Specific HER2 Exon 20 Gly776 Deletion-Insertions in Non-Small Cell Lung Cancer: Structural Analysis and Sensitivity to HER2-Targeted Tyrosine Kinase Inhibitors

Background: HER2 exon 20 insertions remain a subset heterogeneous alterations in lung cancer, with currently unmet need for precision targeted therapy. G776delinsVC, a typical HER2 exon 20 deletion-insertion at codon Gly776, was reported to respond discrepantly to afatinib compared with the predominant insertion A775_G776insYVMA (YVMA). However, it lacks structural evidence to illustrate the possible mechanism and predict the binding activities of its similar variants over YVMA insertion to HER2-targered tyrosine kinase inhibitors (TKIs). Methods: Real-world cohort study was performed to investigate clinical outcomes with HER2-targeted TKI afatinib and pyrotinib, and structural analysis for exon 20 Gly776 deletion-insertions G776delinsVC, G776delinsLC and G776delinsVV, and YVMA by molecular dynamics simulation and cellular kinase inhibition assay were provided for full exploration. Results: Afatinib revealed low objective response rate (ORR) of 0–9.5% and short median progression-free survival (mPFS) of 2.8–3.2 months for YVMA, but with higher ORR of 20–28.6% and longer mPFS of 4.3–7.1 months for G776delinsVC. Pyrotinib presented significantly improved PFS benefit than afatinib for G776delinsVC and YVMA as first-line (median, 6.8 vs. 3.4 months, p = 0.010) or second-line therapy (median, 5.8 vs. 2.8 months, p < 0.001). No significant difference was observed on drug binding pocket and TKI binding activity between G776delinsVC, G776delinsLC and G776delinsVV, and both afatinib and pyrotinib showed favorable binding activity. YVMA insertion significantly affected the loop region with altering HER2 protein secondary structure and forming steric hindrance to binding of afatinib. Pyrotinib showed the best selectivity to HER2, with more favorable activity to YVMA than afatinib indicated by cellular inhibition assay. Conclusion: Both afatinib and pyrotinib showed favorable activity for NSCLC patients with HER2 exon 20 Gly776 deletion-insertions. Pyrotinib revealed more potent activity to A775_G776insYVMA insertion than afatinib due to the steric binding hindrance induced by YVMA.

To date, several studies had reported response heterogeneities to HER2-targeted tyrosine kinase inhibitor (TKI) afatinib among HER2 ex20ins (De Grève et al., 2012;Liu et al., 2018;Nagano et al., 2018;Dziadziuszko et al., 2019;Lai et al., 2019). The prospective NICHE study terminated early with the conclusion that afatinib failed to show the expected benefit for HER2 ex20ins patients, especially for those with YVMA insertion, with a short median progression-free survival (mPFS) of 15.9 weeks and poor objective response rate (ORR) of 7.7% (Dziadziuszko et al., 2019). Similarly, a multicenter retrospective study from China as well issued afatinib just achieved a short mPFS of 3.7 months and ORR of 18.2% (2/11) for YVMA insertion . In addition, afatinib was reported with significantly improved mPFS for non-YVMA insertions (G778_P780dup and G776delinsVC) compared with YVMA (7.6 vs. 1.2 months, p < 0.001), in which no response (ORR, 0/14) was observed in patients with YVMA (Kosaka et al., 2017;Fang et al., 2020). In summary, these studies revealed that YVMA insertion showed relatively much lower response to afatinib than other HER2 ex20ins variants. As a novel pan-ErbB inhibitor, pyrotinib potently inhibits HER2 and has been reported with promising and broader efficacy than afatinib for various HER2 ex20ins variants in both patient-derived xenograft (PDX) model and cohort studies (Wang et al., 2019;Zhou et al., 2020;Yang et al., 2021).
Three-dimensional (3D) modeling has demonstrated that HER2 ex20ins induced a constitutively active conformation and caused steric hindrance of the drug-binding pocket, preventing the binding of the noncovalent HER2-targeted TKIs (Robichaux et al., 2018). As a clinically active inhibitor, poziotinib binds tightly into the sterically hindered drug-binding pocket of YVMA insertion to overcome structural changes and inhibit HER2-mutant NSCLC more potently than afatinib by in vitro evidence (Robichaux et al., 2018). Furthermore, the ZENITH20-2 study of poziotinib showed that the mPFS was 5.5 months and the ORR was 27% in advanced NSCLC patients with HER2 ex20ins (Elamin et al., 2021). In addition, the hypoxiaactivated prodrug tarloxotinib, concentrating high levels of active drug tarloxotinib-E (a potent and covalent pan-ErbB inhibitor) within the tumor microenvironment than normal tissue, has been reported of marked tumor response in HER2-activating mutations in NSCLC observed in the RAIN-701 study and in vitro models (Liu et al., 2020;Estrada-Bernal et al., 2021).
G776delinsVC, another specific ex20ins subtype composed of insertion of amino acids Valine and Cysteine (Val-Cys, VC) instead of original glycine at codon 776 (Gly776), was reported to derive more benefit from afatinib when compared with YVMA insertion, but the sample size was too small, with only 5 cases (Fang et al., 2020). In addition, another study found G776delinsVC displayed very different responses to afatinib . However, currently, it lacks structural research distinguishing the Frontiers in Pharmacology | www.frontiersin.org March 2022 | Volume 13 | Article 806737 molecular heterogeneities between G776delinsVC and its similar variants G776delinsVV and G776delinsLC, especially lacking evidence to predict drug sensitivities to afatinib and pyrotinib for these specific Gly776 deletion-insertion subtypes. It is warranted to give a deeper investigation into their molecular structure to guide precision targeted therapy with anti-HER2 TKIs. In this study, we investigated responses to afatinib and pyrotinib for HER2 Gly776 deletion-insertions from a realworld cohort study, and concomitantly explored to identify their molecular differences over YVMA insertion by structural analysis and cellular kinase inhibition assay.

Patients and Molecular Detection
We retrospectively reviewed 172 patients with histologically confirmed metastatic or recurrent NSCLC harboring certain HER2 ex20ins subtypes of A775_G776insYVMA (c.2324_2325insCACCGTGATGGC), G776delinsVC (c.2326_2327insTGT), G776delinsLC (c.2326delinsTTGT) and G776delinsVV (c. 2327delinsTTGT), who had received at least one systemic therapy regimen from January 2016 to June 2021 recorded by medical database in Chinese Academy of Medical Sciences (CAMS)/Cancer Hospital. Information regarding clinical characteristics, HER2 ex20ins variants, and treatment outcome with afatinib or pyrotinib from medical records were collected. HER2 ex20ins were identified using next-generation sequencing (NGS) based on specimens of tumor tissue or plasma. Molecular testing data of HER2 ex20ins among these patients were both collected from the department of pathology in CAMS/Cancer Hospital, and from patients who performed HER2 ex20ins testing by qualified third-party genetic testing companies which had passed the national quality system certification in China. All of the NGS testing for HER2 ex20ins was performed based on the Illumina sequencing system. This retrospective study was approved by the ethical committee of National Cancer Center, and written informed consent was waived.

Efficacy Evaluation
Tumor response was evaluated using computed tomography images of chest and abdomen and brain magnetic resonance imaging, according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. PFS was defined as time from initiation of HER2-targeted TKIs to date of documented disease progression or death from any cause (whichever occurs first). ORR was defined as the proportion of patients with at least one confirmed complete response (CR) and partial response (PR) after TKI targeted therapy. Disease control rate (DCR) was defined as the proportion of patients with at least one confirmed CR, PR and stable disease (SD) by HER2-targeted TKIs.

Computational Structure and Molecular Dynamics Simulation
The homology models of four HER2 ex20ins variants (A775_G776insYVMA, G776delinsVC, G776delinsLC, G776delinsVV) were computationally constructed based on the crystal structure of human HER2 kinase domain in complex with inhibitor TAK285 by the Schrödinger software (2021-1 Release, Schrödinger Inc., Portland, Oregon) (PDB ID: 3PP0). The protein was prepared using Maestro software (Schrödinger 2021-1 Release) in the Schrödinger modeling package. Compounds were constructed using the 3D-sketcher module in Maestro. Binding free energy (ΔG bind ) was used to evaluate the binding activity of a certain compound, which was calculated by molecular mechanics/Poisson-Boltzmann solvent accessible surface area (MM/PBSA) and molecular mechanics/ Generalized Born solvent accessible surface area (MM/GBSA) methods. The detailed calculation regarding ΔG bind of a proteinligand complex was listed in the Appendix.

Cell Lines and Kinase Inhibition Assay
In order to evaluate the inhibitory ability of compounds synthesized on the proliferation of A775_G776insYVMA and HER2 wild type (WT) Ba/F3 cells (purchased from Beijing Kangyuan Bochuang Biotechnology Co., Ltd.), exponentially growing cells were seeded in the media of RPMI Medium 1640 basic with 1% penicillin-streptomycin (purchased from ThermoFisher, Waltham, MA, United States) and 10% Foetal Bovine Serum (FBS, purchased from Biological Industries) at a concentration of 1000 cells/ml in a 384-well cell culture plate (purchased from CORNING, United States) with 20 µl per well and incubated overnight at 37°C, 5% CO 2 incubator. Compounds were prepared as 12-point, 3-fold serial dilutions in dimethyl sulfoxide (DMSO), beginning at 2 mM. 1 µl of DMSO solutions from the compound stock plates were added to 99 µl of cell culture media (final top concentration of compound in the assay was 10 µM and the final concentration of DMSO was 0.5%). 20 µl of compound solutions in media were added to Ba/F3 cell plates. After adding compound solutions, assay plates were incubated for 3 days at 37°C, 5% CO 2 . Cell viability was measured using the CellTiter-Glo assay kit from Promega (Madison, WI, United States) by quantitating the adenosine triphosphate (ATP) present in the cell cultures. Luminescence was read after 20 min of incubation with the SPARK multiple plate reader from TECAN (Switzerland). The half maximal inhibitory concentrations (IC 50 ) of compounds inhibiting cell viability were determined using a sigmoidal dose-response model (variable slopes, four parameters) in Prism 7 (La Jolla, CA) to evaluate the inhibitory ability of compounds on the proliferation of HER2-WT and YVMA Ba/F3 cells. Each experiment was replicated three separate times to give biological replicates unless indicated otherwise.

Statistical Analysis
The Chi-Square test and Fisher's exact test were applied to compare the significance of baseline differences between subgroups. Statistical analyses were conducted using SAS ® software (version 9.4, SAS Institute Inc, Cary, United States). PFS were analyzed using the Kaplan-Meier method. PFS between different subgroups were compared using the logrank test (two-sided), and corresponding 95% confidence interval (CI) were estimated using Cox proportional  .2) 2 (20.0) 6 (11.0) 3 (12.5) 3 (9.7) G776delinsVV

3D Modelling and TKI Binding Simulation
For human HER2 kinase domain, the exon 20 Gly776 deletioninsertions (G776delinsVC, G776delinsVV and G776delinsLC) and 12-bp insertion A775_G776insYVMA are all located in the ATP-binding kinase domain, especially in the loop region following the C-helix (Figure 2). For structural analysis of G776delinsVC, the deletion-insertion position is in the loop region from residue Ala775 to Arg784, which is flexible in the kinase domain. The two inserted amino acid residues Val776 and Cys777 just slightly affect the loop region, neither with effect on the overall conformation of HER2 kinase domain nor the binding pocket, which less likely affects the binding activity to afatinib ( Figure 3A). Similar results are observed when amino acids Val776 and Val777 insert instead of the original residue Gly776 (G776delinsVV, Figure 3B), or inserting amino acids Leu776 and Cys777 (G776delinsLC, Figure 3C), indicating Gly776 deletion-insertions seldom shifting the drug binding pocket and overall conformation of HER2 kinase domain, thus exerting little influence on binding affinity to afatinib. As was illustrated in Table 2, afatinib revealed good binding activity to these three Gly776 deletion-insertion variants, and pyrotinib revealed lower ΔG bind values compared to afatinib, indicating a more potent binding activity than afatinib. Of note, for the typical Gly776 deletion-insertion subtype G776delinsVC, four kinds of HER2-targeted TKIs (pyrotinib, afatinib, dacomitinib, and poziotinib) all exhibited favorable ΔG bind values, in which pyrotinib showed the most potent binding activity (Table 3).
Notably, the YVMA insertion causes a small segment of C-helix into the flexible loop region, with altering the HER2 protein secondary structure greatly. The C-helix insertion is closer to the small molecules, which may form steric hindrance to TKI binding ( Figure 3D). Molecular dynamics simulation issued that pyrotinib revealed superior binding activity to YVMA insertion than other inhibitors ( Table 4). Afatinib revealed an inferior affinity to A775_G776insYVMA conformation by interaction of H-bond in Nitrogen-Met801 and Halogen bond in Chlorine-Asp863 by drug-binding simulation ( Figure 4A). By comparison, pyrotinib potently bound A775_G776insYVMA conformation by interaction of more H-bonds in imino-Ser728, cyano-Met801, carbonyl-Cys804, imino and carbonyl-Water, and arene-H interaction in benzene-Val734 ( Figure 4B).

Sensitivity to HER2-Targeted TKIs in YVMA Insertion and WT Cell Lines
For detecting cellular ATP, it is calculated as the total light emission amount-relative light unit (RLU). Cell viability assay and IC 50 estimation were performed in HER2 WT and YVMA Ba/F3 cells treated with four different inhibitors including  Frontiers in Pharmacology | www.frontiersin.org March 2022 | Volume 13 | Article 806737 6 pyrotinib, afatinib, dacomitinib, and poziotinib ( Figure 5A).
The RLU values correlated with the amount and viability of cultured cells, and poziotinib demonstrated very high potency against both HER2 WT ( Figure 5B) and YVMA cells ( Figure 5C). Consistent with this finding, afatinib and dacomitinib revealed similarly potent inhibition against WT, but dacomitinib was less active against YVMA cells compared to afatinib. Pyrotinib showed the best selectivity to WT, with the least decreased RLU values, and it as well revealed more favorable activity to YVMA cells than afatinib and dacomitinib. The inhibition ability and IC 50 values of four inhibitors against HER2 WT and YVMA cells were depicted in Figure 5D.

DISCUSSION
To date, we present the first study to provide comprehensive evidence on differences of the binding activity between HER2 exon 20 Gly776 deletion-insertions and A775_G776insYVMA to HER2-targeted TKIs afatinib and pyrotinib based on a realworld cohort investigation, along with structural insights into the mechanism by in silico analysis and cellular inhibition assay to give full exploration. We observed the specific HER2 ex20ins subtype G776delinsVC with its similar variants G776delinsVV and G776delinsLC presented similar and favorable response to afatinib, but the predominant YVMA insertion showed poor response to it. In addition, pyrotinib revealed better efficacy and binding activity for these four insertion variants, with no obvious difference. We identified the specific HER2 exon 20 Gly776 deletion-insertion variants could be as potential genomic modifiers of response to afatinib targeted therapy.
It has been reported that afatinib showed higher ORR (40%) and much longer mPFS (7.6 months) for ex20ins patients with G776delinsVC or G778_P780dup, compared with YVMA insertion (ORR, 0; mPFS of 1.2 months) (Fang et al., 2020). Among five patients with G776delinsVC, two achieved PR to afatinib, and the longest PFS (12.0 months) for afatinib was observed in one patient with G776delinsVC (Fang et al., 2020). Subsequent modeling of G776delinsVC revealed no obvious changes in the structure of drug-binding pocket over HER2 WT. By comparison, structural modeling revealed YVMA conformation contained two bulky side chains, which may induce steric hindrance of the drug-binding pocket and thus preventing the interaction with afatinib (Fang et al., 2020). However, in the above study, the sample of patients with G776delinsVC was small (only five patients), and yet it had not analyzed the structural difference between G776delinsVC and its similar variants G776delinsVV and G776delinsLC, and it lacked molecular dynamics simulation for different HER2-targeted TKIs to provide evidence on the mechanism for binding activity differences.
Currently, there is not enough evidence to guide selective HER2-targeted TKIs for HER2 ex20ins NSCLC patients. Consistent with our findings on afatinib, a series of studies had reported that afatinib showed limited activity for HER2 ex20ins in NSCLC (De Grève et al., 2012;Liu et al., 2018;Nagano et al., 2018;Dziadziuszko et al., 2019;Lai et al., 2019;Fang et al., 2020). In addition, afatinib revealed much worse activity for the most common YVMA insertion compared with other insertion subtypes, with a limited ORR of 0-18.2% and mPFS of 1.2-3.0 months Fang et al., 2020). By contrast, pyrotinib revealed mPFS of 6.9 months and overall ORR of 30% in patients with HER2-mutant advanced lung adenocarcinoma as second-line or above therapy, especially, with a higher ORR of 27.3% for YVMA insertion (Zhou et al., 2020).
In our study, afatinib showed mPFS of 2.9-3.4 months and ORR of 8.3-11.5% as 1L and 2L therapy for overall HER2 ex20ins patients, and it revealed lower ORR of 0-9.5% and short mPFS of  2.8-3.2 months for YVMA insertion in 1L or 2L setting, which issued a much limited activity consistent with previous studies Dziadziuszko et al., 2019;Fang et al., 2020). We also observed that G776delinsVC derived more survival benefit from afatinib than YVMA insertion (1L mPFS, 7.1 vs. 3.2 months; 2L mPFS, 4.3 vs. 2.8 months), in accordance with previous study reported (Fang et al., 2020). By comparison, pyrotinib showed PFS benefit than afatinib in overall patients no matter in 1L (median, 6.8 vs. 3.4 months) or 2L setting (median, 5.8 vs. 2.8 months). In addition, our previous PEARL study as well indicated that pyrotinib showed similar clinical benefit in patients with YVMA and non-YVMA insertions (mPFS, 5.1 vs. 5.2 months, p = 0.98), implying a broad spectrum of activity for distinct HER2 ex20ins subtypes (Yang et al., 2021). We subsequently constructed 3D models of these insertion variants, and observed no difference on drug binding pocket and TKI binding activity between G776delinsVC, G776delinsLC and G776delinsVV by structural analysis. Afatinib and pyrotinib showed favorable binding activities to these three deletioninsertions by molecular dynamics simulation. In our PEARL study, we comprehensively analyzed molecular differences between three HER2 common ex20ins variants YVMA, G776delinsVC, P780_Y781insGSP, and concluded that pyrotinib showed better activity against the most common YVMA insertion, and with a similar activity to G776delinsVC compared with afatinib, dacomitinib, and poziotinib (Yang et al., 2021). Dynamics simulation in PEARL study also confirmed that ΔG bind of pyrotinib was the lowest compared with other pan-ErbB inhibitors for YVMA and G776delinsVC insertions, owing to abundant H-bonds interaction with these insertions (Yang et al., 2021). The PEARL study did not compare G776delinsVC with its similar subtypes G776delinsVV and G776delinsLC, and we are  uncertain if these insertions showed similar activities to HER2targeted TKIs. Our current study makes a supplementary contribution to confirm that these three specific Gly776 deletion-insertions display favorable activity to afatinib and pyrotinib, especially, they are more sensitive to pyrotinib. Of note, in this study, we found YVMA insertion significantly affected loop region following the C-helix with altering HER2 protein secondary structure, thus forming steric hindrance to TKI binding, which was in accordance with our previous findings in PEARL study and other researches (Fang et al., 2020;Yang et al., 2021). As well, in this current study, we demonstrated that pyrotinib revealed superior binding activity to YVMA insertion by more and much potent H-bonds than afatinib, however, afatinib only interacted with YVMA insertion by H-bond in Nitrogen-Met801 and Halogen bond in Chlorine-Asp863 through 3D-based drug-binding simulation. Our PEARL study and this current research together confirmed that afatinib displayed inferior binding activity than pyrotinib for YVMA insertion, but they showed both favorable activities to HER2 exon 20 Gly776 deletion-insertions. The cell viability assay and IC 50 estimation results in this study finally supported our findings above, indicating that pyrotinib showed the best selectivity to HER2 WT, and more potent antitumor activity to YVMA insertion than afatinib and dacomitinib. In our study, poziotinib demonstrated significantly potent antitumor activity in YVMA cells, which confirmed the reported findings in another study issuing that poziotinib was six times more potent than afatinib and dacomitinib in cell lines with HER2 exon 20 mutants in vitro (Robichaux et al., 2018). Whereas, poziotinib as well exhibited high potency against HER2 WT cells, which was correlated with high levels of EGFR-related toxicity, including rash and diarrhea requiring dose reductions in a significant number of patients (Elamin et al., 2021).
Though this real-world study precisely investigated the clinical activity of pyrotinib and afatinib for specific HER2 ex20ins variants in advanced NSCLC, as well as providing valuable evidence from molecular dynamics simulation and cellular kinase inhibition experiment, several limitations must be noted. First, this was a retrospective real-world study that easily appeared selection bias. Moreover, the sample size of patients with HER2 Gly776 deletioninsertions was small, which prevented us from providing further information on the clinical efficacy of HER2-targeted TKIs. Although we explained the potential mechanism on differences of binding activity to afatinib and pyrotinib in terms of specific insertion subtypes, this is an exploratory analysis based on structural modelling and molecular dynamics simulation that cannot fully represent all the possible reasons. More clinical evidence including Gly776 deletion-insertions cell lines and PDX models are warranted to correspond our findings, and so as to draw a clear conclusion.
In conclusion, both afatinib and pyrotinib showed favorable activity for NSCLC patients with HER2 exon 20 Gly776 deletioninsertions. Pyrotinib revealed more potent activity to A775_G776insYVMA insertion than afatinib due to the steric binding hindrance induced by YVMA conformation.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.