From the Cover: PNAS Plus: Mechanism for activation of mutated epidermal growth factor receptors in lung cancer

The initiation of epidermal growth factor receptor (EGFR) kinase activity proceeds via an asymmetric dimerization mechanism in which a “donor” tyrosine kinase domain (TKD) contacts an “acceptor” TKD, leading to its activation. In the context of a ligand-induced dimer, identical wild-type EGFR TKDs are thought to assume the donor or acceptor roles in a random manner. Here, we present biochemical reconstitution data demonstrating that activated EGFR mutants found in lung cancer preferentially assume the acceptor role when coexpressed with WT EGFR. Mutated EGFRs show enhanced association with WT EGFR, leading to hyperphosphorylation of the WT counterpart. Mutated EGFRs also hyperphosphorylate the related erythroblastic leukemia viral oncogene (ErbB) family member, ErbB-2, in a similar manner. This directional “superacceptor activity” is particularly pronounced in the drug-resistant L834R/T766M mutant. A 4-Å crystal structure of this mutant in the active conformation reveals an asymmetric dimer interface that is essentially the same as that in WT EGFR. Asymmetric dimer formation induces an allosteric conformational change in the acceptor subunit. Thus, superacceptor activity likely arises simply from a lower energetic cost associated with this conformational change in the mutant EGFR compared with WT, rather than from any structural alteration that impairs the donor role of the mutant. Collectively, these findings define a previously unrecognized mode of mutant-specific intermolecular regulation for ErbB receptors, knowledge of which could potentially be exploited for therapeutic benefit.The gene encoding the epidermal growth factor receptor (EGFR) tyrosine kinase is somatically mutated in a substantial fraction of patients with lung cancer. The majority of primary activating EGFR mutations occur within the tyrosine kinase domain (TKD). The most frequent of these, which occur with a combined frequency of 90% (1), are exon 19 deletions that eliminate four amino acids (LREA) from the TKD and exon 21 missense mutations that substitute arginine for leucine at position 834 (L834R) (also identified as L858R in an alternative numbering of the human EGFR sequence that includes the 24 residue signal sequence) (2).Exon 19 deletions and L834R substitutions are associated with increased sensitivity to EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, translating to a 70% radiographic response rate in patients (3–5). Unfortunately, all individuals with metastatic disease eventually develop progressive disease after 10–16 mo of treatment with EGFR TKIs. The most common mechanism of acquired resistance is mutation at a second site in the EGFR TKD (the gatekeeper residue), T766M (T790M). This mutation confers resistance by increasing affinity for ATP, with which inhibitors must compete for binding, and also by modestly decreasing intrinsic affinity for TKIs (6).Biochemical and crystallographic studies have shown that activation of the wild-type (WT) EGFR TKD involves formation of an asymmetric dimer in which one molecule allosterically activates its neighbor by promoting the reversal of intramolecular autoinhibitory interactions—acting as a “donor” or “activator” TKD that activates the “acceptor” or “receiver” TKD (7, 8). Crystal structures of individual L834R and T766M EGFR-TKD mutants show that these variants also form asymmetric dimers (6, 9), but whether the double mutant L834R/T766M adheres to the same configuration in the active state is unclear. Biochemical data indicate that the oligomerization potential of mutated EGFRs is enhanced relative to WT. For example, native gel and multiangle light scattering studies showed that the L834R substitution promotes formation of dimers and higher order oligomers of the EGFR TKD (10). Consistent with this observation, cell-based studies have demonstrated a reduced dependence on ligand stimulation for activation of mutated EGFRs. All mutated EGFR TKDs seen in lung cancer show an increase in catalytic efficiency over WT (6, 9, 11, 12). Interestingly, the doubly mutated L834R/T766M EGFR TKD has a two-to fivefold higher catalytic efficiency (k_cat/K_m) than either the singly mutated L834R or T766M mutant TKDs (6).Here, using a biochemical and structural approach, we sought to determine whether mutated EGFRs most commonly associated with primary drug sensitivity and acquired resistance in lung cancer adhere to the well-established asymmetric dimer model. Our biochemical reconstitution data demonstrate that these EGFR mutations do in fact adhere to this model, but with the striking difference that the oncogenic mutants preferentially assume the receiver role when expressed together with WT EGFR. Our crystal structure of active EGFR-L834R/T766M reveals an asymmetric dimer interface essentially the same as that in WT EGFR. We further show that mutated EGFRs can hyperphosphorylate WT EGFR as well as the related family member erythroblastic leukemia viral oncogene (ErbB)2 as a result of their preferential adoption of the acceptor or receiver position in the asymmetric dimer. These findings have important implications for understanding mechanisms of drug sensitivity and resistance in EGFR mutant lung cancers and shed light on the distinct properties of different mutated forms of EGFR.