Co-occurring gain-of-function mutations in HER2 and HER3 modulate HER2/HER3 activation, oncogenesis, and HER2 inhibitor sensitivity

Abstract

Activating mutations in HER2 (ERBB2) drive the growth of a subset of breast and other cancers and tend to co-occur with HER3 (ERBB3) missense mutations. The HER2 tyrosine kinase inhibitor neratinib has shown clinical activity against HER2-mutant tumors. To characterize the role of HER3 mutations in HER2-mutant tumors, we integrate computational structural modeling with biochemical and cell biological analyses. Computational modeling predicts that the frequent HER3^E928G kinase domain mutation enhances the affinity of HER2/HER3 and reduces binding of HER2 to its inhibitor neratinib. Co-expression of mutant HER2/HER3 enhances HER2/HER3 co-immunoprecipitation and ligand-independent activation of HER2/HER3 and PI3K/AKT, resulting in enhanced growth, invasiveness, and resistance to HER2-targeted therapies, which can be reversed by combined treatment with PI3Kα inhibitors. Our results provide a mechanistic rationale for the evolutionary selection of co-occurring HER2/HER3 mutations and the recent clinical observations that HER3 mutations are associated with a poor response to neratinib in HER2-mutant cancers.

Keywords: HER2; HER3; PI3K; Rosetta; breast cancer; molecular dynamics; neratinib; personalized structural biology; precision oncology.

Figure 1.. HER2 and HER3 mutations co-occur in breast and other cancers.

(A) 277 HER2- mutant breast cancers and (B) 1,561 HER2-mutant pan-cancers in the Project GENIE database were interrogated for co-occurring alterations in the indicated genes. HER2 VUS are excluded. (C) Co-occurrence with HER2 mutations was analyzed using cBioPortal. (D) The most common co-occurring HER2/HER3 mutations in breast cancer were determined using databases from Project GENIE, cBioPortal, and Foundation Medicine. See also Figure S1.

Figure 2.. Co-occurring HER2/HER3 mutants enhance HER2/HER3 kinase domain association and HER2 kinase activity.

(A) Rosetta HER2/HER3 heterodimerization binding energy as mean ± standard error across 20 lowest interface energy models. (B) Pairwise sums of per-residue binding energy decomposition for HER2/HER3 heterodimerization. (C) Comparison of the computational structural models of HER2^WT/HER3^WT and HER2^WT/HER3^E928G at the asymmetric dimer interface. HER2 is purple and HER3 is blue. The hydrogen bond distance and angle between G927-O / L790-NH and L790-N / L790-H / G927-O atoms, respectively, are depicted in yellow. (D) Probability density plots of HER2^WT/HER3^WT and HER2^WT/HER3^E928G HER3 G927-O – HER2 L790-N hydrogen bond distance (left), HER2 K716- NZ – HER2 E719-OE1,2 bond distance (middle), and HER2 K716-NZ – HER2 D742-OD1,2 bond distance (right). (E) Activation state conformational free energy landscape of HER2^WT and mutants (see STAR Methods). (F) Quantification of free energy difference between active and inactive states (gray), relative free energy difference compared to HER2^WT (yellow), and integration along the lowest free energy path(s) (green and purple). See also Figures S2 and S3.

Figure 3.. HER3^E928G enhances HER2/HER3 association and PI3K pathway activation.

(A) HEK293 cells were co-transfected with the indicated transgenes. HER2 IP was performed as in STAR Methods. (B) Immunoblot bands from four independent HER2 IP experiments in HEK293 cells were quantified using ImageJ. Data represent the mean ± SEM (n=4). P values, student’s t-test. (C) HEK293 cells were co-transfected with the indicated transgenes, serum-starved overnight, then stimulated ± 10 ng/ml NRG1 for 10 min. (D) MCF10A cells stably expressing the indicated transgenes or GFP control (−/−) were incubated in EGF/insulin-free media + 1% CSS overnight. (E) OVCAR8 cells stably expressing pLX302-GFP (control), HER3^WT, or HER3^E928G were incubated in RPMI + 1% CSS overnight, then subjected to HER2 IP. (F) MCF10A cells stably expressing the indicated transgenes were incubated and lysed as in (D). Where indicated, numbers below bands represent quantification of band intensity by ImageJ; ratios were normalized to WT/WT. See also Figure S4.

Figure 4.. Co-occurring HER2/HER3 mutations enhance oncogenic growth and invasion of breast epithelial cells.

(A) MCF10A cells stably expressing the indicated transgenes were grown in 2D in EGF/insulin-free media + 1% CSS. After 6 d, cell viability was measured by Cell Titer Glo. Data represent the mean ± SEM (n=4). P values, two-way ANOVA + Bonferroni. (B) MCF10A cells were grown in 3D Matrigel in EGF/insulin-free media + 1% CSS and stained with MTT. The total volume of colonies per well was quantified using the Gelcount instrument. Data represent the mean ± SEM (n=3). P values, two-way ANOVA + Bonferroni. (C) MCF10A cells stably the indicated transgenes were grown in 3D Matrigel in EGF-free media + 1% CSS ± 10 ng/ml NRG1 for 7 d. Scale bar, 250 μm. (D) The # of colonies showing invasive branching per field of view (FOV) from (C) was quantified. Data represent the mean ± SEM (n=3). P values, two-way ANOVA + Bonferroni. (E) MCF10A cells stably expressing the indicated genes were seeded on Matrigel-coated chambers. After 22 h, invading cells were stained with crystal violet. Scale bar, 500 μm. (F) Relative invasion (normalized to WT/WT) from two FOVs per well was quantified in ImageJ. Data represent the mean ± SEM (n ≥ 3). P values, two-way ANOVA + Bonferroni. See also Figure S5.

Figure 5.. HER3^E928G promotes resistance to HER2- and HER3-targeting antibodies by retaining HER2/HER3 KD association

(A) Model of HER2/HER3^E928G heterodimer bound to trastuzumab, pertuzumab, or PanHER. (B) MCF10A cells stably expressing the indicated genes were grown in 3D Matrigel in EGF/insulin-free medium +1% CSS and treated with vehicle (PBS), 20 μg/mL PanHER, 20 μg/mL each trastuzumab + pertuzumab for 7 days. Scale bars, 500 μm. (C) The total volume of colonies per well was quantified using the GelCount instrument. Data represent the mean ± SEM (n = 3). (D) MCF10A cells stably expressing the indicated transgenes were treated with vehicle (PBS) or 20 μg/mL each trastuzumab and pertuzumab for 24 h in EGF/insulin-free medium +1% CSS. Following an acid wash to remove bound antibodies, HER2 IP was performed. Line denotes removal of irrelevant lanes; blots are from the same gel/blot. (E) MCF10A cells stably expressing the indicated transgenes were treated with vehicle (PBS), 20 μg/mL each trastuzumab and pertuzumab, or 20 μg/mL PanHER for 24 h in EGF/insulin-free medium + 1% CSS. Line denotes removal of irrelevant lanes; blots are from the same gel/blot. See also Figure S6.

Figure 6.. Co-occurring HER3 mutations modulate neratinib sensitivity in HER2-mutant cells.

(A) MM/GBSA binding affinity estimates of ATP to HER2^WT/HER3^WT and HER2^WT/HER3^E928G. (B) Probability density hinge – ATP H-bond distance in HER2 WT, L755S, V777L, and L869R dimerized with HER3^WT or (C) HER3^E928G. (D) MM/GBSA relative binding affinity estimates of neratinib to HER2 variants heterodimerized with HER3^WT or HER3^E928G. Estimates are reported as mean ± standard error across 3 independent trajectory samples. (E) MCF10A HER2^S310F/HER3^E928G cells were grown in EGF/insulin-free media + 1% CSS and treated with the indicated concentrations of neratinib for 6 d. Cell viability was measured using CellTiterGlo. (F) Neratinib IC50s were determined as in (E). Data represent the mean of 3 independent dose-response curves containing 4 replicates each. (G) MCF10A cells stably expressing the indicated transgenes were grown in 3D Matrigel in EGF-free media + 1% CSS ± 10 nM neratinib and stained with MTT. Data represent the mean ± SEM (n=3). (H) SA493 (HER2^S310F) breast cancer organoids stably expressing HER3^WT, HER3^E928G, or untransduced (parental) were treated with 20 μg/ml each trastuzumab (T) and pertuzumab (P), 10 nM neratinib (N), or the combination. Viability was assessed 6 d later using the 3D CellTiterGlo assay and normalized to vehicle-treated controls. Bars represent the mean ± SEM (n=4). P value, 2-way ANOVA + Bonferroni. See also Figure S7.

Figure 7.. Cancer cells harboring co-occurring mutations in HER2 and HER3 are sensitive to combined inhibition of HER2 and PI3Kα.

(A) MCF10A cells stably expressing the indicated transgenes were treated with vehicle (DMSO), 500 nM alpelisib, 500 nM buparlisib, 50 nM neratinib, or the indicated combinations for 4 h in EGF/insulin-free media + 1% CSS. (B) MCF10A cells stably expressing the indicated genes were grown in 3D Matrigel in EGF/insulin- free media + 1% CSS treated with vehicle (DMSO), 20 nM neratinib, 1 μM alpelisib, or the combination. Scale bar, 250 μm. (C) The number of colonies showing invasive branching per field of view (FOV) from (B) was quantified. Data represent the average ± SD (n=3). (D) CW2 colon cancer cells were treated with vehicle (DMSO), 500 nM alpelisib, 50 nM neratinib, or the combination in serum-free media for 4 h. Lysates were probed with the indicated antibodies. (E) CW2 cells were treated with increasing concentrations of neratinib (0–100 nM) or alpelisib (0–1000 nM) alone or in combination for 72 h. Cell viability was quantified using the CyQuant assay and combination indices were determined using the Chou-Talalay test. Numbers inside each box represent the average % viability relative to untreated controls from two independent experiments. (F) Mice carrying CW2 xenografts were treated with vehicle, 40 mg/kg neratinib, 40 mg/kg alpelisib, or the combination for 14 d, starting when tumors reached ~200 mm³. P values (relative to vehicle), one-way ANOVA + Bonferroni. See also Figure S8.

Figure 8.. Model of HER2/PI3K pathway activation by co-occurring HER2/HER3 mutations.

(A) In the absence of ligand, WT HER3 is in the closed conformation and does not interact with WT HER2. NRG1 treatment promotes HER2/HER3 heterodimerization and a HER2 missense mutation further increases HER3 phosphorylation to recruit the p85 subunit of PI3K and activate PI3K signaling. In the absence of ligand, the HER3^E928G mutation phenocopies NRG1 treatment by increasing HER2/HER3 association via enhanced binding of the HER2/HER3 KDs leading to constitutive activation of PI3K. HER2 insertion mutations alone also increase ligand-independent HER2/HER3 association and PI3K activation. (B) Proposed conformational selection model showing how HER2^missense mutations cooperate with HER3^E928G to enhance receptor heterodimerization and HER2 kinase activation.