Oncogenic mutations at the EGFR ectodomain structurally converge to remove a steric hindrance on a kinase-coupled cryptic epitope

Abstract

Epidermal growth factor receptor (EGFR) signaling is initiated by a large ligand-favored conformational change of the extracellular domain (ECD) from a closed, self-inhibited tethered monomer, to an open untethered state, which exposes a loop required for strong dimerization and activation. In glioblastomas (GBMs), structurally heterogeneous missense and deletion mutations concentrate at the ECD for unclear reasons. We explore the conformational impact of GBM missense mutations, combining elastic network models (ENMs) with multiple molecular dynamics (MD) trajectories. Our simulations reveal that the main missense class, located at the I-II interface away from the self-inhibitory tether, can unexpectedly favor spontaneous untethering to a compact intermediate state, here validated by smallangle X-ray scattering (SAXS). Significantly, such intermediate is characterized by the rotation of a large ECD fragment (N-TR1), deleted in the most common GBM mutation, EGFRvIII, and that makes accessible a cryptic epitope characteristic of cancer cells. This observation suggested potential structural equivalence of missense and deletion ECD changes in GBMs. Corroborating this hypothesis, our FACS, in vitro, and in vivo data demonstrate that entirely different ECD variants all converge to remove N-TR1 steric hindrance from the 806epitope, which we show is allosterically coupled to an intermediate kinase and hallmarks increased oncogenicity. Finally, the detected extraintracellular coupling allows for synergistic cotargeting of the intermediate with mAb806 and inhibitors, which is proved herein. cancer | mutational heterogeneity | structural convergence | intermediate | cryptoepitope

EGFR Oncogenes Expressed In Glioblastoma Are Activated As Covalent Dimers And Paradoxically Stimulated By Erlotinib

“…In EGFR-Viii Tyr270 of the YDK triad is lost, while for EGFR-Vii both Asp587 and Lys609 are lost, thereby preventing adoption of the inactive tethered position and promoting formation of an extended conformation that is poised for dimerization. Indeed, this mechanism has been described for several EGFR mutations expressed in GBM (21). Previous studies have also shown that the oncogenic activation of EGFR-Viii may be attributed to disulfide bond-mediated covalent dimerization of the extracellular domain (22,23).…”

“…Other studies have attributed the oncogenic activation of EGFR-Viii to the disruption of auto-inhibitory tethering of the extracellular domain, as key interactions at the CR1-CR2 contact point that control the inactive tethered conformation will be lost as a result of this truncation (21). Indeed, auto-inhibitory tethering is also likely disrupted for the EGFR-Vvi oncogene as the tethering contact point at the CR2 site is lost.…”

EGFRvIII: An Oncogene with Ambiguous Role

“…Hence, conclusions from studies on EGFR-TKIs/immunotherapies can only be drawn concerning this particular tumor. Finally, as Orellana et al recently suggested the high probability that mutated ectodomain of EGFR vIII induces structural changes in the intracellular kinase domain [183], further research focused on detailed understanding of molecular aspects of EGFR vIII should be expected. On the other hand, considering current standard therapeutic GB regimen, EGFR vIII is associated with prolonged survival of GB patients treated with surgery and radio/chemotherapy [192].…”

“…erefore, the verification whether glioblastoma patients with high frequency of EGFR mutations respond to TKIs is completely justified, even despite different EGFR mutational spectrum. is becomes even more important since Orellana et al showed that ectodomain EGFR mutations including those leading to EGFR vIII may sensitize tumor cells to tyrosine kinase inhibitors [183]. Reports from in vitro studies conducted on EGFR vIII -expressing cell lines tend to be contradictory.…”

Novel EGFR ectodomain mutations associated with ligand-independent activation and cetuximab resistance in head and neck cancer

“…Their contribution to HNSCC pathogenesis and therapy response has received little attention but could have therapeutic implications [7]. It has been demonstrated that EGFR ECD missense mutations can unexpectedly cause spontaneous receptor untethering that removes a restraint on RTK activation and that such mutants can be targeted by specific monoclonal antibodies (mAbs) [11].…”