Identification of Activating Mutations in the Transmembrane and Extracellular Domains of EGFR

The epidermal growth factor receptor (EGFR) is frequently
mutated
in human cancer, most notably non-small-cell lung cancer and glioblastoma.
While many frequently occurring EGFR mutations are known to confer
constitutive EGFR activation, the situation is less clear for rarely
detected variants. In fact, more than 1000 distinct EGFR mutations
are listed in the Catalogue of Somatic Mutations in Cancer (COSMIC),
but for most of them, the functional consequence is unknown. To identify
additional, previously unknown activating mutations in EGFR, we screened
a randomly mutated EGFR library for constitutive EGFR phosphorylation
using a recently developed high-throughput approach termed PhosphoFlowSeq.
Enrichment of the well-known activating mutations S768I, T790M, and
L858R validated the experimental approach. Importantly, we also identified
the activating mutations S442I and L658Q located in the extracellular
and transmembrane domains of EGFR, respectively. To the best of our
knowledge, neither S442I nor L658Q has been associated with an activating
phenotype before. However, both have been detected in cancer samples.
Interestingly, molecular dynamics (MD) simulations suggest that the
L658Q mutation located in the hydrophobic transmembrane region forms
intermolecular hydrogen bonds, thereby promoting EGFR dimerization
and activation. Based on these findings, we screened the COSMIC database
for additional hydrophilic mutations in the EGFR transmembrane region
and indeed detected moderate constitutive activation of EGFR-G652R.
Together, this study demonstrates that unbiased screening for activating
mutations in EGFR not only yields well-established substitutions located
in the kinase domain but also activating mutations in other regions
of EGFR, including the extracellular and transmembrane domains.