New Directions for Advanced Targeting Strategies of EGFR Signaling in Cancer

Abstract

Epidermal growth factor (EGF)–EGF receptor (EGFR) signaling studies paved the way for a basic understanding of growth factor and oncogene signaling pathways and the development of tyrosine kinase inhibitors (TKIs). Due to resistance mutations and the activation of alternative pathways when cancer cells escape TKIs, highly diverse cell populations form in recurrent tumors through mechanisms that have not yet been fully elucidated. In this review, we summarize recent advances in EGFR basic research on signaling networks and intracellular trafficking that may clarify the novel mechanisms of inhibitor resistance, discuss recent clinical developments in EGFR-targeted cancer therapy, and offer novel strategies for cancer drug development.

1. INTRODUCTION—BEYOND THE 60TH ANNIVERSARY OF EPIDERMAL GROWTH FACTOR (EGF)

The first study on EGF was published by Dr. Stanley Cohen in 1962,¹⁾ and the EGF receptor (EGFR) was subsequently identified as a receptor tyrosine kinase (RTK).²⁾ The RAS and mitogen-activated protein kinase (MAPK) cascades are major signaling pathways for cell proliferation leading from cell surface EGFR to nuclear transcription factors.^3–5) Accumulating evidence on the role of the EGF–EGFR pathway in cancer cell proliferation contributed to the development of gefitinib and erlotinib, first-generation EGFR tyrosine kinase inhibitors (TKIs), for the treatment of non-small cell lung cancer (NSCLC) harboring activating mutations in EGFR in the early 2000s.^6–8) Over the last two decades, further advances have been achieved in both basic and clinical aspects. Studies on the basic intracellular signaling network and endosomal trafficking of EGFR have had a positive impact on growth factor signaling research. Although molecularly targeted cancer therapy has been pioneered by the EGFR research, acquired resistance to the targeted agents remain a major challenge.^9–11)

In this review, we provide an overview of recent advances in our basic understanding of the EGFR signaling network, discuss recent advances and challenges in lung and brain cancer therapeutics, and offer exciting insights for future EGFR-targeted drug development.

2. PROGRESS IN THE CHARACTERIZATION OF THE EGFR SIGNALING NETWORK

Significant advances have been achieved in methods and approaches to characterize the EGFR signaling network. The mechanisms by which different types and concentrations of ligands affect the physiological functions of EGFR have been attracting increasing interest.^12–15) Therefore, we now have a more detailed understanding of the non-canonical regulation of EGFR by serine (Ser)/threonine (Thr) phosphorylation and potential novel therapeutic targets (Fig. 1).

Fig. 1. Complexity of EGFR Signaling

Although a vast amount of information has been accumulated about the EGFR signaling pathway, it is not well understood what determines differential responsiveness to ligands: affinity for EGFR, ligand concentration, mutational status of EGFR, and the number of EGFR molecules on the plasma membrane are important factors. How the peak intensity and duration of downstream signals are regulated may be what causes cells to proliferate or differentiate, in which phosphorylation and intracellular trafficking of EGFR may also play an important role. CME, clathrin-mediated endocytosis. CIE, clathrin-independent endocytosis.

2.1. Ligand Diversity and EGFR Signaling

Seven different ligands for EGFR have been identified and divided into two groups based on their affinities to EGFR, with 10- to 100-fold differences. EGF, transforming growth factor-α (TGF-α), heparin-binding EGF, and betacellulin are high-affinity ligands, while epigen (EPGN), epiregulin (EREG) and amphiregulin are low-affinity ligands. The actions of each ligand have been extensively examined. A recent crystal structural analysis revealed that when ligands bind to EGFR on both sides of the dimer, EREG and EPGN induced less stable EGFR extracellular dimers than EGF, in which EGF and EREG formed stable symmetric and unique asymmetric dimers, respectively, in contrast to EPGN, which did not induce dimers.¹⁶⁾ Weakened dimerization with low-affinity ligands elicits more sustained EGFR signaling than EGF stimulation, resulting in cell differentiation rather than proliferation, which warrants further study.¹⁶⁾

The most recent update to the WHO classification of brain tumors established EGFR amplification as a criterion for glioblastoma multiform (GBM).¹⁷⁾ Advanced structural, biophysical, and cellular studies and a large genomic data analysis provided novel insights into the roles of ligands in extracellular EGFR GBM mutations. Wild-type EGFR forms a weak asymmetric dimer with EREG as described above,¹⁶⁾ whereas the EGFR-R84K mutant forms a strong symmetric dimer, similar to EGF binding to wild-type EGFR.¹⁸⁾ The A265V mutation has been shown to strengthen the formation of asymmetric EREG-EGFR dimers by remodeling the key dimerization interface. Since EREG induces cell differentiation in multiple cell types,^19,20) the GBM mutations of EGFR affect cellular responses to EREG and other weak-affinity ligands, making them more EGF-like, which promotes the proliferation of glioblastoma and its progenitor cells.

Another important update was recently reported on the role of the EGFR ligand in the progression of GBM. Even though constitutive EGFR signaling has been shown to enhanced invasion via the activation of the TAB1–TAK1–nuclear factor-kappaB (NF-κB) pathway, EGF-activated EGFR signaling promoted proliferation, but suppressed invasion.²¹⁾ More importantly, an analysis of data from the Cancer Genome Atlas revealed that a low level of EGF ligands correlated with a poor prognosis in EGFR-amplified, but not non-amplified GBM, whereas a high level of EGF ligands improved the prognosis of these patients, supporting the concept that EGFR ligands at higher concentrations change the function of EGFR from oncogenic to tumor suppressive. This may be due to differences in intracellular trafficking routes depending on ligand concentration, as described below, with higher concentrations of ligand leading to the EGFR degradation pathway. Therefore, it is essential to reconsider which ligands act at what concentrations in the tumorigenesis and progression of glioblastoma. Similarly, as described below, EGFR trafficking routes are differentially regulated in a ligand concentration-dependent manner. A single cell analysis demonstrated that approximately 50000 EGFR molecules were expressed on the HeLa cell surface, and that the binding of only 300 EGF molecules to the cell surface was sufficient to trigger a ligand response.²²⁾ We also confirmed that 0.1 ng/mL of EGF activated extracellular signal-regulated kinase (ERK) to the same extent as 100 ng/mL²³⁾; therefore, the concentration of the ligand used in experiments needs to be regarded as a key factor for interpreting the data obtained.

2.2. EGFR Signaling Network

The use of multi-layered omics technologies to obtain a comprehensive understanding of the EGF signaling network has been progressing.^24–26) Proximity labeling applications that map protein–protein interactions and subcellular proteomes in living cells have recently been adopted for the profiling of signaling networks.^27,28) The proximity labeling of EGF-stimulated EGFR provides a comprehensive resource of the time-dependent nanoscale environment of EGFR, thereby opening avenues to discover new regulatory mechanisms of signaling and intracellular trafficking. In fact, Trk-fused gene, an endoplasmic reticulum protein, was identified as a novel regulator of the endosomal sorting of EGFR. EGFR signaling networks have also been analyzed using interactomes and tyrosine phosphoproteomes after the proximity labeling of GRB2, which provided similar findings to the EGFR study; therefore, comprehensive information has been obtained on compartment-dependent changes in receptor-proximal protein networks.²⁹⁾ These analyses highlight the importance of the non-receptor type protein tyrosine phosphatase PTPN11/SHP2. EGF induces the rapid phosphorylation of PTPN11/SHP2 at Y542 and Y580, which are considered to stimulate its phosphatase activity, and mutations in these residues result in clinically similar disorders, namely, Noonan syndrome and LEOPARD syndrome, through the abnormal activation of MAPK.³⁰⁾ It is important to note that allosteric PTPN11/SHP2 phosphatase inhibitors are currently under clinical development for cancer.^31–33) PTPN11 is known to dephosphorylate and inhibit RAS, which would suggest that its inhibitor would rather promote proliferative signals.³⁴⁾ To resolve this discrepancy, the further elucidation of its substrates in EGF-triggered forward/feedback signals and effects on SHP2 signaling complex formation by inhibitors acting on the allosteric site rather than the catalytic site is essential for a more detailed understanding of the role of PTPN11/SHP2 in the activation of MAPK and acquisition of resistance to EGFR-TKIs.³⁵⁾ These studies may contribute to the establishment of promising combination therapy with an anti-EGFR agent and PTPN11/SHP2 inhibitor.^36,37)

3. NON-CANONICAL ENDOCYTOSIS OF EGFR BY SER/THR PHOSPHORYLATION

Since EGFR is a member of the RTK family, EGF initially triggers the tyrosine autophosphorylation of the C-terminal tail of EGFR to transduce the signal. Nevertheless, a phosphoproteomic analysis identified more than forty serine/threonine phosphorylation sites in the intracellular domain of EGFR.^38,39) Therefore, the elucidation of their functions is strongly encouraged because of their potential as therapeutic targets. For example, the ERK-mediated phosphorylation of Thr-669 in the juxtamembrane region is involved in the negative feedback regulation of the tyrosine kinase activity of EGFR in the resistance of melanoma and colorectal cancer cells to a BRAF inhibitor.^40,41) We herein focused on recent findings on a novel intracellular trafficking mechanism by p38.

3.1. Conventional Model of EGFR Trafficking

The EGF-bound EGFR dimer rapidly undergoes endocytosis through clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE).⁴²⁾ Internalization via CME contributes to sustained EGFR signaling and the subsequent induction of cell proliferation, and is the main mechanism for the endocytosis of EGFR in tumors in vivo.⁴³⁾ The endocytosed EGF–EGFR complex via CME or CIE is considered to be sorted to recycling or lysosomal degradation pathways, respectively. A limitation of the conventional model is that it cannot explain why sorting routes differ depending on ligand concentrations. In brief, the majority of endocytosed EGFR is recycled to the plasma membrane when stimulated with EGF at low concentrations, whereas lysosomal degradation increases at higher concentrations. Moreover, the physiological ligand concentration in normal and tumor tissues is not high (<170–340 pM or 1–2 ng/mL). Therefore, we recently proposed an improved model of EGF-induced EGFR trafficking as described below.

3.2. p38-Mediated Endocytosis of EGFR

Cellular stress induces the endocytosis/recycling pathway of ligand-free EGFR monomers via the activation of p38 in a tyrosine kinase-independent manner.^44–46) Ser-1006/Ser-1015/Thr-1017/Ser-1018 surrounding the leucine (Leu)-1010/Leu-1011 clathrin-recognizing sequence have been identified as p38-targeted phosphorylation sites.^23,47) Another feature of stress-induced EGFR-CME is its recycling to the plasma membrane after the inactivation of p38. Cisplatin and temozolomide, chemotherapeutic agents for NSCLC and GBM, also induce EGFR-CME, which is suggested to play a role in resistance to chemotherapeutic agents by cooperating with NF-κB.^48–50) Endocytosed EGFR accumulates in a subset of lysobisphosphatidic acid-rich perinuclear MVBs through a mechanism involving the actin polymerization-promoting protein WASH.⁵¹⁾ Cooperation between EGFR and WASH has also been shown to play a key role in a Drosophila cell competition model, in which the activation of the receptor tyrosine phosphatase PTP10D, a negative regulator of EGFR, by its ligand Sas in loser cells triggered cell elimination through the inactivation of EGFR signaling.^52,53) Therefore, endosomal regulation by p38 is attracting interest in the regulation of EGFR activation.

3.3. Dual-Mode EGFR-CME Model

A long-standing unexplained phenomenon is that EGF at lower physiological concentrations and saturated higher concentrations preferentially trigger different EGFR trafficking routes via the CME/recycling and CIE/degradation pathways, respectively.^54,55) Therefore, we recently proposed a dual-mode EGFR-CME model, in collaboration with Prof. Alexander Sorkin, by incorporating the p38-mediated mechanism into the ligand-induced EGFR trafficking model^15,23,47) (Fig. 2). We demonstrated that EGF at lower physiological concentrations induced the full activation of p38 and triggered the non-canonical p38-mediated CME of ligand-free EGFR monomers in parallel with the canonical CME/CIE of ligand-bound EGFR dimers. It is important to note that non-canonical EGFR-CME is a major component at physiological concentrations of EGF. In other words, despite a ligand stimulation, the majority of endocytosed EGFR is ligand-free, except for a small amount of ligand-bound activated EGFR. In contrast, the majority of surface EGFR are saturated by EGF at higher concentrations. Therefore, ligand-bound EGFR dimers and unbound EGFR monomers are balanced by ligand concentrations, reflecting the initial ligand occupation rate. The stoichiometry of the EGF–EGFR complex also needs to be considered. The first ligand-binding event induces the asymmetric dimerization of extracellular domains, which affects the structure of the counterpart ligand-unoccupied EGFR. This reduces the affinity for binding of the second ligand,⁵⁶⁾ indicating that saturated high ligand concentrations are needed to form the symmetric EGFR dimer with two high affinity ligands as described above. These findings suggest that the dimer with one ligand is preferentially formed upon a stimulation with physiological concentrations of the ligand. Therefore, the endocytosis pathway may be altered by the ligand–receptor ratio, which needs to be verified.

Fig. 2. Dual-Mode Clathrin-Mediated Endocytosis Model

At physiological ligand concentrations, EGFR undergoes endocytosis via both canonical and non-canonical CME, most of which is recycled to the plasma membrane. The EGFR transported by non-canonical CME is a monomer that is phosphorylated via p38. In contrast, at saturating ligand concentrations, EGFR is internalized via canonical CME and CIE and then transported to lysosomes for degradation.

Another study demonstrated that sorting for recycling or degradation was dependent on the recruitment of the clathrin adapter molecule AP-2 or the sorting proteins Eps15/Epsin, respectively, to clathrin-coated pits containing the EGF–EGFR complex, in which the ablation of AP-2 inhibited cell migration.⁵⁷⁾ Since we previously reported that p38 phosphorylated Eps15 at Ser-796,⁵⁸⁾ further studies are warranted to establish whether this phosphorylation is involved in AP-2-independent EGFR-CME for degradation. Moreover, Rab25 participates in the recycling endosomes of endocytosed EGFR triggered by both a ligand stimulation and irradiation, suggesting a common recycling system after p38-mediated EGFR-CME.⁵⁹⁾ Therefore, discrepancies with previous findings need to be considered, which may justify our new model.

4. RECENT ADVANCES IN THE TREATMENT OF EGFR-MUTANT LUNG CANCER

Over the past 20 years, we have obtained a more detailed understanding of the nature of cancer, namely, oncogene addiction and acquired resistance (Fig. 3). The introduction of gefitinib and erlotinib, first-generation reversible ATP-competitive EGFR-TKIs, has markedly improved treatment strategies for NSCLC harboring the exon 19 deletion (Ex19Del) and exon 21 L858R substitution of the EGFR gene.⁸⁾ Although these TKIs are clinically very effective, acquired resistance appears within 1–2 years. The exon 20 T790M secondary EGFR mutation and the activation of bypass pathways, including MET, AXL, HER3, and FGFRs, have been proposed as resistance mechanisms.⁹⁾ Other EGFR-independent resistance also occurs with phenotypic changes due to epithelial-mesenchymal transition,⁶⁰⁾ and CD70, which is highly expressed in such resistant cells, has attracted attention as a therapeutic target.⁶¹⁾

Fig. 3. Progress in EGFR-TKI Development

In 2002, the first generation EGFR-TKI gefitinib was approved in Japan, and now the fourth generation is undergoing clinical trials. Although this has greatly improved the prognosis for lung cancer patients, various resistance mutations have arisen. This continues to broaden the disease concept of EGFR-mutant lung cancer, and the challenge is how to overcome these mutations. The discovery of allosteric inhibitors has had the impact of changing the direction of EGFR-TKI therapy to date and holds promise for combination therapy with the ATP-competitive TKIs that have been developed. In addition, the development of innovative agents for EGFR-mutated cancers is expected.

Further improvements to overcome T790M resistance led to the successful development of osimertinib, a mutant-selective irreversible ATP-competitive third-generation EGFR-TKI that binds covalently to a conserved cysteine residue (C797) in the tyrosine kinase domain.⁶²⁾ FLAURA clinical trials provided strong evidence to show that patient survival was better with osimertinib than with classical EGFR-TKIs in the first-line setting.⁶³⁾ Therefore, osimertinib has now become the standard first-line treatment for EGFR-mutant NSCLC. Although advances have been made in EGFR-targeted therapy, many challenges remain.

4.1. Current Challenges Associated with EGFR-TKI Therapy

Acquired resistance to osimertinib through the additional C797S substitution of the covalent anchor site of osimertinib has already emerged. Regardless of the order of administration, a sequential treatment with clinically available TKIs results in resistance through triple mutations (Ex19Del/T790M/C797S and L858R/T790M/C797S).¹¹⁾ The fourth-generation ATP-competitive inhibitors BLU-945 and BBT-176 are now under clinical trials and expected to overcome osimertinib resistance.^64,65) JIN-A02, another potent EGFR-TKI for various C797S mutants, may also be used to treat the brain metastases of NSCLC due to its high brain penetration.⁶⁶⁾

On-target mutations to osimertinib are rarer (approximately 10%) than T790M (approximately 50%) to conventional TKIs, suggesting that the TKI strategy may gradually prevent evasion by NSCLC cells.⁶⁷⁾ Various bypass mechanisms, including the activation of RTK (the amplification of MET and HER2), activating mutations in the MAPK/phosphatidylinositol 3-kinase (PI3K) pathway (BRAF-V600E, KRAS, and PIK3CA), cell cycle alterations (the amplification of CDK4 and CDK6), transformation into small cell lung cancer, and oncogenic fusions (BRAF, RET, and ALK), have manifested. Furthermore, there are significant barriers to overcoming bypass resistance because these factors are often combined. Glucocorticoids have been shown to suppress bypass RTK signaling pathways via the down-regulation of EGFR inhibition-induced signal transducer and activator of transcription 3 (STAT3), YAP, and NF-κB inflammatory signals.⁶⁸⁾ Although selective targeted agents for these pathways are already available for clinical use to treat other cancers, approaches have not yet been developed to assess their utility against recurrent, subdivided EGFR-TKI-resistant NSCLC.

4.2. Diversity of Major and Minor EGFR Mutations

Although there are more than 20 distinct genomic alterations in Ex19Del, they have been managed as a single disease category in TKI therapy. However, functional differences between distinct Ex19Del variants have been identified.^69,70) ΔE746-S752insV and ΔL747-A750insP display enhanced oncogenic growth and reduced TKI sensitivity, particularly to erlotinib and osimertinib, due to a decreased Km to ATP. Of note, variants with reduced sensitivity to TKIs have a worse prognosis following treatment with erlotinib. Furthermore, approximately 10% of patients with NSCLC harbor uncommon EGFR alterations, typically exon 20 insertions (Ex20Ins) that are resistant to first-/third-generation EGFR-TKIs, due to a similar alignment of the gatekeeper residue T790 to the T790M mutant.^71,72) Nevertheless, advances have been achieved in the development of TKIs for minor mutations. Mobocertinib, an irreversible TKI, was approved for the treatment of patients with Ex20Ins.⁷³⁾ Similar to Ex19Del, sensitivity to Ex20Ins-targeting poziotinib is highly dependent on the insertion location, with near-loop insertions being more sensitive than far-loop insertions.⁷⁴⁾ Moreover, compound mutations that have minor and major mutations result in intrinsic resistance to EGFR-TKIs. These findings expand the framework for therapeutic interventions into a subdivided disease concept by each EGFR variant and increase the difficulty of accumulating clinical evidence on the efficacy of TKIs.

5. NEW DIRECTIONS FOR EGFR-TARGETED THERAPY

5.1. Towards the Clinical Application of Allosteric EGFR-TKIs

The repeated optimization of ATP-competitive EGFR-TKIs has yielded better clinical outcomes; nevertheless, a new wave of EGFR-TKIs is being introduced (Fig. 3). The allosteric, non-ATP-competitive inhibitors, EAI001 and EAI045, which bind to an allosteric site adjacent to the ATP-binding pocket and stabilize its inactive conformation,⁷⁵⁾ have been screened. Advances have been achieved in the optimization of lead allosteric compounds, yielding mutant-selective compounds (JBJ-04-125-02 and JBJ-09-063) that are highly effective as single agents against L858R/T790M and L858R/T790M/C797S.⁷⁶⁾ These inhibitors are also effective against a broad range of osimertinib-resistant EGFR mutants, including L718Q, L792F, and G796S. It is important to note that the combination of conventional ATP-competitive (osimertinib or gefitinib) and allosteric (JBJ-09-063) inhibitors synergize to suppress the growth of lung cancer cells harboring L858R/T790M/C797S mutations in vivo because they simultaneously occupy each binding pocket in the same kinase.⁷⁷⁾ Moreover, this combination therapy has hindered the emergence of acquired resistance. Although not all combinations are effective due to steric hindrance, the dual inhibition strategy is expected to reveal clinical activity and fundamentally change future EGFR-TKI therapy. Furthermore, combinations with cetuximab, an anti-EGFR neutralizing monoclonal antibody, may prevent the emergence of resistance mutations to EGFR-TKIs in patient-derived xenograft models of lung cancer.⁷⁸⁾ Therefore, the combined effects of multiple anti-EGFR agents need to be examined in future clinical trials.

A mutagenesis assay predicted that L747S is an on-target resistance mutation to JBJ-09-063, but not to osimertinib, in Ba/F3 cells expressing EGFR-L858R/T790M.⁷⁶⁾ Structural modeling demonstrated that favorable hydrophobic interactions of the side chain of L747 with the phenyl ring of the allosteric inhibitor were lost in the L747S variant. However, the L747S mutation did not emerge when co-treated with osimertinib, indicating the importance of establishing combination regimens with allosteric and ATP-competitive TKIs. Another challenge is that currently known allosteric inhibitors only target L858R mutants, because L858R, but not 19ExDel, expands the allosteric pocket.⁷⁷⁾ Further studies on their effects on other uncommon/compound mutations, including exon 20 insertions, are needed to expand future clinical applications of allosteric inhibitors. The identification of other allosteric sites is also of importance.

5.2. Innovative Strategies for EGFR-Targeted Therapy

Besides TKI therapy, EGFR-driven tumors are targeted by other innovative strategies (Fig. 3). A bispecific anti-EGFR-MET monoclonal antibody (amivantamab) and antibody-drug conjugate (ADC) with a light-activatable payload (cetuximab-sarotalocan) have been approved for the treatment of patients with advanced NSCLC with EGFR exon 20 insertions and head and neck cancers, respectively.^79,80) Immune checkpoint inhibitors (ICIs), including anti-PD-1 and anti-PD-L1 antibodies, are also attracting attention. EGFR-driven lung cancers generally have a low response to ICIs. However, Prof. Yarden and colleagues recently reported that the depletion of PD-L1 severely impaired EGFR-driven tumorigenesis and metastasis in mice, in which EGFR signaling was enhanced by the recruitment of phospholipase C-γ1 (PLC-γ1) to the cytoplasmic tail of PD-L1.⁸¹⁾ Therefore, targeting the functional relationship between PLC-γ1/PD-L1/EGFR may enhance responses to ICIs. Moreover, the findings of a retrospective study suggest that the levels of p53 and AXL in pretreatment tumors may predict ICI-based therapy outcomes in EGFR-mutant NSCLC patients treated with osimertinib.⁸²⁾

5.3. Potential New Anti-EGFR Therapeutics for GBM

Our increasing understanding of the roles of EGFR alterations, such as amplifications and deletions, has enabled the development of novel potential therapeutics for GBM.¹⁷⁾ However, the targeting of EGFR in GBM patients remains unsuccessful mainly due to the poor blood–brain barrier penetrance of the majority of EGFR-directed agents. Although the percentage of EGFR-amplified GBM that are addicted to its tyrosine kinase activity remains unclear, the development of EGFR-TKIs is still under active investigation. JIN-A02 is expected to be applied to the treatment of GBM due to its high penetration into the brain.⁶⁶⁾

Under these conditions, novel insights have been obtained on the treatment of GBM. A pivotal phase 2 clinical trial on the oncolytic herpes simplex virus G47∆ represented a significant advance in the treatment of recurrent GBM and led to conditional marketing approval in Japan.⁸³⁾ The oncolytic virus strategy is also being applied to EGFR-targeted therapy for GBM. Specific targeting with an oncolytic virus expressing a cetuximab-C-C motif chemokine ligand 5 fusion protein significantly inhibited EGFR signaling in tumors, reduced tumor sizes, and prolonged the survival of GBM-bearing mice by enhancing the migration and activation of natural killer cells, macrophages, and T cells in the tumor microenvironment.⁸⁴⁾ Immunovirotherapy will be effective for EGFR-amplified GBM, regardless of whether they are addicted to EGFR activity.

Another promising therapeutic strategy is the development of EGFR-targeted ADCs. Depatuxizumab mafodotin (depatux-m, ABT-414), the most advanced ADC that is an EGFR antibody conjugated to the tubulin inhibitor monomethyl auristatin F via a stable maleimidocaproyl linker, exhibits potent anti-tumor activity in experimental GBM models.⁸⁵⁾ In a phase III clinical trial on newly diagnosed EGFR-amplified GBM, progression-free survival was longer with depatux-m than with a placebo, particularly in the EGFRvIII-mutant subgroup; however, overall survival (OS) remained unchanged.⁸⁶⁾ Similarly, a phase II clinical trial obtained interesting findings on the combination regimen of depatux-m and temozolomide, an alkylating agent for standard GBM chemotherapy, for EGFR-amplified recurrent GBM, but again did not reach the primary endpoint of OS.⁸⁷⁾ If the efficiency of the intracellular delivery of depatux-m is increased by improving the protocol, it may be possible to achieve therapeutic effects. We recently reported a strategy to synchronously and efficiently deliver EGFR-targeted ADCs into cancer cells via p38-mediated non-canonical endocytosis, in which temozolomide enhanced internalization.⁸⁸⁾ However, since an EGFR–ADC complex is not sorted to lysosomes in p38-mediated non-canonical endocytosis, the redesign of ADC linkers for degradation is also needed in order to further increase the efficacy of anti-EGFR ADCs.

6. CONCLUDING REMARKS AND FUTURE PERSPECTIVES

As summarized in this review, EGFR have attracted much attention as a front-runner in the establishment of a basic intracellular signaling network and clinical molecular targeted cancer therapy; however, they are still expanding. Although these studies have mapped individual signaling and endosome-associated molecular connections, they are still qualitative. Further studies are warranted to obtain a more detailed understanding of individual reactions biochemically. Nevertheless, if we systematize these reactions quantitatively, it may lead to the discovery of novel unknown vulnerabilities in proliferation signals. Furthermore, we need to consider translational research on targeted therapy, such as comparisons of the activation mechanisms of many EGFR mutants identified in NSCLC and GBM with that of the wild type.

As a result of the last 20 years of research, EGFR-mutant lung cancers have become a very diverse disease category due to the large number of activating/resistance/uncommon mutations. In addition, the use of new TKIs, such as allosteric inhibitors, will be accompanied by the emergence of new variants. The structure-based classification of EGFR mutants into small groups may predict patient outcomes following treatment with EGFR-TKIs more accurately than exon-base genetic grouping.⁸⁹⁾ In any case, the establishment of a methodology that manages subdivided disease concepts and reflects them in treatment is urgently needed. It will be challenging to enroll an adequate number of patients; therefore, further discussions on the approaches required for these clinical trials need to be conducted.

Acknowledgments

This work was supported in part by JSPS KAKENHI Grant Number: 22H02763, JST SPRING Grant Number: JPMJSP2145, and Moonshot R&D Grant Number: JPMJMS2021.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Cohen S. Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the new-born animal. J. Biol. Chem., 237, 1555–1562 (1962).
• 2) Lin CR, Chen WS, Kruiger W, Stolarsky LS, Weber W, Evans RM, Verma IM, Gill GN, Rosenfeld MG. Expression cloning of human EGF receptor complementary DNA: gene amplification and three related messenger RNA products in A431 cells. Science, 224, 843–848 (1984).
• 3) Boguski MS, McCormick F. Proteins regulating Ras and its relatives. Nature, 366, 643–654 (1993).
• 4) Nishida E, Gotoh Y. The MAP kinase cascade is essential for diverse signal transduction pathways. Trends Biochem. Sci., 18, 128–131 (1993).
• 5) Cano E, Mahadevan LC. Parallel signal processing among mammalian MAPKs. Trends Biochem. Sci., 20, 117–122 (1995).
• 6) Baselga J, Rischin D, Ranson M, Calvert H, Raymond E, Kieback DG, Kaye SB, Gianni L, Harris A, Bjork T, Averbuch SD, Feyereislova A, Swaisland H, Rojo F, Albanell J. Phase I safety, pharmacokinetic, and pharmacodynamic trial of ZD1839, a selective oral epidermal growth factor receptor tyrosine kinase inhibitor, in patients with five selected solid tumor types. J. Clin. Oncol., 20, 4292–4302 (2002).
• 7) Sandler A. Clinical experience with the HER1/EGFR tyrosine kinase inhibitor erlotinib. Oncology (Williston Park), 17 (Suppl. 12), 17–22 (2003).
• 8) Sharma SV, Bell DW, Settleman J, Haber DA. Epidermal growth factor receptor mutations in lung cancer. Nat. Rev. Cancer, 7, 169–181 (2007).
• 9) Dzul Keflee R, Leong KH, Ogawa S, Bignon J, Chan MC, Kong KW. Overview of the multifaceted resistances toward EGFR-TKIs and new chemotherapeutic strategies in non-small cell lung cancer. Biochem. Pharmacol., 205, 115262 (2022).
• 10) Girard N. New strategies and novel combinations in EGFR TKI-resistant non-small cell lung cancer. Curr. Treat. Options Oncol., 23, 1626–1644 (2022).
• 11) Shi K, Wang G, Pei J, Zhang J, Wang J, Ouyang L, Wang Y, Li W. Emerging strategies to overcome resistance to third-generation EGFR inhibitors. J. Hematol. Oncol., 15, 94 (2022).
• 12) Wilson KJ, Gilmore JL, Foley J, Lemmon MA, Riese DJ 2nd. Functional selectivity of EGF family peptide growth factors: implications for cancer. Pharmacol. Ther., 122, 1–8 (2009).
• 13) Harris RC, Chung E, Coffey RJ. EGF receptor ligands. Exp. Cell Res., 284, 2–13 (2003).
• 14) Ronan T, Macdonald-Obermann JL, Huelsmann L, Bessman NJ, Naegle KM, Pike LJ. Different epidermal growth factor receptor (EGFR) agonists produce unique signatures for the recruitment of downstream signaling proteins. J. Biol. Chem., 291, 5528–5540 (2016).
• 15) Zhou Y, Sakurai H. New trend in ligand-induced EGFR trafficking: a dual-mode clathrin-mediated endocytosis model. J. Proteomics, 255, 104503 (2022).
• 16) Freed DM, Bessman NJ, Kiyatkin A, Salazar-Cavazos E, Byrne PO, Moore JO, Valley CC, Ferguson KM, Leahy DJ, Lidke DS, Lemmon MA. EGFR Ligands differentially stabilize receptor dimers to specify signaling kinetics. Cell, 171, 683–695.e18 (2017).
• 17) Louis DN, Perry A, Wesseling P, Brat DJ, Cree IA, Figarella-Branger D, Hawkins C, Ng HK, Pfister SM, Reifenberger G, Soffietti R, von Deimling A, Ellison DW. The 2021 WHO classification of tumors of the central nervous system: a summary. Neuro-oncol., 23, 1231–1251 (2021).
• 18) Hu C, Leche CA 2nd, Kiyatkin A, Yu Z, Stayrook SE, Ferguson KM, Lemmon MA. Glioblastoma mutations alter EGFR dimer structure to prevent ligand bias. Nature, 602, 518–522 (2022).
• 19) Rizzi M, Pittarella P, Sabbatini M, Renò F. Epiregulin induces human SK-N-BE cell differentiation through ERK1/2 signaling pathway. Growth Factors, 31, 90–97 (2013).
• 20) Riese DJ 2nd, Cullum RL. Epiregulin: roles in normal physiology and cancer. Semin. Cell Dev. Biol., 28, 49–56 (2014).
• 21) Guo G, Gong K, Beckley N, et al. EGFR ligand shifts the role of EGFR from oncogene to tumour suppressor in EGFR-amplified glioblastoma by suppressing invasion through BIN3 upregulation. Nat. Cell Biol., 24, 1291–1305 (2022).
• 22) Uyemura T, Takagi H, Yanagida T, Sako Y. Single-molecule analysis of epidermal growth factor signaling that leads to ultrasensitive calcium response. Biophys. J., 88, 3720–3730 (2005).
• 23) Tanaka T, Zhou Y, Ozawa T, Okizono R, Banba A, Yamamura T, Oga E, Muraguchi A, Sakurai H. Ligand-activated epidermal growth factor receptor (EGFR) signaling governs endocytic trafficking of unliganded receptor monomers by non-canonical phosphorylation. J. Biol. Chem., 293, 2288–2301 (2018).
• 24) Lundby A, Franciosa G, Emdal KB, Refsgaard JC, Gnosa SP, Bekker-Jensen DB, Secher A, Maurya SR, Paul I, Mendez BL, Kelstrup CD, Francavilla C, Kveiborg M, Montoya G, Jensen LJ, Olsen JV. Oncogenic mutations rewire signaling pathways by switching protein recruitment to phosphotyrosine sites. Cell, 179, 543–560.e26 (2019).
• 25) Francavilla C, Papetti M, Rigbolt KTG, Pedersen A-K, Sigurdsson JO, Cazzamali G, Karemore G, Blagoev B, Olsen JV. Multilayered proteomics reveals molecular switches dictating ligand-dependent EGFR trafficking. Nat. Struct. Mol. Biol., 23, 608–618 (2016).
• 26) Uribe ML, Marrocco I, Yarden Y. EGFR in cancer: signaling mechanisms, drugs, and acquired resistance. Cancers (Basel), 13, 2748 (2021).
• 27) Perez Verdaguer M, Zhang T, Surve S, Paulo JA, Wallace C, Watkins SC, Gygi SP, Sorkin A. Time-resolved proximity labeling of protein networks associated with ligand-activated EGFR. Cell Rep., 39, 110950 (2022).
• 28) Ke M, Yuan X, He A, Yu P, Chen W, Shi Y, Hunter T, Zou P, Tian R. Spatiotemporal profiling of cytosolic signaling complexes in living cells by selective proximity proteomics. Nat. Commun., 12, 71 (2021).
• 29) Kong Q, Ke M, Weng Y, Qin Y, He A, Li P, Cai Z, Tian R. Dynamic phosphotyrosine-dependent signaling profiling in living cells by two-dimensional proximity proteomics. J. Proteome Res., 21, 2727–2735 (2022).
• 30) Lauriol J, Kontaridis MI. PTPN11-associated mutations in the heart: has LEOPARD changed its RASpots? Trends Cardiovasc. Med., 21, 97–104 (2011).
• 31) Asmamaw MD, Shi X-J, Zhang L-R, Liu H-M. A comprehensive review of SHP2 and its role in cancer. Cell Oncol. (Dordr.), 45, 729–753 (2022).
• 32) Chen Y-NP, LaMarche MJ, Chan HM, et al. Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases. Nature, 535, 148–152 (2016).
• 33) LaMarche MJ, Acker M, Argintaru A, et al. Identification of TNO155, an allosteric SHP2 inhibitor for the treatment of cancer. J. Med. Chem., 63, 13578–13594 (2020).
• 34) Kano Y, Gebregiworgis T, Marshall CB, Radulovich N, Poon BPK, St-Germain J, Cook JD, Valencia-Sama I, Grant BMM, Herrera SG, Miao J, Raught B, Irwin MS, Lee JE, Yeh JJ, Zhang Z-Y, Tsao M-S, Ikura M, Ohh M. Tyrosyl phosphorylation of KRAS stalls GTPase cycle via alteration of switch I and II conformation. Nat. Commun., 10, 224 (2019).
• 35) Kerr DL, Haderk F, Bivona TG. Allosteric SHP2 inhibitors in cancer: Targeting the intersection of RAS, resistance, and the immune microenvironment. Curr. Opin. Chem. Biol., 62, 1–12 (2021).
• 36) Liu C, Lu H, Wang H, et al. Combinations with allosteric SHP2 inhibitor TNO155 to block receptor tyrosine kinase signaling. Clin. Cancer Res., 27, 342–354 (2021).
• 37) Sun Y, Meyers BA, Czako B, et al. Allosteric SHP2 inhibitor, IACS-13909, overcomes EGFR-dependent and EGFR-independent resistance mechanisms toward osimertinib. Cancer Res., 80, 4840–4853 (2020).
• 38) Tong J, Taylor P, Moran MF. Proteomic analysis of the epidermal growth factor receptor (EGFR) interactome and post-translational modifications associated with receptor endocytosis in response to EGF and stress. Mol. Cell. Proteomics, 13, 1644–1658 (2014).
• 39) Hornbeck PV, Kornhauser JM, Latham V, Murray B, Nandhikonda V, Nord A, Skrzypek E, Wheeler T, Zhang B, Gnad F. 15 years of PhosphoSitePlus^®: integrating post-translationally modified sites, disease variants and isoforms. Nucleic Acids Res., 47 (D1), D433–D441 (2019).
• 40) Lito P, Pratilas CA, Joseph EW, Tadi M, Halilovic E, Zubrowski M, Huang A, Wong WL, Callahan MK, Merghoub T, Wolchok JD, de Stanchina E, Chandarlapaty S, Poulikakos PI, Fagin JA, Rosen N. Relief of profound feedback inhibition of mitogenic signaling by RAF inhibitors attenuates their activity in BRAFV600E melanomas. Cancer Cell, 22, 668–682 (2012).
• 41) Sato K, Shin M-S, Sakimura A, Zhou Y, Tanaka T, Kawanishi M, Kawasaki Y, Yokoyama S, Koizumi K, Saiki I, Sakurai H. Inverse correlation between Thr-669 and constitutive tyrosine phosphorylation in the asymmetric epidermal growth factor receptor dimer conformation. Cancer Sci., 104, 1315–1322 (2013).
• 42) Caldieri G, Malabarba MG, Di Fiore PP, Sigismund S. EGFR trafficking in physiology and cancer. Prog. Mol. Subcell. Biol., 57, 235–272 (2018).
• 43) Pinilla-Macua I, Grassart A, Duvvuri U, Watkins SC, Sorkin A. EGF receptor signaling, phosphorylation, ubiquitylation and endocytosis in tumors in vivo. eLife, 6, e31993 (2017).
• 44) Tan X, Lambert PF, Rapraeger AC, Anderson RA. Stress-induced EGFR trafficking: mechanisms, functions, and therapeutic implications. Trends Cell Biol., 26, 352–366 (2016).
• 45) Singhirunnusorn P, Ueno Y, Matsuo M, Suzuki S, Saiki I, Sakurai H. Transient suppression of ligand-mediated activation of epidermal growth factor receptor by tumor necrosis factor-alpha through the TAK1-p38 signaling pathway. J. Biol. Chem., 282, 12698–12706 (2007).
• 46) Yamagishi N, Takahashi J, Zhou Y, Yokoyama S, Makino T, Shimizu T, Sakurai H. Non-canonical regulation of EGFR by the air pollutant 9,10-phenanthrenequinone. Biol. Pharm. Bull., 45, 1553–1558 (2022).
• 47) Perez Verdaguer M, Zhang T, Paulo JA, Gygi S, Watkins SC, Sakurai H, Sorkin A. Mechanism of p38 MAPK-induced EGFR endocytosis and its crosstalk with ligand-induced pathways. J. Cell Biol., 220, e202102005 (2021).
• 48) Zwang Y, Yarden Y. p38 MAP kinase mediates stress-induced internalization of EGFR: implications for cancer chemotherapy. EMBO J., 25, 4195–4206 (2006).
• 49) Haryuni RD, Tanaka T, Takahashi J-I, Onuma I, Zhou Y, Yokoyama S, Sakurai H. Temozolomide induces endocytosis of EGFRvIII via p38-mediated non-canonical phosphorylation in glioblastoma cells. Biol. Pharm. Bull., 44, 1681–1687 (2021).
• 50) Nishimura M, Shin M-S, Singhirunnusorn P, Suzuki S, Kawanishi M, Koizumi K, Saiki I, Sakurai H. TAK1-mediated serine/threonine phosphorylation of epidermal growth factor receptor via p38/extracellular signal-regulated kinase: NF-{kappa}B-independent survival pathways in tumor necrosis factor alpha signaling. Mol. Cell. Biol., 29, 5529–5539 (2009).
• 51) Tomas A, Vaughan SO, Burgoyne T, Sorkin A, Hartley JA, Hochhauser D, Futter CE. WASH and Tsg101/ALIX-dependent diversion of stress-internalized EGFR from the canonical endocytic pathway. Nat. Commun., 6, 7324 (2015).
• 52) Liu D, Tsarouhas V, Samakovlis C. WASH activation controls endosomal recycling and EGFR and Hippo signaling during tumor-suppressive cell competition. Nat. Commun., 13, 6243 (2022).
• 53) Yamamoto M, Ohsawa S, Kunimasa K, Igaki T. The ligand Sas and its receptor PTP10D drive tumour-suppressive cell competition. Nature, 542, 246–250 (2017).
• 54) Sigismund S, Woelk T, Puri C, Maspero E, Tacchetti C, Transidico P, Di Fiore PP, Polo S. Clathrin-independent endocytosis of ubiquitinated cargos. Proc. Natl. Acad. Sci. U.S.A., 102, 2760–2765 (2005).
• 55) Sigismund S, Argenzio E, Tosoni D, Cavallaro E, Polo S, Di Fiore PP. Clathrin-mediated internalization is essential for sustained EGFR signaling but dispensable for degradation. Dev. Cell, 15, 209–219 (2008).
• 56) Alvarado D, Klein DE, Lemmon MA. Structural basis for negative cooperativity in growth factor binding to an EGF receptor. Cell, 142, 568–579 (2010).
• 57) Pascolutti R, Algisi V, Conte A, et al. Molecularly distinct clathrin-coated pits differentially impact EGFR fate and signaling. Cell Rep., 27, 3049–3061.e6 (2019).
• 58) Zhou Y, Tanaka T, Sugiyama N, Yokoyama S, Kawasaki Y, Sakuma T, Ishihama Y, Saiki I, Sakurai H. p38-Mediated phosphorylation of Eps15 endocytic adaptor protein. FEBS Lett., 588, 131–137 (2014).
• 59) Zhang L, Xie B, Qiu Y, Jing D, Zhang J, Duan Y, Li Z, Fan M, He J, Qiu Y, Tan R, Li JJ, Sun L-Q. Rab25-mediated EGFR recycling causes tumor acquired radioresistance. iScience, 23, 100997 (2020).
• 60) Chung J-H, Rho JK, Xu X, Lee JS, Yoon H, Lee CT, Choi YJ, Kim HR, Kim CH, Lee JC. Clinical and molecular evidences of epithelial to mesenchymal transition in acquired resistance to EGFR-TKIs. Lung Cancer, 73, 176–182 (2011).
• 61) Nilsson MB, Yang Y, Heeke S, et al. CD70 is a therapeutic target upregulated in EMT-associated EGFR tyrosine kinase inhibitor resistance. Cancer Cell, 41, 340–355.e6 (2023).
• 62) Cross DAE, Ashton SE, Ghiorghiu S, et al. AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer. Cancer Discov., 4, 1046–1061 (2014).
• 63) Soria J-C, Ohe Y, Vansteenkiste J, et al. FLAURA investigators. osimertinib in untreated EGFR-mutated advanced non-small-cell lung cancer. N. Engl. J. Med., 378, 113–125 (2018).
• 64) Eno MS, Brubaker JD, Campbell JE, et al. Discovery of BLU-945, a reversible, potent, and wild-type-sparing next-generation EGFR mutant inhibitor for treatment-resistant non-small-cell lung cancer. J. Med. Chem., 65, 9662–9677 (2022).
• 65) Lim SM, Ahn JS, Hong M-H, Kim TM, Jung H-A, Jung H-A, Ou S-HI, Jeong S, Lee Y-H, Yim E, Jung S, Lee S-Y, Kim D-W. MA07.09 BBT-176, a 4th generation EGFR TKI, for progressed NSCLC after EGFR TKI therapy: PK, safety and efficacy from phase 1 study. J. Thorac. Oncol., 17, S70–S71 (2022).
• 66) Yun MR, Yu MR, Duggirala KB, Lee K, Jo A, Seah E, Kim C, Cho BC. MA07.08 JIN-A02, a highly effective 4th generation EGFR-TKI, targeting EGFR C797S triple mutation in NSCLC. J. Thorac. Oncol., 17, S69–S70 (2022).
• 67) Leonetti A, Sharma S, Minari R, Perego P, Giovannetti E, Tiseo M. Resistance mechanisms to osimertinib in EGFR-mutated non-small cell lung cancer. Br. J. Cancer, 121, 725–737 (2019).
• 68) Gong K, Guo G, Beckley NA, Yang X, Zhang Y, Gerber DE, Minna JD, Burma S, Zhao D, Akbay EA, Habib AA. Comprehensive targeting of resistance to inhibition of RTK signaling pathways by using glucocorticoids. Nat. Commun., 12, 7014 (2021).
• 69) Brown BP, Zhang Y-K, Kim S, Finneran P, Yan Y, Du Z, Kim J, Hartzler AL, LeNoue-Newton ML, Smith AW, Meiler J, Lovly CM. Allele-specific activation, enzyme kinetics, and inhibitor sensitivities of EGFR exon 19 deletion mutations in lung cancer. Proc. Natl. Acad. Sci. U.S.A., 119, e2206588119 (2022).
• 70) van Alderwerelt van Rosenburgh IK, Lu DM, Grant MJ, Stayrook SE, Phadke M, Walther Z, Goldberg SB, Politi K, Lemmon MA, Ashtekar KD, Tsutsui Y. Biochemical and structural basis for differential inhibitor sensitivity of EGFR with distinct exon 19 mutations. Nat. Commun., 13, 6791 (2022).
• 71) Kohsaka S, Nagano M, Ueno T, Suehara Y, Hayashi T, Shimada N, Takahashi K, Suzuki K, Takamochi K, Takahashi F, Mano H. A method of high-throughput functional evaluation of EGFR gene variants of unknown significance in cancer. Sci. Transl. Med., 9, eaan6566 (2017).
• 72) Yasuda H, Kobayashi S, Costa DB. EGFR exon 20 insertion mutations in non-small-cell lung cancer: preclinical data and clinical implications. Lancet Oncol., 13, e23–e31 (2012).
• 73) Wang J, Lam D, Yang J, Hu L. Discovery of mobocertinib, a new irreversible tyrosine kinase inhibitor indicated for the treatment of non-small-cell lung cancer harboring EGFR exon 20 insertion mutations. Med. Chem. Res., 31, 1647–1662 (2022).
• 74) Elamin YY, Robichaux JP, Carter BW, et al. Poziotinib for EGFR exon 20-mutant NSCLC: clinical efficacy, resistance mechanisms, and impact of insertion location on drug sensitivity. Cancer Cell, 40, 754–767.e6 (2022).
• 75) Jia Y, Yun C-H, Park E, et al. Overcoming EGFR(T790M) and EGFR(C797S) resistance with mutant-selective allosteric inhibitors. Nature, 534, 129–132 (2016).
• 76) To C, Beyett TS, Jang J, et al. An allosteric inhibitor against the therapy-resistant mutant forms of EGFR in non-small cell lung cancer. Nat. Cancer, 3, 402–417 (2022).
• 77) Beyett TS, To C, Heppner DE, Rana JK, Schmoker AM, Jang J, De Clercq DJH, Gomez G, Scott DA, Gray NS, Jänne PA, Eck MJ. Molecular basis for cooperative binding and synergy of ATP-site and allosteric EGFR inhibitors. Nat. Commun., 13, 2530 (2022).
• 78) Marrocco I, Romaniello D, Vaknin I, Drago-Garcia D, Oren R, Uribe ML, Belugali Nataraj N, Ghosh S, Eilam R, Salame T-M, Lindzen M, Yarden Y. Upfront admixing antibodies and EGFR inhibitors preempts sequential treatments in lung cancer models. EMBO Mol. Med., 13, e13144 (2021).
• 79) Yun J, Lee S-H, Kim S-Y, Jeong S-Y, Kim J-H, Pyo K-H, Park C-W, Heo SG, Yun MR, Lim S, Lim SM, Hong MH, Kim HR, Thayu M, Curtin JC, Knoblauch RE, Lorenzi MV, Roshak A, Cho BC. Antitumor activity of amivantamab (JNJ-61186372), an EGFR-MET bispecific antibody, in diverse models of EGFR exon 20 insertion-driven NSCLC. Cancer Discov., 10, 1194–1209 (2020).
• 80) Tahara M, Okano S, Enokida T, Ueda Y, Fujisawa T, Shinozaki T, Tomioka T, Okano W, Biel MA, Ishida K, Hayashi R. A phase I, single-center, open-label study of RM-1929 photoimmunotherapy in Japanese patients with recurrent head and neck squamous cell carcinoma. Int. J. Clin. Oncol., 26, 1812–1821 (2021).
• 81) Ghosh S, Nataraj NB, Noronha A, et al. PD-L1 recruits phospholipase C and enhances tumorigenicity of lung tumors harboring mutant forms of EGFR. Cell Rep., 35, 109181 (2021).
• 82) Morimoto K, Yamada T, Sawada R, Azuma K, Goto Y, Harada T, Shiotsu S, Tamiya N, Chihara Y, Takeda T, Hiranuma O, Hasegawa I, Tanaka S, Yoshimura A, Iwasaku M, Tokuda S, Kim YH, Takayama K. Predictive value of p53 and AXL immunostaining for the efficacy of immune checkpoint inhibitor-based therapy after osimertinib treatment in patients with epidermal growth factor-mutant non-small cell lung cancer. Cancer Immunol. Immunother., 72, 1699–1707 (2023).
• 83) Todo T, Ito H, Ino Y, Ohtsu H, Ota Y, Shibahara J, Tanaka M. Intratumoral oncolytic herpes virus G47∆ for residual or recurrent glioblastoma: a phase 2 trial. Nat. Med., 28, 1630–1639 (2022).
• 84) Tian L, Xu B, Chen Y, Li Z, Wang J, Zhang J, Ma R, Cao S, Hu W, Chiocca EA, Kaur B, Caligiuri MA, Yu J. Specific targeting of glioblastoma with an oncolytic virus expressing a cetuximab-CCL5 fusion protein via innate and adaptive immunity. Nat. Cancer, 3, 1318–1335 (2022).
• 85) von Achenbach C, Silginer M, Blot V, Weiss WA, Weller M. Depatuxizumab mafodotin (ABT-414)-induced glioblastoma cell death requires EGFR overexpression, but not EGFRY1068 phosphorylation. Mol. Cancer Ther., 19, 1328–1339 (2020).
• 86) Lassman AB, Pugh SL, Wang TJC, et al. Depatuxizumab mafodotin in EGFR-amplified newly diagnosed glioblastoma: a phase III randomized clinical trial. Neuro-oncol., 25, 339–350 (2023).
• 87) Clement PMJ, Dirven L, Eoli M, et al. Impact of depatuxizumab mafodotin on health-related quality of life and neurological functioning in the phase II EORTC 1410/INTELLANCE 2 trial for EGFR-amplified recurrent glioblastoma. Eur. J. Cancer, 147, 1–12 (2021).
• 88) Takahashi J-I, Nakamura S, Onuma I, Zhou Y, Yokoyama S, Sakurai H. Synchronous intracellular delivery of EGFR-targeted antibody-drug conjugates by p38-mediated non-canonical endocytosis. Sci. Rep., 12, 11561 (2022).
• 89) Robichaux JP, Le X, Vijayan RSK, et al. Structure-based classification predicts drug response in EGFR-mutant NSCLC. Nature, 597, 732–737 (2021).