The paradigm of cancer has evolved in the last years and conveyed into the concept of cancer “epigenome”, strictly linked to cancer “genome” (1). Many epigenetic regulatory players are involved in the transcription modulation of multiple tumor suppressor genes (TSG); DNA methylation, histone modifications, aberrant expression of microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) participate in many neoplastic steps, such as dysregulation of cell growth, malignant cell transformation, invasion and metastatization (2–5).

Among all epigenetic alterations, DNA methylation represents one of the most studied chemical modifications in human disease. It occurs when methyl groups are covalently attached to the carbon at 5’ position of the cytosine residue of DNA by the DNA methyltransferase (DNMT) enzymes (6, 7).

The recent implementation of high-throughput approaches for methylation analysis gives a more detailed and dynamic overview of the DNA methylation perturbation in human disease and provides new important insights on the understanding of both temporal and spatial related gene expression modification and chromatin remodeling (8–10). This enhances, by consequence, to better explain the role of this class of epigenetic changes in cancer biology, unveiling novel epigenetic predictive and prognostic molecular biomarkers for neoplastic disease monitoring and outcome prediction in patients (11, 12).

The human cancer cells are characterized by the presence of a complex aberrant methylation signature, which takes place either as a hypo- or hyper-methylation events at single interspersed CpGs and/or CpG islands located both in the promoter and intergenic regions of genes. These epigenetic modifications may represent an early event in cancer development and progression, as well as they could cooperate with genetic lesions to guide the heterogeneity of response/resistance to therapies in patients (13, 14).

In lung cancer, the association between aberrant methylation profiles and resistance to anti-EGFR therapy is still understudied. More attention is required, since changes in methylation levels may help to explain the observed heterogeneity of lung tumor response to multiple targeted therapies (14). Here we detailed and updated the most recent advances in DNA methylation modifications linked to TKI resistance mechanisms in EGFR-mutated patients and their related cellular pathways (15, 16).

Scientific evidences on the role of miRNA signature alterations as players in TKI of EGFR resistance was also briefly discussed. All available scientific evidences about the prognostic value of epigenetic alterations as primary/intrinsic and secondary/acquired mechanisms of resistance were summarized. Publications in English language, peer-reviewed international journals were identified on PubMed. All scientific knowledges were updated until October 2022.

One of the most frequent distinctive outcome of NSCLC patients is linked to the activation of EGFR mutations. Somatic mutations at exons 19-21, codifying for the tyrosine kinase domain, actually represent the main molecular condition to predict a good EGFR-TKIs response in upfront therapy (17, 18). First-generation TKIs, erlotinib and gefitinib, can compete in a reversible manner with adenosine triphosphate (ATP) at EGFR binding site, whereas the second-generation (e.g. afatinib, neratinib and dacomitinib) and third generation (e.g. osimertinib) TKIs can irreversibly block the ATP pocket of EGFR receptor, thereby inhibiting its phosphorylation and downstream signal transduction activity by covalently binding the ATP binding pocket mutations. As consequence, EGFR-TKI administration frequently allows a higher overall response rate (ORR) and progression-free survival (PFS) in EGFR-mutated metastatic patients compared with upfront chemotherapy (19, 20). In addition, osimertinib also received in recent years the approval for the administration in patients who acquired p.T790M mutation of EGFR as first/second-TKI generation resistance mechanism (21, 22).

In all cases, however, all therapies administered to inhibit oncogenic kinases activity are unable to completely eradicate tumors, so the EGFR-mutated patients invariably develop acquired resistance after 9-12 months of treatment initiation or they do not respond to TKIs treatment at all (23, 24). Several biological mechanisms of resistance have been reported to date, such as secondary EGFR mutations, bypass track signaling pathways and histologic transformation, not all strictly related to TKIs affinity (23, 25). All just reported mechanisms can be classified as primary or acquired resistance events, even if some of these, such as the co-expression of other ErbB receptors or the constitutive activation of other downstream pathways, remained ambiguous and are unlikely to be located in one of the two types of resistance.

Intrinsic or primary resistance refers to patients who either do not achieve stable disease or who progress within 6 months after an initial clinical response, according to the RECIST (Response Evaluation Criteria in Solid Tumor) criteria, with a consequent worsening of clinical conditions as well as response rate and disease control rates (LDCR), approximately in 20-30% EGFR-TKIs treated patients (26). Host-related mechanisms, such as defective immune system activity, rapid metabolism, or poor absorption, are predominantly responsible for intrinsic/primary resistances. Moreover, non-sensitive EGFR mutations, which contribute to an inconsistent drug activity, such as the naïve threonine-to-methionine substitution at the “gatekeeper” amino acid position 790 (p.T790M) in exon 20 and some mutations in exon 19 (p.L747S/p.D761Y), p.T854A or p.L868R in exon 21 of EGFR, can be included in this category (27, 28).

Apart from these, other molecular mechanisms could be the activation of different pathways by mutations in HGF (hepatocyte growth factor) gene (29), IGF1R (insulin growth factor 1 receptor) gene (30), MET (MET proto-oncogene, receptor tyrosine kinase) gene (31), and/or PI3K/AKT (phosphatidylinositol-3-kinase and protein kinase B) pathway genes (32, 33). All above listed mechanisms of primary resistance generally arise after the administration of first- and second-generation TKIs in patients with NSCLC. There is also an emerging literature on primary resistance to the third-generation TKI osimertinib used in up-front therapy in EGFR mutated NSCLC, although data are actually in progress. The most compelling studies came from intrinsic resistance to osimertinib as second-line option: KRAS (Kirsten rat sarcoma virus) p.G12D mutation (co-occurring with the loss of PTEN, Phosphatase and tensin homolog, gene), BRAF (B-Raf Proto-Oncogene, Serine/Threonine Kinase mutation) mutations, ALK (Anaplastic lymphoma kinase) gene translocation, HER2 (Human epidermal growth factor receptor-2) and MET (tyrosine-protein kinase Met) amplifications were reported (15, 34).

Acquired or secondary resistance to TKIs typically occurs in lung cancer patients after an initial response or stable disease to EGFR-TKIs (≥ 6 months), according to the RECIST criteria. In 50-60% of NSCLC patients who developed resistance to first/second-generation TKIs, the occurrence of p.T790M in exon 20 of EGFR is a fixed point for lung cancer management. In NSCLC patients with a pre-existent EGFR activating mutation, this last condition confers resistance to TKIs by sterically blocking the binding of drugs to the receptor pocket, thus giving an advantage to cancer cells by activating signaling pathways associated with tumor progression and metastasis (35). In this specific context, the administration of osimertinib as second line of treatment in patients harboring p.T790M mutation can re-block the tumor expansion, until additional resistance mechanisms occur as a result of the loss of the p.T790M mutation and the acquisition of novel resistance to second-line osimertinib, such as p.C797S mutation at exon 20 of EGFR (15, 34).

Other secondary mutations, in addition to the already mentioned p.T790M which are involved in EGFR-TKI acquired resistance, are represented by p.D761Y or p.L747S (exon19 of EGFR), and p.T854A (exon 21 of EGFR) (36). Uncommon and combined EGFR mutations, intratumoral heterogeneity beyond EGFR alterations, drug inefficacy due to adaptive mechanisms exploited by cancer to convey resistance, such as histological transformation of lung adenocarcinoma into small cell lung cancer (SCLC) (37), squamous cell carcinoma (SCC), as well as the activation of alternative pro-oncogenic signaling pathways are also reported (38). The epithelial-to-mesenchymal transformation (EMT) can be also included among these resistance mechanisms, as well as the consequent loss of cell adhesion and polarity and promote the formation of tumor stem cells and decreasing the EGFR signaling addiction (39).

Variations in methylation levels and deregulation of miRNA and lncRNA machinery are widely associated with neoplastic transformation, carcinogenesis, and cancer progression. Anyway, the fluctuations of cancer methylome, both at DNA and RNA levels, remain the less investigated epigenetic changes in the context of target therapy resistance and TKI resistance in NSCLC ( Figure 1 ).

Starting from the molecular profiling of epigenetic marks across the genome, a new focus on the methylome evolution of lung cancer may help to more clearly understand how cell biology contributes to TKI drugs resistance in NSCLC patients (38, 40). The most interesting findings in this field are summarized in Table 1 and detailed below. A representative scheme that depicts methylated genes and their associations with TKI resistance in NSCLC is also illustrated in Figure 2 .

MicroRNAs (miRNAs) are 18-25 nucleotides single-stranded non-coding RNAs which are able to turn on and off the expression of their targeted genes (81). The deregulation of miRNA machinery is linked to the acquisition of cancer hallmarks that lead to genome instability and impact on tumor growth, invasion and metastatization (82). Recent findings have shown that miRNAs could modulate as post-transcriptional regulators the response to EGFR TKIs in overcoming resistance in NSCLC patients, as summarized in Table 2 (92, 93). The first evidence of this derived from Hashida S and colleagues’ work, whom investigated the EGFR-TKI resistance mechanisms in a total of 10 afatinib-resistant cell lines from parental NSCLC cells with activating EGFR mutations (83). In particular, they found that these EGFR-mutant lung cancer cell lines mainly acquired MET amplification as a mechanism of resistance and become sensitive to afatinib plus crizotinib. The acquired MET amplification was co-occurrent with miR200c epigenetic silencing and the acquisition of EMT and stem cell-like features. Moreover, it was demonstrated that the acquisition of EMT and other associated features was also due to a downregulation of epithelial markers as well as E-cadherin that was observed in this group of afatinib-resistant cell lines. These findings were consistent with previous observations by Shien et al. (84), who reported a downregulation of miR200c by methylation in a group of NSCLC cell lines gefitinib resistant having acquired MET amplification and stem cell-like features (83).

Many other reports are providing evidences about the contribution of miRNAs in the complex and heterogeneous mechanisms of EGFR-TKI drug resistance in NSCLC. A role in EMT promotion linked to acquired osimertinib resistance in NSCLC was also attributed to Let-7c (85), whereas miR-7 was demonstrated to enhance TKI-induced cytotoxicity by gefitinib through the suppression of both IGF1R (Insulin like growth factor1 receptor) and EGFR signaling pathways (86).

A critical role of miR-130a in overcoming acquired resistance to EGFR-TKIs via MET signaling was highlighted in two separate studies. A first study demonstrated that the overexpression of miR-130a enhanced apoptosis and suppressed NSCLC cells proliferation after gefitinib treatment. Otherwise, a down-regulation of this miRNA triggered cell apoptosis with rapid proliferation in both gefitinib-sensitive and resistant NSCLC cell lines. It was also demonstrated that miR-130a binds to the 3’-UTR of MET and significantly suppresses its expression, so the overexpressing of MET could rescue the functions of miR-130a regarding cell apoptosis and proliferation after cells are treated with gefitinib (87). These results are in line with those published in 2015, when it has been shown that the decrease of cell invasion and migration due to miR-200a overexpression leds to gefitinib resistance in NSCLC cells via EGFR and MET signaling (88).

Bisagni A et al. was able to prove how the increase expression levels of miR-133b significantly correlated with a better PFS and OS and allowed to better discriminate NSCLC responder patients to erlotinib from non-responder. Similarly, they found a direct relationship between the upregulation of miR-200c and improved gefitinib sensitivity in NSCLC (89).

Finally, increased miR-497 level was seen to have a significant impact in enhancing sensitivity to EGFR-TKI in NSCLC cells via IGF1R targeting and AKT activation (90), whereas in vitro experiments and in vivo models were useful to demonstrate that the combination of gefitinib plus a microRNA mimic, miR-30a-5p, could overcome acquired EGFR-TKI resistance in NSCLC via a direct regulation of IGF1R and HGFR (hepatocyte growth factor receptor) signaling (91).

Worldwide, lung cancer represents one of the most common types of cancer and, by far, the leading cause of cancer deaths (94, 95). Among these cases, 80-85% have an NSCLC histology, which includes adenocarcinoma, squamous cell carcinoma, and large cell carcinoma subtypes (96, 97). Changes in DNA methylation may largely underpin lung cancer in several processes as well as the capability of growth, invasion and spreading of cancer cells (98, 99). A special attention is now drowning toward DNA methylation patterns of drug-treated tumor cells that could change and support the acquisition of resistance to treatments.

The double interaction between epigenetic alterations and therapy resistance of tumors is progressively emerging and is looking for answers as to why it happens. In solid tumors and in EGFR-TKI treated NSCLC patients there are little translational evidences about the occurrence and role of DNA methylation at different regions of single/multiple genes (promoter and other regulatory regions) or CpG islands (14, 100).

To bridge the gap in this specific field is actually demanded. Specifically, it is necessary to understand how methylome could be modified and which methylated regions or specific CpG sites are affected in order to translate relevant epigenome associations into clinically personalized treatment in lung cancer patients (101). Next to this, the definition of methodological high-throughput approaches to study changes in methylation will improve the advances in this field, since measuring global or single CpG methylation will help to construct a more robust and integrated algorithm to predict and monitor disease evolution in a non-invasive manner for lung cancer patients (102–104). In recent years, there has been a growing interest in liquid biopsy to identify and monitor epigenomic drivers, also in the context of primary and acquired resistance in lung cancer (105). There are many reasons why circulating tumor DNA (ctDNA) methylation may rapidly emerge in this clinical settings. The first point leads to the fact that DNA methylation occurred as an early event in the etiology and progression of lung carcinogenesis, besides it could be strictly dependent both on tissue location and type of cancer (106). Second, it should be taken into consideration that DNA methylation profiling provides a deep characterization of ctDNA which contains important information about longitudinal changes in CpG islands across genomic regions (107). Third, the heterogeneity of CpG methylation patterns in different regions of multiple genes (promoter and other regulatory regions) is significantly associated with a poor outcome that might allow for accurate discrimination among lung cancer subtypes in liquid biopsies samples (108). Moreover, starting from the track of tumor evolution in serial ctDNA, it could be possible to identify minimal residual disease and manage early cancer progression, overcome temporally and spatially intratumor heterogeneity aiming at stratifying lung cancer patients according to recurrence risk and response to therapy (109). Although it remains to define several methodological strategies as well as optimize ctDNA extraction step to ensure a high-quality cfDNA or establish the gold standard for setting a better sensitivity and specificity of ctDNA methylation assay detection (110), a systematic analysis of liquid biopsy samples could provide important insights into the heterogeneity of TKI resistance mechanisms in NSCLC patients, thus providing essential information to better predict resistance and help the selection of subsequent treatments.

Ongoing research studies are also focused on single-cell DNA-methylation profiling that may contribute to the examination of cell-of-origins and cancer cell type heterogeneity by which becomes possible to clarify the correlation between DNA methylation and the expression fluctuations of cancer driver genes sets in different subtypes.

Conceptualization, FPF and LM. Data curation, FPF and AS. Writing—original draft preparation, FPF. Writing—review and editing, LM. Visualization, AS. Supervision, LM. All authors contributed to the article and approved the submitted version.