Journal: iScience

Article Title: Novel mutant KRAS addiction signature predicts response to the combination of ERBB and MEK inhibitors in lung and pancreatic cancers

doi: 10.1016/j.isci.2023.106082

Figure Lengend Snippet: ERBB signaling constitutes the core of KRAS dependency (A) PPI network of the 30 genes generated with STRING and visualized with Cytoscape. Colors indicate log 2 FC (nodes’ size indicates absolute log 2 FC) in gene expression between 20 most mt KRAS-dependent and 20 most mt KRAS-independent cell lines. (B) Results from GO enrichment analysis of the 30 genes using ShinyGO. Only terms with FDR<0.01 are plotted. Expression of genes from each category was averaged among cell lines with smallest and highest KDS30 scores. ‘Any (L)’ represents mt KRAS cancer cell lines with 10 lowest KDS30 scores. ‘Any (H)’ represents mt KRAS cancer cell lines with 10 highest KDS30 scores. ‘Panc (L)’ and ‘Panc (H)’ represent mt KRAS pancreatic cancer cell lines with 4 lowest and 4 highest KDS30 scores, respectively. ‘Lung (L)’ and ‘Lung (H)’ represent mt KRAS lung cancer cell lines with 4 lowest and 4 highest KDS30 scores, respectively. (C and E) EGFR/ERBB2 (in blue), MEK (in red) and RAF (in green) inhibitors that were found to selectively kill high KDS30 over low KDS30 PAAD (in C) or LUAD (in E) cancer cells, considering max top 40 drugs. PRISM_1ry: primary PRISM Repurposing screen, PRISM_2ry: secondary PRISM Repurposing screen, CTRP: Cancer Therapeutics Response Portal v2. Y-axis: difference in mean FC or mean AUC, see main text. Only inhibitors of EGFR/ERBB2/MEK/RAF with p<0.05 are shown. (D and F) Results from drug sensitivity data analysis for mt KRAS pancreatic cancer cells included in the PRISM 2ry screen (D) and mt KRAS lung cancer cell lines included in the CTRP screen (F). Mean AUC of high KDS30 cells was subtracted from mean AUC of low KDS30 cells and plotted on X-axis. See main text for thresholds. Student’s t - test was applied to determine the statistical significance to observe the difference and plotted on Y-axis. MEK inhibitors are highlighted in red. EGFR/ERBB2 inhibitors are highlighted in blue. NERA: neratinib; SELU: selumetinib; TRAM: trametinib; PD31: PD-318088; AFAT: afatinib; GEFI: gefitinib; AZD8: AZD8931; DACO: dacomitinib; TUCA: tucatinib; CANE: canertinib; COBI: cobimetinib; AS70: AS-703026; OSIM: Osimertinib; ERLO: erlotinib . See also Figure S3 .

Article Snippet: pHER2/ErbB2 (Tyr-1221/1222) , Cell Signaling Technology , catalog no. 2243S.

Techniques: Generated, Expressing

Journal: iScience

Article Title: Novel mutant KRAS addiction signature predicts response to the combination of ERBB and MEK inhibitors in lung and pancreatic cancers

doi: 10.1016/j.isci.2023.106082

Figure Lengend Snippet: mt KRAS addicted lung cancer lines are highly sensitive to the combination of EGFR/ERBB2 inhibitors with MEK inhibitors High KDS30 H358 and low KDS30 LU99 lung cancer cells were treated with vehicle control (Ctrl), an EGFR/ERBB2 inhibitor and a MEK inhibitor, either alone or in combination as indicated on each figure panel, for 48 h and processed for western blotting as described in . pEGFR, pErbB2, pERK: phosphorylated EGFR, ErbB2, and ERK, respectively. c-Casp3 and c-PARP: cleaved Casp3 and PARP, respectively. Actin: loading control. Single ∗ and double ∗∗ indicate gels each protein is blotted from. See also Figure S4 .

Article Snippet: pHER2/ErbB2 (Tyr-1221/1222) , Cell Signaling Technology , catalog no. 2243S.

Techniques: Western Blot

Journal: iScience

Article Title: Novel mutant KRAS addiction signature predicts response to the combination of ERBB and MEK inhibitors in lung and pancreatic cancers

doi: 10.1016/j.isci.2023.106082

Figure Lengend Snippet:

Article Snippet: pHER2/ErbB2 (Tyr-1221/1222) , Cell Signaling Technology , catalog no. 2243S.

Techniques: Recombinant, Expressing, CRISPR, Software

Journal: Breast Cancer Research : BCR

Article Title: Fatty acid synthase phosphorylation: a novel therapeutic target in HER2-overexpressing breast cancer cells

doi: 10.1186/bcr2777

Figure Lengend Snippet: Association of FASN and HER2 in breast cancer cells . SKBR3 (A) and BT474 (B) cells were treated for one hour with: 1) no agent (control); 2) 50 ng/mL HRG; 3) 1 μM lapatinib or 4) 50 ng/mL HRG plus 1 μM lapatinib. The immunoprecipitated FASN complexes were subjected to Western blotting for FASN, HER2 and PT66. (C) Whole-cell lysates from SKBR3 (left panel) and BT474 cells (right panel) were subjected to Western blotting for pHER2 (Y1248), pHER3 (Y1289), pAkt (S473), pErk1/2, HER2, HER3, Akt, Erk1/2, FASN and β-actin.

Article Snippet: The following antibodies were used for Western blot analysis: mouse monoclonal antibodies detecting tyrosine phosphorylation (PT66) and β-actin (Sigma-Aldrich); rabbit polyclonal antibodies detecting pHER2 (Tyr 1248), pHER3 (Tyr 1289), pAkt (S473), pErk1/2, Akt, Erk1/2 and HER3 (Cell Signaling Technology, Inc., Danvers, MA, USA); mouse monoclonal antibodies detecting FASN, HER2 and HER3 (Santa Cruz) and an Alexa Flour 680 goat anti-mouse immunoglobulin (IgG) antibody, Alexa Flour 680 goat anti-rabbit IgG antibody and Alexa Flour 680 donkey anti-goat IgG antibody (Invitrogen Corporation, Carlsbad, CA, USA).

Techniques: Immunoprecipitation, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: Fatty acid synthase phosphorylation: a novel therapeutic target in HER2-overexpressing breast cancer cells

doi: 10.1186/bcr2777

Figure Lengend Snippet: FASN phosphorylation in breast cancer cells treated with C75 . SKBR3 (A) and BT474 (B) cells were pretreated with 10 μM C75 for five hours and then treated for one additional hour with 1) no agent (control); 2) 50 ng/mL HRG; 3) 10 μM C75; or 4) 50 ng/mL HRG plus 10 μM C75. The immunoprecipitated FASN complexes were assessed for FASN, HER2 and PT66 by Western blotting. (C) Whole-cell lysates from SKBR3 and BT474 cells were subjected to Western blotting for pHER2 (Y1248), pHER3 (Y1289), pAkt (S473), pErk1/2, HER2, HER3, Akt, Erk1/2, FASN and β-actin.

Article Snippet: The following antibodies were used for Western blot analysis: mouse monoclonal antibodies detecting tyrosine phosphorylation (PT66) and β-actin (Sigma-Aldrich); rabbit polyclonal antibodies detecting pHER2 (Tyr 1248), pHER3 (Tyr 1289), pAkt (S473), pErk1/2, Akt, Erk1/2 and HER3 (Cell Signaling Technology, Inc., Danvers, MA, USA); mouse monoclonal antibodies detecting FASN, HER2 and HER3 (Santa Cruz) and an Alexa Flour 680 goat anti-mouse immunoglobulin (IgG) antibody, Alexa Flour 680 goat anti-rabbit IgG antibody and Alexa Flour 680 donkey anti-goat IgG antibody (Invitrogen Corporation, Carlsbad, CA, USA).

Techniques: Immunoprecipitation, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: Direct inhibition of PI3K in combination with dual HER2 inhibitors is required for optimal antitumor activity in HER2+ breast cancer cells

doi: 10.1186/bcr3601

Figure Lengend Snippet: PIK3CA mutation uncouples phosphoinositide 3-kinase signaling from HER2 inhibition by lapatinib. (A) BT474 and SKBR3 cells infected with wild-type, E545K or H1047R constructs were treated with lapatinib at the indicated doses, and lysates were analyzed by immunoblotting with the indicated antibodies. (B) Lysates from PIK3CA wild-type or mutant expressing cells treated with a range of lapatinib doses (0.0016 to 5 μM) were analyzed by ELISA for pHER2, pAkt and pS6. Half-maximal concentration (IC 50 ) values were calculated, and the mean log IC 50 ± SEM values for three replicate dose–inhibitor curves are shown. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001. (C) HER2+ cell lines with wild-type PIK3CA (BT474 or SKBR3) or with a PIK3CA mutation (MDA361, HCC1954, SUM190 or UACC893) were treated with varying lapatinib doses and analyzed as described in (B) . Mean log IC 50 values from three replicates ± SEM are shown. Mean IC 50 data are shown in Table .

Article Snippet: Antibodies from the following sources were used for analysis: pHER2 Y1248 (R&D Systems, Minneapolis, MN, USA); Y877 pHER2 (Epitomics, Burlingame, CA, USA); Y1221/2 pHER2, Y11197 and Y1289 pHER3, S473 pAkt, Akt, S240/44 pS6, pErk1/2, Erk, and p110α PI3K (Cell Signaling Technology, Danvers, MA, USA); p85 N-terminal Src homology 2 (SH2) domain (EMD Millipore); HA (Covance, Princeton, NJ, USA); actin (Sigma-Aldrich, St Louis, MO, USA); HER2 (Thermo Fisher, Pittsburgh, PA, USA); and glyceraldehyde 3-phosphate dehydrogenase (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Mutagenesis, Inhibition, Infection, Construct, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay