Targeting the EGFR signaling pathway in cancer therapy: What’s new in 2023?

Abstract

Introduction:

Epidermal growth factor receptor (EGFR) is frequently amplified, overexpressed, and mutated in multiple cancers. In normal cell physiology, EGFR signaling controls cellular differentiation, proliferation, growth, and survival. During tumorigenesis, mutations in EGFR lead to increased kinase activity supporting survival, uncontrolled proliferation, and migratory functions of cancer cells. Molecular agents targeting the EGFR pathway have been discovered, and their efficacy has been demonstrated in clinical trials. To date, fourteen EGFR-targeted agents have been approved for cancer treatments.

Areas covered:

This review describes the newly identified pathways in EGFR signaling, the evolution of novel EGFR-acquired and innate resistance mechanisms, mutations, and adverse side effects of EGFR signaling inhibitors. Subsequently, the latest EGFR/panEGFR inhibitors in preclinical and clinical cancer studies have been summarized. Finally, the consequences of combining immune checkpoint inhibitors and EGFR inhibitors have also been discussed.

Expert opinion:

As new mutations are threatened against EGFR-tyrosine kinase inhibitors (TKIs), we suggest the development of new compounds targeting specific mutations without inducing new mutations. We discuss potential future research on developing EGFR-TKIs specific for exact allosteric sites to overcome acquired resistance and reduce adverse events. The rising trend of EGFR inhibitors in the pharma market and their economic impact on real-world clinical practice were discussed.

1. Introduction

Epidermal growth factor receptor (EGFR) family proteins are the most widely investigated and aberrantly expressed transmembrane receptors in cancer studies. EGFR family members consist of EGFR/HER1, HER2, HER3, and HER4, belong to receptors tyrosine kinase superfamily with receptors composed of an extracellular ligand-binding domain with approximately ~ 620 amino acids (AA), a single transmembrane domain (‘22 AA), and a C-terminal region (an intracellular region) with tyrosine kinase region (~982–1210 AA) with five ligand-dependent autophosphorylations (tyrosine residues) sites. The extracellular domain (ECD) of EGFR can further be subdivided into four segments. The first and third segments of EGFR ECD are rich in leucine amino acids, which play a crucial role in ligand interaction, while the second and fourth ECD segments, rich in cysteine residues, form bonds with the ligands. The oncogenic function of EGFR family members’ genes and the corresponding translated protein anomalies were demonstrated to drive cancer initiation, progression, and metastasis in lung, breast, pancreatic, prostate, glioblastoma, head and neck, and brain cancers. The activity of the EGFR family receptor depends on the associated ligands such as epidermal growth factor (EGF), Epiregulin (EPR), Amphiregulin (AREG), Heparin-binding EGF (HB-EGF), TGF-α, Betacellulin (BTC) and Neuregulins (Neu), etc. These ligands are regarded as growth factors that have a high affinity for their cognate receptors and play a key role in cell proliferation, differentiation, and growth.

The epidermal growth factor (EGF) and EGF-like family of ligands share a common motif with a conserved cysteine residue that specifically recognize cysteine residues in the extracellular portion of the EGF receptor to form disulfide bonds (6). When the ligand binds to EGFR family receptors, it either induces homo or heterodimerization of EGFR family members, resulting in autophosphorylation of tyrosine residues present in the C-terminal domain of EGFR-member. Further, the phosphorylated tyrosine residues function as docking sites for Src homology 2 (SH2) and phosphotyrosine-binding proteins leading to activation of downstream intracellular signal transduction leading to downstream activation of transcription factors such as AP-1, Elk-1, c-Fos promoting expression of mitogen-activated protein kinase/ERK, JNK, c-Jun N-terminal kinase, and phosphatidylinositol-3-kinase/AKT signaling that facilitate proliferation, growth, migration, and metastasis. The HER2 lacks ligand binding ability, and HER3 lacks kinase activity. Hence both HER2 and HER3 prefer to heterodimerize with other family members to elicit their downstream signaling. The trigger to potentiate activation of EGFR family members occurs outside the cell membrane in the form of a ligand binding or induction with radiation [1,2]. This is transmitted through a cascade of oncogenic signaling in the cytoplasm and ends up regulating diverse transcription factors in the nucleus to promote cancer cell proliferation, survival, and invasion [3]. To counteract the EGF-EGFR family proteins mediated dysregulation, several molecular targeted agents such as small molecule inhibitors, monoclonal antibodies, and antibody-drug conjugates have been evaluated in various cancers.

2. Updates on EGFR signaling mechanism (s) in cancer

EGF was first discovered by Nobel laureate Dr. Stanley Cohen in 1959, and functional characterization studies revealed that EGF can stimulate the expression of 3172 genes and 596 proteins in mammary epithelial cells [4]. Another study in Hela cells demonstrated phosphorylation of 2244 proteins at 6600 sites globally upon EGF treatment [5]. Several reviews have been published to demonstrate EGF/EGFR signaling resulting in MAPK- ERK, PI3K-AKT, JAK-STAT, JNK, and PKC-PLCγ1 pathways [2,6]. This section will describe novel EGFR signaling mechanisms recently identified in various cancers. Recently the role of the EGFR/JAK/STAT3 axis was identified in head and neck squamous cell carcinoma (HNCC). EGFR forms a complex with transmembrane protein 16A (TMEM16A) which forms a Ca^2+-activated Cl-channel on the plasma membrane leading to the stimulation of NF-kB and JAK/STAT3 pathways, thereby promoting tumorigenesis [7]. In colorectal cancer (CRC), EGF, on binding with EGFR, induces PI3K/AKT/mTOR signaling that supports tumor cell survival, growth, and invasion. [8]. Currently, an extracellular matrix protein ASPORIN (ASPN) is getting attention in cancer signaling. ASPN belongs to a small leucine-rich proteoglycan (SLRP) family and is a vital component of the extracellular matrix [9]. It is known to interact with EGFR and activate signaling. ASPN interacts with EGFR, activating the PI3K/AKT signaling cascade and supporting the proliferation and metastasis in breast, pancreatic, gastric, colorectal, and prostate cancer. It has been recently reported by Zhan et. Al., that an interplay of the ASPN/HER2/SRC/EGFR axis increases the expression of epithelial-mesenchymal transition-activating transcription factors (EMT-TFs) by inducing MAPK signaling [10]. In addition, cell surface mucins have also been demonstrated to interact with EGFR. EGFR interacts with mucin 13 (MUC13), which is highly expressed in several cancers. Specifically, in human intrahepatic cholangiocarcinoma (iCCA), MUC13/EGFR interaction induced PI3K/AKT signaling by EGFR dimerization, avoiding its internalization and leading to metastasis [11]. Another transmembrane mucin MUC4 was also shown to interact with EGFR through its EGF-like domains. Further, CRISPR knockout of Muc4 in the background of mutant Kras (G12D) and Trp53 (R172H) in autochthonous pancreatic cancer mouse model decreased phosphorylation of EGFR and ERK [12], and these data are similar to the studies observed with MUC4/HER2 in pancreatic and ovarian cancer [13,14]. TSPAN8, a membrane-bound protein, was shown to translocate to the nucleus whenever it is activated through EGFR signaling. Upon EGFR activation, AKT is phosphorylated and further phosphorylates TSPAN8 to interact with STAT3 in the nucleus to transcriptionally promote oncogenes such as MYC, BCL2, and MMP9 in breast cancer [15]. Seshacharyulu et al. were the first to provide evidence that panEGFR inhibitors canertinib and afatinib could downregulate MUC4 mucin by blocking EGFR/STAT1 axis in pancreatic cancer [16]. Subsequently, Kaushik et al. reported that the panEGFR inhibitor afatinib inhibits pancreatic cancer metastasis and stemness by blocking EGFR/ERK/FOXA2/SOX9 signaling [17]. Furthermore, it was shown that afatinib treatment in HNSCC cells showed radio-responsiveness by abrogating cancer stemness through blocking pEGFR/ALDH1 and upregulating DNA damage and response signaling protein γH2AX [18]. The M3 muscarinic acetylcholine receptor (M3R) acts as an upstream molecule for EGFR activation through activation of c-Src and β-arrestin-1, which leads to activation of downstream MEK/ERK and PI3K/AKT signal pathways to initiate proliferation and migration of NSCLC [19]. Endogenous thrombopoietin, a hematopoietic cytokine, is overexpressed in NSCLC tissue and enhances EGFR signaling by interacting with EGFR protein and delaying ligand-induced endosomal degradation [20]. Shao et al. showed that EGFR activation promotes NSCLC cell migration through NEDD4-mediated cathepsin B secretion. NEDD4 is an E3 ubiquitin ligase activated by EGFR signaling and is mediated by increased cytoplasmic calcium and interacts with the secretory lysosome to secrete Cathepsin B, which in turn degrade extracellular matrix protein and cell-to-cell-junctional-protein-to-initiate-cell-migration [21]. PARK2 (Parkin) is another E3 ubiquitin ligase, which is frequently altered in cancers and attenuates both in vitro and in vivo proliferation of glioma cells through inhibition of EGFR-AKT signaling. RNA interference experiments with PARK2 revealed that PARK2 regulates EGFR expression and suppresses downstream AKT phosphorylation at the Ser473 position [22]. Lastly, protein phosphatase enzymes also regulate EGFR signaling in cancer cells. Two independent groups have shown that the ectopic introduction of protein tyrosine phosphatase PTPN12 cDNA decreased the proliferation and migration of cancer cells by negative regulation of EGFR levels [23,24]. Similarly, activation of another phosphatase known as protein phosphatase 2A (PP2A) using an LB100 small molecule inhibitor has shown to be responsible for enhancing the tumor-responsive effects of gefitinib that targets EGFR [25]. Newly identified mechanisms in the EGFR pathway in several cancers are illustrated in Figure 1.

Figure 1. EGFR family members in various cancer signaling and their inhibition through EGFR-TKIs.

(A). EGFR signaling participates in cancer cell survival, proliferation, invasion, and metastasis in lung cancer through PI3K/AKT/mTOR, MEK1/ERK1/2, and NEDD4/Cathepsin B signaling pathways. Active EGFR signaling increases intracellular Ca2+ levels to activate NEDD4; further, active NEDD4 induces exocytosis of cathepsin B, which degrade extracellular matrix protein and cell-to-cell junction protein to initiate metastasis. In head and neck cancer (HNSCC), both EGFR and HER2 involve in cancer progression. Additionally, radiation therapy stimulates both receptors and repairs radiation-mediated cell damage by increasing the expression of BRCA1. Other tumorigenic signalings, such as JAK/STAT3 and PI3K/AKT pathways, support the proliferation and migration of HNSCC cells. In glioma, cancer progresses through activating FAK, JAK2, cMET, phosphoAKT and cancer stemness (Oct3/4 and Nanog). Prolongation of EGFR signaling by retarding EGFR proteasomal degradation involved in glioma metastasis. PARK2 prolongs EGFR signaling by inhibiting EGFR proteasomal degradation. Also, a zinc finger CCCH-type containing 15 protein ZC3H15 is highly elevated and associated with glioblastoma progression. (B). EGFR signaling promotes colorectal tumorigenesis by activating PI3K/AKT/mTOR signaling. In breast cancer, cellular growth was controlled by oncogenic signaling activating TSPAN8 and STAT3 and translating downstream targets such as MYC, BCL2, and MMP9. Apart from that, HER2 also induces apoptosis in breast cancer cells by activating FOXO3 and expressing Bim1. In ovarian cancer, HER2-mediated AKT signaling promotes metastasis and cell growth. MUC4 mucin stimulates orphan receptor HER2 to activate AKT and express FOXO3 to initiate cell cycle progression and inhibit apoptosis. Both HER2 and EGFR play a significant role in pancreatic cancer progression. EGFR signaling activates STAT1 to translate MUC4. MUC4 stimulates FAK/MAPK and p38/MEF2C/MMP10 to trigger cancer cell proliferation and invasion. EGFR also signals through EKR/FOXA2/SOX9 to cause pancreatic cancer survival, growth, and metastasis upon inhibition with afatinib, reducing pancreatic cancer metastasis by reducing Sox9-associated stemness. Like other cancers, PI3K/AKT signaling is influenced by EGFR mucin interactions to promote cholangiocarcinoma metastasis. TPO= Thrombopoietin; M3R = M3 muscarinic acetylcholine receptor.

3. Limitations of EGFR signaling blockade

In the past decade, even though EGFR signaling inhibition through small molecule inhibitors or antibody-mediated cytotoxicity has led to their use in multiple cancers, resistance almost invariably develops, leading to the loss of efficacy of these agents. This section focuses on the most recently identified new bypass signaling mechanisms, new mutations, and previous un-identified resistance mechanism (s) of first, second, and third-generation EGFR TKIs.

3.1. Novel resistance mechanism (s) against EGFR-targeted agents

Resistance against first- and second-generation EGFR-TKI emerges with the development of secondary mutations in EGFR. The initial response to first-generation gefitinib and erlotinib is appreciable. However, most of the patients who respond to EGFR-TKIs developed resistance against those therapy within 10–14 months after initiation of treatment. In this section, we discuss the resistance mechanism of acquired EGFR-TKIs under four subsections; (a) EGFR secondary mutations, (b) acquired mutations in genes other than EGFR family members, (c) EGFR downstream signaling activation, and (d) histological changes in the cancer phenotype.

3.2. EGFR secondary mutations.

EGFR and KRAS are the most common driver mutations in NSCLC. Most EGFR primary mutations will occur either at exons 19 or 21. We briefly reviewed EGFR mutation and its therapeutic implication in our previous paper [2]. We also discussed EGFRvI, EGFRvII, and EGFRvIII mutations in the extracellular domain and several point mutations in the intracellular kinase domain, such as T790M EGFR mutation, which is the most common in acquired resistance against the first and second generation of EGFR-TKIs. Resistance develops rapidly over nine to thirteen months of TKI treatment [26]. Common EGFR mutations seen following osimertinib therapy include mutations in C797, G796, L792, L718, G724, G719, V948, and in exon 20 [27]. Currently, fourth-generation TKI, EAI001, and EAI045 are under investigation to combat EGFR T790M/C797S/V948R mutations. Recent studies discovered several other EGFR mutations located in exon 18–21 that play a significant role in cancer progression and therapy resistance, which are elaborately illustrated in Figure 2.

Figure 2. EGFR TKI-associated primary and acquired resistance mutations as the target for third and fourth-generation EGFR-TKIs.

The Human EGFR gene is composed of 28 exons. Point mutations emerged due to acquired resistance against first and second-generation EGFR-TKIs and were denoted as colored lollipops. Human EGFR protein comprises approximately 1186 amino acids with a molecular weight of around 134kDa. Its first 16 exons translate an extracellular domain with around 620 amino acids and two intact ligand-binding domains. It has a single transmembrane domain having 22 amino acids. EGFR intracellular region contains tyrosine kinase and autophosphorylation domain. Insertion, deletion, and point mutation usually occur in the tyrosine kinase domain. The third-generation EGFR-TKI, like Osimertinib, upon treatment induces L718Q point mutation at exon 18, whereas Dacomitinib can successfully target this mutation. Some other common point mutations are S768I, V769M, T790M, C797S, L858R and L861Q. Conventional first- and second-generation EGFR-TKI cannot target these mutations. However, Osimertinib can target T790M, and L858R, Erlotinib can target V769M, EA1045 can target C797S, and Dacomitinib can target L718Q and L858R effectively. Apart from point mutation, Osimertinib and CH7233163 (fourth generation EGFR-TKI) can target exon 19 deleted EGFR. Poziotinib and Mobocertinib can target exon 20 insertion mutation of EGFR.

3.3. TKI-acquired mutations other than EGFR family members.

In recent years new discoveries of the mechanism of EGFR-TKIs resistance were identified to be through mutations/amplifications in other oncogenes such as BRAF (3%), HER2 (1–2%), and MET (7–15%) or as a bypass mechanism. Another important mechanism of acquired drug resistance in NSCLC is the bypass activation involving HER2 and MET mutations. An important mechanism for osimertinib resistance is MET amplifications. Capmatinib and tepotinib are approved in patients harboring MET amplification or MET exon 14 skipping mutations in NSCLC [28]. A recent study identified the potency of savolitinib in selectively reduced MET along with osimertinib in blocking the MET-dependent resistance to EGFR-TKIs in NSCLC [29]. Similarly, alteration in HER2 also leads to bypass activation-mediated acquired drug resistance in NSCLC by inducing uncontrolled cell proliferation by the activation of MAPK/PI3K/STAT signaling [30].

3. 4. EGFR downstream signaling activation.

Apart from acquired mutations in the EGFR and other proteins, several molecular expression alterations lead to EGFR-targeted therapy resistance. Through screening of 55 protein kinase inhibitors along with EGFR inhibitor gefitinib and next-generation sequencing, Yi et al. identified the IKK/NF-κB as a potential target to overcome resistance against the EGFR inhibitor, gefitinib to treat TNBC [31]. Another study in breast cancer cells showed loss of enhanced pro-apoptotic protein Bim and PARP activation due to overexpression of protein tyrosine kinase 6 (PTK6) protein due to resistance mechanism against lapatinib. Downregulation of PTK6 reverses lapatinib resistance by activating Bim expression via p38 MAPK [32]. Recently, AXL, a receptor tyrosine kinase belonging to the TAM family, has been reported to play a significant role in acquired resistance to EGFR-TKIs in NSCLC [33]. Likewise, the plasminogen activator urokinase receptor (PLAUR) expression is significantly high in EGFR-TKI (gefitinib)-resistant NSCLC patients and confers this resistance by stimulating EGFR/p-AKT/survivin signaling pathway [34]. Chaudhary et al. described that significant co-expression of EGFR and Cyclin D1 showed enrichment of glycolytic and EMT-related genes in HNSCC [35]. Supportively, Wee et al. summarized the contribution of EGFR signaling in cancer cell proliferation through the induction of cyclin D overexpression [6]. Similarly, a distinct resistance against second-generation TKI afatinib has been observed in EGFR-mutant lung cancer cells. Long-term afatinib treatment leads to Kras amplification (Wild-type) with increased expression and signaling associated with insulin-like growth factor-binding protein 3 [36].

3. 5. Histological changes in the cancer phenotype.

Lastly, the phenotypic conversion of NSCLC to small cell carcinoma (SCLC) or squamous cell carcinoma (3–10%) is another way of acquiring resistance by the tumor to EGFR-TKIs. SCLC is a lethal neuroendocrine tumor resulting from EGFR-TKIs resistance acquired by the malignant cells. The NSCLC developed into combined SCLC, which is the combination of SCLC and NSCLC with mixed histology. The clinical transformation of SCLC from NSCLC is not clear. The most common mutations associated with SCLC include Rb (58%), TP53 (79%), and PI3KCA (27%), which results in central nervous system metastasis (64%) [37]. A whole genome sequencing of progressively collected lung tumors showed that clonal evolution of therapy-induced SCLC occurs at a very early stage of lung cancer initiation, even before EGFR-TKI treatment [38]. Specifically, Rb and p53 loss are regarded as pre-requisite of SCLC phenotype. A study by Ahn et al, revealed that both initial NSCLC and later transformed SCLC harbored Rb and p53 genes are completely inactivated, suggesting that Rb and p53 assessment could be a predictor for SCLC phenotype transformation [39]. Further, studies have shown that Achaete-scute homolog-1 (ASCL1) has been identified as a transcription factor for SCLC development in a mouse model and is important in neuroendocrine differentiation. Delta-like protein-3 (DLL3) is an actionable target of ASCL1, and therapeutic targeting of DLL3 was found to be more efficient in high ASCL1 SCLC [40]. Overall, the resistance mechanisms described in this section were bypass signals against EGFR targeting, leading to the transformation of advanced phenotype in lung cancer and can be extrapolated to the brain, breast, colon, and pancreatic cancers where EGFR-TKIs can potentially be used.

4. Next-generation sequencing (NGS) reveals the evolution of new mutations in EGFR family members

Several fundamental cellular mechanisms and molecular breakthroughs in cancer studies have evolved due to the advantage of technological advancements in the NGS-based approach. Usually, EGFR-mutated patients respond better to EGFR TKIs than patients harboring “uncommon mutations”. Patients with the most common mutations, such as T790M, L858R, and exon 19 deletions, respond better to first and second-generation EGFR-TKIs. However, patients with uncommon EGFR mutations such as G719X, L861Q, and S768I are not good responders to TKIs. Thus, comprehensive and unique mutation profile identification through NGS-based analysis would identify a subgroup of EGFR TKI non-responsive patients. It is predicted that T790M EGFR mutation will be present in 50% of patients pre-treated with EGFR-TKIs as a mechanism of acquired resistance. A meta-analysis of eleven lung cancer studies reported the specific occurrence of EGFR mutations in females, non-smokers, and of Asian origin. The researchers also concluded that lung adenocarcinoma patients also commonly mutated at exon 19 deletion (45%) and point mutation (L858R) in exon 21 (35%), favoring TKIs efficacy [41]. The discovery of NGS has led to the identification of oncogenic driver mutations in HER2, along with mutations in EGFR, ROS, and ALK translocation in NSCLC [42]. A study by Tuononene et al. demonstrated that targeted NGS performance is superior in detecting insertion, deletion, and nonsynonymous single-nucleotide variation in EGFR than conventional real-time PCR in NSCLC patient tissues [43]. In Triple-negative breast cancer (TNBC) patients, Kim et al. performed pyrosequencing and identified EGFR mutation frequencies in exon 21 (L861Q and L858R) and exon 19 (deletion). The authors concluded that EGFR mutations occur at the rarest frequency in TNBC. Still, it needs to be investigated based on ethnicity since previous reports from China [44,45] and Korea [46,47] demonstrated EGFR gene mutation at about 10%, 11.4%, and 1–2% of EGFR mutation in exons 19 and 21, respectively [48]. Next-generation EGFR-TKIs targeting newly identified acquired mutations are depicted in Figure 2. Thus, next-generation sequencing should be performed along with molecular agent treatment to understand the sequential emergence of new mutations in EGFR and other family members. This will help the physician and patient decision-making process much easier to apply specific targeted agents against specific mutations to increase clinical outcomes.

5. Newly identified germline mutations and variants of EGFR

Germline mutation in EGFR (T790M) is associated with multiple primary lung cancer (MPLC). In this report, testing of the unaffected relative of an MPLC patient revealed germline EGFRT790M mutation. This individual was later diagnosed with metastatic lung cancer [49]. Recently, Li et al. reported on unique EGFR mutation in patients with MPLC compared with sporadic lung adenocarcinoma patients. The well-known T790M mutation was first identified in MPLC patients’ families of European origin, and later it was found to be associated with therapy resistance. The authors conducted PCR-based Sanger sequencing in 162 MPLC patients. They reported the prevalence of germline mutations in exons 19, 20, and 21 in MPLC family members with a high frequency and a substantial number of mutations in Exon 20 [50]. In another study by Hellmann et al., a novel germline variant of EGFR V769M in exon 20 was found to occur along with somatic mutation in the same exon 20 with EGFRS768I. Functionally, introducing the EGFR V769M variant using site-directed mutagenesis into full-length EGFR wild-type HEK293T cells resulted in the activation of AKT and ERK pathways. The authors also evaluated the sensitivity of EGFR V769M mutant introduced cells against erlotinib treatment. The EGFR TKI demonstrated intermittent sensitivity in cells overexpressed with the EGFR V769M variant, suggesting that this variant might predict EGFR TKI response [51].

6. New EGFR/pan-EGFR signaling targeting agents in cancer therapy

Understanding the protein structure of EGFR and its family members and their tyrosine kinase domain structures has led to the development of kinase inhibitors. Most kinase receptors hold highly conserved ATP-binding pockets, and EGFR-TKIs were designed to target these ATP-binding sites in the EGFR tyrosine kinase catalytic domain. The first EGFR tyrosine kinase inhibitor (TKI), gefitinib, was approved in 2003 for NSCLC patients as a monotherapy [52]. Later in 2004, FDA approved a second EGFR-TKI, erlotinib, based on the survival advantage in NSCLC patients over placebo [53]. Earlier review from our group had discussed the role of EGFR targeting (cetuximab, panitumumab; gefitinib, erlotinib) and dual/pan EGFR family targeting (lapatinib, canertinib) in multiple cancers [2]. This section will discuss and update the third and fourth-generation EGFR inhibitors in preclinical and clinical studies.

6.1. EGFR signaling inhibitors approved for clinical use.

Dacomitinib- (PF299804/Vizimpro):

Dacomitinib is an orally active aniline-quinazoline-based highly selective second-generation pan EGFR TKI developed to overcome EGFR, and HER2 mutations emerge due to the first generation (gefitinib and erlotinib) EGFR-TKIs [54]. Specifically, dacomitinib binds to all EGFR family receptors with irreversible affinity. In 2018, the FDA approved dacomitinib for treating metastatic NSCLC patients with EGFR mutation based on two major randomized and multicenter clinical trials (ARCHER 1050 and NCT01774721). ARCHER 1050 was a phase III clinical trial of newly diagnosed advanced NSCLC patients with an EGFR mutation. In this study, dacomitinib at 45mg/day significantly extended progression-free survival (14.7 vs. 9.2 months) compared to gefitinib (250 mg/day) [55,56]. Likewise, the results of previous ARCHER 1009 and A7471028 also validate the superiority of dacomitinib over erlotinib in NSCLC patients with either exon 19 deletion or L858R substitution in EGFR exon 21 [57]. These results were further validated in the sub-population of Japanese NSCLC patients harboring EGFR-activating mutations, comparing the safety and efficacy of first-line dacomitinib versus gefitinib [58]. Osimertinib, a third-generation EGFR tyrosine kinase inhibitor, successfully treats EGFR mutation-positive NSCLC. However, one of the resistance mechanisms is the development of EGFR L718Q mutation. Dacomitinib has been shown to be active against this mutation [59]. Also, dacomitinib is efficacious in EGFR-mutated brain metastatic NSCLC patients with low-grade toxicity [60,61]. It also showed some clinical benefits in glioblastoma patients harboring EGFR mutation, and around 40% of the glioblastoma patient show EGFR amplification, and 50% of cases harbor a mutation in EGFR. Hence, it demonstrated a remarkable effect in preclinical models, but due to unacceptable side effects, it has yet to pass phase II clinical trials [62].

Neratinib-(HKI-272/Nerlynx):

Neratinib is a quinazoline-based, orally available small molecule receptor tyrosine kinase inhibitor that binds irreversibly to both EGFR and HER2. It shows potential antitumor activity to HER2 and EGFR overexpressing tumors by inducing cell cycle arrest, apoptosis, and decreasing cellular proliferation. The US FDA approved neratinib as extended adjuvant therapy for advanced or metastatic breast cancer in July 2017. The NALA (NCT01808573) study was a randomized, active-controlled, phase III trial comparing the efficacy of neratinib compared to lapatinib in combination with capecitabine in HER2-positive metastatic breast cancer patients. Neratinib (240 mg daily) showed a significant improvement in progression-free survival and onset of central nervous system-related (CNS) disease for those patients [63]. Another multicenter, randomized, double-blind, phase III trial with 2840 patients (ExteNET) validated its efficacy in improving invasive disease-free survival (iDFS) of early-stage HER-2 positive and hormone receptor-positive breast cancer patients [64]. Recently, preclinical studies have shown that neratinib, combined with c-MET inhibitor cabozantinib, can inhibit primary breast cancer cell growth and breast cancer metastasis to the brain [65]. A recent study found that hyperactivation of the mTORC1 signaling cascade can lead to resistance to neratinib therapy [66].

Icotinib (BPI-2009H /Conmana):

Icotinib is an orally bioavailable quinazoline-based EGFR TKI that selectively blocks wild-type and several mutants of EGFR tyrosine kinase with a potent antineoplastic effect. In China, it was approved by SFDA in 2011, but the US FDA has yet to approve it due to a lack of clinical studies. In 2014, US FDA issued a “May Proceed” permission to conduct a phase I clinical trial with icotinib to treat EGFR-positive NSCLC patients. The dose of icotinib has been determined as 125mg thrice a day for 6 weeks for locally advanced cervical cancer patients [67]. Icotinib was reported to be the highly antiproliferative, anti-apoptotic, and anti-EMT agent in NSCLC cells in both time and concentration-dependent manner. It inhibits EMT by regulating EMT-related proteins, like E-cadherin, N-cadherin, Vimentin, and fibronectin [68]. Apart from its effect in NSCLC, it is found to be effective in cervical cancer, enhancing the radiotherapy effect in childhood nasopharyngeal carcinoma, and improving radiosensitivity of biliary tract cancer and hepatocellular carcinoma [67,69–71]. A study of 1321 advanced-stage NSCLC patients confirmed the long-term treatment effect, safety, and tolerability of icotinib [72].

Afatinib (BIBW 2992/Gilotrif):

Afatinib is an orally active anilino-quinazoline derivative EGFR-TKI which binds and inhibits selectively and irreversibly to EGFR, HER2, and HER4. It was approved by US FDA and European Medical Agency (EMA) in 2013 to treat advanced metastatic EGFR mutation harboring NSCLC [73]. This is also under investigation as monotherapy to treat HER2-positive breast cancer patients who have progressed to trastuzumab [74]. Our group has extensively investigated the effect of afatinib on multiple cancers. Recently, we found a synergistic effect of afatinib with gemcitabine in reducing pancreatic cancer (PC) progression and metastasis by impeding cancer stem cell (CSC) growth using preclinical organoid and mouse models [17]. Afatinib has a synergistic effect with temozolomide in inhibiting the growth, proliferation, and tumorigenesis ability of glioblastoma cells modulated to express EGFR (wild type), mutant EGFRvIII, and EGFR dead kinase [75]. Furthermore, our group demonstrated afatinib’s anti-proliferation, migration, and anti-survival action in pancreatic cancer cells [16]. We have also reported that afatinib shows an anti-tumorigenic effect and radio-sensitizes head and neck squamous cell carcinoma (HNSCC) cells by eradicating the CSC population [18].

Osimertinib (AZD9291/Tagrisso):

Osimertinib is a member of aminopyrimidines class of compounds that binds and selectively inhibits numerous mutant forms of EGFR, such as T790M, L858R, and exon 19 deletions. It is orally bioavailable and induces cell death and inhibits tumor growth. As it does not bind to wild-type EGFR and only binds selectively to a mutation-positive form of EGFR, it has lower toxicity. In 2018, US-FDA approved osimertinib as the first line of treatment for NSCLC patients harboring specific EGFR mutation based on the FLAURA trial, which showed longer progression-free survival upon treatment with osimertinib to NSCLC patients [76]. Osimertinib is the only third-generation EGFR TKI that can effectively treat EGFR T790M mutation-positive NSCLC patients. However, triple mutations in EGFR against osimertinib (L858R/T790M/C797S or Del19/T790M/C797S) were recently reported [77]. Based on the new EGFR mutations identified against Osimertinib, Kashima et al. identified and developed a selective EGFR TKI CH7233163 from a library of drugs and assessed its efficacy through in vitro and in vivo lung cancer models [77]. Osimertinib is also effective in sensitizing EGFR T790M mutation harboring NSCLC patients who later develop central nervous system metastasis.

Vandetanib (ZD6474/Caprelsa):

Vandetanib is a 4-anilinoquinazoline, which is orally bioavailable. It inhibits VEGFR, EGFR, and RET kinase. In 2011, US FDA approved vandetanib for indolent, asymptomatic, unresectable, locally advanced, or metastatic medullary thyroid cancer [78]. It inhibits tumor cell proliferation, migration, and angiogenesis. It also effectively shows the antitumor effect in EGFR overexpressing cell lines and preclinical models of head and neck squamous cell carcinoma (HNSCC) [79]. Researchers have also demonstrated its effectiveness and synergy with cisplatin in neuroblastoma, irinotecan in pancreatic Cancer, and apoptosis inducer in breast cancer [80–82].

Pyrotinib (SHR-1258):

Pyrotinib is an orally active dual kinase inhibitor that binds and inhibits both EGFR and HER2. It shows potent antineoplastic activity by inhibiting tumor growth and angiogenesis in EGFR and HER2 overexpressing tumors. Pyrotinib is currently approved in China in combination with capecitabine as a second-line treatment for HER2-positive metastatic breast cancer. Currently, pyrotinib is under clinical investigation in a randomized, double-blind, multicenter study evaluating its safety along with trastuzumab plus carboplatin and docetaxel on HER2-positive breast cancer patients (NCT03756064). Pyrotinib is also under clinical trial evaluating its safety and efficacy versus docetaxel in advanced non-squamous NSCLC patients harboring exon 20 mutations on HER2 (NCT04447118) [83].

Mobocertinib (TAK-778, Exkivity):

Mobocertinib is the first US FDA-approved drug to treat locally advanced or metastatic NSCLC patients harboring EGFR exon 20 insertion mutation who are resistant to platinum-based chemotherapy. US FDA approved this orally active EGFR TKI in September 2021 based on the outcome of a phase I/II clinical trial (NCT02716116) [84], which recruited 114 patients with progressive diseases even after platinum-based chemotherapy. The study demonstrated a 28% objective response rate (ORR) and 17.5 months duration of response [85]. However, patients with progressive brain disease showed limited efficacy. Another major limitation is gastrointestinal toxicity, which subsided upon the dosage reduction strategy.

Amivantamab:

Amivantamab is a monoclonal antibody with dual specificity against EGFR and MET receptors. US FDA has approved it for the treatment of EGFR exon 20 NSCLC. It is approved based on CHRYSALIS non-randomized and open-label clinical trial, which recruited 81 patients with progressive diseases or after platinum-based chemotherapy. The recommended dosage of amivantamab is 1050 mg (once weekly) for patients with body weight < 80 Kg. For patients with >80 kg, amivantamab will be given at the dose of 1400 mg/weekly once. Side effects of amivantamab include rash, musculoskeletal pain, nausea, vomiting, fatigue, dyspnea, stomatitis, edema, constipation, and infusion-related adverse reactions.

Apart from tyrosine kinase-specific small molecule inhibitors, monoclonal antibodies targeting EGFR were coupled with nanoparticles for exact delivery to the tumors as well to reduce unnecessary toxicity due to overdosing. The latest nanoparticle labeled EGFR inhibitors were summarized in Table. 1.

Table. 1.

Nanoparticle-based EGFR therapy in cancer.

Nanoparticles	Encapsulated agent	Agent type	Treated/ diagnosed cancer	Function	References
Au NPs (Gold Nanoparticles)	Cetuximab	Anti-EGFR McAB	Glioblastoma	Inhibits EGFR autophosphorylation	[116]
			Tiple-negative breast cancer	Effective delivery of the drug into the cancer cell	[117]
Ag NPs (Silver Nanoparticles)	Cetuximab (Erbitux; C225)	Anti-EGFR McAB	Nasopharyngeal carcinoma	Sensitize tumor to radiotherapy	[118]
Se NPs (Selenium Nanoparticles)	HSNM-siRNA	Anti-EGFR Si-RNA	NSCLC	Increase apoptosis by impeding the cell cycle	[119]
IONPs (Iron Oxide Nanoparticles)	Cetuximab	Anti-EGFR McAB	Glioma	Induces apoptosis and suppresses tumor growth	[120]
			Nasopharyngeal carcinoma	Sensitize tumor to radiotherapy	[121]
			Colorectal cancer	Increase therapeutic efficacy up to 29-fold	[122]
PLGA-ZnS NPs (PLGA- Zinc sulfide Nanoparticles)	Cetuximab	Anti-EGFR McAB	Breast and lung cancer cell line	Increase drug uptake by the cell	[123]
InP/ZnS QD Nanoparticles	Cetuximab	Anti-EGFR McAB	TNBC cell line	Effective tumor regression	[124]
CuS-Ab NPs	Cetuximab	Anti-EGFR McAB	Breast cancer cell line	Inhibits angiogenesis and decreases xenograft tumor growth	[125]
Silica nanorattles (SNs)	EGFRAb	Anti-EGFR McAB	TNBC cell line	Shows low toxicity and better antitumor activity	[126]
Porous silicon nanoparticles (pSiNP)	Cetuximab	Anti-EGFR McAB	TNBC cell line	Inhibits breast cancer bone metastasis	[127]
Mesoporous silica nanoparticle (MP-SiO2 NP)	Cetuximab	Anti-EGFR McAB	Lung cancer cell line and xenograft	Alter drug resistance and inhibits tumor progression	[128]

PLGA= poly (lactic-co-glycolic acid); QD= Quantum Dots; InP= Indium phosphate core; McAB= Monoclonal antibody.

6.2. Investigational unexplored EGFR-targeted agents in the pipeline for preclinical studies

Most identified, validated, and US FDA-approved EGFR inhibitors belong to pyrimidine heterocycle compounds. The pyrimidine-fused dinitrogenous penta-heterocyclic compounds remain the core structure and a vital portion of anti-EGFR therapies. Recent advances in pyrimidine fused heterocycles and their structural-activity relationship (SAR) have demonstrated the importance of heterocycles and substituents of pyridopyrimidine derivatives in maximizing the inhibitory activity against EGFR. Recently, Gaber et al. identified a 1H-pyrazolo [3, 4-d] pyrimidine derivative as an anti-EGFR inhibitor targeting both wild-type EGFR and mutant EGFR (T790M) and evaluated its potency in lung cancer cells for its apoptosis induction, cell cycle arrest at both G2/M and S phase and anti-proliferation effect [86]. Earlier, the same research group also assessed similar compounds or pyrimidine derivatives for their anticancer and anti-proliferation activities against three cancer cells originating from breast, hepatocellular, and lung by inhibiting either EGFR wild-type or EGFR mutant (T790M) forms [87]. Elmetwally et al. synthesized a thienol [2, 3-d] pyrimidine derivative as a dual EGFR/HER inhibitor and evaluated its apoptosis-inducing effect coupled with G2/M cell cycle arrest properties in breast, lung, and hepatocellular carcinoma cells with erlotinib as the reference standard. In addition, molecular docking studies also showed a prominent binding pattern to EGFR and mutant EGFR (T790M) [88]. Recently, thienotriazolopyrimidines and thienol [3, 2-d] pyrimidine-derived compounds exhibited anticancer properties in luminal A subtype MCF-7 and triple-negative subtype MDA-MB-231 breast cancer cells. Specifically, the compounds showed apoptosis-inducing effects by enhancing Caspase-9 expression and downregulating phospho ERK and phospho AKT proteins by affecting EGFR [89]. Similarly, new 2-anilinopyrimidine derivatives showed promising EGFR inhibition in breast cancer cells by inducing apoptosis via caspase-9 activation. Apart from EGFR inhibition, the compounds also showed excellent binding activity with aromatase enzymes in breast cancer cells [90]. Based on the drug discovery process coupled with virtual pharmacokinetic analysis, eight different pyrimidine derivatives were synthesized, and the eightieth compound, R8, showed promising antitumor activities in breast cancer cells. These results were validated with molecular docking studies showing bonding interactions of R8 with ATP binding pockets of wild-type EGFR [91]. Specifically, its fused form with heterocycles such as pyrrole, furan, thiophene, pyrazole, pyran, pyridine, acridine, piperidine, and azepine has shown to exhibit good cytotoxicity by selectively inhibiting EGFR, HER2, EGFR T790M, EGFR T790M/L858R wild-type and mutant forms in cancer cells [92]. Recently, researchers have discovered novel 4-arylamino-quinazoline derivatives specific for both EGFR^WT and EGFR mutant (L858R/T790M) harboring cells. As per the report, they exhibit excelled cell cycle arrest at the S phase and G1 phase with enhanced apoptosis-inducing effect when evaluated in non-small cell lung cancer (NSCLC) cells [93]. Zhang et al. have designed, synthesized, and assessed the biological function of 2-aryl-4-amino substituted quinazoline derivative in the double mutant (L858R/T790M) and triple mutant (L858R/T790M/C797S) NSCLC cell lines [94]. A 4-hydroxy substituted quinazoline derivatives will be fourth-generation irreversible EGFR inhibitors if their pharmacokinetic action is evaluated in preclinical models. Similarly, 9-heterocyclyl substituted 9H-purine derivatives were found to be more potent in exhibiting antiproliferative activity against mutant EGFR (L858R/T790M/C797S) in HCC827 and H1975 lung adenocarcinoma cell lines [95]. Later, acrylamide-linked anilinoquinazoline derivatives were developed, and they displayed excellent anti-proliferation activity in gefitinib-resistant NSCLC cell lines. In addition, the derivatives also showed lower toxicity when evaluated in the non-tumorigenic HepG2 human hepatoma cell line [96]. Su et al. designed and synthesized indole derivatives to selectively overcome EGFR triple mutant (19D/T790M/C797S)-mediated acquired resistance [97]. These studies indicate that increasing the substituents and modification of the R group will influence the potency of the EGFR TKIs. The derivatives mentioned above must be assessed in animal models to reduce tumor growth and metastasis in EGFR-mutated and amplified cancers.

7. Lesson learned through combing Immune checkpoint inhibitors with EGFR targeting agents in cancer.

Immune checkpoint inhibitor treatment often results in an effective and elevated immune response in immunologically inflamed cancers like NSCLC and melanoma. NSCLC and melanoma, being associated with dysfunctional EGFR, can be meted out with combinatorial treatments of ICB and EGFR inhibitors. Although the initial phases of clinical trials of immunotherapy in EGFR-related cancers manifest promises, the use of IO (Immunotherapy) in a clinical context is often debatable. NCCN guidelines recommend not to endorse immunotherapy in the context of EGFR-mutated cancers owing to the risk of toxicities and lack of clinical benefits. A meta-analysis by Lee et al. showed that ICB alone significantly extended the overall survival of advanced NSCLC patients versus docetaxel. A major limitation is that the ICB does not improve OS in EGFR mutant patients but provides significant survival benefits to the sub-group of the EGFR wild-type population [98]. Hence, we need to examine the combinatorial treatments of ICB and EGFR inhibitors in the context of acquired mutations-carrying patients versus EGFR wild-type harboring patients. EGFR is a major regulator for the transition between immunosuppressive and immune-activated phenotype in breast cancer and its inhibition by EGFR-TKIs potentiates IO. However, it is unsure whether this stands true for all other EGFR-related cancers. Hence, it is obligatory to explore EGFR cancer TME, how it is modified in response to TKI therapy and its consequent downstream physiological manifestations. Jia et al. studied EGFR-driven autochthonous lung cancer mouse models to decipher the in vivo changes caused to the tumor-immune microenvironment by EFGR-TKIs. It was observed that EGFR-TKIs caused tumor size diminution accompanied by infiltration of cytotoxic T-cells (CTLs) and dendritic cells (DCs). They also showed that treatment with osimertinib and gefitinib overcome the immunosuppressive tumor microenvironment (TME) by inhibiting M2 polarization and eradicating Foxp3+ T-regs. However, MDSCs (myeloid-derived suppressor cells) levels remain unaltered throughout, which is justified for mediocre results obtained when EGFR-TKIs are combined with immunotherapeutic agents [99]. Immune checkpoint inhibitors (ICIs) are a powerful tool for boosting immune response and cancer therapy in NSCLC and other cancers. ICI atezolizumab (IMpower150 clinical trial) operates through binding with PD-L1 on cancer cells and also showed improved OS when combined with bevacizumab (targeting VEGF), carboplatin, and paclitaxel as compared with bevacizumab, carboplatin, and paclitaxel combination in chemotherapy naïve metastatic nonsquamous NSCLC patients. Similarly, another ICI, durvalumab targeting PD-L1 in ATLANTIC clinical trial, showed an excellent safety profile similar to other ICIs in EGFR-/ALK- patients compared with EGFR+/ALK+ NSCLC patients. Unfortunately, some sub-group of patients (40/444 (9%)) receiving durvalumab showed grade 3 or 4 adverse events of pneumonitis, enhanced transaminase, aspartate aminotransferase, γ-glutamyltransferase, diarrhea, fatigue, and vomiting. However, encouragingly clinical outcomes were achieved in ATLANTIC and Impower150 clinical trials using durvalumab in EGFR+ and PD-L1 expressing NSCLC patients [100,101]. However, the question of how mutant EGFR contrives to escape immunotherapy has still not been answered fully. Furthermore, the tumor-immune microenvironment varies across patients with wild-type EGFR and mutant EGFR. Subtle changes in mutations can also cause a differential response of ICB [102]. The association of EGFR mutation and PD-L1 expression is also disputable. First, EGFR mutant lung cancer patient manifests poor sensitivity to therapeutic regimens targeting PD1 or PD-L1. Additionally, it is accompanied by hepatotoxicity in patients treated with pembrolizumab along with gefitinib. Akbay et al. reported [103] that mutant EGFR-driven lung cancers are positively correlated with activated PD-1 pathways. The effectiveness of ICB varies across EGFR mutational landscapes, and differential tumor mutation burden could provide a basis for identifying patients with EGFR mutations who are likely to be benefitted from ICB therapy [102]. Especially in patients with uncommon EGFR mutations like G719A in NSCLC, Yoshida and colleagues observed a longer PFS (progression-free survival) when treated with nivolumab [104]. Acquired resistant mutation like T790M can also be a determining factor for efficacious response of ICI (Immune checkpoint inhibitor) treatment. A study by Haratani et al. showed that NSCLC patients who do not have T790M are amenable to ICB treatment after EGFR-TKI treatment and showed a better PFS compared to patients exhibiting T790M mutation as the mutational status negatively correlated with PD-L1 expression [105]. Also, nivolumab causes an increased immune infiltration in both cohorts irrespective of the T790M mutational status [105]. Nevertheless, ICI combined with chemotherapy has proved to be advantageous in locally advanced and metastatic NSCLC patients who acquired TKI resistance in a study by Sun et al. for a cohort of 22 patients [106]. Contrarily, Lee et al. found no overall survival benefit for EGFR mutant patients and recommended the usage of ICI only after the complete failure of TKI to invoke an effective response [107]. ICI should not be restrained to PD-L1/PD-1 inhibitors as immunosuppression depends on various molecular factors. PD-L1 expression is regulated by CTLA4 and using inhibitors for other immunosuppressive markers can help overcome the futility of ICB in the context of EGFR-related cancers. A phase Ib clinical trial for a combination of durvalumab and tremelimumab in NSCLC patients showed promising results for the optimized dose of anti-CTLA4 and anti-PD-L1 and has reached Phase III clinical trial [108]. These studies encourage combinatorial therapies for ICB. The tumor immune microenvironment exhibits a high degree of heterogeneity. Apart from PD-L1, it is characterized by other disparate immunosuppressive elements like LAG-3 and TIM-3 that predominates the tumor immune microenvironment. Exosomes containing EGFR causes DCs to produce IDO (Indoleamine 2,3-dioxygenase), a pertinent enzyme that can convert T-helper cells into immunosuppressive T-regs [109]. Zhou et al. outlined the correlation of checkpoint protein LAG-3 upregulation with TKI failure in advanced NSCLC patients with EGFR mutations. They also noted TIM-3 as a significantly corresponding factor associated with poor PFS to first-line EGFR-TKI [110]. Therefore, it is imperative to devise alternative strategies and identify therapeutic regimens that can effectively modulate the immune response in EGFR-related cancers. This can be instrumental in cases where conventional PD-L1/PD-1 targeted therapy deems to be futile in invoking appropriate immune response. Tumor immune microenvironment factors regulation in response to EGFR-TKIs and their associated crosstalk mechanism is shown in Figure 3. This provides a basis for understanding the fact that monotherapy is never a solution and tumor microenvironment comprises of multiple immunosuppressive factors that demand targeting. Prior to that, it is imperative to delve deep into the molecular mechanism that leads to tumor suppressive microenvironment caused by mutated EGFR.

Figure 3. Activated and constitutive EGFR signaling in tumors leads to an immunosuppressive tumor microenvironment.

The use of EGFR inhibitors exacerbates the immunosuppressive nature of the tumor. (a) EGFR activation leads to subsequent phosphorylation of downstream PI3K/AKT/mTOR pathway to induce PD-L1 expression to help immune evasion. Also, tumor intrinsic CTLA4 regulates PD-L1 expression. Parallelly, EGFR-mediated Ras activation also supports PD-L1 expression on the tumor surface via Raf/MEK/ERK signaling (b) Upregulation of PI3K/PKC induces ROS-mediated STAT3 phosphorylation in dendritic cells that results in the production of Indoleamine 2,3-Dioxygenase (IDO) in dendritic cells. Thus, activated EGFR causes activation of STAT3 in dendritic cells that cause the production of IDO, making the tumor microenvironment immunosuppressive. Further, IDO prevents dendritic cells from maturing, causing them to be tolerogenic DCs. Also, tumor secretomes comprising IL-10 and IL-6 cytokines make the tumor immunologically cold. (c) EGFR/STAT3-activated IDO molecules are packed as cargo in exosomes that approach T-helper cells to propagate T-regs proliferation and expansion. Amphiregulin (AREG) secreted by the tumor cells leads to the expansion of T-regs by the GSK-3β pathway and impends Foxp3 transcription. (d) Tumor-derived exosomes disable the cytolytic T-cell and NK cell activity. (e) There are various other ways by which EGFR signaling can manipulate immune cells to its advantage. It increases the expression of CD73/CD39 on EGFR-positive tumors and results in adenosine accumulation, further inhibiting the cytolytic activity of T-cells and recruitment of pro-tumorigenic MDSCs. (f) Accumulated adenosine in the tumor-immunome impairs NK cell activity. Hence, targeting EGFR alone won’t enhance survival benefits, but combining chemo/radio and immunotherapy could be strategized to overcome disabled immunological responses. The red inhibition symbol indicates the feasible mechanisms that could be exploited to devise effective therapeutic regimes to overcome TKI-induced acquired resistance and immune tolerance.

8. Expert Opinion: EGFR inhibitors in cancer and beyond

Numerous studies have shown that EGF stimulation led to the activation of EGFR downstream signaling driving the cellular phenotype of epithelial cells. Treatment of non-epithelial and cancerous epithelial cells using EGFR inhibitors demonstrated an inhibited network of EGFR signaling events. EGFR family members’ overexpression was associated with poor clinical outcomes in all cancers. EGFR gene expression and protein expression in various cancers were summarized in Figure 4 and Table 2. Over several years of research, our research community has identified several targeted therapies against activated and mutated EGFR, HER2, HER3, and HER4 (panEGFR inhibitors). These agents were approved by USFDA, European Medicines Agency (EMA), and National Medical Products Administration (NMPA) China for early, late, and advanced-stage cancer patients. More importantly, EGFR-targeted inhibitors are the first molecular agents to be approved and applied for several advanced cancers. They are the first targeted agents to be used as monotherapy with superior efficacy over conventional chemotherapy in cancer patients.

Figure 4. Global transcriptome analysis of EGFR family members among various cancers compared to normal tissue.

(a) Box plots demonstrate the differential pattern of EGFR among various cancer and normal tissues. Relative to respective normal counterparts, significant EGFR upregulation was found in kidney, lung, pancreas, brain, stomach, head and neck, and esophageal cancer. EGFR downregulation in the ovary, breast, blood, liver, adrenal, testis, colorectal, uterus, bladder, urothelial, and prostate cancer compared to normal. However, no significant differential expression was observed in biliary, skin, thyroid, and cervical cancer. (b) Box plots show HER2 gene upregulation (significant and relative to normal) in the lung, breast, thyroid, liver, pancreas, testis, brain, stomach, colorectal, esophagus, and biliary cancers. HER2 downregulation was observed in kidney, blood, and adrenal cancer. However, no significant differential expression was observed in the ovary, cervix, prostate, skin, head and neck, uterus, and bladder and urothelial cancer. (c) Box plots depict a significant increase in HER3 mRNA in cancers of the kidney, ovary, skin, lung, breast, thyroid, liver, pancreas, testis, stomach, colorectal, cervix, uterus, esophagus, prostate, biliary tract and downregulated only in blood and head and neck cancers. No significant variation in expression was shown in bladder, urothelial, adrenal, and brain cancers. (d) HER4 upregulation in the ovary, breast, adrenal, brain, uterus and downregulated in the kidney, lung, thyroid, blood, pancreas, testis, stomach, colorectal, head and neck, and prostate cancer were shown as boxplots. Like other family members, no significant difference or variation is found between normal and cancers of the liver, biliary tract, skin, cervix, bladder, and urothelial and esophagus.

Table: 2.

Differential expression analysis of EGFR family members during initiation and progression of cancer.

Organ/site	Cancer/ Sample type	EGFR family member	% of overexpression	References
Breast	Primary	EGFR	66%	[129]
	TNBC	EGFR	(45.3%) 136/300	[130]
	Pre-cancerous tissues		(21.7%) 26/120
	TNBC	EGFR	2.7% High; 16% Low	[131]
	TBNC	EGFR	61.2% (60/98)	[132]
	Non-TNBC		22.9% (25/109)
	TNBC	EGFR	71.4%	[133]
	Primary	HER2	7.1% strong expression 15.1% moderate expression 13.5%-week expression	[134]
	Primary	HER2	30%	[135]
	Primary breast carcinomas	HER3	51.9% (160/308)	[136]
Lung	NSCLC	EGFR	52.3%	[137]
		HER3	82.7%
	NSCLC	HER2	3%	[138]
	NSCLC	EGFR	52.3% in primary tumors 40.0% in lymph nodes 62.7% of brain metastasis	[137]
		HER3	82.7% in primary tumors, 86.6% in lymph nodes and 91.2% of brain metastasis
	Primary	EGFR	100% (31% High expression and 69% Low expression)	[139]
	NSCLC	HER2	16%	[140]
	Adenocarcinoma		35%
	Large cell carcinoma		20%
	Squamous cell carcinoma		1%
	Adenocarcinoma	EGFR	65% of primary and 97% metastatic tumor	[141]
	Primary	HER2	1.9% (High protein expression)	[142]
	NSCLC	HER2	21.62%	[143]
Colorectal	Primary	EGFR	97% (80% High and 20% Low)	[144]
	Stages iii and iv	EGFR	84.1%	[145]
	Primary	EGFR	64%	[146]
		HER2	66%
		HER3	85%
	Lymph node metastases	EGFR	56.8%
		HER2	46%
		HER3	76%
	mCRC	HER2	4.8% (429/8887)	[147]
		HER3	1.7% (148/8887)
	Colon	HER2	14%	[148]
Pancreatic	PDAC	EGFR	48.9% (64/131)	[149]
	Primary	EGFR	53.66% High; 34.15% Low	[150]
		HER2	56.09% High; 36.58% medium/ low
	Primary	EGFR	77% (192/249)	[151]
		HER2	19% (56/287)
	PDAC	HER2	22% (10/45)	[152]
		HER3	27% (12/44)
	Primary	EGFR	41.4%	[153]
		HER2	60%
		HER3	24.3%
		HER4	65.7%
Prostate	Acinar adenocarcinoma	EGFR	18.7%	[154]
	Primary	EGFR	8%	[155]
	Primary	EGFR	14% High expression	[156]
	Castration-resistant bone metastatic		29% High expression
	Prostatic adenocarcinoma	EGFR	40.9%	[157]
		HER2	1.5%
	Without ADT	HER2	85.7%	[158]
	Underwent ADT		1%
	Primary	HER2	17.2%	[159]
	Metastatic CRPC	HER2	63% (CTCs)	[160]
	Gleason score 6	EGFR	3% high; 27% low	[161]
	Gleason score 7		18% high; 49% low
	Gleason score 8		8% high; 4% low
	Gleason score 9		61% high; 18% low
	Gleason score 10		11% high; 2% low
	Primary	EGFR	41.7% (5/12)	[162]
		HER2	33% (4/12)
		HER3	75% (9/12)
	Lymph node metastases	EGFR	50% (6/12)
		HER2	66.7% (8/12)
		HER3	58.3% (7/12)
Gastric	Primary site	EGFR	30.4% (17/56)	[163]
		HER2	8.3% High; 31.8% Low	[164]
		HER2	9.38% (18/192)	[165]

8.1. What do current EGFR-targeted agents teach in the context of clinical methodologies and adoptions?

Based on the earlier clinical trials, it is predicted that EGFR TKI’s effectiveness is primarily based on the presence or absence of activating EGFR tyrosine kinase (TK) mutations in patients with lung cancer. These acquired EGFR mutations will occur after 9–14 months of treatment initiation. EGFR-positive and non-smokers had significant survival benefits upon erlotinib treatment. Similarly, gefitinib’s clinical response is good in female patients with a history of nonsmoking habits. Later, it was identified that EGFR-TKI’s effectiveness is higher in females (gender) and Asian origin (ethnicity) due to more participation of Asian women with EGFR TK mutation-positive NSCLC patients. A major limitation of EGFR-TKI is the Starting from first-generation erlotinib (T790M), second-generation Afatinib, and third-generation osimertinib (L858R/T790M/C797S or Del19/T790M/C797S) were shown to induce acquired EGFR mutations. Hence, all these EGFR family members targeting agents exhibited modest benefits in clinical settings to cancer patients. 50% of cancer patients will show acquired EGFR mutation at T790M in EGFR. Thus, there is a critical need to progress towards identifying and validating EGFR mutant-specific EGFR-TKIs without causing additional mutations. Some of the acquired mutations are the same in two different cancers. For e.g., EGFR mutations at exon 21 (L858R) and exon 19 (deletion) are also observed among TNBC (although rare) like NSCLC patients. In such cases, the newly identified compounds should be evaluated regardless of cancer and its subtype.

In the future, multiple molecular agents can be combined to obtain superior tumor cell killing and extend the survival of cancer patients. For example, NSCLC tumors found a way to tackle osimertinib by developing acquired resistance against it, especially via B-Raf proto-oncogene (BRAF) mutation, which is a downstream signaling molecule of EGFR. BRAF is a component of RAS/MAPK signaling which engages in cell growth and transformation. BRAF V600 involves the exchange of valine to glutamate at 600^th residue on exon 15, which is a constitutive active BRAF mutation and further leads to tumor cell growth, invasion, and reducing apoptosis. In an anecdotal report of a patient who developed a BRAF V600 mutation, triple therapy involving dabrafenib, trametinib, and osimertinib targeting EGFR/BRAF/MEK pathway showed clinical improvement and partial tumor suppression [111].

8.2. What are the major challenges and weaknesses in EGFR-targeted agents and their associated combination therapies?

Toxicity is another major limitation of EGFR-targeted agents starting from the first to the fourth generation of EGFR-TKIs. The most common adverse events against EGFR-TKIs are rash, dry skin, aches, diarrhea, nausea, and vomiting. Very rarely interstitial lung disease (1%) will be observed in patients treated with gefitinib. Further, to resolve the issue of acquired resistance, we need to develop and optimize EGFR-TKIs targeting the exact allosteric site to avoid resistance development and toxicity.

EGFR mutants exhibited disappointing outcomes in clinical trials with low to modest responses to ICB. ICBs are often responsible for inflammatory side-effects conventionally termed immune-related adverse events (irAEs) that manifest as multi-organ failure, pneumonitis, colitis, and hepatotoxicity. Therefore, in a clinical context, the implementation of ICB should be cautiously considered in patients receiving EGFR-TKIs. However, the reason for the dismal consequences is still unknown and involves multiple cellular processes and disparate physiological pathways. Therefore, selecting the optimal immune checkpoint inhibitor or immune component to target as a part of the immune therapy is a critical factor that demands in-depth evaluation. Additionally, the outcome of ICB also varies based on EGFR mutation; hence it is vital to understand the heterogeneity in EGFR mutation to extend the benefits of PD-1, PD-L1, and CTLA4-mediated ICB effects along with anticancer therapies to cancer patients. It is extremely critical to improving our knowledge about the ICB failure mechanism (s) against EGFR-TKIs using preclinical mouse models to enhance the ICBs effectiveness.

8.3. How can current problems with EGFR-targeted agents be solved using technical and technological innovations?

Other than EGFR mutations, amplification, and overexpression in cancer, it is essential to recognize the abnormalities due to HER2, HER3, and HER4 in NSCLC, breast, CRC, HNSCC, and Glioblastoma, in which EGFR targeting agents are the major player in disease control. For example, a recent study demonstrated four types of exon 20 insertion mutations in HER2 and their association with partial response to pyrotinib [83]. Furthermore, patients who harbor aberration in other EGFR family members and do not have EGFR mutation should be identified and treated differently to improve overall survival. A study has shown that KRAS missense mutations in codons 12, 13, and 61 are predictors of antitumor treatment resistance against monoclonal antibodies targeting EGFR, such as cetuximab, panitumumab, and EGFR TKIs. In CRC, cetuximab is approved for EGFR-wild-type patients with restricted access to KRAS-mutated patients. A recent study demonstrated that hereditary CRC patients with KRAS G13D mutation could benefit from the combination of cetuximab and chemotherapy, and the degree of clinical benefit is similar to that of KRAS wild-type patients [112]. The team also showed that RAS-selective lethal 3 (RSL3), a small molecule that can trigger ferroptosis, a non-apoptotic cell death, by blocking GPX4 and when administered in combination with cetuximab in KRAS-mutated CRC cells. Further, RSL3/cetuximab-mediated ferroptosis suppresses nuclear factor erythroid 2-related factor 2 (Nrf2) via p38 MAPK activation. Nrf2 regulates antioxidant transcription factors and prevents lipid peroxidation and apoptosis by raising the transcription of cytoprotective enzymes such as haem-oxygenase-1 (HO-1) [112]. Overall, the above-referred literature provides us with enlightenment and hope to evaluate EGFR-TKIs in the context of KRAS mutation beyond EGFR mutations. Also, further research holds on detecting the emergence of new driver mutations or existing mutations in other oncogenic drivers other than EGFR family members using next-generation sequencing to predict treatment effect and response.

Innovatively, a personalized approach was taken by a group of researchers in HNSCC, who delineated a PaSSS-based approach (Patient-specific signaling-based approach) to overcome the emergence of alternative signaling and tumor heterogeneity limitations against anti-EGFR, ICB, and chemo-radiotherapy [113]. By this approach, we can identify the central hub proteins to target and potentially avoid unnecessary trials on patients who will never benefit from a particular drug combination.

8.4. How do economics impact real-world research and advancements?

The global EGFR targeted agents’ market is projected to grow by US$ 6.75 billion with a progressing compound annual growth rate (CAGR) of 8.6% from 2023–2028. The out-of-pocket cost of EGFR TKIs increased several folds from $198/month in 2011 to $7800 for 12 months of treatment in 2019. A recent analysis demonstrated that due to the heavy cost burden, there could be a chance of “missing doses” and being “less likely to adhere to the treatment schedule,” leading to the reduced overall survival of cancer patients [114]. Thus, socioeconomic status is the major factor influencing cancer therapy, and overall progression-free survival is an objective response of the drug. This could be prevented by simplifying the accessibility of anti-cancer/EGFR-targeted agents to patients.

8.5. What are the new avenues that EGFR-targeted agents hold in the future?

Beyond cancer, HER2 mutations have been identified in Alzheimer’s disease. Neuregulin 1 (NRG1) is associated with Alzheimer’s and schizophrenia. Apart from antineoplastic activity, EGFR-TKIs exhibit a protective role in other diseases such as (a) Neratinib is found to protect pancreatic beta cells in the diabetic mouse model [115] and (b) another study in mice model found potential role of icotinib in cardiac remodeling and improvements in pulmonary hypertension by blocking EGFR-AKT/ERK signaling mechanism [72]. Hence, drug-repurposing strategies should be applied to extend the utility of EGFR signaling blockade apart from cancer studies.

Article highlights.

1. The emergence of EGFR and HER2 activating mutations harboring cancer patients are the candidates for the fourth generation EGFR-TKIs.

2. The genomic era opens next-generation platforms to monitor mutation evolution after TKI treatment timely.

3. The inevitable limitation of first to fourth-generation TKI is toxicity.

4. EGFR signaling blockade helps Alzheimer’s disease patients and cardiac remodeling.

Abbreviations

EGFR

Epidermal growth factor receptor

CRC

Colorectal cancer

PDAC

Pancreatic ductal adenocarcinoma

PCa

Prostate cancer

BC

Breast cancer

TNBC

Triple-negative breast cancer

NSCLC

Non-small cell lung cancer

HCC

Hepatocellular carcinoma

ICB

Immune checkpoint blockades

ICI

Immune checkpoint inhibitor

DC

Dendritic cells

TKI

Tyrosine kinase inhibitor

CTC

Circulating Tumor Cells

EMT

Epithelial to Mesenchymal Transition

CSCs

Cancer Stem Cells

TME

Tumor Microenvironment

CRT

Chemoradiotherapy

PD-1

Programmed cell death protein 1

PD-L1

Programmed death-ligand 1

CTLA-4

Cytotoxic T-lymphocyte associated protein 4

WT

Wild-Type

OS

Overall Survival

CTLs

Cytotoxic T lymphocyte cells

US FDA

United States Food and Drug Administration