Journal: Scientific Reports

Article Title: Induction of MET Receptor Tyrosine Kinase Down-regulation through Antibody-mediated Receptor Clustering

doi: 10.1038/s41598-018-36963-3

Figure Lengend Snippet: Antibody-mediated MET receptor crosslinking leads to the receptor clustering and patch formation on the plasma membrane through activation of MET receptor. ( A ) Live U373-MG cells, which were serum starved for three hours, were incubated with the APC-conjugated mouse monoclonal anti-MET antibody (1 μg/ml) (red) or its isotype control, APC-conjugated, mouse IgG (1 μg/ml) for 7 minutes. Cells were then washed, fixed and co-immunostained with an anti-MET antibody pre-conjuagted to Alexa Fluor 488, which detects the total MET protein (green). Cells were imaged by confocal microscopy. Clusters (or patches) of the MET proteins on the plasma membrane are indicated by arrows. ( B ) Live serum-starved U373-MG cells were treated with biotin-conjugated goat anti-MET antibodies (2 μg/ml) or biotin-conjugated normal goat IgG antibodies (2 μg/ml) (control) for 7 minutes. Cells were then washed, fixed and immunostained with the Streptavidin-conjugated to Brilliant Violet 421 (BV421) to detect the biotin-conjugated antibodies (purple), a rabbit anti-Y1234/1235-phosphorylated MET antibody for the activated MET protein (secondary antibody conjugated with Alexa Fluor 647, red), and an anti-MET antibody pre-conjugated to Alexa Fluor 488 for total MET protein (green). ( C ) Serum-starved cells were left untreated or stimulated with HGF (50 ng/ml) for 7 minutes. Cells were fixed and immunostained with a rabbit antibody against Y1234/Y1235-phosphorylated MET for the activated MET protein (red) and with an anti-MET antibody pre-conjugated with the Alexa Fluor 488 to detect total MET (green). ( D ) Serum-starved cells were treated with the biotin-conjugated anti-MET antibody (2 μg/ml) and fixed at the various time points as indicated. Cells were immunostained with strepavidin-conjugated to BV421 as in panel B. Scale bars in ( A–C ), 20 μm.

Article Snippet: Rabbit antibodies against AXL, MET, Y1234/Y1235-phosphorylated MET, phosphorylated AKT (at S473, recognizing all AKT isoforms, Cat. #9272), and anti-MET rabbit monoclonal antibody (clone D1C2, Alexa Fluor 488 conjugate, Cat. #8494) were obtained from Cell Signaling Technology.

Techniques: Clinical Proteomics, Membrane, Activation Assay, Incubation, Control, Confocal Microscopy

Journal: Scientific Reports

Article Title: Induction of MET Receptor Tyrosine Kinase Down-regulation through Antibody-mediated Receptor Clustering

doi: 10.1038/s41598-018-36963-3

Figure Lengend Snippet: The MET receptor is activated by HGF, or APC- or Biotin-conjugated anti-MET antibodies. ( A ) Serum-starved U373-MG cells were treated either with HGF (50 ng/ml) for 7 minutes or left untreated (-HGF) as indicated. The cells were directly lysed in SDS buffer and proteins were detected by Western blotting with antibodies for the Y1234/Y1235-phosphorylated MET (p-MET, Y1234/5), total MET, phosphorylated AKT (p-AKT), total AKT, or actin (loading control), as indicated. ( B ) Serum-starved U373-MG cells were left untreated, or treated with APC-conjugated mouse IgG control antibody or APC-conjugated mouse monoclonal anti-MET antibody (each at 1 μg/ml) for 7 minutes. Cells were directly lysed in SDS buffer and proteins were detected by Western blotting with antibodies as in panel A. ( C ) The same as panel B except that the Biotin-conjugated goat control IgG or Biotin-conjugated goat anti-MET antibodies (each at 2 μg/ml) were used. ( D ) Serum-starved human glioblastoma cells T98G, LN229, and LN18 cells were treated with HGF or biotin-conjugated IgG or biotin-conjugated anti-MET antibodies as in panel A and C, or left untreated. The proteins were detected by Western blotting with antibodies as in panel A.

Article Snippet: Rabbit antibodies against AXL, MET, Y1234/Y1235-phosphorylated MET, phosphorylated AKT (at S473, recognizing all AKT isoforms, Cat. #9272), and anti-MET rabbit monoclonal antibody (clone D1C2, Alexa Fluor 488 conjugate, Cat. #8494) were obtained from Cell Signaling Technology.

Techniques: Western Blot, Control

Journal: Scientific Reports

Article Title: Induction of MET Receptor Tyrosine Kinase Down-regulation through Antibody-mediated Receptor Clustering

doi: 10.1038/s41598-018-36963-3

Figure Lengend Snippet: Activation of the MET receptor by HGF or anti-MET antibodies requires the MET RTK activity. ( A ) Serum-starved U373-MG cells were treated with the MET kinase inhibitor, crizotinib (1 μM) (lane 4–6) or DMSO (lane 1–3) for 3 hours. The cells were then treated either with biotin-conjugated anti-MET antibodies or biotin-conjugated IgG (each at 2 μg/ml) for 7 minutes, or left untreated, as indicated. The cells were directly lysed in SDS buffer and proteins were detected by Western blotting with antibodies for the Y1234/Y1235-phosphorylated MET (p-MET, Y1234/5), total MET, phosphorylated AKT (p-AKT), total AKT, and actin (loading control), respectively, as in Fig. . ( B ) The same as in panel A except that the APC-conjugated mouse monoclonal anti-MET antibodies or mouse IgG control antibodies (each at 1 μg/ml) were used. ( C ) Serum-starved U373-MG cells were treated with dimethyl sulfoxide (DMSO) vehicle control (lane 1, 2), or with the MET kinase inhibitors crizotinib (1 μM) (lane 3, 4) or JNJ-38877605 (1 μM) (lane 5,6) for 3 hours. Cells were then treated with or without HGF (50 ng/ml) for 7 minutes as indicated. The cells were directly lysed in SDS buffer and proteins were detected by Western blotting with indicated antibodies. ( D ) Serum-starved U373-MG cells were treated with the MET kinase inhibitors, crizotinib, JNJ-38877605 or DMSO as in panel C. Cells were stimulated with or without HGF (50 ng/ml) for 7 minutes and were subsequently fixed and stained with rabbit antibody against Y1234/Y1235-phosphorylated MET (red) for the activated MET protein and with the Alexa Fluor 488 pre-conjugated anti-MET antibody for total MET protein (green), and bright field for the contrast phase. Cells were also counter stained with DAPI for nuclei. MET receptor clusters (patches) are indicated by arrows. ( E ) Serum-starved U373-MG cells were treated with the MET inhibitors crizotinib, JNJ-38877605 or dimethyl sulfoxide (DMSO) as in panel C. The live serum-starved U373-MG cells were then treated with biotin-conjugated goat anti-MET antibodies (2 μg/ml) or biotin-conjugated goat IgG antibodies (2 μg/ml) (control) for 7 minutes. The cells were then fixed and stained with streptavidin (labeled with Brilliant Violet 421) for biotin-conjugated antibodies (purple) and the rabbit anti-Y1234/1235-phosphorylated MET antibodies (followed by secondary antibodies conjugated to the Alexa Fluor 647) for the activated MET protein (red), and with the Alexa Fluor 488 pre-conjugated anti-MET antibody for total MET protein (green). MET receptor clusters (patches) are indicated by arrows. Scale bars in ( D,E ), 20 μm.

Article Snippet: Rabbit antibodies against AXL, MET, Y1234/Y1235-phosphorylated MET, phosphorylated AKT (at S473, recognizing all AKT isoforms, Cat. #9272), and anti-MET rabbit monoclonal antibody (clone D1C2, Alexa Fluor 488 conjugate, Cat. #8494) were obtained from Cell Signaling Technology.

Techniques: Activation Assay, Activity Assay, Western Blot, Control, Staining, Labeling

Journal: Cellular and Molecular Life Sciences

Article Title: An oncogene addiction phosphorylation signature and its derived scores inform tumor responsiveness to targeted therapies

doi: 10.1007/s00018-022-04634-2

Figure Lengend Snippet: Phenotypic and protein phosphorylation responses of EGFR-positive cancer cell lines towards EGFR targeting. A Representative pictures (left) and quantification (right) of viability of a panel of EGFR-positive cells towards EGFR inhibition by gefitinib (EGFRi) or AZD9291 (EGFRi*), IR and their combination. Viability assays were performed to assess sensitivity of EGFR-positive models to EGFRi exerted by gefitinib, IR and the combinatorial treatment; pMET-positive models were exposed to METi, alone and in combination with IR, and the T790M-harboring mutation cells were exposed to third generation EGFR TKI AZD9291, alone and in combinatorial regime with IR. Statistical analysis was performed by GraphPad Prism. p values were calculated by Student t -test (* P < 0.05; ** P < 0.01; *** P < 0.001, **** P < 0.0001). B Heat map displaying shared phosphorylations occurring in the EGFR-addicted systems HCC827 (HCC827 ) and PC-9 upon exposure to the EGFR inhibitor gefitinib (EGFRi) and their modulation upon EGFR inhibition, IR and their combination 1 or 8 h post IR across the EGFR-positive cell line cohort. In addition to the gefitinib treatment (H1975 1 ), the H1975 cell line has been exposed also to EGFR inhibition by AZD9291 (EGFRi*; H1975 ). Apart of the gefitinib treatment (HCC827 ), MET inhibition by tepotinib was tested in MET- and EGFR-expressing HCC827 cells (HCC827 1 ). Blue, upregulated phosphopeptides. Red, downregulated phosphopeptides. Dot, adjusted p -value < 0.05. C Modulation of DDR-related phosphopeptides in the EGFR-positive cell line panel that were identified in MET-addicted systems as significantly differently regulated between IR and METi + IR condition (Fig. C). The heat map displays differences between the treatment by IR and EGFRi + IR assessed in the EGFR-positive cell line panel. To demonstrate the difference between EGFR and MET targeting, effects of the treatments including either METi or EGFRi have been assessed for the HCC827 cell line (HCC827 1 and the HCC827 , respectively)

Article Snippet: Monoclonal antibodies used in this study were directed against phospho-MET (Tyr1234/1235) (Cell Signaling Technology), MET (clone D1C2, Cell Signaling Technology), phospho-EGFR (Tyr845) (Cell Signaling Technology), phospho-ALK (Tyr1064) (Cell Signaling Technology), phospho-ATM (Ser1981) (Cell Signaling Technology), phospho-c-Raf (Ser259) (Cell Signaling Technology), phospho-AKT (Ser473) (Cell Signaling Technology), phospho-MAPK (Thr202/Tyr204) (Cell Signaling Technology), phospho-H2AX (Ser139) (Upstate), Ki67 (Cell Signaling Technology) and ß-actin (Millipore Chemicon).

Techniques: Phospho-proteomics, Inhibition, Mutagenesis, Expressing

Journal: Cellular and Molecular Life Sciences

Article Title: An oncogene addiction phosphorylation signature and its derived scores inform tumor responsiveness to targeted therapies

doi: 10.1007/s00018-022-04634-2

Figure Lengend Snippet: Oncogene targeting-induced responses in ALK- and BRAF-addicted models. A Viability of the BRAF V600E-expressing melanoma cell line model G361 upon BRAF inhibition (vemurafenib), IR and their combination (upper panel—representative pictures, lower panel—crystal violet quantification (Student t -test, ** P < 0.01; *** P < 0.001). B Viability of EML4-ALK translocated NSCLC cell line H3211 upon ALK inhibition by crizotinib, IR and their combination (upper panel—representative pictures, lower panel—crystal violet quantification (Student t -test, *** P < 0.001, **** P < 0.0001). C Heat map of changes in abundance of phosphopeptides that compose the MET oncogene addiction phosphosignature in G361 and H3211 cells upon inhibition of BRAF and ALK, respectively, IR and the combination of the two modalities. Blue , upregulated phosphopeptides. Red, downregulated phosphopeptides. Dot, adjusted p-value < 0.05. D BRAF and ALK inhibitor-induced modulation (in G361 and H3211 cells, respectively) of DDR-related phosphopeptides that were identified in MET-addicted systems as significantly differently regulated between IR and METi + IR condition (Fig. C). The heat map shows a comparison of the phosphorylation levels between BRAFi + IR versus IR alone in the G361 cells (left part) and ALKi + IR versus IR in the H3211 cell line (right panel)

Article Snippet: Monoclonal antibodies used in this study were directed against phospho-MET (Tyr1234/1235) (Cell Signaling Technology), MET (clone D1C2, Cell Signaling Technology), phospho-EGFR (Tyr845) (Cell Signaling Technology), phospho-ALK (Tyr1064) (Cell Signaling Technology), phospho-ATM (Ser1981) (Cell Signaling Technology), phospho-c-Raf (Ser259) (Cell Signaling Technology), phospho-AKT (Ser473) (Cell Signaling Technology), phospho-MAPK (Thr202/Tyr204) (Cell Signaling Technology), phospho-H2AX (Ser139) (Upstate), Ki67 (Cell Signaling Technology) and ß-actin (Millipore Chemicon).

Techniques: Expressing, Inhibition, Comparison, Phospho-proteomics

Journal: Cellular and Molecular Life Sciences

Article Title: An oncogene addiction phosphorylation signature and its derived scores inform tumor responsiveness to targeted therapies

doi: 10.1007/s00018-022-04634-2

Figure Lengend Snippet: Oncogene addiction phosphorylation signature (OAPS) and the OAPS-derived score. A Map of phosphorylation changes elicited by oncogene inhibition in MET-, EGFR-, ALK- and BRAF-addicted cell systems. The 8 phosphopeptides-containing signature shared by all of these models of addiction ( oncogene addiction phosphorylation signature , OAPS) is visualized in the last row. (Dark red box, downregulation of phosphorylation detected upon inhibition of the respective addicting oncoprotein, white box, significant phosphorylation change not observed.) B Upper panel: visualisation of the OAPS-included proteins and their main GO terms cellular functions within the network of all tested phosphopeptides. (GO terms: blue—cell cycle, orange—DNA damage, green—kinase activity, red—apoptosis, gray—other) Lower panel: OAPS phosphosites’s upstream kinases. C The viability suppression upon inhibition highly correlates with the signature-derived score computed from phosphoproteomics in cell line systems (correlation 0.94, p -value 3 × 10 –10 ). The solid line and the shaded area show the linear regression result (intercept − 0.02, slope 0.46, p -value 3 × 10 −9 ) and its uncertainty (95% CI)

Article Snippet: Monoclonal antibodies used in this study were directed against phospho-MET (Tyr1234/1235) (Cell Signaling Technology), MET (clone D1C2, Cell Signaling Technology), phospho-EGFR (Tyr845) (Cell Signaling Technology), phospho-ALK (Tyr1064) (Cell Signaling Technology), phospho-ATM (Ser1981) (Cell Signaling Technology), phospho-c-Raf (Ser259) (Cell Signaling Technology), phospho-AKT (Ser473) (Cell Signaling Technology), phospho-MAPK (Thr202/Tyr204) (Cell Signaling Technology), phospho-H2AX (Ser139) (Upstate), Ki67 (Cell Signaling Technology) and ß-actin (Millipore Chemicon).

Techniques: Phospho-proteomics, Derivative Assay, Inhibition, Activity Assay

Journal: Cellular and Molecular Life Sciences

Article Title: An oncogene addiction phosphorylation signature and its derived scores inform tumor responsiveness to targeted therapies

doi: 10.1007/s00018-022-04634-2

Figure Lengend Snippet: Validation of the oncogene addiction phosphorylation signature (OAPS) in NSCLC tissues. A Schematic workflow of the OAPS analysis by SRM in NSCLC tumor biopsies combining PDX models and ex vivo organotypic cultures. B Heat maps displaying OAPS changes in NSCLC patient tissues following 16 h of EGFR (gefitinib; upper left panel), MET (tepotinib; lower left panel), and ALK (crizotinib; right panel) inhibition. ( LFC —log fold change in phosphorylation upon inhibition of addicting oncoprotein; cross —phosphopeptide not detected) C OAPS-derived score computed from phosphoproteomics in patient tissues (the score for up to three different inhibitors has been assessed for each patient). D Representative pictures of immunohistochemical staining ( pEGFR —EGFR activation; Ki67 —cell proliferation marker) of selected gefitinib-treated patient tissues (baseline—tissues stained prior start of the treatment, control—untreated tissues (OTCs) collected and stained at day 3, gefitinib—gefitinib-treated OTCs collected and stained at day 3 (48 h upon the start of EGFR inhibition)

Article Snippet: Monoclonal antibodies used in this study were directed against phospho-MET (Tyr1234/1235) (Cell Signaling Technology), MET (clone D1C2, Cell Signaling Technology), phospho-EGFR (Tyr845) (Cell Signaling Technology), phospho-ALK (Tyr1064) (Cell Signaling Technology), phospho-ATM (Ser1981) (Cell Signaling Technology), phospho-c-Raf (Ser259) (Cell Signaling Technology), phospho-AKT (Ser473) (Cell Signaling Technology), phospho-MAPK (Thr202/Tyr204) (Cell Signaling Technology), phospho-H2AX (Ser139) (Upstate), Ki67 (Cell Signaling Technology) and ß-actin (Millipore Chemicon).

Techniques: Biomarker Discovery, Phospho-proteomics, Ex Vivo, Inhibition, Derivative Assay, Immunohistochemical staining, Staining, Activation Assay, Marker, Control