • Fig. 5 Afatinib rapidly destabilizes endogenous TRIB2 and specifically induces caspase 3 cleavage and U937 cytotoxicity.

    (A) Endogenous TRIB2 is destabilized in human U937 cells in a dose-dependent manner after a 4-hour exposure to afatinib. Cells were incubated with either 0.1% (v/v) DMSO or the indicated concentrations of afatinib, lapatinib (10 μM), erlotinib (10 μM), or the proteasome inhibitor bortezomib (10 μM) for 4 hours before lysis and immunoblotting of endogenous TRIB2, pERK, or cleaved caspase 3. GAPDH served as a loading control. (B) Endogenous TRIB2 is destabilized after exposure to afatinib for 24 hours, concomitant with reduced AKT phosphorylation at Ser473. After cell collection and lysis, whole-cell extracts were immunoblotted with the indicated antibodies. AKT and GAPDH served as loading controls; erlotinib and bortezomib were used at 10 μM. (C) The cytotoxicity of a panel of TRIB2-destabilizing small molecules (neratinib) was compared to EGFR inhibitors or the TRIB2 stabilizer TAK-285. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays were performed after 72 hours of compound exposure, with bortezomib used as a positive control. IC50 values (nM, mean ± SD) were derived from N = 3 independent experiments, each performed in triplicate. Statistical analysis confirmed a significant difference in cytotoxicity between afatinib and erlotinib (Student’s t test; P = 0.0104), between bortezomib and erlotinib (P = 0.0072), and between neratinib and erlotinib (P = 0.0104). Blots are representative of three independent experiments.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/549/eaat7951/DC1

    Fig. S1. Discovery of multiple PKIS compounds as TRIB2-binding compounds.

    Fig. S2. Chemical structure of preclinical and clinical compounds evaluated in this study.

    Fig. S3. Validation of DSF assay using TRIB1 and a PKAc counterscreen.

    Fig. S4. Thermal melting profiles of TRIB2 using DSF.

    Fig. S5. Analysis of C104Y mutation in TRIB2.

    Fig. S6. MS-based analysis of the covalent TRIB2:afatinib complex.

    Fig. S7. MST assay.

    Fig. S8. Molecular docking analysis.

    Fig. S9. A new TRIB2 antibody for quantitative analysis of TRIB2 expression levels and stability.

    Fig. S10. Analysis of TRIB2 dephosphorylation in cell extracts.

    Fig. S11. TRIB2 binding to afatinib induces destabilization relative to DMSO in a whole-cell thermal shift assay (CETSA).

    Fig. S12. Comparative protein expression analysis of stable HeLa FLAG-TRIB2 cell lines.

    Fig. S13. Lack of effect of the noncovalent TRIB1- and TRIB2-destabilizing compound GW804482X on TRIB2 stability in TRIB2-expressing HeLa cells.

    Table S1. PKIS compound screening data for full-length TRIB2.

  • This PDF file includes:

    • Fig. S1. Discovery of multiple PKIS compounds as TRIB2-binding compounds.
    • Fig. S2. Chemical structure of preclinical and clinical compounds evaluated in this study.
    • Fig. S3. Validation of DSF assay using TRIB1 and a PKAc counterscreen.
    • Fig. S4. Thermal melting profiles of TRIB2 using DSF.
    • Fig. S5. Analysis of C104Y mutation in TRIB2.
    • Fig. S6. MS-based analysis of the covalent TRIB2:afatinib complex.
    • Fig. S7. MST assay.
    • Fig. S8. Molecular docking analysis.
    • Fig. S9. A new TRIB2 antibody for quantitative analysis of TRIB2 expression levels and stability.
    • Fig. S10. Analysis of TRIB2 dephosphorylation in cell extracts.
    • Fig. S11. TRIB2 binding to afatinib induces destabilization relative to DMSO in a whole-cell thermal shift assay (CETSA).
    • Fig. S12. Comparative protein expression analysis of stable HeLa FLAG-TRIB2 cell lines.
    • Fig. S13. Lack of effect of the noncovalent TRIB1- and TRIB2-destabilizing compound GW804482X on TRIB2 stability in TRIB2-expressing HeLa cells.
    • Table S1. PKIS compound screening data for full-length TRIB2.

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