Specific mutations in the tyrosine kinase domain of EGFR
are associated with sensitivity to either gefitinib or erlotinib, but mechanisms of acquired resistance have not yet been reported. Based upon analogous studies in other diseases with another kinase inhibitor, imatinib, a single amino acid substitution from threonine to methionine at position 790 in the wild-type EGFR kinase domain was predicted to lead to drug resistance, even before the association of exon 19 and 21 mutations of EGFR
with drug responsiveness in NSCLC was reported. The T790M mutation was shown in vitro in the context of wild-type EGFR to confer resistance to gefitinib [21
] and a related quinazoline inhibitor, PD153035 [22
We show here, through molecular analysis of tumor material from three patients and one NSCLC cell line, as well as additional biochemical studies, that acquired clinical drug resistance to gefitinib or erlotinib is indeed associated with the T790M mutation. Importantly, we find that the T790M mutation confers drug resistance not just to wild-type EGFR but also to mutant EGFRs associated with clinical responsiveness to EGFR tyrosine kinase inhibitors [1
]. Our results further demonstrate that an analogous mechanism of acquired resistance exists for imatinib and EGFR tyrosine kinase inhibitors (), despite the fact that the various agents target different kinases in distinct diseases.
Analogous Mutations in Four Kinases Associated with Resistance to Kinase Inhibitors
In tumors from patients not treated with either gefitinib or erlotinib, the 2369 C→T mutation (T790M) appears to be extremely rare. We have not identified this mutation in 155 tumors (see above), and among nearly 1,300 lung cancers in which analysis of EGFR
exons 18 to 21 has been performed [1
], only one tumor (which also harbored an L858R mutation) was reported to contain the T790M mutation. Whether the patient from which this tumor was resected had received gefitinib or erlotinib is unclear, and the report did not note an association with acquired resistance to either drug [5
How tumor cells bearing the T790M mutation emerge within gefitinib- or erlotinib-treated patients is a matter of investigation. Subclones bearing this mutation could arise de novo during treatment.
However, based upon analogous studies in CML, it is also possible that NSCLC subclones bearing this secondary mutation pre-exist within the primary tumor clone in individual patients, albeit at low frequency [23
]. In either scenario, treatment with gefitinib or erlotinib subsequently allows these resistant subclones to become apparent, because most cells bearing sensitivity-conferring mutations die, while cells with the T790M mutation persist.
From analysis of the crystal structure of the EGFR kinase domain bound to erlotinib, it is has been shown that the wild-type threonine residue at position 790 is located in the hydrophobic ATP-binding pocket of the catalytic region, where it forms a critical hydrogen bond with the drug [24
]. The related compound, gefitinib, is predicted to interact with this threonine residue as well. Substitution of the threonine at position 790 by a larger residue like methionine would probably result in steric clash with the aromatic moieties on these two drugs [25
]. By contrast, ATP would likely not depend on the accessibility of the same hydrophobic cavity and is therefore probably not affected by the incorporation of a bulky methionine side chain [25
]. Consistent with this, the T790M mutation has been shown not to abrogate the catalytic activity of wild-type EGFR [22
The T790M mutation could also affect the kinase activity or alter the substrate specificity of mutant EGFRs, such that a proliferative advantage would be conferred upon cells bearing the mutation. Consistent with this, the H1975 NSCLC cell line reported here to contain both T790M and L858R did not to our knowledge undergo any prior treatment with gefitinib or erlotinib; the doubly mutated cells must have become dominant over time through multiple passages in vitro. This scenario could explain the seemingly contradictory report by others who found the H1975 cell line to be highly sensitive to gefitinib [18
]; our H1975 cells could represent a subclone that emerged over time. Analysis of earlier passages of H1975 cells for the T790M mutation would be informative in this regard.
Recently, new small-molecule inhibitors have been identified that retain activity against the majority of imatinib-resistant BCR-ABL mutants. The new drugs bind to ABL in an “open” conformation, as opposed to imatinib, which binds ABL in a “closed” conformation [12
]. Analogously, it may be possible to find EGFR tyrosine kinase inhibitors that bind to the EGFR kinase domain in different ways than gefitinib and erlotinib. For example, the crystal structure of another EGFR inhibitor, lapatinib (GW572016), was recently solved bound to EGFR [26
]. This study revealed that the quinazoline rings of erlotinib and lapatinib interact differently with the EGFR kinase domain, suggesting that while the T790M mutation may affect inhibition by erlotinib and gefitinib, it may not affect inhibition of EGFR by compounds similar to lapatinib. To our knowledge, no NSCLC patient who initially responded to but then progressed on either gefitinib or erlotinib has yet been treated with lapatinib.
In some of the patient specimens analyzed, the actual sequencing peaks demonstrating the T790M mutation were smaller than originally anticipated. These results differ from those of acquired resistance mutation in CML [10
], GIST [15
], and HES [16
]. However, in contrast to all of these diseases, in which tumor cells are readily accessible, lung-cancer-related tumors are more difficult to access, as illustrated by the limited manner in which we were able to obtain tumor cells from various sites of disease (see ). Moreover, re-biopsy of patients with lung cancer is not routinely performed. The use of position emission tomography scans to identify the most metabolically active lesions for biopsy could possibly circumvent this factor in the future, as long as such lesions are resectable. Additionally, as more molecularly tailored treatment options become available for lung cancer, re-biopsy of progressive sites of disease should become a standard procedure, especially for patients on clinical trials of targeted agents.
Since tumor specimens from three additional patients with acquired resistance to EGFR tyrosine kinase inhibitors did not demonstrate the T790M mutation, this specific lesion does not account for all mechanisms of acquired resistance to gefitinib or erlotinib. Given the paradigm established with imatinib, other drug-resistance mutations in EGFR,
either within or outside the tyrosine kinase domain, are likely to exist. It is also possible that EGFR
amplification itself plays a role in acquired resistance, since imatinib-resistant clones have been shown to lack resistance mutations but contain amplified copies of BCR-ABL
]. Nonetheless, studies presented here provide a basis for the rational development of “second generation” kinase inhibitors for use in NSCLC.