The mechanisms of on-target EGFR resistance consist of genetic alterations in EGFR occurring during progression under 3rd G EGFR-TKIs. In clinical studies, EGFR-dependent resistance is related to additional somatic EGFR mutations and to gene amplification (9, 27). EGFR point mutations occur in the kinase domain and affect the osimertinib covalent binding residue (C797S/G, exon 20), the EGFR solvent-front (G796S/R, exon 20), the hinge region (L792H/F), and residues inducing steric interaction (L718Q/V, G719C/S/A and G724S, exon 18) (62, 66, 67).

In preclinical models, EGFR amplification was reported in an established rociletinib-resistant cell line ( Table 1 ) and sensitivity to rociletinib was restored by cetuximab, a specific anti-EGFR monoclonal antibody, or by afatinib (20). Somatic EGFR mutations, however, have not been identified, which could be explained by the efficacy of 3rd G EGFR-TKIs in inhibiting EGFR protein. Thus, to better understand the involvement of EGFR aberrations in the induction of resistance, studies have been limited to ectopic overexpression of the wild-type (25) or mutated protein or to site-directed mutagenesis replicating mutations described in relapsed patients. The C797S mutation, engineered with a deletion within exon 19 in Ba/F3 cells, conferred significant resistance against osimertinib compared to other EGFR variants such as L718V, L792F/H, and G724S. However, when associated with L858R, C797S/G and L718Q/V conferred comparable resistance, which was greater than in a Del19 background. L792F/H, in contrast, induced significantly less resistance with L858R (68). This indicates that the initial activating mutation may play a role in the potency of resistance to osimertinib. Consistent with this finding, it was reported in the FLAURA study that patients with the L858R mutation have a worse prognosis than those with Del19 (7). Importantly, as observed in the clinic (69–72), earlier-generation EGFR-TKIs may be effective against osimertinib resistance; this may depend not only on the position of the mutation but also on its allelic context. Afatinib inhibited EGFR phosphorylation and cell growth in osimertinib-resistant Ba/F3 cells that exogenously express the G724S mutation, alone or with Del19 (67). The S724 variant induced conformational changes that are incompatible with EGFR-TKIs 3rd G and 1st G binding but not with 2nd G (67). Ba/F3 cells expressing EGFR^L858R/C797S by N-ethyl-N-nitrosurea mutagenesis were found to be sensitive to gefitinib (73). A recent study showed that sensitivity and response to EGFR-TKIs are also heterogeneous within the same EGFR exon and proposes a new classification rather based on the structure function of the mutation to determine potential future therapeutic approaches (74).

In clinical studies, emergence of the EGFR^C797X mutation is the most common mechanism of resistance to EGFR-dependent osimertinib regardless of treatment line. It was detected in 15% of patients progressing to second-line osimertinib therapy (75) and in only 7% of disease progression when osimertinib is administrated in first-line therapy (76). C797S occurred more frequently in association with Del19 than with L858R mutation (77, 78). Otherwise, C797S was observed in less than 3% of cases in rociletinib-resistant patients (25) and was not observed in patients who progressed after AC0010 treatment (79), suggesting that the resistance mechanism might be drug dependent.

Resistance to osimertinib mediated by EGFR-independent mechanisms can be acquired through activation of alternative bypass pathways, aberrant downstream signaling or histologic transformation. Figure 1 illustrates the involved pathways described in the review.

MAPK and AKT are common downstream modulators of receptor tyrosine kinases (RTKs). Activation of the MAPK pathway via ERK activation is a common feature of nearly all preclinical models of resistance to 3rd G EGFR-TKIs. However, alterations of other upstream effectors have also been identified as a driving event in the occurrence of resistance. Copy number gains of MAPK1, CRKL, NRAS, and KRAS as well as single mutations in NRAS (G12V/R, Q61K and E63K) were identified in resistant populations established from PC9 and H1975 cells after chronic exposure to osimertinib or WZ4002 ( Table 1 ). Combination with MEK inhibitor Selumetinib prevented the emergence of resistant PC9 cells after 34 days at which time resistant cells appeared in the presence of osimertinib alone, and delayed resistance in H1975 from 17 days in the presence of osimertinib alone to 40 days in the presence of selumetinib. Interestingly, combination therapy induced regression in osimertinib-resistant tumors in transgenic mice carrying EGFR^L858R/T790M (21). NRAS amplification was also reported, in a subsequent study, in naquotinib-resistant PC9 cells harboring the T790M mutation ( Table 1 ). Either selumetinib or trametinib, a second MEK inhibitor, resensitized naquotinib-resistant cells (22). Single KRAS mutations have also been described in resistance to 3rd G EGFR-TKIs. First, to assess the relevance of KRAS^G12S identified in a patient with acquired osimertinib resistance, Ortiz-Cuaran et al. showed that exogenous expression of KRAS^G12S in PC9 and HCC827 cells reduced sensitivity to osimertinib and rociletinib, indicating that expression of an activated KRAS allele is sufficient to drive resistance to 3rd-generation TKI (10). In a second study, KRAS^G13D mutation was reported as a potential resistance mechanism in osimertinib-resistant PC9 cells (20) ( Table 1 ). Another variant, HRAS^G13R, in association with increased MET expression was reported in osimertinib-resistant PC9 cells (23) ( Table 1 ). Moreover, BRAF^G469A mutation occurred in osimertinib-resistant PC9 clones ( Table 1 ). Similar to the models mentioned, dual therapy using selumetinib or trametinib with osimertinib was effective in overcoming resistance and enhancing cell death in mutated cells (24). To our knowledge, no NRAS alteration has been reported in clinical studies. Alterations in KRAS, however, are widely reported in clinical data. The KRAS^G12S mutation was identified in the lymph node biopsy, collected after relapse of osimertinib treatment, but not in plasma sample. Interestingly, EGFR^C797S variant, which in turn appeared after relapse, had been found in the patient’s plasma but not in the lymph node biopsy, indicating that different resistance mechanisms may develop at the same time (10). In another study, the KRAS^G12D mutation detected in patient’s plasma relapsing on first-line osimertinib therapy was associated with the CTNNB1^S37F mutation. Notably, the initial Del19 has not been detected in the post-therapy plasma sample (11). Clearance of the Del19 subpopulation may be due to different sources of the pre- and post-therapy samples or to selection of EGFR^WT cells during the development of resistance. The KRAS^Q61R variant has also been reported in the occurrence of osimertinib resistance (78). KRAS G12A, Q61H, and A146T variants were found in patients treated with rociletinib that were not detected in their pre-treatment plasma specimens (25). KRAS amplification has also been involved in osimertinib resistance. Molecular profiling of patients who relapsed on osimertinib therapy showed one case of KRAS/MDM2/CDK4 co-amplification (78). Finally, the BRAF^V600E mutation has been reported as a mechanism of resistance to osimertinib treatment, both alone (80) and in combination with MET amplification (81). The variant was also identified in a liquid biopsy sample from a patient undergoing treatment with ASP8273, a Japanese 3rd G EGFR-TKI (82). Combination therapies with 3rd G EGFR-TKIs and MEK inhibitors have been developed for lung cancers with EGFR mutations (NCT02143466).

As with ERK, maintained AKT activation is shown in the majority of preclinical models resistant to 3rd G TKIs and its restoration to normal status often required dual therapy. Shigenari et al. reported Src-AKT pathway activation, through Integrin β1 overexpression, as a resistance mechanism in established osimertinib- and rociletinib-resistant H1975 cells ( Table 1 ). Either dasatinib or bosutinib, both Src inhibitors, suppressed Src phosphorylation and restored sensitivity to 3rd G EGFR-TKIs. Finally, to understand if the co-occurring of PIK3CA^G106V with CTNNB1^S37F mutations observed in a patient that had progressed on rociletinib treatment had a role in acquired resistance, HCC827 cells were engineered with single or both mutations. While PIK3CA^G106V expression promoted invasion and migration, CTNNB1^S37F activated the wnt/beta-catenin pathway, promoted cellular invasion and suppressed apoptosis in response to rociletinib. Authors suggested a non-redundant cooperation of CTNNB1 with PI3CA alterations to promote tumor metastasis or limit EGFR inhibitor response (83). β-catenin has been shown to play an important role in acquired resistance to EGFR-TKIs and EMT in NSCLC cells (84, 85).

In patients, PIK3CA mutations, including E542K, E545K, and E418K, are frequently observed in resistance to 3rd G EGFR-TKIs (25, 62, 78, 83). They accounted for 7% of the resistance mechanisms to first-line osimertinib therapy (76).

Activation of C-mesenchymal–epithelial transition factor (c-MET) appears to be a common resistance mechanism to third-generation EGFR-TKIs, and has been found to be associated with resistance to osimertinib, to rociletinib (10), and to AC0010 (86). In preclinical studies, MET activation is due to gene copy gain or protein overexpression. No MET point mutations have been reported as observed in patients. In an preclinical, in vivo, model, rociletinib-resistant tumors were collected from mice bearing PC9 tumors ( Table 1 ). Genomic and biochemical analysis revealed MET amplification and activation as the only mechanism of acquired resistance. The combination of rociletinib with crizotinib, a kinase inhibitor with multiple targets including MET, reduced significantly the viability of cell lines derived from rociletinib-resistant tumors. Dual therapy effectively decreased growth on mice of patient-derived xenograft tumors harboring L858R and MET copy gain (25). In a second original model, established by Wanhong et al., AC0010-resistant H1975 cells were generated in two steps first, in vivo, and then after selection, in vitro ( Table 1 ). The derived cells were cross-resistant to EGFR-TKIs of other generations and showed upregulation of MET. Consistent with the above mentioned, double inhibition of EGFR and MET with AC0010 and crizotinib, respectively, prevented colony formation and suppressed MET activation in resistant cell lines and reduced significantly tumor growth in xenograft, compared with single therapy (86). MET activation has also been reported in cell line-based models generated by exposure to progressively increasing concentrations of 3rd G EGFR-TKI. Increased MET copy number was observed in osimertinib-resistant HCC827 cells ( Table 1 ), which were also cross-resistant to rociletinib and the 1st-generation EGFR-TKI, erlotinib. MET inhibitors such as crizotinib, ARQ179, or SGX523 sensitized osimertinib-resistant cells (26). In a second model, increased MET phosphorylation was reported to be responsible for acquired resistance in naquotinib-resistant PC9 cells ( Table 1 ). Interestingly, dual therapy by naquotinib with crizotinib or SGX-523 had a limited effect on bulk resistant cells, while drastically reducing the proliferation of monoclonal resistant cells, suggesting that heterogeneity may underlie the resistance to a specific TKI target. Notably, the one clone that overexpressed MET protein showed an increase in MET copy number, which was not observed in the resistant parental cells arguing for clonal evolution in the development of resistance. In addition, naquotinib administrated with crizotinib induced robust regression in mice bearing monoclonal resistant cell tumors without apparent cytotoxicity (22).

In clinical studies, MET copy number gains are the most common alternative bypass mechanisms for osimertinib resistance, regardless of treatment line (76, 78). In a recent report, MET amplification was found in 66% (n = 6/9) of patients treated with first-line osimertinib (12). Besides osimertinib, MET amplification has also been reported in tumor biopsies from patients with lung adenocarcinoma who developed resistance to rociletinib (10). Point mutations, such as MET P97Q, I865F, and H1094Y, have also been identified in patients with lung cancer progressing on osimertinib (62, 77). The efficacy of MET inhibitors alone or in combined therapy with 3rd G EGFR-TKIs has been reported in clinical studies (87, 88). Recently, the feasibility of combining osimertinib with savolitinib, a potent and selective MET inhibitor, has been tested in clinical trials (89).

In NSCLC, it has been reported that anexelekto (AXL) plays a role in resistance to many anti-cancer drugs including EGFR inhibitors (90). Indeed, Tanguichi et al. showed that PC9 cells do not have basal AXL activity as it is the case for MET and HER3 and that AXL phosphorylation appears shortly (4 h) after exposure to osimertinib and increases throughout the exposure period. They also observed a concomitant increase in MET, HER3, and EGFR phosphorylation, suggesting that AXL activation may accelerate the emergence of tolerant cells (59). In addition, drug-tolerant cells isolated from PC9, HCC4011, or H1975, 9 days after high-dose osimertinib treatment (3 μmol/L) expressed a higher level of AXL than parental cells and were highly sensitive to the AXL inhibitor, ONO-7475, in contrast to parental cells (28). Moreover, initial combined treatment with osimertinib and ONO-7475 had more effective effect on tumor regression PC9-derived xenograft when used as the initial treatment than as an alternative therapy once resistance to osimertinib developed (28). Since PC9 cells are enriched in AXL, this indicates that AXL expression level may be a predictor of response to osimertinib. Moreover, primary PE2988 cells, established from the pleural effusion of a patient who developed resistance to osimertinib showed high level of total and phosphorylated AXL and of stanniocalcin (STC2) and responded to the combination of AXL inhibitors and osimertinib (29). STC2 is involved in EGFR-TKIs resistance and was found upregulated in established gefitinib-resistant PC9 and osimertinib-resistant H1975 cells ( Table 1 ). Indeed, exogenous overexpression of STC2 activated AXL and increased c-jun level and phosphorylation. In fact, c-jun forms with c-Fos the transcription factor, activation protein-1 (AP-1), which binds to AXL promoter (91), suggesting the involvement of the STC2-JUN-AXL axis in EGFR-TKI resistance (29, 92). Another mechanism of resistance to osimertinib and gefitinib, involving AXL, was described by Kim et al. in osimertinib-resistant HCC827 cells ( Table 1 ). The model showed increased expression level of GAS6, a ligand of AXL, and prolonged protein degradation rates in parallel with AXL overexpression. Thus, YD-mediated AXL degradation synergized with osimertinib to restore osimertinib sensitivity in vitro and in vivo (30). AXL activation was identified as the mechanism of resistance in different established osimertinib-resistant cell lines, despite different EGFR mutational profiles ( Table 1 ). It has been observed alone, with an EMT phenotype, or with MET amplification (31–33). Cabozantinib, a multiple TKI including AXL (31), or CB469, a dual inhibitor of MET and AXL, with osimertinib (32) overcame resistance. Notably, resistant clones generated with AXL upregulation lost the T790M subpopulation and some the Del19 population, indicating that clonal evolution leads to heterogeneity in resistance mechanisms. In testing a library of drugs, foretinib, a type II inhibitor targeting a panel of RTKs including MET and AXL, showed the lowest IC₅₀ in resistant cell lines, which were T790M-negative (33), indicating that clonal heterogeneity is very likely to impair the efficacy of targeted therapy.

In clinical data, high AXL expression was associated with low RR to osimertinib in patients with EGFR-mutated NSCLC (59). Furthermore, PFS and ORR were inversely correlated with AXL mRNA expression in patients with EGFR-mutated NSCLC (93). Results of phase 1 clinical trials to assess the safety and tolerability of DS-1205c, a specific AXL inhibitor, when combined with osimertinib in metastatic or unresectable subjects with EGFR-mutant NSCLC (NCT03255083) are not yet published.

Insulin-like growth factor receptor (IGF-R) activation is involved in EGFR-TKIs resistance in NSCLC cell lines (94) and patients (95). Regarding its implication in resistance to 3rd G EGFR-TKIs, it was shown that drug-tolerant cells obtained 72 h after osimertinib treatment expressed elevated levels of total and phosphorylated IGF1-R without changes in the expression of its ligands, IGF1 and IGF2. This activation in the presence of osimertinib was due to epigenetic activation of its own transcription, mediated by the transcription factor FOX1. Moreover, osimertinib treatment enhanced the association of IGF1-R with its adaptor proteins Gab1 and IRS1, thereby promoting cell survival (58). Indeed, WZ4002-resistant PC9 cells ( Table 1 ) showed activated IGF1-R associated with IGFBP3 downregulation. Chemical inhibition of IGF1-R with AG-1024 or the blocking monoclonal anti-IGF1/2 antibody, BI836845, restored sensitivity to WZ4002, in vitro, and in xenograft mice (34). Loss of IGFBP3 was shown to induce activation of IGF1-R signaling and enhance resistance to WZ4002 in gefitinib-resistance cell line. Reciprocally, addition of recombinant IGFBP3 was sufficient to restore sensitivity to the 3rd G EGFR-TKI (96). In osimertinib-resistant cell line models, IGF1-R activation was observed in the presence (35) or absence of T790M ( Table 1 ). Indeed, a protein phosphorylation array performed in PC9 osimertinib cells ( Table 1 ) detected high activity of the “p-Y-IRS1 p-IRS2 bind PI3K” pathway, which is involved in IGF1-R signaling. Interestingly, the pathway was not activated in gefitinib- or erlotinib-resistant PC9, suggesting a mechanism specific to third-generation EGFR inhibitors. In contrast to the above models, the resistant cells did not show IGFBP3 downregulation but did show increased IGF2 expression. The IGF1-R inhibitor linsitinib overcame osimertinib resistance in resistant cell lines and in the patient-derived KOLK43 cells [established from pleural effusion of a erlotinib- and osimertinib-resistant patient with high IGF1-R phosphorylation (36)]. Increased phosphorylation of IGF1-R was observed, by immunohistochemistry, in the tumor sample of an EGFR‐mutated NSCLC patient who acquired resistance to osimertinib (35).