Journal: Genome Medicine

Article Title: The expressed mutational landscape of microsatellite stable colorectal cancers

doi: 10.1186/s13073-021-00955-2

Figure Lengend Snippet: Correlation between TP53 and RAS / BRAF V600E mutation expression levels and sensitivity to targeted anticancer agents in pre-clinical models. a Sensitivity to the EGFR inhibitor erlotinib, the MEK inhibitor trametinib, and the MDM2 inhibitor idasanutlin in a panel of 29 unique CRC cell lines plotted according to RAS / BRAF V600E or TP53 mutation status, as indicated (mut, mutated; wt, wild-type; color codes are shown in c ). Higher DSS indicates stronger sensitivity. p -value is from Welch’s t -test of wild-type versus mutated samples. b , c Upper panels show the mutation status for RAS / BRAF V600E and TP53 in each of the 7 selected cell lines and 8 patient-derived organoids (PDOs). Scatter plots show the DSS of matched drugs versus mutant allele expression levels (color-coded as indicated). Spearman’s correlations in blue are for KRAS -mutated PDOs only (excluding the single NRAS -mutated sample). d Scatter plot of RNA-level versus DNA-level MAFs of RAS and TP53 in matched primary and metastatic tumor samples from each of four patients (three with RAS mutations and two with TP53 mutations). Patient 2 showed higher relative expression of the RAS mutant allele in the metastasis

Article Snippet: The MDM2-TP53 inhibitor idasanutlin (MedChemExpress, Monmouth Junction, NJ, USA), three EGFR inhibitors (afatinib: Selleck Chemicals; erlotinib: MedChemExpress; and lapatinib: LC Laboratories, Woburn, MA, USA) and two MEK inhibitors (binimetinib and trametinib, ChemieTek, Indianapolis, IN, USA) were included in the screens at five and nine different concentrations over a 10,000-fold concentration range each (typically 1–10,000 nmol/L) in the cell lines and PDOs, respectively.

Techniques: Mutagenesis, Expressing, Derivative Assay

Journal: Cells

Article Title: EGFR-D770>GY and Other Rare EGFR Exon 20 Insertion Mutations with a G770 Equivalence Are Sensitive to Dacomitinib or Afatinib and Responsive to EGFR Exon 20 Insertion Mutant-Active Inhibitors in Preclinical Models and Clinical Scenarios

doi: 10.3390/cells10123561

Figure Lengend Snippet: Ba/F3 system isogenic preclinical models of EGFR exon 20 insertions mutations to probe EGFR-TKIs. ( A ) Therapeutic window of different EGFR-TKIs to a set of EGFR exon 20 mutants. Cells were plated at a density of 10,000 cells per well (96-well plates) and grown over 3 days after treatment. Logarithm of the 50% inhibitory concentration (IC 50 ) of EGFR exon 20 mutants compared to EGFR-WT is plotted with 3 separate experiments used to generate IC 50 . Values below zero (0) indicate sensitivity, while values above 0 indicate resistance to EGFR-TKIs. The therapeutic window of Ba/F3 cells with EGFR -D770>GY are contrasted with other exon 20 insertion mutations. ( B ) Dose-response proliferation assays (the proportional percent viability) of dacomitinib and afatinib for cells with EGFR -D770>GY compared with those with EGFR -A767_V769dupASV. Three separate experiments were used to generate IC 50 , and standard deviations are depicted in vertical bars. For afatinib, the 95% confidence intervals (95%CIs) did not overlap for both mutants with values of IC 50 31.10 nM (95%CI 20.37–47.47) and 0.10 nM (95%CI 0.07–0.15). For dacomitinib, the 95% CIs did not overlap for both mutants with values of IC 50 86.90 nM (95%CI 65.43–115.40) and 0.005 nM (95%CI 0.002–0.016). ( C ) Western blotting of Ba/F3 cells driven by EGFR-D770>GY and EGFR-A767_V769dupASV mutants. Cells were treated with the EGFR-TKI dacomitinib for 8 h at the indicated ascending concentrations. pEGFR, phosphorylated EGFR at position 1068, total EGFR and β-actin as a loading control are displayed in the graphical compilation.

Article Snippet: Erlotinib, afatinib, osimertinib (LC Laboratories Woburn, MA, USA), dacomitinib (Selleckchem [Houston, TX, USA]), poziotinib (AdooQ BioScience Irvine, CA, USA) and mobocertinib (MedChemExpress, Monmouth Junction, NJ, USA) were dissolved in dimethyl sulfoxide and stored at −80 °C.

Techniques: Concentration Assay, Western Blot, Control

Journal: Cells

Article Title: EGFR-D770>GY and Other Rare EGFR Exon 20 Insertion Mutations with a G770 Equivalence Are Sensitive to Dacomitinib or Afatinib and Responsive to EGFR Exon 20 Insertion Mutant-Active Inhibitors in Preclinical Models and Clinical Scenarios

doi: 10.3390/cells10123561

Figure Lengend Snippet: Outcomes of reported patients with advanced/metastatic lung cancers harboring EGFR exon 20 insertion mutations encompassing G770 equivalence.

Article Snippet: Erlotinib, afatinib, osimertinib (LC Laboratories Woburn, MA, USA), dacomitinib (Selleckchem [Houston, TX, USA]), poziotinib (AdooQ BioScience Irvine, CA, USA) and mobocertinib (MedChemExpress, Monmouth Junction, NJ, USA) were dissolved in dimethyl sulfoxide and stored at −80 °C.

Techniques: Mutagenesis

Journal: Heliyon

Article Title: Activating mutations in EGFR and PI3K promote ATF4 induction for NSCLC cell survival during amino acid deprivation

doi: 10.1016/j.heliyon.2023.e14799

Figure Lengend Snippet: Involvement of the mTOR pathway in ATF4 regulation downstream of PI3K a, b. Immunoblot analyses with the indicated antibodies in NCI–H1975 and HCC827 cells transfected with siRNA against RPTOR (a) or RICTOR (b) for 48 h followed by treatment with 1 U/mL ASNase for 6 h. β-Actin was used as an internal control. c–f. Immunostaining for ATF4 in NCI–H1975 and HCC827 cells treated with the indicated concentration of rapamycin (c, e) or PP242 (d, f) in the presence or absence of ASNase (c, d) or His (NCI–H1975 2 mM, HCC827 1 mM; e, f) for 6 h. Bar graphs show the quantification of nuclear ATF4 signal intensities (mean ± S.D., n = 3, **; p < 0.005, ****; p < 0.0001, and ns; not significant). The non-adjusted, full images for immunoblots are shown in Fig. S8.

Article Snippet: Osimertinib, alpelisib, buparlisib, copanlisib, GSK2636771, MK2206, trametinib, rapamycin, PP242, halofuginone (HF) (Selleckchem, Houston, TX, USA), GCN2iB (MedChemExpress, Monmouth, NJ, USA), and silvestrol (ChemScene, Monmouth, NJ, USA) were dissolved in dimethyl sulfoxide (Wako) as a stock solution. l -Asparaginase (ASNase; Kyowa Kirin Co., Ltd., Tokyo, Japan), L-Histidinol dihydrochloride (His), and puromycin (Sigma-Aldrich, Tokyo, Japan) were dissolved in sterilized distilled water.

Techniques: Western Blot, Transfection, Control, Immunostaining, Concentration Assay

Journal: Molecular cancer research : MCR

Article Title: TAS6417/CLN-081 is a pan-mutation-selective EGFR tyrosine kinase inhibitor with a broad spectrum of preclinical activity against clinically-relevant EGFR mutations

doi: 10.1158/1541-7786.MCR-19-0419

Figure Lengend Snippet: Types, frequency and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor approval for EGFR kinase domain mutations in lung cancer ( 3 ).

Article Snippet: Osimertinib, poziotinib, afatinib, and erlotinib were purchased from CHEMSCENE, LLC, MedChem Express, Selleck Chemicals, and LC Laboratories, respectively.

Techniques: Mutagenesis

Journal: Molecular cancer research : MCR

Article Title: TAS6417/CLN-081 is a pan-mutation-selective EGFR tyrosine kinase inhibitor with a broad spectrum of preclinical activity against clinically-relevant EGFR mutations

doi: 10.1158/1541-7786.MCR-19-0419

Figure Lengend Snippet: A-F, Dose inhibition curves and SDs of three independent experiments for erlotinib (blue), afatinib (red), osimertinib (green), poziotinib (purple), and TAS6417 (orange) in HCC827 (A), PC-9 (B), H1975 (C), H820 (D), BEAS-2B (E), and NHEK-Neo (F) cells. G, Western blot analysis showing the effects of increasing concentration of osimertinib, poziotinib, and TAS6417 in BEAS-2B and H1975 cells. H, Selectivity indexes showing mutation-selectivity calculated as ratios of the IC50 values of NHEK-Neo to those of each cell line. I, Selectivity indexes calculated as ratios of the IC50 values Ba/F3 expressing WT EGFR to those of Ba/F3 cells expressing each mutant EGFR.

Article Snippet: Osimertinib, poziotinib, afatinib, and erlotinib were purchased from CHEMSCENE, LLC, MedChem Express, Selleck Chemicals, and LC Laboratories, respectively.

Techniques: Inhibition, Western Blot, Concentration Assay, Mutagenesis, Expressing

Journal: Molecular cancer research : MCR

Article Title: TAS6417/CLN-081 is a pan-mutation-selective EGFR tyrosine kinase inhibitor with a broad spectrum of preclinical activity against clinically-relevant EGFR mutations

doi: 10.1158/1541-7786.MCR-19-0419

Figure Lengend Snippet: A-E, Dose inhibition curves and SDs of three independent experiments for erlotinib (blue), afatinib (red), osimertinib (green), poziotinib (purple), and TAS6417 (orange) in Ba/F3 cells expressing EGFR G719A (A), S768I+V769I (B), L861Q (C), G719A+T790M (D), L861Q+T790M (E). F, Selectivity indexes of TAS6417 and other EGFR-TKIs tested were calculated the WT/mut ratio of the IC50 values.

Article Snippet: Osimertinib, poziotinib, afatinib, and erlotinib were purchased from CHEMSCENE, LLC, MedChem Express, Selleck Chemicals, and LC Laboratories, respectively.

Techniques: Inhibition, Expressing

Journal: Molecular cancer research : MCR

Article Title: TAS6417/CLN-081 is a pan-mutation-selective EGFR tyrosine kinase inhibitor with a broad spectrum of preclinical activity against clinically-relevant EGFR mutations

doi: 10.1158/1541-7786.MCR-19-0419

Figure Lengend Snippet: A-D, Dose inhibition curves and SDs of three independent experiments for erlotinib (blue), afatinib (red), osimertinib (green), poziotinib (purple), and TAS6417 (orange) in BID007 (A), BID019 (B), H1975-InsSVD (C), and LXF2478L cells (D). E, Western blot analysis showing the effects of increasing concentration of osimertinib, poziotinib, and TAS6417 in BID007 and BID019 cells. F, Selectivity indexes showing mutation-selectivity calculated as ratios of the IC50 values of NHEK-Neo to those of each cell line. G, Selectivity indexes calculated as ratios of the IC50 values Ba/F3 expressing WT EGFR to those of Ba/F3 cells expressing each ins20 mutant EGFR.

Article Snippet: Osimertinib, poziotinib, afatinib, and erlotinib were purchased from CHEMSCENE, LLC, MedChem Express, Selleck Chemicals, and LC Laboratories, respectively.

Techniques: Inhibition, Western Blot, Concentration Assay, Mutagenesis, Expressing

Journal: Molecular cancer research : MCR

Article Title: TAS6417/CLN-081 is a pan-mutation-selective EGFR tyrosine kinase inhibitor with a broad spectrum of preclinical activity against clinically-relevant EGFR mutations

doi: 10.1158/1541-7786.MCR-19-0419

Figure Lengend Snippet: A, Nude mice bearing H1975 cells (n=6 per group) were treated with either vehicle, TAS6417 at 50 mg/kg, 100 mg/kg, or 200 mg/kg, or osimertinib at 25 mg/kg. The administration was orally once daily for 15 consecutive days. B, Nude mice bearing NIH/3T3 EGFR G719A allografts (n=6 per group) were treated with either vehicle, TAS6417 at 50 mg/kg, 100 mg/kg, or 200 mg/kg, or afatinib at 20 mg/kg. One mouse of control group was euthanized on day 11 due to overgrowth of tumor. C, Nude mice bearing BID007 (n=6 per group) were treated with either vehicle, TAS6417 at 50 mg/kg, 100 mg/kg, or 200 mg/kg, or poziotinib at 1 mg/kg. One mouse treated with poziotinib died on day 10 due to extensive skin rash, and the dose was reduced to 0.5 mg/kg as two mice showed body weight loss greater than 20%. D, Nude mice bearing H1975 EGFR D770_N771insSVD xenografts (n=6 per group) were treated with either vehicle, TAS6417 at 100 mg/kg or 200 mg/kg, or poziotinib at 0.5 mg/kg or 1 mg/kg. E. Body weight changes in mice described in (D). Data are presented as mean tumor volume ± SEM or mean body weight change ± SEM in each group.

Article Snippet: Osimertinib, poziotinib, afatinib, and erlotinib were purchased from CHEMSCENE, LLC, MedChem Express, Selleck Chemicals, and LC Laboratories, respectively.

Techniques: Control

Journal: Cancer Science

Article Title: Altered intracellular signaling by imatinib increases the anti‐cancer effects of tyrosine kinase inhibitors in chronic myelogenous leukemia cells

doi: 10.1111/cas.13442

Figure Lengend Snippet: K562 cells continuously exposed to imatinib ( IM ) become sensitive to tyrosine kinase inhibitors ( TKI ). A, BrdU incorporation into K562 cells continuously cultured with 0.1 μmol/L IM for 0‐4 weeks was evaluated. ** P < .01 and N.S., not significant, compared with cells not treated with IM . B, Death rate of K562 cells continuously exposed to 0.1 μmol/L IM for 0‐4 weeks was evaluated. C, Total cell lysates prepared from K562 cells continuously cultured with 0.1 μmol/L IM for 0‐3 weeks and treated with 1 μmol/L IM for 6 hours were subjected to western blotting with anti‐phospho‐ BCR , anti‐ BCR , and anti‐ GAPDH antibodies. D, K562 cells were cultured for the indicated periods with 0.1 μmol/L IM and then treated with 15 μmol/L IM for 72 hours. Cell death was evaluated. * P < .05 and ** P < .01, compared with cells not treated with IM . E, Parental K562 and K562‐ IM 3w cells were treated with the following agents at the indicated concentrations for 72 hours, and cell death was evaluated: dasatinib, nilotinib, bosutinib, and ponatinib (all TKI ), and methotrexate, cytarabine, cisplatin, and vincristine (all classical anti‐cancer agents). * P < .05 and ** P < .01

Article Snippet: Imatinib (Phoenix Pharmaceuticals, Belmont, CA, USA), 2‐deoxy‐ d ‐glucose (Wako, Osaka, Japan), CQ (Sigma, St Louis, MO, USA), metformin hydrochloride (LKT Laboratories, St Paul, MN, USA), rapamycin (Adooq Bioscience, Irvine, CA, USA), dasatinib (Cayman Chemical, Ann Arbor, MI, USA), bosutinib (KareBay Biochem, Ningbo, China), nilotinib (Cayman Chemical), ponatinib (Adooq Bioscience), methotrexate (Wako), cytarabine (LKT Laboratories), cisplatin (Sigma), vincristine sulfate (LKT Laboratories), STAT5 inhibitor (Calbiochem, San Diego, CA, USA), LY294002 (LC Laboratories, Woburn, MA, USA), and LY3900120 (Medchem Express, Princeton, NJ, USA).

Techniques: BrdU Incorporation Assay, Cell Culture, Western Blot

Journal: Molecular Cancer Research

Article Title: TANKYRASE Inhibition Enhances the Antiproliferative Effect of PI3K and EGFR Inhibition, Mutually Affecting β-CATENIN and AKT Signaling in Colorectal Cancer

doi: 10.1158/1541-7786.mcr-17-0362

Figure Lengend Snippet: Figure 4. Effect of G007-LK, BKM120, and erlotinib treatment on WNT signaling pathway components. Regulations of TNKS1/2, AXIN1, AXIN2 (A, COLO320DM; B, HCT-15), total and inactive GSK3b (phospho serine 9 GSK3b; P-GSK3b), N-terminal phosphorylated b-CATENIN (phospho-serine 33/37/threonine 41 b-CATENIN; P-b-CATENIN), total b-CATENIN and non-phospho (active) b-CATENIN (ABC) protein levels in total cell extracts (C, COLO320DM; D, HCT- 15, top), and nuclear protein levels of AXIN2 and ABC proteins (C and D, bottom) upon incubation with inhibitors as indicated. Relative AXIN2 expression levels normalized to internal GAPDH level are indicated in E (COLO320DM) and F (HCT-15). Data represent mean relative expression values (SD) of 3 independent experiments, each with 3 technical replicates. , P < 0.05 by two- tailed t test. Complete statistical analysis is found in Supplementary Statistics.

Article Snippet: The following inhibitors were used: G007-LK (ChemRoyal), BKM120 (NVP-BKM120; Chemietek), API-2 (TOCRIS), and GDC-0941 (Selleckchem), erlotinib (Chemietek), gefitinib (Selleckchem), and GDC-0973 (MedChem Express).

Techniques: Incubation, Expressing, Two Tailed Test

Journal: Molecular Cancer Research

Article Title: TANKYRASE Inhibition Enhances the Antiproliferative Effect of PI3K and EGFR Inhibition, Mutually Affecting β-CATENIN and AKT Signaling in Colorectal Cancer

doi: 10.1158/1541-7786.mcr-17-0362

Figure Lengend Snippet: Figure 5. Effect of G007-LK, BKM120, and erlotinib treatment on AKT/mTOR and EGFR signaling pathway components. Protein levels of total and active AKT (phospho-serine 473 AKT; P-AKT) in COLO320DM (A) and HCT-15 (B) cells. Protein levels of total and active P70S6K (phospho-tyrosine389-P70S6K; P-P70S6K), and total and active S6RP (phospho-serine240/244-S6RP; P-S6RP) in COLO320DM (C) and HCT-15 (D) cells. Protein levels of total and active EGFR (phospho-tyrosine 1068 EGFR; P-EGFR) protein levels in COLO320DM (E) and HCT-15 (F) cells, and total RAS in COLO320DM (G) and HCT-15 (H) cells. Fifteen to 20 mg protein was probed with the indicated antibodies, and 40 mg for P-EGFR in the bottom panel in E. Data represent one experiment of at least two.

Article Snippet: The following inhibitors were used: G007-LK (ChemRoyal), BKM120 (NVP-BKM120; Chemietek), API-2 (TOCRIS), and GDC-0941 (Selleckchem), erlotinib (Chemietek), gefitinib (Selleckchem), and GDC-0973 (MedChem Express).

Techniques:

Journal: Molecular Cancer Research

Article Title: TANKYRASE Inhibition Enhances the Antiproliferative Effect of PI3K and EGFR Inhibition, Mutually Affecting β-CATENIN and AKT Signaling in Colorectal Cancer

doi: 10.1158/1541-7786.mcr-17-0362

Figure Lengend Snippet: Figure 6. Effect of G007-LK, BKM120, and erlotinib treatment on TNKS/b-CATENIN complex formation. Representative images of immunofluorescent staining of b-CATENIN (left), TNKS1/2 (middle) and merge (right) of b-CATENIN (green), TNKS1/2 (red) and DAPI (blue) in COLO320DM (A), and HCT-15 (B) cells treated with inhibitors as indicated. A colocalization mask displaying TNKS1/2/ b-CATENIN colocalization in white was superimposed on the merged images. Arrows indicate b-CATENIN puncta (left image), TNKS1/2 puncta (middle image), and TNKS/b-CATENIN colocalization (right image). Scale bar; 10 mm.

Article Snippet: The following inhibitors were used: G007-LK (ChemRoyal), BKM120 (NVP-BKM120; Chemietek), API-2 (TOCRIS), and GDC-0941 (Selleckchem), erlotinib (Chemietek), gefitinib (Selleckchem), and GDC-0973 (MedChem Express).

Techniques: Staining