Journal: bioRxiv

Article Title: Met-HER3 crosstalk supports proliferation via MPZL3 in MET -amplified cancer cells

doi: 10.1101/2021.07.27.454013

Figure Lengend Snippet: Gene expression analysis identifies MPZL3 as a HER3-regulated transcript in MET -amplified cells. (A) Western blot analysis of the phosphorylation of Akt (Ser 473 ), ERK1/2 (Thr 202 /Tyr 204 ) and STAT3 (Tyr 705 ) in the indicated cell lines stably transduced with control (pLKO) or shRNA targeting HER3. (B) Volcano plot of RNA-sequencing data. Genes with significant increase or decrease in expression (log 2 FC ≥ |1|, FDR < 0.05) between control (pLKO) and HER3 depleted cells are labeled. (C-D) Measurement of the relative amounts of ERBB3, MPZL3, NYNRIN, BHLHE41 and LGR6 genes in the indicated cell lines by RT-qPCR. (C) Genes downregulated under steady state conditions upon stable HER3 depletion in EBC1 cells. (D) Genes downregulated under steady state conditions upon stable HER3 depletion in H1993 cells. Data are means (bars) of three individual replicates. Two-way ANOVA with Dunnett’s test for multiple comparisons; *p≤ 0.05; **p ≤ 0.01; ***p≤ 0.001; ****p≤ 0.0001. Error bars indicate SEM.

Article Snippet: Split intein mediated protein ligation (SIMPL) Gateway destination vectors were generously gifted by the Stagljar lab. Gateway entry vector for ERBB3 was obtained through Addgene (pDONR223-ERBB3, #23874) (Johannesen et al. , 2010).

Techniques: Gene Expression, Amplification, Western Blot, Phospho-proteomics, Stable Transfection, Transduction, Control, shRNA, RNA Sequencing, Expressing, Labeling, Quantitative RT-PCR

Journal: Clinical Cancer Research

Article Title: PI3Kα Inhibitor and Degrader Inavolisib Can Co-opt FGFR2 to Enhance Responses in Patients with PIK3CA -Mutated Solid Tumors and in Preclinical Models

doi: 10.1158/1078-0432.CCR-25-1459

Figure Lengend Snippet: Inavolisib sensitivity depends on high FGFR2 expression. A, Legend related to panels in B–I . B, Cell lines were treated with 0.03 μmol/L of the FGFR2i or 2-μmol/L lapatinib for 1 hour followed by immunoblotting with the antibodies indicated at left. Representative results from experiments ( n = 2). C, Cell lines were treated with inavolisib or the FGFR2i at various concentrations for 1 hour. Cell lysates were immunoprecipitated with an antibody against p85β, followed by immunoblotting with the antibodies indicated at left. Representative results from experiments ( n = 2). IB, immunoblotting. D, Following RAS-GTP pulldown, cell lines were treated with the FGFR2i or lapatinib for different durations and immunoblotted with the antibodies indicated at left. Representative results from experiments ( n = 2). E, Cell lysates from cells treated with inavolisib alone or in combination with the FGFR2i or lapatinib for 4 hours were immunoprecipitated with RAS antibody and blotted with p110α antibody. F, Mechanistic model of the effects of FGFR2 and HER2 inhibition on HER3 and RAS activity. FGFR2-high–expressing cell lines induced PI3K signaling through both HER3 and WT RAS activity (top) compared with HER2-induced PI3K signaling through HER3 but not RAS activity (bottom). G, FGFR2-high–expressing cell lines, MFM223 and SUM52PE, were treated with inavolisib, FGFR2i, or lapatinib for 1 hour. Membrane fractions were analyzed by reciprocal co-IP with one another using HER3 or FGFR2 antibody and Western blotting with FGFR2, HER3, RAS, and p85β antibody. Representative results from experiments ( n = 2). H, SUM52PE, MFM223, and MFE280 cells were treated with inavolisib single-agent or in combination with the FGFR2i or lapatinib for 6 hours. Ubiquitinated proteins were pulled down from the membrane fraction with TUBE1 reagent and blotted with p110α antibody. Representative results from experiments ( n = 2). I, Western blots of the inhibitor response in PI3K signaling (pHER3 and pAKT) in PIK3CA mutant MFM223 and PIK3CA WT SUM52PE; cell lines were treated with 0.5-μmol/L inavolisib or 1-μmol/L alpelisib for different durations. Representative results from experiments ( n = 2). J, Ratio of inavolisib and alpelisib GR 50 values in FGFR2-high ( n = 12) vs. FGFR2-low ( n = 9) expressing cell lines harboring PIK3CA mutations, as assessed in a 5-day viability assay. Data are represented as median (center line) ± IQR (25th to 75th percentile, box) and ± full range (minimum to maximum, whiskers). P value was calculated using Wilcoxon rank-sum test. Representative results from experiments ( n = 2). WB, Western blotting.

Article Snippet: For HER3 and FGFR2 IP, lysates were incubated overnight with a biotin-conjugated HER3 or FGFR2 antibody, respectively (Invitrogen, cat. No. MA5-13037, RRID: AB_10983790; and OriGene, cat. No. TA502917AM), which was followed by the addition of 50 μL of streptavidin agarose beads to each sample and an additional 2 hours of incubation.

Techniques: Expressing, Western Blot, Immunoprecipitation, Inhibition, Activity Assay, Membrane, Co-Immunoprecipitation Assay, Mutagenesis, Viability Assay

Journal: Cancer cell

Article Title: Unbiased Combinatorial Screening Identifies a Bispecific IgG1 that Potently Inhibits HER3 Signaling via HER2-Guided Ligand Blockade.

doi: 10.1016/j.ccell.2018.04.003

Figure Lengend Snippet: Figure 3. Selectivity of PB4188 for HER2/HER3 Signaling (A–D) PathHunter assays to measure ligand-induced dimerization of EGFR/HER2 (A), HER2/HER4 (B), HER2/HER3 (C), and EGFR/HER3 (D). Cells were stim- ulated with an EC80 concentration of EGF (A) or HRG (B–D) and titrations of the indicated antibodies. (E) Human stem cell-derived cardiomyocytes were incubated with the indicated antibodies in combination with doxorubicin. Cell viability was determined by measuring cellular ATP. (F) SKBR-3 cells cultured in serum-free medium were supplemented with 12.5 nM HRG and the indicated antibodies. Twenty-four hours later, protein lysates were analyzed with PathScan array. (G) Phosphorylated (p prefixed) and total levels of signaling proteins after HRG stimulation (12.5 nM) in N87 cells analyzed by western blot. T, trastuzumab; P*, pertuzumab*; T + P*, equimolar combination of trastuzumab plus pertuzumab*. Data in (A–D) are represented as means ± SEM. Boxes in (E) show the middle quartile (25%–75%); horizontal bars represent the median; whiskers show the maximum and minimum values in the dataset.

Article Snippet: AM2016-4759 Chemicals, Peptides, and Recombinant Proteins Human NRG1-beta 1/HRG1-beta 1 EGFlike domain R&D Systems Cat#396-HB Human NRG1-beta 1/HRG1-beta 1 EGFlike domain Prospec Bio Cat#Cyt-733 Human EGF DiscoverX Cat#92-1113 Human HER2 Fc chimera R&D Systems Cat#1129-ER Human HER3 Fc chimera R&D Systems Cat348-RB Human HER4 Fc chimera R&D Systems Cat#1131-ER Human EGFR Fc chimera R&D Systems Cat#344-ER Human HER2 ectodomain Bender MedSystems Cat#362 Critical Commercial Assays Alamar BlueTM Invitrogen Cat#DAL1100 Click-iTTM EdU AlexaFluorTM 488 Imaging Kit Life Technologies Cat#C10337 PathScan RTK Signaling Antibody Array Kit Cell Signaling Cat#7949 PathScan Intracellular Signaling Array Kit Cell Signaling Cat#7323 PathHunter U2OS Dimerization Cell Line DiscoverX N/A Human IgG ELISA ZeptoMetrix Cat# 0801182 VeraTag Assay Monogram Biosciences N/A Cell Signaling Multiplex Assays Millipore Cat# 48-618MAG, 46-645MAG, 46-645M-1K ADCC Reporter Bioassay Promega Cat#G7011, G9790, G7941 Epitope mapping Integral Molecular N/A Deposited Data HER2-ECD:MF3958 structure This paper PDB: 5O4G HER3-ECD:MF3178 structure This paper PDB: 5O4O, 5O7P Experimental Models: Cell Lines Human: JIMT-1 DSMZ Cat#ACC-589; RRID:CVCL_2077 Human: SKBR-3 ATCC Cat#HTB-30; RRID:CVCL_0033 Human: BT-474 DMSZ Cat#ACC-64; RRID:CVCL_0179 Human: MCF-7 DMSZ Cat#ACC-115; RRID:CVCL_0031 Human: MDA-MB-468 Cell Line Service N/A (Continued on next page) e2 Cancer Cell 33, 922–936.e1–e10, May 14, 2018

Techniques: Concentration Assay, Derivative Assay, Incubation, Cell Culture, Western Blot