Targeting C797S mutations and beyond in non-small cell lung cancer—a mini-review

Introduction

Non-small cell lung cancer (NSCLC) represents over 80% lung cancer cases and still has a huge mortality worldwide. In China, it is also the most common cancer and is one of the major causes of cancer-related deaths (1). Targeting epidermal growth-factor receptor (EGFR) alterations has provided a paradigm shift in the treatment of NSCLC, and NSCLC harbouring EGFR mutations are detectable in 10–20% of all lung cancer cases in Europe and the USA over 45% in Asian patients with NSCLC (2). Deletions of exon 19 and exon 21 L858R mutations are the most common alterations, accounting for approximately up to 90% of mutations in NSCLC; these are termed “classic” (common) mutations and result in high sensitivity to tyrosine kinase inhibitor (TKI) treatment.

Of note, NSCLC patients with exon 19 deletions and exon 21 L858R mutations have a longer progression-free survival (PFS) when treated with TKIs compared with platinum-based chemotherapy. Other EGFR mutations are termed “uncommon” mutations, and account for up to 18% of all EGFR mutations (3) (Figure 1).

Figure 1 EGFR mutations within exons 18–21 of the tyrosine kinase domain of the gene. Common and uncommon EGFR mutation are depicted by orange and yellow background, respectively. *, TKI-naïve NSCLC samples (4). EGFR, epidermal growth-factor receptor; TKI, tyrosine kinase inhibitor; NSCLC, non-small cell lung cancer.

Uncommon EGFR mutations

Uncommon EGFR mutations were found to show a variable efficacy to EGFR-targeted drugs depending on the molecular alterations within exons 18–21 (4,5), which are still not fully investigated to date. The mutations of G719X in exon 18, L861Q in exon 21, S768I in exon 20, and exon 20 insertions (e.g., S768_D770dup, A767_V769dup, N771_H773dup, P772_H773insV, p.A767delinsASVD) are generally accepted to be the most frequent alterations within the group of uncommon mutations (6).

The exon 20 insertion mutations are known to be resistant to previously approved EGFR-targeted drugs (7) and treatment with platinum-based chemotherapy and immune checkpoint inhibitors are currently standard of care in NSCLC patients with uncommon EGFR mutations. Wu et al. (8) provided the first evidence that NSCLC patients harbouring exon 20 insertions had a significantly shorter PFS than those with exon 19 deletions and exon 21 L858R mutations (1.4 vs. 8.5 months, P<0.001), which adds weight to the proposal that the development of novel drugs targeting uncommon mutations is a high unmet medical need.

Role of EGFR mutations in other cancers

EGFRs have been studied in great detail as novel targets for drugs in adult cancers (9), but rarely in paediatric indications. EGFR pathway alterations (e.g., EGFR amplification or overexpression) has been found in various paediatric tumours (10), however, treatment outcomes of recurrent diseases remain poor (11). In addition, studies of EGFR-targeting TKIs in paediatric patients have shown almost no activity (12), potentially due to bypassing oncogenic signal transduction linked to other than EGFRs (e.g., c-MET) (13).

EGFRs are frequently mutated in human cancers (14) and represent a valid therapeutic target in NSCLC patients harbouring specific EGFR alterations. EGFR mutations predominantly occur in the intracellular tyrosine kinase domain of the receptor and thereby directly promote its activity.

During the last two decades some reports have provided evidence that specific EGFR mutations or amplifications can be detected in paediatric brain tumours (e.g., glioblastomas), however, these mutations are exclusively extracellular (15-17) and their role as driver mutations is far from being clear. They can represent both, small in-frame insertions within exon 20 (intracellular domain) or missense mutations (exon 7 extracellular ligand-binding domain) that can be found even without accompanying gene amplification (18). Among these reported EGFR mutations, the most common one is EGFRvIII. This mutation is characterised by a deletion of 267 amino acids in the extracellular domain which forms a mutant receptor that is unable to bind the EGF ligand and is permanently active (19). EGFRvIII and other extracellular variants are regarded to be a late event, mainly occurring after amplification of the EGFR-wildtype (20).

Fourth-generation EGFR TKIs

BDTX-1535 (Figure 2) is an orally bioavailable, brain-penetrating, mutation-selective, irreversible EGFR inhibitor with significant antitumour activity in NSCLCs and glioblastomas (21,22). It is a fourth-generation EGFR inhibitor that was found to overcome resistance to osimertinib in preclinical models and has shown promising activity in NSCLC patients harbouring C797S mutations (22). Of note, BDTX-1535 has been found to easily cross the blood-brain-barrier and, therefore, might be of great benefit for the treatment of central nervous system (CNS) metastases in NSCLC. In addition, BDTX-1535 has been shown to have a high activity in glioblastomas harbouring mutations in the extracellular domain (23).

Figure 2 Chemical structure of BDTX-1535.

In experimental models BDTX-1535 was found to inhibit all common EGFR mutations and more than 50 of uncommon mutations including T790M, C797S, L718X, E709X, S784F, V834L and A289V, however, exon 20 insertions are inhibited to a much lesser extent (22). Moreover, mutations in the extracellular domain of the EGFR (e.g., EGFRvII, III, IV; Figure 3) can be blocked as well (24).

Figure 3 Mutation-associated development of TKI resistance in NSCLC. First-generation TKIs: gefitinib, erlotinib, icotinib; second-generation TKIs: afatinib, dacomitinib; third-generation TKIs: osimertinib, lazertinib, furmonertinib, nazartinib; fourth-generation TKIs: BDTX-1535, EA1045. NSCLC, non-small cell lung cancer; EGFRmut, epidermal growth factor receptor mutated; Del, deletion; ins, insertions; TKIs, tyrosine kinase inhibitors.

With the substitution of reversible EGFR TKIs (first-generation) by novel third-generation TKIs (e.g., osimertinib) in the first-line setting for patients with NSCLC expressing common EGFR mutations, the appearance of acquired resistance mutations is now associated with a decline of the T790M mutations and the emergence and growing incidence of C797S mutations (5–15% of EGFR-mutated patients) (22). It should be noted that many NSCLC patients harbouring L858R mutations are also harbouring noncommon mutations which might explain, at least in part, the reduced activity for osimertinib in patients harbouring L858R mutations versus exon 19 deletions. For the latter one co-expression of uncommon mutations is less frequent, however, these patients more often develop C797S mutations as an acquired resistance mechanism (25).

BDTX-1535 is currently in a phase I/II dose expansion for NSCLC and dose escalation for glioblastomas (NCT05256290). Patients with exon 20 insertions were not eligible to be enrolled in this trial. Preliminary results from 27 NSCLC patients revealed that BDTX-1535 (100 mg once daily, oral administration) was active in NSCLC patients across almost all EGFR mutations including T790M, C797S, L747P, L718Q, as well as compound mutations (24). The drug was found to have only mild or moderate adverse events even at the highest dose level (200 mg daily). The overall response rate (ORR) was found to be 36% (22 evaluable patients) in this heavily pre-treated population (prior treatment lines: 1–9) with durable responses seen in most patients. In addition, 6 patients had stable disease at doses at or above 100 mg once daily [disease control rate (DCR) 85%] (26). A total of 19/22 was found to be resistant to osimertinib before enrolment. Of those 19 patients, the ORR was reported to be 42%, and 14/19 patients with documented resistance to osimertinib still remained on treatment as of data cutoff. The most common adverse events were rash (70%, two patients with grade 3, no grade 4) and diarrhea (35%, no grade ≥3 seen) (24,26).

To date several anti-EGFR TKIs and monoclonal antibodies for treatment of NSCLCs have been approved [European Medical Association (EMA), Food and Drug Administration USA (FDA)] or are in late-stage clinical development (Table 1). Amongst them, only two have been found to target the C797S mutation in clinical trials at low inhibitory concentration 50% (IC₅₀) values: tigozertinib (formerly known as BLU-945) (IC₅₀: 2.9–4.4 nM) and BDTX-1534 (IC₅₀: <10 nM) (Table 2). The development of tigozertinib was stopped in January 2024, the clinical development of its sister compound BLU-701 has also been discontinued due to futility (Table 2). EAI-045 is the first of allosteric kinase inhibitors; the molecule does not bind to Cys797 because its residue is outside the allosteric binding pocket, however, the drug was found to have a much higher IC₅₀ (0.33 µM) and has not been evaluated clinically so far (41,42).

In contrast, BDTX-1535 is a pan-EGFRmut TKI targeting common and uncommon EGFR mutations (including C797S) as well as extracellular variants and EGFR amplifications. Of particular interest, however, is the observation that BDTX-1535 is the first molecule in clinical development that can also target extracellular EGFR mutations/variants and amplifications which could be beneficial for other tumour entities (e.g., glioblastomas). Interestingly, despite its broad kinase spectrum BDTX-1535 appears to have only weak activity against NSCLC patients harbouring exon 20 insertions (21), and, as a result, these patients were not enrolled in the ongoing clinical trials. Since exon 20 insertions comprise a very heterogeneous and diverse group of EGFR alterations with frameshift and non-frameshift gene mutations, it is, therefore, conceivable that no specific binding side for BDTX-1535 exists in these patients.

EGFR: exon 20 insertions

Only a few compounds are currently available (approved or in late-stage development) for treating NSCLC patients harbouring exon 20 insertions (Table 1). Amongst them, amivantamab, firmomertinib, sunvozertinib, and zipalertinib are the most advanced compounds in clinical development. Amivantamab, a monoclonal bispecific antibody targeting EGFR and c-MET, is approved by EMA for second-line therapy following platinum-based therapy and by FDA for first-line therapy in combination with platinum and pemetrexed or second-line after prior platinum-based chemotherapy. Upon binding the antibody-receptor complex is internalised and degraded by the proteasome (including the intracellular domain containing the T790M, C797S and other mutations) (47,48).

In this context a future clinical trial combining a specific TKI targeting exon 20 insertions combined with BDTX-1535 may, therefore, be beneficial in certain cohorts of NSCLC patients.

C797S mutation resensitizing approaches

The C797S mutation in the EGFR gene has been identified to mediate resistance to all currently available third-generation TKIs. C→S mutations (i.e., C481S) are also known to confer resistance to ibrutinib (49) suggesting that the C→S mutation may be a recurring and critical mutation that can impede inhibitor binding to a large range of tyrosine kinases. It is likely that after a longer treatment time with these new compounds, again resistant clones will occur. It remains to be seen whether novel EGFR mutations will then be detected or a bundle of different mutations will confer resistance, comparable, at least in part, with the resistance development of anaplastic lymphoma kinase (ALK) inhibitors (50). Finally, it is also conceivable that the C797S inhibition might increasingly spark off-target resistance mechanisms suggesting that these patients will then only be eligible for chemotherapy (Figure 3).

Recently published work has provided increasing evidence that the allelic relationship of the EGFR mutations T790M and C797S appears to be critical for the response to EGFR-targeting TKIs (51-53). In this regard, it has been shown that NSCLC patients harbouring concomitant T790M and C797S in trans (on different DNA strands) remain sensitive to a combination of a first- and third-generation EGFR TKI (e.g., gefitinib and osimertinib). However, if these mutations are in the cis allelic relationship (on the same DNA strand), no TKI alone or in combination can suppress the resistant clone suggesting that the clonal progression of C797S from in trans to in cis may be indicative of a new resistance mechanism. It is conceivable, therefore, that clonal heterogeneity may play an important role and may exist simultaneously in osimertinib-resistant tumours (53). On the other hand, the detection of the in trans allelic relationship may offer a tailored opportunity for NSCLC patients with C797S-based osimertinib resistance.

Resistance beyond C797S

It should be noted that in up to 50% of all NSCLC patients who progress following osimertinib therapy no underlying resistance mechanism can be identified suggesting that non-mutational signal transduction pathways may also be operative (54,55). In this regard, Haratake et al. (56) most recently have provided the first evidence that the oncogenic protein MUC1-C (MUCIN) is an important driver of acquired osimertinib resistance in NSCLC patients which adds weight to the proposal that acquired osimertinib resistance is very pleotropic and thus making the development of therapeutic strategies to overcome this clinically observed resistance challenging. MUC1-C is known to act with several oncogenes and is thereby driving cancer stem cells, DNA damage resistance, and immune invasion (57). Using the osimertinib-resistant cell line H1975-OR and MGH700-2D cells (derived from an osimertinib-resistant NSCLC patient), Haratake et al. could demonstrate that the osmertinib-resistance was associated with increased MUC1-C levels, and inhibition of MUC1-C with GO-203 restored sensitivity in human xenograft models (56). Similar results have been provided by Hu et al. (58) who demonstrated that osimertinib-resistance in patient-derived mice xenografts was associated with an upregulation of the neuroendocrine lineage transcription factor achaete-scute homolog 1 (ASCL1). ASCL1 is essential for the survival of a majority of lung cancers with neuroendocrine features by initiating an epithelial-to-mesenchymal gene expression programme which was then, in turn, found to be responsible for the development of osimertinib resistance.

Tumour heterogeneity and EGFR TKI resistance

Tumour heterogeneity and the co-expression of several mechanisms of resistance may represent a huge hurdle to overcome the clinically observed osimertinib resistance apart from targeting C797S mutations.

Moreover, intratumoural heterogeneity is regarded to have a major impact on EGFR TKI resistance in NSCLC patients (59). It is well established that many tumour subclones exits in an individual tumour. For NSCLC patients, drug-tolerant persister (DTP) cell clones are known to have the ability to accelerate the development of drug treatment resistance through neutral selection (59).

Intratumoural heterogeneity is defined by the observation that a few clones of TKI-resistant NSCLC cell populations may remain, which then, in turn, re-start proliferation and lead to relapse or progression of NSCLCs even if the majority of TKI-sensitive cells respond following treatment (60). In particular, it consists of three main mechanisms: (I) DTP cells, (II) chromosomal instability, and (III) extrachromosomal extracellular DNA (ecDNA). It is generally accepted that ecDNA is a major contributor to tumour genetic heterogeneity and can be detected in 50% of all NSCLCs (61). Genetic alterations other than EGFR mutations may also contribute to the DTP state (59).

It is also known that NSCLC cells undergo various changes to adapt to the new tumour microenvironment (TME) caused by TKI treatment. Therefore, DTP cells are regarded to play a critical role in this process and may be a key player for the observed resistance mechanisms (62). Moreover, it has been assumed that specific acquired chromosomal alterations (e.g., T790M, C797S, c-MET amplifications etc.) are obtained during the DTP state (62). The importance of DTPs for tumour drug resistance is underlined by the observation that DTP cell survival cells is granted by H3K9-methylation and by H3K27-methylation (63,64). In addition, polycomb repressive complex 2 (PRC2), involved in DNA damage repair, has been shown to methylate H3K27 via enhancer of zeste homolog 2 (EZH2; enzyme subunit of PRC2) suggesting that inhibiting of EZH2 activity can suppress the DTP cell clones in lung cancers (65).

Conclusions

The development of fourth-generation EGFR TKIs has provided major interest as these drugs have the potential to inhibit resistance mutations (e.g., C797S) often seen in NSCLC patients with clinically observed resistance to third-generation EGFR TKIs. However, despite promising clinical activity, the development of these compounds still faces several caveats in sufficiently targeting on-target resistance mechanisms. One of the major hurdles is the occurrence of TKI resistance, which can result from off-target mutations or alternative signaling pathway bypass activation. Moreover, the ideal strategy to combine fourth-generation EGFR TKIs with other therapy modalities (e.g., immunotherapy, or other EGFR TKIs) is yet to be established and warrants additional preclinical and clinical research to better understand their ability to manage intratumoural heterogeneity. Addressing treatment resistance beyond the C797S mutation will be a major objective, and incorporating our knowledge of the impact of DTP cells and ecDNA on intratumoural heterogeneity could lead to more effective strategies for NSCLC patients. The use of comprehensive genomic profiling is now a clinical standard, and a deeper understanding of resistance mechanisms can be accelerated through the advancement of precision medicine and artificial intelligence. Therefore, an improved understanding of the molecular factors involved in NSCLC resistance may pave the way for the development of novel, personalised, and molecularly-targeted drugs for the treatment of NSCLCs. The development of novel drugs that can overcome TKI resistance in NSCLC patients harbouring the C797S mutation and beyond is, therefore, eagerly warranted. If the current results can be confirmed in larger randomised trials, this will have major clinical implications for the treatment of NSCLC patients in the future.

Acknowledgments

Funding: None.

Footnote

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-24-690/prf

Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-24-690/coif). The authors have no conflicts of interest to declare.

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