Fig 2: Combinations of mAbs and EGFR‐specific TKIs downregulate in vitro several receptors for survival factors and effectively block the ERK and AKT pathways NSCLC expressing single‐site mutants of EGFR, PC9 (3 × 106) or H3255 (8 × 106), were seeded in 10‐cm dishes. On the next day, complete media were replaced with media containing serum (1%) and the cells were treated for 24 h with different EGFR‐specific TKIs (erlotinib, 50 nM; osimertinib, 50 nM, or afatinib, 10 nM), either alone or in combination with 2XmAbs (cetuximab and trastuzumab, 5 μg/ml each). Thereafter, cells were washed with cold saline and extracted. Proteins were separated using gel electrophoresis and transferred onto nitrocellulose membranes. After blocking, membranes were incubated overnight with the indicated primary antibodies, followed by incubation with horseradish peroxidase‐conjugated secondary antibodies (60 min), and treatment with Clarity™ Western ECL Blotting Substrates (Bio‐Rad). ECL signals were detected using the ChemiDoc™ Imaging System (Bio‐Rad) and images were acquired using the ImageLab software. Signals (relative to Control) were quantified and normalized to the signals of GAPDH (numbers shown below each lane).PC9 cells (1 × 106) were seeded in 6‐well plates and treated as in (A). After washing with acidic buffer (glycine 100 mM, pH 3.0), cells were incubated with fluorescently labeled antibodies against EGFR, HER2, and HER3, and surface expression levels of each receptor were analyzed using flow cytometry. The normalized fluorescence intensity is shown as averages + SEM of four experiments. Significance was assessed using two‐way ANOVA followed by Dunnett’s multiple comparisons test. Note that non‐significant comparisons are not shown. See P‐values in Appendix Table S2.PC9 cells were seeded on coverslips and treated for 24 h as in (A). Cells were washed in acidic buffer, fixed in paraformaldehyde (4%), and incubated with specific primary antibodies, followed by an Alexa Fluor 555‐conjugated secondary antibody (pseudo colored in green). DAPI (blue) was used to stain nuclei. Images were captured using a confocal microscope (40× magnification). Scale bars, 40 μm. Source data are available online for this figure.

Fig 3: ErbB receptors activation. Western blot of SCOCs revealed an increased phosphorylation ratio of ErbB2 (A), ErbB3 (B), and ErbB4 (C) receptors upon rhNRG1 treatment, that was totally or partially blocked by co-addition of LP. Data are shown as mean ± SEM with n = 3–5 values per treatment. **p < 0.01; *p < 0.05. (D) Microphotographs of ErbB2 and ErbB4 protein labeling with DAB and cresyl violet in control SCOCs at 14 DIV. Inset show MNs labeled against ErbB receptor at higher magnification. Scale bar = 100 μm.

Fig 4: Association of PI3K Subunits with HER3 Regulated by NRG1 and HER2 Targeting Drugs(A) Heatmap of detection counts from mass spectrometry proteomic analysis of HER3 immunoprecipitation from AU565 cell lysate following 48 hr of treatment with 500 nM lapatinib, 50 ng/mL NRG1β, 25 µg/mL pertuzumab, and selected combinations. Each count is normalized to the detected quantity of HER3 in each treatment sample. Scale shows the ratio of individual protein count to HER3 count (BR = 3).(B) A simplified model of HER2 and HER3 dimerization on the cell surface when activated by NRG1β. This structure alters the conformation of the ATP-binding pocket on the HER2 cytoplasmic kinase domain to one to which lapatinib cannot readily bind.(C) Pertuzumab inhibits cis-phosphorylation of HER3 via separation of the cytoplasmic kinase domains. This structure returns the HER2 kinase domain conformation to one to which lapatinib can bind. Note that receptors remain linked under pertuzumab treatment.(D) Higher-order receptor structures overcome inhibition by pertuzumab via trans-phosphorylation of HER3 (orange dot).(E) The proposed mechanism for how pertuzumab and lapatinib combine to overcome cis- and trans-phosphorylation of HER3, respectively.

Fig 5: L-HER2+ and HER2E Lines Differ in Their Resistance Mechanisms to HER2 Inhibition(A) Mean cell count and SEM (n = 3) of eight HER2+ cell lines treated for 72 hr with combinations of 500 nM lapatinib, 500 nM crizotinib, 30 µg/mL pertuzumab, 50 ng/mL NRG1β, 50, and ng/mL HGF. PBS, DMSO, and human IgG isotype control were used as controls for growth factors, TKIs, and pertuzumab, respectively. Addition of NRG1β to lapatinib results in significantly increased cell counts in each L-HER2+ line compared with drug alone (****AU565, p < 0.0001; ***SKBR3, p =0.0003; ***BT474, p =0.0002; ****EFM192A, p< 0.0001). Addition of HGF to neratinib results in significantly increased cell count in each HER2E line compared with drug alone (**HCC1954, p = 0.0037; ***HCC-3153, p = 0.0002; ***JIMT1, p = 0.0001; **21-MT1, p = 0.0045). Addition of pertuzumab to lapatinib plus NRG1β results in significantly decreased cell counts in each L-HER2+ line compared with lapatinib plus NRG1β alone (***AU565, p = 0.0005; ***SKBR3, p = 0.0008; **BT474, p = 0.0013; **EFM192A, p = 0.0001). Addition of crizotinib to neratinib plus HGF results in significantly decreased cell counts in each HER2E line compared with neratinib plus HGF alone (***HCC1954, p = 0.0006; ****HCC-3153, p < 0.0001; ****JIMT1, p < 0.0001; ****21-MT1, p < 0.0001).(B) Maximum projection fluorescent images of SKBR3 and HCC1954 cells treated for 48 hr with combinations of 500 nM lapatinib and 12.5 ng/mL NRG1β. Cell nuclei imaged with DAPI (blue), β-tubulin (green), and HER2-HER3 heterodimers (red) imaged by PLA.(C) Mean and SEM of PLA spot counts for SKBR3 (n = 79, 93, 150, 54) and HCC1954 (n = 52, 60, 54) treated for 48 hr with DSMO, 12.5 ng/mL NRG1β, 500 nM lapatinib, and the combination. p value shows unpaired t test of significance. NS, not significant.