An alternative EGFR activation by patient-derived R252C mutation promotes cancer progression

Domain: chemistry
Published: 2026-01-21
Editorial outcome: major_revision

Abstract
Mutations in the extracellular or intracellular domains of epidermal growth factor receptor (EGFR) are implicated in the development of various cancers. While the intracellular mutations of EGFR have been extensively studied, the function of extracellular mutations remains poorly understood. In this study, we identify an EGFR mutant (EGFR R252C) in a patient with multifocal lung cancer and glioma, in which arginine (R) 252 is mutated to cysteine (C) in the EGFR extracellular domain. This mutation promotes C252-C252 disulfide-mediated EGFR dimerization and induces a conformational change of EGFR, leading to absent autophosphorylation and enhanced direct interaction between EGFR and extracellular signal-regulated protein kinase 1/2 (ERK1/2). Importantly, EGFR directly phosphorylates ERK1/2 at threonine (T) 202/tyrosine (Y) 204 and activates ERK1/2, thereby promoting tumor cell proliferation and tumor growth in vivo. Afatinib, a second-generation EGFR tyrosine kinase inhibitor, effectively suppresses primary tumor growth and extends progression-free survival in the patient with multifocal lung cancer and glioma driven by EGFR R252C. Our finding elucidates the activation mechanism of this extracellular EGFR mutation and demonstrates the efficacy of afatinib in treating lung cancer or glioma patients with this variant.

Introduction
Epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase protein that has been implicated in tumor cell proliferation, angiogenesis, tumor invasion, metastasis, and inhibition of apoptosis. EGFR mutations are observed in approximately 5% of all cancer patients, 14% of non-small cell lung cancer (NSCLC) patients, and 26% of glioma patients1–3. In lung cancer, EGFR mutations predominantly occur in the intracellular kinase domain (KD); these mutations enhance receptor activation by stabilizing the active conformation of the KD, thereby facilitating the dimerization process. While in glioma, EGFR mutations mainly occur in the extracellular domain (ECD)1,4. EGFRvIII, the most commonly identified mutation in glioma, features an in-frame deletion between residues 6 and 273 within the ECD, which impedes EGF binding to the receptor while maintaining its kinase activity5. Furthermore, point mutations in the ECD of EGFR, such as arginine (R) 108 to lysine (K), alanine (A) 289 to valine (V)/aspartate (D)/threonine (T), glycine (G) 598 to V, and others, have been identified in glioma samples6. Mutations in both the EGFR KD and ECD are common in cancer patients and hold significant importance. However, compared to the EGFR KD, research on mutations in the ECD remains relatively scarce. Therefore, further studies on EGFR ECD mutations are urgently needed.

Given the prevalence of EGFR mutations observed in numerous cancer patients, several generations of EGFR TKIs have been developed, with ongoing research efforts persisting in this field. EGFR mutations occurring at different positions within the KD exhibit varying sensitivities to different generations of EGFR TKIs. For instance, point mutations at exon 21 (leucine 858 to arginine) show favorable responses to both first- and third-generation EGFR TKIs. However, mutations such as G719X, S768I, and L861Q demonstrate greater sensitivity to second-generation TKIs1. In contrast to the well-established research and therapeutic advancements for EGFR KD mutations, progress for EGFR ECD mutations has lagged behind. Although EGFR ECD mutations also alter the structure and function of EGFR, leading to aberrant activation of the EGFR signaling pathway, existing EGFR TKIs demonstrate limited efficacy against these mutations. This limited response may be attributed to the conformational changes or differences in the signaling mechanisms induced by ECD mutations4,6. Currently, no approved treatment strategies exist for cancers driven by EGFR mutations within ECD1. Therefore, investigating novel therapies for these mutations is not only urgent but also essential. Only through further in-depth exploration of the mechanisms and biological characteristics of ECD mutations can we develop new treatments that improve patient outcomes and provide effective treatment options for a broader population of EGFR-mutant patients.

In this study, we identified an EGFR mutation within the ECD from a patient with multifocal lung cancer and glioma. We conducted a comprehensive analysis of the activation mechanisms and functional properties associated with this mutation and proposed that second-generation EGFR-TKI afatinib effectively inhibited tumor progression driven by this alteration. Notably, treatment with afatinib led to a substantial reduction in tumor burden and prolonged progression-free survival in the patient with multifocal lung cancer and glioma harboring this mutation.

Results
EGFR R252C promotes tumor cell proliferation and tumor growth in vivo
We admitted a patient with malignant tumor lesions detected on both lung CT and brain MRI (Fig. 1a). The patient was initially treated with four cycles of platinum-based chemotherapy combined with pemetrexed, with CT imaging demonstrating a partial response (PR). To further suppress tumor progression, a combination of chemotherapy and bevacizumab was administered, but the patient experienced tumor enlargement. Consequently, KEYTRUDA immunotherapy was then initiated, but the patient showed no response. The treatment timeline of this patient was shown in Fig. 1b. To explore potential treatment options for the patient, we investigated the underlying causes of the disease. Circulating tumor DNA (ctDNA) sequencing of the patient’s peripheral blood was conducted to identify clinically actionable mutations. No pathogenic variants were detected in common hotspots (NRAS Q61/G13/G12, PIK3CA H1047, EGFR G719/L858, BRAF V600, or KRAS Q61/G13/G12). Notably, comprehensive genomic profiling uncovered a rare EGFR R252C mutation. This suggests that the EGFR R252C mutation may have oncogenic activity and induce tumor growth in the patient.

Considering the presence of tumor lesions in both the lungs and brain of the patient, we searched the Cancer Genome Atlas (TCGA) database for the EGFR R252C mutation. We found that this mutation occurs in 0.4 % of glioma patients, but it was not detected in lung cancer patients. However, in an unpublished database from China, among 4000 lung cancer patients, we identified one patient harboring this mutation. To further confirm the function of EGFR R252C, we adopted CRISPR-Cas9 technique to generate EGFR R252C knock-in U87 and U251 human glioblastoma cells and H1299 human non-small cell lung cancer cells. Successful gene knock-in was validated by genetic sequencing (Fig. 1c). EGFR R252C did not obviously influence EGFR expression (Fig. 1d, e). As shown in Fig. 1f, g, EGFR R252C expression markedly increased the proliferation and colony formation in these tumor cells compared to EGFR WT.

To validate our findings in a clinically relevant GBM model, we utilized the patient-derived GSC387 glioma stem cell line, which maintains key molecular and phenotypic characteristics of primary glioblastoma7. Using lentiviral-mediated gene delivery, we established GSC387 cell lines stably expressing either HA-tagged EGFR WT or the R252C mutant. The expression of EGFR R252C significantly enhanced GSC387 proliferation compared to EGFR WT (Supplementary Fig. 1a, b). Furthermore, we intracranially injected luciferase-expressing U87 cells with or without EGFR R252C mutation into randomized athymic nude mice. Bioluminescence imaging of mice showed that mice bearing intracranial tumors with the EGFR R252C mutation demonstrated a more rapid growth rate compared to those implanted with tumor cells expressing EGFR WT (Fig. 1h). Additionally, we injected luciferase-expressing H1299 cells harboring EGFR WT or R252C into the left lung of athymic nude mice via orthotopic lung injection. Similarly, the results indicated that the growth of lung tumors in mice with H1299 cells expressing EGFR mutation exhibited a faster growth rate compared to those with EGFR WT cells (Fig. 1i). In summary, EGFR R252C mutation promotes cell proliferation and tumor growth in vivo.

EGFR R252C phosphorylates and activates ERK1/2
To explore how EGFR R252C promotes tumor cell proliferation, we first examined the activation of EGFR and its downstream signaling pathways in U87, U251, and H1299 cells expressing EGFR WT or R252C. Strikingly, EGFR R252C expression did not influence the levels of phosphorylated (p) EGFR tyrosine (Y) 1068 (EGFR pY1068), phosphorylated MEK serine (S) 217/221 (MEK pS217/221), phosphorylated AKT threonine (T) 308 (AKT pT308) and phosphorylated STAT3 tyrosine 705 (STAT3 pY705), while it markedly increased the levels of phosphorylated ERK threonine 202 and tyrosine 204 (ERK1/2 pT202/Y204) (Fig. 2a). Then, we wondered whether the kinase activity of EGFR R252C is required for ERK1/2 activation. We incubated tumor cells expressing EGFR WT or R252C with afatinib, the EGFR inhibitor, which showed that EGFR R252C-activated ERK1/2 activation was suppressed by afatinib treatment (Fig. 2b). A similar result was obtained in GSC387 cells (Supplementary Fig. 2a).

ERK1/2 are the final components of the mitogen-activated protein kinase (MAPK) phosphorylation cascade, serving as a crucial module in various signaling pathways that shape cell behavior and fate, including cell proliferation8. To examine whether EGFR R252C enhanced cell proliferation through ERK1/2, we treated U87, U251, and H1299 cells expressing EGFR WT or R252C with ERK1/2 inhibitor, ravoxertinib. ERK1/2 inhibition abrogated EGFR R252C-promoted tumor cell proliferation (Fig. 2c), demonstrating that ERK1/2 activation serves as a critical downstream effector of EGFR R252C oncogenic signaling.

ERK1/2 activation is initiated through a sequential phosphorylation cascade involving receptor tyrosine kinase (RTK)-mediated RAS activation, subsequent RAF kinase recruitment, and ultimately MEK-dependent dual phosphorylation of ERK1/2 at their activation loop8. To investigate whether EGFR R252C-induced ERK1/2 activation is dependent on the canonical RAS/RAF/MEK pathway, we treated U251 cells harboring either EGFR WT or EGFR R252C with RAS inhibitor (tipifarnib), RAF inhibitor (dabrafenib), or MEK inhibitor (trametinib), respectively. As shown in Supplementary Fig. 2b–d, inhibition of RAS, RAF, or MEK failed to block EGFR R252C-induced ERK1/2 activation. The efficacy of the above inhibitors was assessed under EGF stimulation (Supplementary Fig. 2e). Above results indicated that EGFR R252C-induced ERK1/2 activation is independent of the canonical RAS/RAF/MEK pathway. This conclusion was further validated by MEK1/2 depletion experiments in both U251 and H1299 cell lines (Supplementary Fig. 2f, g).

To elucidate how EGFR R252C activates ERK1/2 independent of MEK1/2 activation, we performed co-immunoprecipitation (co-IP) experiments in EGFR WT and EGFR R252C cells. We found that EGFR directly interacted with ERK1/2 and R252C enhanced the interaction between EGFR and ERK1/2 in U87, U251, or H1299 cells (Fig. 2d). It was further evidenced by the in vitro pulldown assay (Fig. 2e). To test whether EGFR R252C directly phosphorylates ERK1/2, we performed the in vitro kinase assays by mixing recombinant His-ERK1 or GST-ERK2 with recombinant Flag-EGFR WT or R252C. It was shown that EGFR directly phosphorylated ERK1/2, while more ERK1/2 were phosphorylated by EGFR R252C (Fig. 2f, g). Taken together, these results suggest that EGFR R252C directly phosphorylates T202/Y204 to activate ERK1/2, there by promoting the proliferation of tumor cells.

Structural basis of EGFR R252C-dependent ERK1/2 activation
Upon binding with its ligand, EGFR forms either homodimers or heterodimers, subsequently activating the intracellular kinase domain and initiating downstream signaling pathways9. To determine whether R252C affects ERK1/2 phosphorylation by affecting the dimerization of EGFR, we performed co-IP experiments and observed that EGFR R252C forms more dimers than EGFR WT (Fig. 3a).

Next, we investigated the structural basis of EGFR R252C-enhanced EGFR-ERK1/2 interaction by performing molecular dynamics (MD) simulation for the ligand-free EGFR R252C ECD (PDB code 3NJP10). The conformations sampled from independent trajectories based on the clustering analysis11 showed that the ligand binding site gap between domain I and III still maintained in the ligand-free state of EGFR R252C (Supplementary Fig. 3a). The main reason for the binding site gap existing was that EGFR R252C formed the disulfide bond between the two monomers, which stabilized the dimeric ligand-free conformation.

To confirm whether R252C mutation stabilizes a unique ECD dimer of EGFR and whether the dimer formation is dependent on the C252-C252 disulfide, we transfected H1299 cells with HA-tagged EGFR WT, R252C, or R252S. The cells were harvested and analyzed by non-reducing SDS-PAGE to preserve disulfide bonds. As shown in Supplementary Fig. 3b, R252C mutation indeed promoted the dimer formation of EGFR, while R252S mutation failed to do so. The result confirmed that R252C stabilizes a unique EGFR ECD dimer specifically through C252-C252 disulfide bond formation. This conclusion was further validated by glutaraldehyde-mediated crosslinking experiments (Supplementary Fig. 3c). Besides, we transfected H1299 cells with HA-tagged EGFR WT, R252C, or R252S. The cells were harvested and analyzed by SDS-PAGE. As shown in Supplementary Fig. 3d, compared to EGFR WT, R252C mutation enhanced ERK1/2 activation, while EGFR R252S mutation failed to do so. These results confirmed that R252C mutation promotes the dimerization of EGFR to activate ERK1/2, which is dependent on the C252-C252 disulfide bond.

Moreover, in ECD of EGFR R252C dimer, the distance between C terminal (Cα of residue 638, dcc) of the two subunits was more similar to that of ligand-bound EGFR WT rather than ligand-free EGFR WT (Supplementary Fig. 3e). Meanwhile, the angle formed by I214 and P228 (in the same subunit) and P228 (in the other subunit) was also more similar to that of ligand-bound EGFR WT (Supplementary Fig. 3f). As shown in Supplementary Fig. 3a, the ligand-free EGFR R252C, like the ligand-bound EGFR WT, maintained a staggered conformation, which contrasted with the ligand-free EGFR WT that exhibited a flush conformation. Therefore, in the ligand-free EGFR R252C, the extracellular dimer presented a “V shape” and staggered conformation, which was similar to the extracellular dimer of the ligand-bound EGFR WT, but with a smaller dcc.

The transmembrane (TM) segment links the extracellular and intracellular domains. Because of the dcc of the EGFR R252C extracellular dimer conformation was similar to that of the ligand-bound EGFR WT, the N-terminal TM dimer was more suitable for the EGFR R252C in the absence of a ligand. The GxxxG motifs often form the dimerization interfaces of the TM helices12–14. In EGFR R252C, the distance between the center of mass of the two motif in helix was about 7.5 Å, which was consistent with the results of ligand-bound EGFR WT obtained by Arkhipov et al.12 (6 Å), but was significantly different from the ligand-free EGFR WT conformation (4.5 Å) (Supplementary Fig. 3g). The contact probability map of ligand-free EGFR R252C showed that the TM dimer maintained N-terminal contact, similar to that of the ligand-bound EGFR WT (Supplementary Fig. 3h).

In intact EGFR, juxtamembrane (JM) dimers directly connected the TM dimers and the KD. It has been previously reported that the JM dimers form an antiparallel helix in the active state of EGFR12. We found that the JM dimer of EGFR R252C was also antiparallel helix and stabilized along the simulations (Supplementary Fig. 4a). Hence, the conformation of the extracellular domain, TM, and JM dimers of EGFR mutant was similar to that of the ligand-bound EGFR WT.

Due to the smaller dcc of the EGFR R252C ECD and the antiparallel helix structure of the JM, the active site in the asymmetric KD was exposed. The final conformation was shown in Fig. 3b. The distribution of the center of mass between the two KDs in the simulated ECD-TM-JM-KD dimer of EGFR R252C showed that significantly larger than ligand-bound EGFR WT (Fig. 3c). The PCA results revealed that in the EGFR R252C, the two KD regions behaved differently: the residues in one KD remain nearly stationary, while the residues in the upper and lower parts of the other KD region move in opposite directions, causing the KD to twist and exposing the active site (Supplementary Fig. 4b). Although the active site faced the interior of the cell in both the ligand-free EGFR R252C and the ligand-bound EGFR WT, the PCA results indicate that the orientation of the active site was altered in the ligand-free EGFR R252C, causing it to deviate from the other KD (Supplementary Fig. 4b).

To corroborate our simulation results, we performed a NanoBiT assay, where two non-fluorescent subunits of the luciferase enzyme (LgBiT and SmBiT) are fused to EGFR WT or R252C. The experimental data indicated that compared to EGFR WT treated with EGF, EGFR R252C exhibited reduced fluorescence intensity, which is likely due to the increased distance between the two KD dimers in the EGFR R252C (Fig. 3d). Due to the change in the active site orientation in the ligand-free EGFR R252C, the distance between the KD of the two subunits increases, resulting in absent autophosphorylation of EGFR R252C, compared to that in the activated state of EGFR WT (Supplementary Fig. 4e, f).

Thus, we evaluated the binding affinities between ERK1 and KD dimer of the ligand-free EGFR R252C and ligand-bound EGFR WT using MM-GBSA calculations. The stronger total binding free energies of KD dimer of ligand-free EGFR R252C with ERK1 is −85.34 kcal/mol (Fig. 3e), which was significantly stronger than the ligand-bound EGFR WT with ERK1 (−48.53 kcal/mol). The dominant residues favoring for EGFR R252C KD dimer binding with ERK1 were R986, E804, E1004, L718, L1001, M1002, F795, R803, P919, S720 and K716 of ligand-free EGFR R252C (Fig. 3f). Through the analysis of native contact, combined with the possible electrostatic interaction analysis, the residues E922 and I926 were also consider as the possible dominate residues of EGFR R252C.

To identify the dominant residues involved in the binding of EGFR R252C with ERK1/2, we generated mutations at R803, E804, P919, E922, I926, and R986 in the EGFR R252C and conducted immunoprecipitation assays, which identified P919 as a potential binding site between the EGFR R252C KD dimer and ERK1/2 (Fig. 3g and Supplementary Fig. 4g, h). Additionally, cell proliferation and soft agar colony formation assays demonstrated that the P919G mutation significantly abrogated the proliferative capacity of EGFR R252C cells (Fig. 3h, i and Supplementary Fig. 4i). Our experiments identified P919 as a critical binding site within the EGFR R252C for ERK1/2, providing evidence that it mediates the functional effects of EGFR R252C.

In summary, the simulated EGFR R252C model contains an active ligand-free extracellular dimer conformation, similar to the ligand-bound active EGFR WT extracellular dimer, including the N-terminal TM, antiparallel JM dimer, and active asymmetric KD dimer. The active site of the KD dimer was oriented toward the cell interior; however, the ligand-free EGFR R252C exhibits an increased distance between the KDs and an altered domain orientation. These changes are the primary reasons for the absent autophosphorylation levels observed in EGFR R252C compared to ligand-bound EGFR WT. The binding free energy between the EGFR mutant and ERK1 is stronger than that between EGFR WT and ERK1 due to the conformational change, leading to a dramatic increase in ERK1/2 phosphorylation levels.

Afatinib inhibits EGFR R252C-driven tumor cell proliferation and tumor growth in vivo
Gefitinib and erlotinib are first-generation EGFR-TKIs that reversibly bind the ATP-pocket of EGFR, mainly targeting common mutations (ex19del/L858R). Afatinib, a second-generation inhibitor, irreversibly blocks EGFR with broader mutation coverage but higher toxicity. Osimertinib, the leading third-generation TKI, irreversibly inhibits both classic mutations and T790M resistance mutations while sparing wild-type EGFR, reducing side effects15. Studies have shown that mutations in different regions of EGFR elicit varying responses to EGFR TKIs. Mutations in the KD preferentially respond to the first-generation EGFR TKIs, whereas mutations in the ECD exhibit greater sensitivity to second-generation EGFR TKIs4.

We next evaluated the sensitivity of EGFR R252C to four EGFR-TKIs (including afatinib, gefitinib, erlotinib, and osimertinib) by examining EGFR R252C-induced ERK1/2 activation and tumor cell proliferation. Immunoblotting analysis revealed that afatinib inhibited R252C-induced ERK1/2 phosphorylation in a dose-dependent manner, while gefitinib, erlotinib, and osimertinib exhibited minimal effects on phospho-ERK1/2 levels (Supplementary Fig. 5a, b). Supplementary Fig. 5c provides validation of the therapeutic efficacy of afatinib, gefitinib, erlotinib, and osimertinib. Consistent with the signaling data, afatinib exhibited stronger inhibition on R252C-driven proliferation than gefitinib and erlotinib, while osimertinib showed no anti-proliferative activity (Fig. 4a, b and Supplementary Fig. 6a). Collectively, these results suggested EGFR R252C mutation confers sensitivity to afatinib.

We then treated GSC387 cells stably expressing HA-EGFR WT or R252C with afatinib. Similarly, afatinib fully suppressed R252C-driven proliferation in GSC387 cells (Supplementary Fig. 6b). In addition, colony formation assays also demonstrated that U87, U251 and H1299 cells harbored EGFR R252C mutation had much less colonies formation after afatinib treatment than untreated cells (Fig. 4c). These results showed that afatinib can effectively suppressed the proliferative capacity of EGFR R252C-mutated cells.

To directly test the ability of afatinib to reduce tumor growth, we administered luciferase-expressing U87 cells with or without the EGFR R252C mutation, via intracranial injection into randomized athymic nude mice, and subsequently treated these mice with afatinib. Bioluminescence imaging of these mice showed that afatinib inhibited tumor growth in both EGFR WT and R252C tumor cells. Importantly, it showed a more pronounced inhibitory effect on tumor growth in cells with EGFR R252C (Fig. 4d). Meanwhile, we orthotopically injected luciferase-expressing H1299 cells with or without EGFR R252C mutation into the left lung of athymic nude mice and gave afatinib treatment. Similarly, the results indicated that afatinib more effectively inhibited the growth of lung tumors in mice with H1299 cells expressing EGFR R252C compared to EGFR WT cells, suggesting that afatinib can inhibit tumor growth induced by the EGFR R252C mutation in vivo (Fig. 4e). Furthermore, in U87 engraftment mouse models, tumors expressing EGFR R252C exhibited significantly reduced overall survival compared to WT controls (median survival: 52 vs. 59 days; p < 0.001), aligning with their aggressive proliferative phenotype. Notably, afatinib treatment prolonged the survival of R252C-bearing mice. These findings were recapitulated in H1299 xenograft models, where EGFR R252C expression similarly correlated with worse survival outcomes (median survival: 39 vs. 49 days for WT; p = 0.002). Afatinib again demonstrated therapeutic efficacy, increasing the median survival of EGFR R252C-bearing mice (Supplementary Fig. 6c).

The patient with multifocal lung cancer and glioma, harboring the EGFR R252C mutation as previously described, showed disease progression despite multiple treatment regimens, including platinum-based chemotherapy (cisplatin), chemotherapy combined with bevacizumab, and immunotherapy. Based on our prior experimental findings that afatinib inhibits the growth of EGFR R252C mutant tumor cells, we decided to administer afatinib therapy. Notably, following afatinib treatment, the patient exhibited remarkable clinical improvement, with CT and MRI scans revealing a marked reduction in lung and brain tumor burden (Fig. 4f). This clinical benefit persisted for approximately four years, ultimately leading to the patient’s discharge (Fig. 4g). These findings suggest that afatinib is an effective therapeutic option for patients harboring this mutation and provide compelling evidence supporting its clinical utility in the treatment of EGFR ECD mutations.

Methods
Ethics statement
Ethical approval for the human participant was approved by the Ethics Committee in Clinical Research (ECCR) of the First Affiliated Hospital of Wenzhou Medical University and the study was performed in accordance with the approved protocol. Informed consent was obtained from the patient’s family for both research and publication of de-identified data included in this article. No compensation was provided to the participant.

All animal experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the Shanghai Institute of Biochemistry and Cell Biology (approval number: SIBCB-S355-2312-40) and complied with all relevant ethical regulations.

Mice
Six-week-old female BALB/c athymic nude mice were purchased from Lingchang Biotech (Shanghai, China). Littermates were randomly assigned to experimental groups. Sex was not considered as a biological variable in the study design. All experiments were conducted with female mice to limit complications of male territorial behavior and fighting during long-term cancer experiments. Mice were housed under specific pathogen-free conditions with a standard chow diet and maintained at a room temperature of 20–26°C with 40–70% humidity on a 12-h light/12-h dark cycle. In accordance with the approved IACUC protocol, in vivo experiments were terminated at predefined time points or when mice reached humane endpoints, including a >20% body weight loss or signs of severe distress (such as lethargy or impaired mobility). Mice were monitored daily, and euthanasia was performed according to IACUC-approved protocols once the endpoint criteria were met. No mouse exceeded these ethical limits during the study.

Antibodies
Rabbit monoclonal antibodies against HA (3724S), phospho-MEK1/2 (9154S), MEK1/2 (8727S), phospho-AKT (4056S), STAT3 (63585S), phospho-EGFR Y845 (6963S), phospho-EGFR Y1173 (4407S), EGFR (4267S) and rabbit polyclonal antibodies against phospho-EGFR Y992 (2235S), phospho-EGFR Y1045 (2237S), phospho-EGFR Y1086 (2220S), phospho-EGFR Y1148 (4404S), and phospho-EGFR Y1068 (2234S) were obtained from Cell Signaling Technology. Mouse monoclonal antibodies against GST (66001-2-Ig), His-tag (66005-1-Ig) and rabbit monoclonal antibody against Flag (20543-1-AP) were obtained from Proteintech Group. Mouse monoclonal antibody against phospho-ERK1/2 (sc-81492) and rabbit polyclonal antibody against ERK1/2 (sc-94) were purchased from Santa Cruz Biotechnology. Rabbit polyclonal antibodies against EGFR (A11351), AKT (A11016) and phospho-STAT3 (AP0070) were brought from Abclonal Technology. Mouse monoclonal antibody against Tubulin (T5201) was purchased from Millipore Sigma. The following secondary antibodies were used: goat-anti-mouse IgG second antibody (31160, Thermo); goat-anti-rabbit IgG second antibody (31210, Thermo). The primary antibodies were used at a 1:1000 dilution and the secondary antibodies were used at 1:3000 dilution for immunoblotting.

Reagents
Puromycin (540411-100MGCN) was bought from Merck/Millipore (Darmstadt, Germany). DNA transfection reagent Hieff Trans Liposomal Transfection Reagent (H17520) was purchased from Yeasen Biotechnology (Shanghai, China). Flag peptide was obtained from Abclonal (Wuhan, China). ATP was purchased from InvivoGen (CA, USA). EGFR inhibitors (afatinib, gefitinib, erlotinib and osimertinib) and ERK1/2 inhibitor (ravoxertinib) was bought from MedChemExpress (New Jersey, USA). EGF and bFGF were purchased from Novoprotein (Suzhou, China). B27, sodium pyruvate and GlutaMAX were bought from Thermo Fisher Scientific.

DNA constructs and mutagenesis
PCR-amplified human EGFR, ERK1, ERK2 were cloned into pCDH-Flag, pCDH-HA, pET28a-sumo, and pCold-GST vectors. Mutations were constructed by using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA).

The pGIPZ shNT was generated with the control oligonucleotide 5′-GCTTCTAACACCGGAGGTCTT-3′. pGIPZ human MEK1 shRNA was generated with 5′-CTAGATGTTTAACAAATCT-3′ oligonucleotide. pGIPZ human MEK2 shRNA was generated with 5′-CCGAGAGAA GCACCAGATC-3′ oligonucleotide.

Immunoprecipitation and immunoblotting analysis
Extraction of proteins with a modified buffer from cultured cells was followed by immunoprecipitation and immunoblotting with corresponding antibodies, as described previously26.

Cell culture
HEK293T (GNHu17), H1299 (TCHu160), U87 (TCHu138), and U251 (TCHu 58) were obtained from the cell library of the Chinese Academy of Science and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS and streptomycin–penicillin. Glioma stem cell line GSC387 was a gift from Prof. Huairui Yuan at CAS Center for Excellence in Molecular Cell Science. GSC387 cells were maintained in Neurobasal medium supplemented with B27, 20 ng/ml recombinant human EGF, 20 ng/ml recombinant human bFGF, sodium pyruvate, GlutaMAX and streptomycin-penicillin. Cells were incubated in 5% CO2 at 37°C. All cell lines were authenticated using the short tandem repeat method and tested negative for mycoplasma.

Transfection
U87, U251, and H1299 cells were seeded in 35 mm, 60 mm, or 100 mm plates and transfected with the indicated plasmids using Hieff Trans Liposomal Transfection Reagent according to the manufacturer’s instructions.

Cell proliferation assay
1×103 H1299, U87, or U251 cells in DMEM with 10% FBS were seeded per well in a 96-well plate. After culturing for 1, 3, 5, and 7 days, cells were collected. Then, cells were fixed with trichloroacetic acid and stained with SRB solution, followed by rinsing the plates four times with 1% acetic acid and solubilizing the protein-bound dye with 10 mM Tris base solution. The absorbance was measured at 510 nm.

Generation of EGFR R252C knock-in cells
To establish EGFR R252C knock-in cells, the pX330-mCherry plasmid containing EGFR-sgRNA and the pCDH-Puro plasmid containing the donor fragment were co-transfected into U87, U251, and H1299 cells, respectively. 48 h after transfection, the cells were treated with 5 μg/ml puromycin for another 48 h. The cells were then sorted using a Sony MA900 sorter into a 96-well plate to isolate mCherry-positive single-cell clones. Two weeks later, genomic DNA was extracted from each clone, and successful knock-in was confirmed by PCR amplification followed by sequencing. The gRNA was designed based on the sequence: 5′-CCTTGCACGTGGCTTCGTCT-3′.

In vitro kinase assay
In brief, the bacterially purified recombinant His-ERK1 or GST-ERK2 proteins were heated at 95°C for 10 min. Flag-EGFR WT or R252C proteins were immunoprecipitated from 293T cells using anti-Flag agarose beads. Then 1 μg of His-ERK1 or GST-ERK2 proteins were incubated with Flag-EGFR WT or R252C proteins in kinase buffer (50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.1 mM Na3VO4, and 1 mM DTT). 20 μM ATP was added to the kinase buffer to start the reaction. The reactions were performed in a total volume of 50 μl at 37°C for 30 min and then terminated by adding SDS-PAGE loading buffer.

Purification of recombinant proteins
His-ERK1 or GST-ERK2 was expressed in bacteria and purified. Briefly, the vectors expressing His-ERK1 or GST-ERK2 were used to transform BL21/DE3 bacteria. Then, 0.5 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) was used to induce protein expression at 16°C for 20 h. Cell pellets were collected and sonicated in PBS with the addition of proteasome inhibitors before centrifugation at 12,000×g for 30 min at 4°C. Cleared lysates were then bound to Ni-NTA resin (GenScript) with rolling at 4°C for 4 h. Beads were washed extensively before eluting for 1 h in elution buffer (PBS, pH 7.4, plus 500 mM imidazole). Eluted proteins were then dialyzed against PBS.

Colony formation assay
For colony formation assay, 4000 cells per well were plated in a 6-well plate in triplicate and cultured for 14 days before staining viable colonies with crystal violet.

Non-reducing SDS-PAGE
Non-reducing SDS-PAGE was performed to analyze protein samples under denaturing but non-reducing conditions. Cells were lysed in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100) supplemented with protease inhibitors. Protein extracts were mixed with 2× non-reducing loading buffer (50 mM Tris-HCl pH 6.8, 10% SDS, 10% glycerol, 0.1% bromophenol blue). Electrophoresis was carried out using 8% polyacrylamide gels with standard Bis-Tris running buffers. Following electrophoresis, proteins were transferred to PVDF membranes for subsequent immunoblotting analysis.

Protein cross-linking analysis
Protein cross-linking analysis was performed by treating cells with 0.1% glutaraldehyde in PBS for 2 min at room temperature to stabilize protein-protein interactions, followed by quenching with 100 mM glycine for 5 min. Cells were then lysed in lysis buffer (50 mM Tris-HCl pH 7.5, 1% SDS) containing protease inhibitors. The cross-linked protein samples were mixed with 4× SDS loading buffer (100 mM Tris-HCl pH 6.8, 4% SDS, 20% glycerol, 1.4% β-mercaptoethanol, 0.1% bromophenol blue) and analyzed by 8% SDS-PAGE, followed by immunoblotting analysis.

Intracranial injection
2×105 (in 5 μl of DMEM per mouse) luciferase-expressing U87 cells with or without EGFR R252C mutation were injected into randomized 6-week-old female athymic nude mice by intracranial injection. After 35 days post-inoculation, mice were treated with afatinib for two weeks. Afatinib (10 mg/kg body weight) or saline was administered orally by gavage, 5 days per week. Five mice per group in each experiment were included. Bioluminescence imaging of mice injected with U87 cells was performed 49 days after implantation.

For the mouse survival analysis, the observation period was terminated upon the death of the last mouse in the vehicle-treated R252C group. At this endpoint, the surviving mice in the other group remained in good condition, with no significant weight loss or other adverse effects observed. These surviving mice were right-censored to uphold ethical standards and statistical integrity, as the Kaplan-Meier method appropriately handles censored data27–29.

Intrapulmonary injection
2×106 (in 50 μl mixture of 25 μl of DMEM and 25 μl of Matrigel per mouse) luciferase-expressing H1299 cells with or without EGFR R252C mutation were injected into randomized 6-week-old female athymic nude mice by orthotopic pulmonary injection. After 21 days post-inoculation, mice were treated with afatinib for two weeks. Afatinib (10 mg/kg body weight) or saline was administered orally by gavage, 5 days per week. After 35 days of inoculation, bioluminescence imaging of these mice was performed.

For the mouse survival analysis, the observation period was terminated 10 days after the death of the last mouse in the vehicle-treated R252C group. At this endpoint, the surviving mice in other group remained in good condition, with no significant weight loss or other adverse effects observed.

Bioluminescence imaging
Mice were anaesthetized with isoflurane inhalation and were subsequently intraperitoneally injected with 200 μl of 7.5 mg/ml D-luciferin. Bioluminescence imaging was initiated 10 min post-injection using a Tanon-5200 Chemiluminescent Imaging System (Tanon), with an exposure time of 5 min. All bioluminescent data were collected and analyzed using the Tanon-5200 Chemiluminescent Imaging System (version 1.0).

NanoBiT assay
EGFR WT or R252C-SmBiT plasmid, EGFR WT or R252C-LgBiT plasmid, and control Renilla plasmid were used to transfect HEK293T cells in 12-well plates using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. After 36 h, the cells were harvested for the measurement of luciferase activities. The relative levels of luciferase activity of NanoLuc were normalized to the levels of luciferase activity of control Renilla.

Molecular dynamics simulations
Atomistic molecular dynamics simulations of initial models were carried out in the AMBER18 program using AMBER14SB force field for protein30,31, Lipid17 force field for lipid32,33, and the parameters for the nucleotides (ATP) were obtained from the parameters reported previously34.

The simulations adopted the divide and conquer strategy, followed the strategy used by Arkhipov et al.12 for constructed the active and inactive EGFR WT dimer model. The whole EGFR R252C model was divided into ECD, TM, JM, and KD, where the division was the same as in the active and inactive dimer construction12, then the components were assembled into larger models. The disulfide bond covalently links the sulfur atoms between the close spatial proximity cysteine residues, and the C252 from each monomer formed the disulfide bond, which cross-linked the two monomers. The simulations were performed in following order: ECD, ECD+TM, ECD+TM+JM, ECD+TM+JM+KD models. And the model lipid consisted of 100% POPC lipids in extracellular leaflets, and 70% POPC and 30% negatively charged POPS lipids in intracellular leaflets, which the model was widely used in the membrane protein35. Based on the conformation of the EGFR R252C KD dimer, two initial molecular models were obtained: one of the ligand-free EGFR R252C KD dimer and ERK1, and the other of the ligand-bound EGFR WT KD dimer with ERK1, which was used as the control example. The models were generated from the results of ZDOCK36.

The protein systems contain the dimer ECD (EGFR WT, EGFR R252C) system and dimer KD with ERK1 (EGFR R252C). Each protein system was neutralized with a number of sodium ions and then immersed in a solvent box filled with TIP3P water molecules37, to warrant a distance of at least 10 Å between the surface of each protein model and the water box edge. The protein systems were subject to energy minimization in three stages to remove the bad contacts. Firstly, the solvent and the neutralized ions were minimized by holding the protein and ligand using a restraint with a strength of 100 kcal/(mol Å2), and then the minimization was performed by holding the protein and ligand using a constraint of 10 kcal/(mol Å2). Finally, the systems were minimized by removing any constraints. Each stage was performed using the steepest descent minimization of 1000 steps, followed by a 9000-step conjugate gradient minimization. NVT (constant Number of atoms, Volume and Temperature) simulations were carried out by heating the whole system linearly with time, gradually from 100 to 300 K in the first 300 ps, and the Berendsen thermostat38 was used to maintain the temperature of the system. Subsequently, the system was equilibrated at a temperature of 300 K for 1 ns, followed by a NPT (constant Number of atoms, Pressure, and Temperature) production run. During the heating stage, all the protein and ligands were restrained by a restraint of 100 kcal/(mol Å2), and under the equilibration stage, the restraint strength was decreased to 10 kcal/(mol Å2). During the NPT production run, the Berendsen barostat39 was used to control the pressure at 1 atm, and the Langevin thermostat was employed to control the temperature of the systems at 300 K. Five independent 6 μs (total 30 μs) production simulations of ligand-free R252C ECD were performed to obtain the stable conformation of the extracellular dimer.

The lipid systems contain ECD+TM, ECD+TM+JM, and ECD+TM+JM+KD. The semi-isotropic pressure coupling was employed using the Monte Carlo barostat, which controlled the pressure at 1 bar with a coupling constant of 5 ps when the production run was performed. To maintain the stability of the lipid system, all simulations were performed above the experimental liquid-crystalline phase transition temperature (∼315 K for pure dipalmitoyllecithin)40 with a time constant of 1 ps.

All bonds associated with hydrogen atoms were constrained by employing the SHAKE algorithm41, and the Hydrogen Mass Repartitioning (HMR) method was adopted, such that the integration time step of 4 fs could be used. A cutoff value of 12 Å was set for nonbonded interactions, and the Particle Mesh Ewald method42 was employed for treating electrostatic interactions. For each system, five independent molecular dynamic simulations were carried out using different velocities that were randomly generated at the beginning of the simulations and a long production run, totally up to 80 μs, only for ligand-free EGFR R252C model.

The last 500 ns of each simulation were used for analysis, and the analysis of distance, angle, and native contacts, etc., was performed by the cpptraj module in AMBER31.

Principal component analysis (PCA)
PCA provides orthogonal eigenvectors and eigenvalues according to covariance matrix diagonalization30. The elements Cij in the matrix are defined as:
Cij = <Δri · Δrj> / ( <Δri2>1/2 <Δrj2>1/2 ) (1)
where Δri/Δrj indicated the deviation of the Cα atom of the ith residue from its mean position. The value of Cij fluctuates between −1 and 1. A positive Cij value indicated the correlation motion between the ith residue and the jth residue, while a negative Cij value represented the inverse correlation motion.

The eigenvalues indicated the magnitude of motions along a direction. In general, the first few principal components (PCs) described the most important slow system modes related to the functional motions of a biomolecular system.

MM-GBSA calculation
To understand the interaction between the two dimers, the binding free energies were calculated using the MM-GBSA method. For each complex, 500 snapshots were extracted from the last 100 ns along the molecular dynamic trajectory at an interval of 200 ps. The MM-GBSA method43 was performed to compute the binding free energies. The binding free energy (ΔG) can be represented as:
ΔG = ΔEMM + ΔGsol (2)
where ΔEMM is the difference of molecular mechanic energy between the complex and each binding partner in the gas phase, ΔGsol is the solvation free energy contribution to binding and TΔS. ΔEMM is further divided into two parts:
ΔEMM = ΔEele + ΔEvdW (3)
where ΔEele and ΔEvdW are described as the electrostatic interaction and van der Waals energy in the gas phase, respectively. The solvation free energy is expressed as:
ΔGsol = ΔGgb + ΔGnp (4)
where ΔGgb and ΔGnp are the polar and non-polar contributions to the solvation free energy, respectively.

Statistics and reproducibility
All the results were expressed as the mean ± s.d. No statistical method was used to predetermine sample size. The exact sample size (n) for each experimental group was indicated in the figure legends. No data were excluded from the analyses. All statistical analyses were performed using GraphPad Prism 8. For comparisons between two unpaired independent groups, the two-tailed Student’s t test was used. Statistical significance was defined as p < 0.05. Randomization was applied to the allocation of experimental groups to minimize selection bias. Throughout the experiments and data analysis, investigators were blinded to group assignments to prevent any potential bias during outcome assessment.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Zhang et al. present a compelling study describing the identification and mechanistic characterization of a novel EGFR extracellular domain mutation, R252C, found in a patient with multifocal lung cancer and glioma. Using CRISPR-engineered isogenic models and orthotopic xenografts, the authors demonstrate that R252C enhances tumor proliferation via a non-canonical mechanism that bypasses typical EGFR autophosphorylation and upstream MAPK activation. Instead, R252C stabilizes an atypical EGFR dimer that directly phosphorylates ERK1/2, a mechanism supported by structural modeling and biochemical assays. The work is strengthened by the use of multiple model systems, elegant integration of computational and experimental data, and clinically relevant findings, including durable therapeutic response to afatinib in the index patient.\nHowever, several limitations temper the overall conclusions. Key mechanistic claims—such as direct MEK-independent ERK activation and disulfide-stabilized dimerization—lack definitive experimental validation. Comparisons to other EGFR TKIs are missing, limiting interpretation of afatinib’s selectivity. Quantitative analyses, especially for immunoblots and NanoBiT assays, are inconsistently applied, and survival data for xenograft models are absent. Additionally, concerns about CNS drug penetration and lack of target engagement data in GBM models raise questions about translational applicability.\nOverall, this study offers a novel and potentially paradigm-shifting insight into EGFR biology, with clear therapeutic relevance, but would benefit from additional mechanistic and pharmacologic validation to fully support its conclusions.\nSpecific Comments:\n1) Mechanistic validation of ERK activation: A major claim is that EGFR mediated ERK activation is direct and therefore bypasses canonical RAS-RAF-MEK signaling. However, this claim needs experimental validation. To improve on the mechanistic claims relating to direct EGFR mediated activation of ERK, the authors should test whether MEK/RAF/RAS inhibitors (e.g., trametinib, dabrafenib, tipifarnib) impact R252C-driven ERK1/2 phosphorylation as a way to directly assess MEK independence. The authors should also include genetic perturbation experiments (e.g., siRNA or CRISPR knockdown of MEK1/2) to further confirm bypass of canonical MAPK signaling.\n2) Broaden analysis of EGFR autophosphorylation: To determine whether activation patterns of EGFR are site specific to Y1068, the authors should assess a broader panel of EGFR phosphorylation sites (e.g., Y845, Y992, Y1045, Y1086, Y1148, Y1173), all of which can differentially impact binding of adaptor/effector proteins. This analysis should be done -/+ EGF stimulation to assess dynamic regulation.\n3) Direct evidence for disulfide dimerization. While the molecular dynamics simulations offer a compelling mechanistic hypothesis in which R252C stabilizes a unique ECD dimer, more evidence is needed to support this conclusion. The authors should consider non-reducing PAGE or quantitative mass-spectrometry to directly demonstrate C252-C252 disulfide formation in cells through demonstration of an intact dimer under non-liganded conditions (PAGE) and/or to directly identify the relevant C252-C252 dimer peptide via MS. Moreover, these data could be complemented by a loss-of-function variant (e.g., C225S mutant), which presumably would lose disulfide formation capability, dimer stability (via NanoBiT or crosslinking assays), and fail to phosphorylate ERK1/2.\n4) Confirmation of ERK docking site on EGFR. The authors claim the EGFR P919 residue is responsible for binding ERK1/2 in the R252C mutant. To support this finding, the authors could generate a single P919G mutant in the WT background to compare to the double R252C/P919G mutant.\n5) Evaluation of sensitivity against other EGFR TKIs. Mutations in ECD vs KD can impact EGFR TKI sensitivity (Vivanco et. al., Cancer Discovery). The authors should compare afatinib with other clinically relevant EGFR TKIs (gefitinib, erlotinib, osimertinib) in dose-response experiments using proliferation and signaling readouts (e.g., pERK1/2 levels). Importantly, they should include side-by-side comparison of TKI efficacy in WT vs. R252C cell lines to better define mutation-specific sensitivity.\n6) Lack of Relevant Models: While the U87MG cell line can be beneficial for biochemical investigations, it does not represent GBM in a substantive manner. The authors should put in considerable effort to include more clinically relevant models (such as patient-derived gliomaspheres) to validate their findings. If they are unable to locate such models, the authors should clearly state this as a limitation of this study.\nMinor points\n1) Mouse survival should be included as an endpoint\n2) Afatinib has extremely low CNS penetration and has failed in the clinic (Reardon et al. Neuro-Oncology. 2014). The authors should either provide direct evidence that these mutations are different from those previously targeted with afatinib in GBM or substantially state this as a limitation of their study, and to propose other drugs (see point 5) that have greater CNS exposures but can target this mutation effectively relative to WT EGFR\n3) Improvement to data analysis and interpretation: A. Quantify immunoblots and NanoBiT assay results using appropriate densitometry or luminescence normalization, and include statistical analyses. B) Add expression/loading controls (e.g., total protein or luciferase levels) to validate NanoBiT assay comparisons. C) Clarify discrepancies in afatinib efficacy between WT and R252C cells (e.g., Fig. 4A) with fold-change metrics or normalized growth curves. D) Raw uncropped blots should be provided for review.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "This manuscript describes a novel EGFR mutation in a patient with lung cancer and glioma and characterises the molecular mechanisms of EGFR signalling exerted by this EGFR mutant. The R252C mutation leads to enhanced dimer formation of the receptor, direct phosphorylation of ERK1/2 and enhanced proliferative responses in cell lines and xenograft tumor growth. The effects can be reversed by afatinib, suggesting a potential therapeutic strategy.\nThe paper is well written, presents a coherent story and uses state of the art technologies. In addition to the experimental work, there are structural molecular simulations to support the hypothesis and key validation experiments to substantiate the claims.\nI have some minor comments to improve the quality of the manuscript:\n1. The abstract states that the R252C mutation results in reduced auto-phosphorylation, but this is not substantiated by the results.\n2. I would have liked to see more references in the introduction. Many statements are not backed up by the correct references.\n3. Apart from the R252C mutation: were there other mutations in other genes observed? Was the status of other key oncogenes tested?\n4. There are some minor typos throughout the text. Please run a spell checker over the manuscript.\n5. Figures 2a and 2b need quantification from three independent experiments.\n6. Is the Grb2-SOS-Kras axis not required for phosphorylation of ERKs? I wonder if a quick siRNA or inhibitor experiment could be done to verify that the direct binding of ERKs is sufficient for signalling by the R252C mutant.\n7. Figure 3a: a negative control is missing."}, "author_response": "Response to the referees’ comments:\nReviewer #1 (Remarks to the Author):\nZhang et al. present a compelling study describing the identification and mechanistic\ncharacterization of a novel EGFR extracellular domain mutation, R252C, found in a patient\nwith multifocal lung cancer and glioma. Using CRISPR-engineered isogenic models and\northotopic xenografts, the authors demonstrate that R252C enhances tumor proliferation via a\nnon-canonical mechanism that bypasses typical EGFR autophosphorylation and upstream\nMAPK activation. Instead, R252C stabilizes an atypical EGFR dimer that directly\nphosphorylates ERK1/2, a mechanism supported by structural modeling and biochemical\nassays. The work is strengthened by the use of multiple model systems, elegant integration of\ncomputational and experimental data, and clinically relevant findings, including durable\ntherapeutic response to afatinib in the index patient.\nHowever, several limitations temper the overall conclusions. Key mechanistic claims—such as\ndirect MEK-independent ERK activation and disulfide-stabilized dimerization—lack definitive\nexperimental validation. Comparisons to other EGFR TKIs are missing, limiting interpretation\nof afatinib’s selectivity. Quantitative analyses, especially for immunoblots and NanoBiT assays,\nare inconsistently applied, and survival data for xenograft models are absent. Additionally,\nconcerns about CNS drug penetration and lack of target engagement data in GBM models raise\nquestions about translational applicability.\nOverall, this study offers a novel and potentially paradigm-shifting insight into EGFR biology,\nwith clear therapeutic relevance, but would benefit from additional mechanistic and\npharmacologic validation to fully support its conclusions.\nResponse: Thank you for the constructive comments and insightful suggestions. To address the\nconcerns raised, we have conducted additional experiments and expanded the discussion in the\nDiscussion section. All changes we have made in the manuscript have been highlighted in\nyellow.\nSpecific Comments:\n1) Mechanistic validation of ERK activation: A major claim is that EGFR mediated ERK\nactivation is direct and therefore bypasses canonical RAS-RAF-MEK signaling. However, this\nclaim needs experimental validation. To improve on the mechanistic claims relating to direct\nEGFR mediated activation of ERK, the authors should test whether MEK/RAF/RAS inhibitors\n(e.g., trametinib, dabrafenib, tipifarnib) impact R252C-driven ERK1/2 phosphorylation as a\nway to directly assess MEK independence. The authors should also include genetic perturbation\nexperiments (e.g., siRNA or CRISPR knockdown of MEK1/2) to further confirm bypass of\ncanonical MAPK signaling.\nResponse: Thank you for the constructive comment and suggestion. To investigate whether\nEGFR R252C-induced ERK1/2 activation is dependent on the canonical RAS/RAF/MEK\npathway, we treated U251 cells harboring either EGFR WT or EGFR R252C with RAS\ninhibitor (tipifarnib), RAF inhibitor (dabrafenib), or MEK inhibitor (trametinib), respectively.\nAs indicated by the result of Supplementary Fig. 2b-d, inhibition of RAS, RAF, or MEK failed\nto block EGFR R252C-induced ERK1/2 activation, indicating that EGFR R252C-induced\nERK1/2 activation is independent of the canonical RAS/RAF/MEK pathway. This conclusion\nwas further validated by MEK1/2 depletion experiments in both U251 and H1299 cell lines\n(Supplementary Fig. 2e,f). The results were described in line 154.\n2) Broaden analysis of EGFR autophosphorylation: To determine whether activation patterns\nof EGFR are site specific to Y1068, the authors should assess a broader panel of EGFR\nphosphorylation sites (e.g., Y845, Y992, Y1045, Y1086, Y1148, Y1173), all of which can\ndifferentially impact binding of adaptor/effector proteins. This analysis should be done -/+ EGF\nstimulation to assess dynamic regulation.\nResponse: Thank you for the constructive comment and suggestion. To determine whether\nR252C-induced activation patterns of EGFR are site-specific to Y1068, we harvested U251\ncells harboring either EGFR WT or EGFR R252C and U251 cells harboring EGFR WT treated\nwith EGF (10 ng/ml). Immunoblotting analysis revealed that both EGFR WT and EGFR R252C\ncells exhibited comparable basal phosphorylation levels at Y845, Y992, Y1045, Y1086, Y1148,\nand Y1173 residues. In contrast, EGF-treated U251 cells exhibited increased phosphorylation\nlevels at Y845, Y992, Y1045, Y1086, Y1148, and Y1173 residues (Supplementary Fig. 4e). The\nabove results suggest that R252C-induced activation patterns of EGFR are not site-specific to\nY1068. The results were described in line 243.\n3) Direct evidence for disulfide dimerization. While the molecular dynamics simulations offer\na compelling mechanistic hypothesis in which R252C stabilizes a unique ECD dimer, more\nevidence is needed to support this conclusion. The authors should consider non-reducing PAGE\nor quantitative mass-spectrometry to directly demonstrate C252-C252 disulfide formation in\ncells through demonstration of an intact dimer under non-liganded conditions (PAGE) and/or\nto directly identify the relevant C252-C252 dimer peptide via MS. Moreover, these data could\nbe complemented by a loss-of-function variant (e.g., C252S mutant), which presumably would\nlose disulfide formation capability, dimer stability (via NanoBiT or crosslinking assays), and\nfail to phosphorylate ERK1/2.\nResponse: Thank you for the insightful suggestion. To determine whether R252C mutation\nstabilizes a unique ECD dimer of EGFR and whether the dimer formation is dependent on the\nC252-C252 disulfide, we transfected H1299 cells with HA-tagged EGFR WT, R252C, or\nR252S. The cells were harvested and analyzed by non-reducing SDS-PAGE to preserve\ndisulfide bonds. As shown in Supplementary Fig. 3b, R252C mutation indeed promoted the\ndimer formation of EGFR, while R252S mutation failed to do so. The result confirmed that\nR252C stabilizes a unique EGFR ECD dimer specifically through C252-C252 disulfide bond\nformation. This conclusion was further validated by glutaraldehyde-mediated crosslinking\nexperiments (Supplementary Fig. 3c). Besides, we transfected H1299 cells with HA-tagged\nEGFR WT, R252C, or R252S. The cells were harvested and analyzed by SDS-PAGE. As shown\nin Supplementary Fig. 3d, compared to EGFR WT, R252C mutation enhanced ERK1/2\nactivation, while R252S mutation failed to do so. These results confirmed that R252C mutation\npromotes the dimerization of EGFR to activate ERK1/2, which is dependent on C252-C252\ndisulfide bond. The results were described in line 187.\n4) Confirmation of ERK docking site on EGFR. The authors claim the EGFR P919 residue is\nresponsible for binding ERK1/2 in the R252C mutant. To support this finding, the authors could\ngenerate a single P919G mutant in the WT background to compare to the double R252C/P919G\nmutant.\nResponse: Thank you for the constructive comment and suggestion. To confirm the role of\nP919 in mediating EGFR R252C-induced ERK1/2 activation and cell proliferation, we\nintroduced the P919G mutation into both EGFR WT and R252C backgrounds. Co-\nimmunoprecipitation assay demonstrated that the P919G mutation significantly abrogated the\nenhanced interaction between EGFR R252C and ERK1/2, concurrently suppressing R252C-\ndriven ERK1/2 phosphorylation (Supplementary Fig. 4h). Notably, the P919G mutation had no\ndiscernible effect on either the EGFR-ERK1/2 interaction or basal ERK1/2 phosphorylation in\nthe wild-type EGFR background (Supplementary Fig. 4h). Consistently, both cell proliferation\nand soft agar colony formation assays confirmed that the P919G mutation significantly\nattenuated EGFR R252C-driven proliferation (Fig. 3h,i and Supplementary Fig. 4i). The results\nwere described in line 256.\n5) Evaluation of sensitivity against other EGFR TKIs. Mutations in ECD vs KD can impact\nEGFR TKI sensitivity (Vivanco et. al., Cancer Discovery). The authors should compare afatinib\nwith other clinically relevant EGFR TKIs (gefitinib, erlotinib, osimertinib) in dose-response\nexperiments using proliferation and signaling readouts (e.g., pERK1/2 levels). Importantly,\nthey should include side-by-side comparison of TKI efficacy in WT vs. R252C cell lines to\nbetter define mutation-specific sensitivity.\nResponse: Thank you for the constructive comment and suggestion. To evaluate the sensitivity\nof EGFR R252C to EGFR-TKIs, U251 and H1299 cells expressing EGFR WT or R252C were\ntreated with increasing concentrations (0, 1, 5, 10 μM) of four EGFR inhibitors: afatinib,\ngefitinib, erlotinib, or osimertinib. Immunoblotting analysis revealed that afatinib inhibited\nERK1/2 phosphorylation in a dose-dependent manner, while gefitinib, erlotinib, and\nosimertinib exhibited minimal effects on phospho-ERK1/2 levels (Supplementary Fig. 5a,b).\nSupplementary Fig. 5c provides validation of the inhibitory efficacy of gefitinib, erlotinib, and\nosimertinib. Consistent with the signaling data, afatinib exhibited stronger inhibition on\nR252C-driven proliferation than gefitinib and erlotinib, while osimertinib showed no anti-\nproliferative activity (Supplementary Fig. 6a). Collectively, these results suggested that EGFR\nR252C mutation confers sensitivity to afatinib. The results were described in line 282.\n6) Lack of Relevant Models: While the U87MG cell line can be beneficial for biochemical\ninvestigations, it does not represent GBM in a substantive manner. The authors should put in\nconsiderable effort to include more clinically relevant models (such as patient-derived\ngliomaspheres) to validate their findings. If they are unable to locate such models, the authors\nshould clearly state this as a limitation of this study.\nResponse: Thank you for the constructive comment. To validate our findings in a clinically\nrelevant GBM model, we utilized the patient-derived GSC387 glioma stem cell line, which\nmaintains key molecular and phenotypic characteristics of primary glioblastoma. Using\nlentiviral-mediated gene delivery, we established stable GSC387 cell lines expressing either\nHA-tagged EGFR WT or the R252C mutant. As demonstrated in Supplementary Fig. 1a,b,\nexpression of EGFR R252C significantly enhanced GSC387 proliferation compared to EGFR\nWT. Mechanistically, immunoblot analysis revealed that the R252C mutation markedly\nincreased ERK1/2 phosphorylation. Importantly, this mutant-enhanced ERK1/2 activation was\neffectively suppressed by afatinib treatment (Supplementary Fig. 2a). Consistent with these\nmolecular observations, the proliferation advantage conferred by EGFR R252C was completely\nabolished upon afatinib exposure (Supplementary Fig. 6b). These results collectively\ndemonstrate that the EGFR R252C mutation drives GSC proliferation through enhanced\nERK1/2 activation, and that this oncogenic phenotype is therapeutically targetable by afatinib.\nThe results were described in line 115, line 142 and line 292.\nMinor points\n1) Mouse survival should be included as an endpoint.\nResponse: We appreciate this insightful suggestion. In our revised study, we have incorporated\nmouse survival as a critical endpoint to complement the tumor growth and molecular analyses.\nSpecifically, in U87 engraftment mouse models, log-rank analysis revealed that tumors\nexpressing EGFR R252C exhibited significantly reduced overall survival compared to WT\ncontrols (median survival: 52 vs. 59 days; p < 0.001), aligning with their aggressive\nproliferative phenotype. Notably, afatinib treatment prolonged the survival of R252C-bearing\nmice. These findings were recapitulated in H1299 xenograft models, where EGFR R252C\nexpression similarly resulted in worse survival outcomes (median survival: 39 vs. 49 days for\nWT; p = 0.002). Afatinib again demonstrated therapeutic efficacy, increasing the median\nsurvival of R252C-bearing mice (Supplementary Fig. 6c). The results were described in line\n309.\n2) Afatinib has extremely low CNS penetration and has failed in the clinic (Reardon et al.\nNeuro-Oncology. 2014). The authors should either provide direct evidence that these mutations\nare different from those previously targeted with afatinib in GBM or substantially state this as\na limitation of their study, and to propose other drugs (see point 5) that have greater CNS\nexposures but can target this mutation effectively relative to WT EGFR.\nResponse: We appreciate the reviewer’s insightful comment regarding afatinib’s CNS\npenetration and clinical performance in GBM. Indeed, the limited efficacy of EGFR-targeted\ntherapies in GBM was attributed to poor blood-brain barrier (BBB) permeability and diverse\nresistance mechanisms (PMID: 37805108). As reported by Reardon et al., afatinib\ndemonstrated minimal single-agent activity in unselected recurrent GBM cohorts, and its\ncombination with temozolomide failed to improve PFS-6 rates or median PFS (PMID:\n25140039). However, our study revealed a distinct pharmacological profile of afatinib against\nthe EGFR R252C mutation. Specifically, in vitro, afatinib exhibited superior inhibitory potency\nand selectivity for EGFR R252C compared to other EGFR-TKIs (Supplementary Fig. 6a); in\nvivo, afatinib treatment significantly suppressed R252C-driven tumor growth and extended\nsurvival in xenograft models (Fig. 4d,e, and Supplementary Fig. 6c). Importantly, afatinib\neffectively suppressed tumor growth and extended progression-free survival in a patient with\nmultifocal lung cancer and glioma driven by EGFR R252C (Fig. 4f,g). These results could be\nattributed to the EGFR R252C mutation potentially altering BBB permeability or increasing\nafatinib-EGFR binding affinity, thereby creating a distinct therapeutic scenario that differs from\nconventional GBM paradigms. While the clinical implications of these observations require\nvalidation in larger patient cohorts, our data support reconsideration of afatinib’s therapeutic\nutility in molecularly defined GBM subtypes harboring this mutation.\nOf note, several case reports have also documented prolonged responses to afatinib in\nglioblastoma patients (PMID: 26423602; PMID: 34787778), suggesting further investigation\nof afatinib in GBM, particularly through biomarker-guided patient selection. We have added\nthis discussion to the Discussion section (line 375).\n3) Improvement to data analysis and interpretation: A. Quantify immunoblots and NanoBiT\nassay results using appropriate densitometry or luminescence normalization, and include\nstatistical analyses. B) Add expression/loading controls (e.g., total protein or luciferase levels)\nto validate NanoBiT assay comparisons. C) Clarify discrepancies in afatinib efficacy between\nWT and R252C cells (e.g., Fig. 4A) with fold-change metrics or normalized growth curves. D)\nRaw uncropped blots should be provided for review.\nResponse: We appreciate the constructive comments. In the revised manuscript, we have made\nrevisions based on the suggestions of the reviewers. Specifically: (A) For immunoblots, we\nhave performed densitometric analysis using ImageJ, normalized all signals to their\ncorresponding loading controls, and added statistical comparisons (unpaired t-tests, n=3\nbiological replicates) in revised figures. For NanoBiT assays, we have added Renilla luciferase\nco-transfection as an internal control and normalized luminescence values to Renilla luciferase\nactivity. Statistical comparisons (unpaired t-tests, n=3 biological replicates) were also\nperformed. (B) We have added expression controls to validate NanoBiT assay comparisons\n(Supplementary Fig. 4c,d). (C) Regarding afatinib efficacy comparisons, we have now\nquantified the growth curves in Fig. 4a using normalized growth rates (relative to DMSO\ncontrols) and calculated fold-change differences in EGFR WT or R252C group at day 7 (Fig.\n4b). (D) For blot transparency, all uncropped western blots with molecular weight markers have\nbeen compiled in the Source data.\nReviewer #3 (Remarks to the Author):\nThis manuscript describes a novel EGFR mutation in a patient with lung cancer and glioma and\ncharacterises the molecular mechanisms of EGFR signalling exerted by this EGFR mutant. The\nR252C mutation leads to enhanced dimer formation of the receptor, direct phosphorylation of\nERK1/2 and enhanced proliferative responses in cell lines and xenograft tumor growth. The\neffects can be reversed by afatinib, suggesting a potential therapeutic strategy.\nThe paper is well written, presents a coherent story and uses state of the art technologies. In\naddition to the experimental work, there are structural molecular simulations to support the\nhypothesis and key validation experiments to substantiate the claims.\nResponse: We thank the reviewer for the positive comments and insightful suggestions. We\nperformed additional experiments to address the concerns of the reviewer. All changes we have\nmade in the manuscript have been highlighted in yellow.\nMajor points:\nI have some minor comments to improve the quality of the manuscript:\n1. The abstract states that the R252C mutation results in reduced auto-phosphorylation, but this\nis not substantiated by the results.\nResponse: Thank you for the constructive comment. We have included the results in the revised\nmanuscript. To examine auto-phosphorylation levels of EGFR, we harvested U251 cells\nharboring either EGFR WT or EGFR R252C and U251 cells harboring EGFR WT treated with\nEGF (10 ng/ml). Immunoblotting analysis revealed that both EGFR WT and R252C cells\nexhibited comparable basal phosphorylation levels at Y845, Y992, Y1045, Y1086, Y1148, and\nY1173 residues. In contrast, EGF-treated U251 cells exhibited increased phosphorylation levels\nat Y845, Y992, Y1045, Y1086, Y1148, and Y1173 residues (Supplementary Fig. 4e),\ndemonstrating the reduced auto-phosphorylation of EGFR R252C, compared to the ligand-\nbound EGFR WT. The results were described in line 243.\nNotably, in Fig. 3, we found that the simulated EGFR R252C model contains an active\nligand-free extracellular dimer conformation, similar to the ligand-bound active EGFR WT\nextracellular dimer. However, the ligand-free EGFR R252C exhibits an increased distance\nbetween the KDs and an altered domain orientation. These changes are the primary reasons for\nthe reduced auto-phosphorylation levels observed in EGFR R252C compared to ligand-bound\nEGFR WT.\n2. I would have liked to see more references in the introduction. Many statements are not backed\nup by the correct references.\nResponse: We appreciate this helpful suggestion and have now expanded the introduction with\nadditional references to better support our key statements.\n3. Apart from the R252C mutation: were there other mutations in other genes observed? Was\nthe status of other key oncogenes tested?\nResponse: Thank you for the comment. Comprehensive mutation profiling of key oncogenic\nloci, including NRAS Q61/G13/G12, PIK3CA H1047, EGFR G719/L858, BRAF V600, and\nKRAS Q61/G13/G12, were examined and revealed no detectable pathogenic variants. The sole\nmutation identified was EGFR R252C. We have added this information in the revised\nmanuscript and can be found at line 97.\n4. There are some minor typos throughout the text. Please run a spell checker over the\nmanuscript.\nResponse: We sincerely appreciate your careful reading. We have now thoroughly proofread\nthe manuscript using both spell-check software and manual review to correct all typographical\nerrors.\n5. Figures 2a and 2b need quantification from three independent experiments.\nResponse: This point was well taken. We have now added quantitative analyses with statistical\ncomparisons in the revised figure.\n6. Is the Grb2-SOS-Kras axis not required for phosphorylation of ERKs? I wonder if a quick\nsiRNA or inhibitor experiment could be done to verify that the direct binding of ERKs is\nsufficient for signalling by the R252C mutant.\nResponse: Thank you for the constructive comment and suggestion. To investigate whether\nEGFR R252C-induced ERK1/2 activation is dependent on Grb2-SOS-Kras, we treated U251\ncells harboring either EGFR WT or EGFR R252C with Ras inhibitor (tipifarnib). As indicated\nby the result of Supplementary Fig. 2b, inhibition of RAS failed to block EGFR R252C-induced\nERK1/2 activation, indicating that EGFR R252C-induced ERK1/2 activation is independent of\nGrb2-SOS-Kras pathway. The results were described in line 154.\n7. Figure 3a: a negative control is missing.\nResponse: This point was well taken. We have repeated the experiment with the addition of a\nnegative control. As shown in the new Fig. 3a, a similar result was obtained."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Zhang et al. have addressed the major concerns from our first review and have added substantial new data that strengthen the central claim that EGFR R252C directly drives oncogenic ERK activation and is therapeutically targetable by afatinib. The revised manuscript and reviewer response detail our requested mechanistic experiments (disulfide-dependent dimerization, NanoBiT/crosslinking, P919 mutational tests), RAS/RAF/MEK inhibitor and genetic MEK depletion controls, and side-by-side TKI comparisons and in vivo survival analyses. Overall, the revisions are convincing and the authors should be commended for their effort and rigor that went into addressing the comments. However, a few revisions/additions are requested to further strengthen and clarify their conclusions\n1. Major: Provide final updated survival data and Kaplan-Meier analyses with the publication. The survival endpoints of afatinib treatment strengthen the authors’ translation claim. If data remains censored, please provide censoring details and an explanation of why full curves could not be presented.\n2. Minor: Clarify why afatinib shows apparent mutation-selective efficacy and fails to target wild-type EGFR. Despite its known biochemical potency against wild-type EGFR (~0.5 nM; Li et al., Oncogene 2008, in Supplemental Fig. 2a, afatinib does not show a significant impact on pEGFRWT and in Supplemental Fig. 5a neither afatinib nor other wild-type targeting EGFR TKIs (erlotinib, gefitinib) significantly suppress ERK phosphorylation, even at high concentrations (>1 μM).", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "I would like to thank the authors for the additional data that has been provided and that addresses some of the concerns that were raised. However, there are still some aspects that require further clarification.\n1. I find the legends in the supplementary figures too short to be able to understand what was done. This makes it very difficult to understand the figures. For example, in Supplementary figure 2: has this been done in full media with FBS, have the cells been stimulated, etc. In the new supplementary figure 2, it is thus not obvious to understand whether the inhibitors actually work. One would need to stimulate the cells with EGF in order to see whether the Ras, Raf and MEK inhibitor actually work at the concentration used (what is the concentration anyway? – not mentioned in the legend).\n2. I still do not see a reduced auto-phosphorylation for R252C. Supplementary Figure 4e does not show this. In unstimulated cells, there is no phosphorylation visible in both wt and R252C cells. Also, R252C should be shown with EGF stimulation. One cannot compare unstimulated R252C to stimulated wt cells."}, "author_response": "1 Reviewer #1 (Remarks to the Author):\n3 new data that strengthen the central claim that EGFR R252C directly drives oncogenic ERK\n4 activation and is therapeutically targetable by afatinib. The revised manuscript and reviewer\n5 response detail our requested mechanistic experiments (disulfide-dependent dimerization,\n7 depletion controls, and side-by-side TKI comparisons and in vivo survival analyses. Overall,\n8 the revisions are convincing and the authors should be commended for their effort and rigor\n9 that went into addressing the comments. However, a few revisions/additions are requested to\n10 further strengthen and clarify their conclusions.\n12 have conducted additional experiments and revised the manuscript accordingly. We believe\n13 these changes have strengthened our conclusions. All changes we have made in the manuscript\n14 have been highlighted in yellow.\n15 1. Major: Provide final updated survival data and Kaplan-Meier analyses with the publication.\n17 remains censored, please provide censoring details and an explanation of why full curves could\n18 not be presented.\n20 the observation period was terminated upon the last death of mouse in the vehicle-treated\n22 with no significant weight loss or other adverse effects observed. These surviving mice were\n23 right-censored to uphold ethical standards and statistical integrity, as the Kaplan-Meier method\n24 appropriately handles censored data (PMID: 30015653, PMID:27713848, PMID: 21367692).\n26 and line 672).\n27 2. Minor: Clarify why afatinib shows apparent mutation-selective efficacy and fails to target\n28 wild-type EGFR. Despite its known biochemical potency against wild-type EGFR (~0.5 nM;\n30 on pEGFRWT and in Supplemental Fig. 5a neither afatinib nor other wild-type targeting EGFR\n32 concentrations (>1 μM).\n34 clarify the point. To active EGFR, U251 cells were treated with EGF. As shown in new\n36 induced phospho-ERK1/2 in EGFR WT cells upon EGF stimulation. The inhibitory efficacy of\n37 afatinib was comparable between EGFR WT cells (with EGF stimulation) and EGFR R252C\n38 mutant cells (Supplemental Fig. 5a and 5c). These results demonstrate that all three EGFR\n39 inhibitors are functional in EGFR WT cells upon EGF stimulation. The results were described\n40 in line 289.\n42 phospho-ERK1/2 levels in U251 and H1299 cells. This may be because basal ERK1/2\n43 activation in these cell lines is not primarily mediated by EGFR signaling.\n45 assay using purified EGFR protein. In contrast, our results are derived from cellular assays\n46 conducted in U251 or H1299 cell lines. Such discrepancies in drug efficacy can be expected\n47 when comparing biochemical and cellular systems.\n48\n51 addresses some of the concerns that were raised. However, there are still some aspects that\n52 require further clarification.\n54 have conducted additional experiments and revised the manuscript accordingly. We believe\n55 these changes have strengthened our conclusions. All changes we have made in the manuscript\n56 have been highlighted in yellow.\n57 1. I find the legends in the supplementary figures too short to be able to understand what was\n58 done. This makes it very difficult to understand the figures. For example, in Supplementary\n59 figure 2: has this been done in full media with FBS, have the cells been stimulated, etc. In the\n60 new supplementary figure 2, it is thus not obvious to understand whether the inhibitors actually\n61 work. One would need to stimulate the cells with EGF in order to see whether the Ras, Raf and\n63 not mentioned in the legend).\n65 supplementary figure legends to explicitly describe the experimental conditions, such as the use\n66 of full media and EGF stimulation. The concentrations of all inhibitors have also been clearly\n67 indicated.\n69 cells were serum-starved for 24 hr and then treated with RAS, RAF, or MEK inhibitors for 6 hr\n70 prior to EGF stimulation for 30 min. The immunoblotting results, now included as\n72 described in line 160.\n73 2. I still do not see a reduced auto-phosphorylation for R252C. Supplementary Figure 4e does\n74 not show this. In unstimulated cells, there is no phosphorylation visible in both wt and R252C\n75 cells. Also, R252C should be shown with EGF stimulation. One cannot compare unstimulated\n78 tyrosine residues (Y845, Y992, Y1045, Y1086, Y1148, and Y1173) in U251 cells expressing\n79 either EGFR WT or the R252C mutant, under both unstimulated and EGF-stimulated conditions.\n82 residues compared to those expressing EGFR WT (Supplementary Fig. 4e and 4f). The results\n83 were described in line 245."}, {"round": 3, "reviewer_comments": {"reviewer_3": "REVIEWER#3\nWell, I still have an issue with the auto-phosphorylation of the EGFR, but maybe this is just a misunderstanding. There is no auto-phosphorylation by wt nor the mutant receptor (i.e. phosphorylation without EGF stimulation, see supplementary figure 4e). But maybe what the authors mean to say is ABSENT auto-phosphorylation?"}, "author_response": "Response: We apologized for the confusion. Indeed, as shown in Supplementary Fig. 4e, no\nphosphorylated band was detected in the R252C group without EGF stimulation, indicating that\nthe EGFR R252C mutant does not undergo auto-phosphorylation in the absence of EGF. This\nphenomenon is attributed to an increased distance between the kinase domains and an altered\ndomain orientation in the R252C mutant. We have revised our manuscript to replace “reduced”\nwith “absent”."}]

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