Actionable Activating Oncogenic ERBB2/HER2 Transmembrane and Juxtamembrane Domain Mutations

Abstract

Deregulated HER2 is a target of many approved cancer drugs. We analyzed 111,176 patient tumors and identified recurrent mutations in HER2 transmembrane domain (TMD) and juxtamembrane domain (JMD) that include G660D, R678Q, E693K, and Q709L. Using a saturation mutagenesis screen and testing of patient-derived mutations we found several activating TMD and JMD mutations. Structural modeling and analysis showed that the TMD/JMD mutations function by improving the active dimer interface or stabilizing an activating conformation. Further, we found that HER2 G660D employed asymmetric kinase dimerization for activation and signaling. Importantly, anti-HER2 antibodies and small-molecule kinase inhibitors blocked the activity of TMD/JMD mutants. Consistent with this, a G660D germline mutant lung cancer patient showed remarkable clinical response to HER2 blockade.

Keywords: ERBB2 activation; ERBB2 structure; ERBB2/HER2; HER2 germline mutation; HER2 kinase inhibitors; HER2 somatic mutation; anti-HER2 antibodies; juxtamembrane (JMD) domain mutation; transmembrane domain (TMD) mutation.

Figure 1.. HER2 mutations in patient tumors.

(A) Mutations observed in HER2 shown across a diagram of the HER2 protein. (B) Mutational hotspots observed in HER2 TMD/JMD regions across different cancers. Only mutations found in at least 4 samples are shown. (C) Amino acid composition of the TMD in WT HER2 (PDB ID: 2JWA) and in V659E, G660D, or G660R mutants, highlighting the relative arrangement of side chain atoms of polar (oxygen (red) and nitrogen (blue) atoms shown as spheres) and apolar (carbon atoms (green) shown as sticks) residues. See also Tables S1 and S2.

Figure 2.. Saturation mutagenesis of HER2 TMD/JMD.

(A) Schematic of the mutagenesis screen. (B) Bar plot representing the allele frequency of HER2 mutations identified in the screen 4 days following IL-3 removal. Screen was done without or with co-expressed WT HER2. HER2 protein sequence where mutations were observed in patient tumors is shown within shaded boxes below the bar plot. See also Table S1.

Figure 3.. HER2 TMD/JMD mutants promote cell survival.

(A) Relative survival of BaF3 cells expressing indicated HER2 mutants in the absence or presence of WT HER2. Residues mutated in patient tumors in the TMD and JMD are shown in shaded boxes below the bar plot. Solid green bars indicate mutants that showed statistically significant (two-tailed Student t-test; Bonferroni adjusted p value < 0.05) survival when compared to WT HER2 alone expressing BaF3 cells. Grey bars indicate mutants that did not show significant survival compared to WT HER2 alone. Data are presented as mean ± Standard Deviation (SD) of relative cell survival of twelve replicates and representative of three independent experiments. Circles inset with the single letter amino acid code representing the mutants below the bar plot shows the amount of pHER2 observed in BaF3 cells expressing the mutants. (B,C) Western blots showing the expression of total HER2, pHER2 and β-actin in the indicated stable BaF3 cells in the absence (B) or presence (C) of WT HER2. (D) MCF10A cell lines expressing the indicated HER2 mutants tested in the absence or presence of WT HER2. Scale bar shown at the bottom of each image represent 400 μm. Assay was performed in the absence of exogenous EGF or other growth factors. EV- empty vector. See also Figure S1 and Table S2.

Figure 4.. Conformational analysis of HER2 TMD/JMD mutants.

(A) Representative TMD/JMD germline and somatic mutations mapped onto a structural model of full-length HER2 composed using the crystal structures of the HER2 extracellular domain (PDB ID: 1N8Z), HER2 transmembrane domain (PDB ID: 2JWA) and HER2 kinase domain (PDB ID: 3PP0). (B) Sequence alignment of the HER family receptors. (C) MD simulations were performed using the coordinates of the WT HER2 TMD dimer (PDB ID 2JWA, residues 649 – 679) as a starting model. (D) Overview of the final state obtained at the end of a 100 ns simulation of the WT HER2 TMD dimer (left), a heterodimer between a WT TMD and an activating C-terminal R678Q TMD mutant (WT/R678Q) (middle) and a homodimer of the activating N-terminal G660D TMD mutant (G660D/G660D) (right). (E) Overlay of the final states observed for five independent MD simulations of the G660D/G660D HER2 TMD dimer (sim1-sim5). (F) Surface representation of the C-lobe of the HER2 kinase domain bound to a juxtamembrane latch binding region to depict interactions mediated by Q709 and L709. The interface was modeled by aligning structures of the HER2 kinase domain (PDB ID: 3PP0) on both activator and receiver kinases in the structure of the HER3/EGFR asymmetric dimer (PDB ID: 4RIW). See also Figure S2.

Figure 5.. G660D HER2 activation involves asymmetric dimerization of the KD.

(A) Active HER2 asymmetric dimer model. Residues involved in the asymmetric kinase domain interactions in HER2 deduced from EGFR structural studies are shown (inset). (B) IL-3 independent cell survival of BaF3 cells stably expressing the indicated kinase, activator or receiver mutants. Data are presented as mean ± Standard Deviation (SD) of relative cell survival of twelve replicates and representative of three independent experiments. (C) Cartoon representation of the proposed mechanism of activation of HER2 G660D. The number shown below corresponds to the lane number in (B). See also Figure S3.

Figure 6.. HER2 inhibitory drugs are effective in blocking HER2 TMD/JMD mutants.

(A) Cartoon of HER2 depicting binding of trastuzumab and pertuzumab to ECD domain IV and domain II, respectively. (B) Effect of indicated anti-HER antibodies and the Fab portion of pertuzumab on survival of BaF3 cells expressing HER2 G660D mutant. (C-F) Effect of trastuzumab (C), pertuzumab (D), neratinib (E), and afatinib (F) on IL-3 independent survival of BaF3 cell expressing the indicated HER2 mutants. Data shown in (B-F) are mean ± SEM, where SEM is standard error of mean of four technical replicates and representative of experiment repeated three independent times. RLU = Relative luciferase units; EV = empty vector; WT = wild-type HER2. (G) Kaplan-Meier survival curves for cohorts of mice (n = 10) implanted with HER2 G660D expressing BaF3 cells that were either untreated or treated with anti-Ragweed (control) or trastuzumab. WT HER2 expressing BaF3 cells served as controls. See also Figures S4, S5 and Tables S3, S4.

Figure 7.. Germline HER2 G660D lung cancer patient responds to therapy.

(A) Pedigree of a family in which multiple members were diagnosed with lung cancer. Solid black and grey circles (females) and squares (males) indicate affected individuals. Blood samples were obtained from affected individuals represented by solid black circle or squares. Slash mark indicates deceased individuals. (B) Flowchart depicting the exome analysis. (C) Chest CT scan of the patient before and after 12 weeks of treatment with afatinib. See also Figures S6, S7 and Table S5.