Journal: The Journal of Biological Chemistry

Article Title: Activation of Gcn2 by small molecules designed to be inhibitors

doi: 10.1016/j.jbc.2023.104595

Figure Lengend Snippet: Gcn2iB dose response for modulation of Gcn2 activity. A , HEK293 cells were pretreated with the indicated concentrations of Gcn2iB for 30 min and then treated with 12.5 nM HF or vehicle (No HF) for 6 h. Protein lysates were collected and the amounts of p-Gcn2 (T899), total Gcn2, p-eIF2α (S51), total eIF2α, Atf4, and actin were measured by immunoblot. Protein levels were quantitated by densitometry and are presented below each lane of their respective blots. Phospho-Gcn2 and p-eIF2α were first normalized to their corresponding total protein blots and p-Gcn2/Gcn2, p-eIF2α/eIF2α, and Atf4 are presented relative to vehicle-treated samples. A representative example from n = 2 independent experiments is shown. B , HEK293 cells expressing P (AAREx6) -Luc were pretreated with the indicated concentrations of Gcn2iB for 30 min, followed by treatment with 12.5 nM HF or vehicle (Gcn2iB only) for 6 h. Lysates were collected and luciferase activity was measured. Error bars represent the SD of three biological samples. Result shown is a representative example of n = 2 independent experiments. C , schematic of Gcn2 and ISR regulation by small molecules. Halofuginone (HF) and borrelidin inhibit aminoacylation of tRNA Pro and tRNA Thr , respectively, triggering increases in uncharged tRNAs that lead to activation of Gcn2. Low doses of Gcn2iB and other kinase inhibitors neratinib, dovitinib, and dabrafenib can activate Gcn2, leading to increased phosphorylation of p-eIF2α and enhanced expression of the downstream ISR effector, Atf4. By contrast, high doses of Gcn2iB and A-92 inhibit Gcn2 despite activation by uncharged tRNAs and lower p-eIF2α and Atf4 expression and activity. ISR, integrated stress response.

Article Snippet: Express #HY-125021), dabrafenib (SelleckChem #52807), borrelidin (Tocris #4706), neratinib (Med.

Techniques: Activity Assay, Western Blot, Expressing, Luciferase, Activation Assay, Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: Activation of Gcn2 by small molecules designed to be inhibitors

doi: 10.1016/j.jbc.2023.104595

Figure Lengend Snippet: Activation of Gcn2 by gatekeeper substitution and kinase inhibitor binding. A , Atf4-Luc activity was measured in Gcn2 KO HEK293 cells expressing M802V or M802F mutant versions of Gcn2. Cells were treated with the indicated concentration of Gcn2iB or vehicle for 6 h. Atf4-Luc is expressed as a percentage relative to untreated cells. Error bars represent the SD of three biological replicates. B , Levels of Atf4-Luc were measured in cells expressing WT, M802V, or M802F versions of Gcn2. WT Gcn2 expressing cells were pretreated with the indicated concentration of A-92 for 30 min and then 25 nM HF was added to the medium for an additional 6 h. In parallel, cells expressing Gcn2 M802F or M802V were treated with only A-92 for 6 h. Levels of Atf4-Luc are presented as a percentage relative to untreated cells (for M802F and M802V) or cells treated with HF only (for WT Gcn2). Error bars represent the SD of three biological replicates. The IC 50 values for A-92 were calculated using variable slope (four parameter) nonlinear regression. Values for IC 50 are displayed in the table below the graph. C , Gcn2 KO HEK293 cells were transfected with empty vector (−), WT, K619A, T899A, or M2 Gcn2. Cells were treated with vehicle, 50 nM Gcn2iB, 1 μM neratinib, 2 μM dovitinib, or 2 μM dabrafenib for 6 h and then Atf4-Luc activity was measured and presented normalized to vehicle-treated cells with empty vector (−). Error bars represent the SD of three biological replicates, which are each represented by points. Significant changes in Atf4-Luc activity were measured by two-way ANOVA comparing each treatment to the vehicle for that genotype using Dunnett correction for multiple hypothesis testing: ∗ = Adj. p < 0.05, ∗∗∗ = p < 0.001, ∗∗∗∗ = p < 0.0001. D , HEK293 cells containing the integrated P (AAREx6) -Luc reporter were treated with the indicated concentrations of Dovitinib or Neratinib for 6 h, and luciferase activity was measured and is presented as a percentage of Veh-treated luciferase activity. Error bars represent the SD of three biological samples. The overlaid curves were generated using Bell-shaped nonlinear regression in GraphPad Prism, where x is concentration and y is luciferase activity. E , cells expressing endogenous Gcn2 and P (AAREx6) -Luc were pretreated with the indicated concentrations of A-92 for 30 min and then treated with 12.5 nM halofuginone, 1 μM borrelidin, 31.25 nM Gcn2iB, 1 μM neratinib, 2 μM dovitinib, or 2 μM dabrafenib for 6 h. Luciferase activity was measured and is expressed as a percentage relative to cells treated with agonist only. Error bars represent the SD of three biological replicates. IC 50 values for inhibition by A-92 were calculated using variable slope (four parameter) nonlinear regression and are displayed in the table to the right. HF, Halofuginone.

Article Snippet: Express #HY-125021), dabrafenib (SelleckChem #52807), borrelidin (Tocris #4706), neratinib (Med.

Techniques: Activation Assay, Binding Assay, Activity Assay, Expressing, Mutagenesis, Concentration Assay, Transfection, Plasmid Preparation, Luciferase, Generated, Inhibition

Journal: Cancers

Article Title: Integrative In Vivo Drug Testing Using Gene Expression Signature and Patient-Derived Xenografts from Treatment-Refractory HER2 Positive and Triple-Negative Subtypes of Breast Cancer

doi: 10.3390/cancers11040574

Figure Lengend Snippet: In vivo efficacy of PX-478 and neratinib against PDX models from PT9 (HR − /HER2 + subtype). ( A ) PT9 treatment history. See text for details. Scale bar, one year. ( B ) Tumor volumes (F3) were determined in female mice ( n = 3–4) administered PX-478 (10 mg/kg), neratinib (20 mg/kg), or their combination three times a week for 40 days by gavage; mice administered saline served as controls. Tumor volumes at 40 days are presented as means ± SD ( p ≤ 0.05 for control vs. PX-478 and control vs. PX-478 + neratinib; p = 0.28 for control vs. neratinib; unpaired t -test). ( C ) MRI images of tumors 30 days after administration of drugs. Red arrows indicate necrosis and green arrows are traces of blood flow. ( D ) H&E staining and immunohistochemical analyses of HER2, Ki-67, VEGFA and HIF1A in PDX tumors from PT9 after treatment with drugs. Scale bar, 200 μm.

Article Snippet: The following drugs were used for in vivo treatment of tumor-bearing mice (low passage PDXs, ≤F4): The HIF1-inhibitor, PX-478 (10 mg/kg, by oral gavage, three times a week for 2 months; Selleckchem, Houston, TX, USA); the EGFR/ERBB2 inhibitor, neratinib (20 mg/kg, orally, three times per week for 2 months; Selleckchem, Houston, TX, USA); docetaxel (3 mg/kg, by intraperitoneal administration, twice weekly for 4 weeks; Selleckchem, Houston, TX, USA); the anti-VEGF (vascular endothelial growth factor) monoclonal antibody, bevacizumab (5 mg/kg, by intraperitoneal administration, twice weekly for 4 weeks; Roche, Basel, Switzerland); the VEGFR/PDGFR/Raf inhibitor, sorafenib (120 mg/kg, via oral gavage, daily for 3 weeks; Bayer, Leverkusen, Germany); and the mTOR inhibitor, everolimus (20 mg/kg, by oral gavage, daily for 3 weeks; Novartis, Basel, Switzerland).

Techniques: In Vivo, Saline, Control, Staining, Immunohistochemical staining

Journal: Cancers

Article Title: Integrative In Vivo Drug Testing Using Gene Expression Signature and Patient-Derived Xenografts from Treatment-Refractory HER2 Positive and Triple-Negative Subtypes of Breast Cancer

doi: 10.3390/cancers11040574

Figure Lengend Snippet: In vivo efficacy of PX-478 and neratinib against PDX models from PT10 (HR − /HER2 + subtype). ( A ) PT10 treatment history. See text for details. Scale bar, one year. ( B ) Tumor volumes (F3) were determined in female mice ( n = 2–3) given PX-478 (10 mg/kg), neratinib (20 mg/kg), or their combination three times a week for 30 days by peroral administration; mice administered saline served as controls. Tumor volumes at 30 days are presented as means ± SD ( p ≤ 0.05 for control vs. PX-478 and control vs. PX-478 + neratinib; p = 0.32 for control vs. neratinib; unpaired t -test). ( C ) MRI images of tumors at 30 days after administration of drugs. Red arrows represent necrosis and green arrows are traces of blood flow. ( D ) H&E staining and immunohistochemical analyses of HER2, Ki-67, VEGFA and HIF1A in PDX tumors from PT10 after treatment with drugs. Scale bar, 200 μm.

Article Snippet: The following drugs were used for in vivo treatment of tumor-bearing mice (low passage PDXs, ≤F4): The HIF1-inhibitor, PX-478 (10 mg/kg, by oral gavage, three times a week for 2 months; Selleckchem, Houston, TX, USA); the EGFR/ERBB2 inhibitor, neratinib (20 mg/kg, orally, three times per week for 2 months; Selleckchem, Houston, TX, USA); docetaxel (3 mg/kg, by intraperitoneal administration, twice weekly for 4 weeks; Selleckchem, Houston, TX, USA); the anti-VEGF (vascular endothelial growth factor) monoclonal antibody, bevacizumab (5 mg/kg, by intraperitoneal administration, twice weekly for 4 weeks; Roche, Basel, Switzerland); the VEGFR/PDGFR/Raf inhibitor, sorafenib (120 mg/kg, via oral gavage, daily for 3 weeks; Bayer, Leverkusen, Germany); and the mTOR inhibitor, everolimus (20 mg/kg, by oral gavage, daily for 3 weeks; Novartis, Basel, Switzerland).

Techniques: In Vivo, Saline, Control, Staining, Immunohistochemical staining

Journal: Molecular Cancer Therapeutics

Article Title: Identification of Predictive ERBB Mutations by Leveraging Publicly Available Cell Line Databases

doi: 10.1158/1535-7163.mct-20-0590

Figure Lengend Snippet: Figure 6. Sensitivity of EGFR Y1069C and ERBB2 E936K to ERBB TKIs. Ba/F3 cells expressing the indicated EGFR (A) or ERBB2 (B) variants were cultured for 72 hours in the presence or absence of increasing concentrations of the ERBB TKIs erlotinib, lapatinib, afatinib, or neratinib. Cells expressing ERBB receptors were analyzed in the absence of IL3 and in the absence or presence of an activating ligand, 10 ng/mL EGF or 20 ng/mL NRG-1. Vector control cells were cultured in the presence of IL3. Cell viability was measured using MTT assays. The mean and SD are shown for the dose-response curves. IC50 values for the drug responses were calculated from three to six independent analyses after fitting the dose-response curves with four-parameter log-logistic function (LL.4; R). Data were statistically analyzed to determine the difference in IC50 in comparison to cells expressing the respective wild-type ERBB control using unpaired two-sample t test. P values <0.05 indicating enhanced sensitivity as compared with wild-type receptor are indicated in bold.

Article Snippet: The following day ERBB TKIs erlotinib, lapatinib, afatinib, or neratinib (all from Santa Cruz Biotechnology) were applied in a volume of 75 mL/well to reach a final concentration ranging from 0 to 10 mmol/L.

Techniques: Expressing, Cell Culture, Plasmid Preparation, Control, Comparison

Journal: British Journal of Cancer

Article Title: Evidence into practice: a national cohort study of NICE-recommended oncological drug therapy utilisation among women diagnosed with invasive breast cancer in England

doi: 10.1038/s41416-023-02439-z

Figure Lengend Snippet: Overview of NICE technology appraisal guidance with a positive recommendation published for invasive breast cancer.

Article Snippet: Neratinib (Nerlynx, Pierre Fabre) , HER2-targeting therapy , Extending adjuvant treatment of hormone receptor-positive, HER2-positive early breast cancer where trastuzumab was completed within the previous year. , November 2019 (TA612) [ ] , 2017 , 2018 , Provided according to the commercial agreement..

Techniques: Adjuvant