A driving test for oncogenic mutations

Abstract

Activating mutations in protein kinases are a frequent cause of cancer, and selecting drugs that act on these oncogenic kinases can lead to effective therapies. Targeted or whole-genome sequencing of tumor samples can readily reveal the presence of mutations, but discerning previously uncharacterized activating "driver" mutations that will respond to drug treatment from much more abundant but inconsequential "passenger" mutations is problematic. Chakroborty et al. apply a screening approach that leverages error-prone PCR and a proliferating cell model to identify such gain-of-function mutants in the epidermal growth factor receptor (EGFR) kinase. The screen is validated by the identification of known cancer-promoting mutations and reveals a previously unappreciated oncogenic EGFR mutation, A702V, demonstrating its power for discovery of driver mutations.

Figure 1.

Identification of novel EGFR-activating mutations. A, screening workflow for identifying EGFR activating mutations. Single-nucleotide variants are generated by amplifying WT EGFR with error-prone PCR followed by cloning and retroviral transduction of the random DNA mutations into Ba/F3 cells. Activating mutations are selected by withdrawing IL-3, and surviving cells are sequenced. This method revealed well-known EGFR mutations as well as a novel A702V activating mutation postulated to reinforce the active asymmetric EGFR kinase dimer. B, molecular model generated with active EGFR crystal structures (Protein Data Bank code 2GS6) shows activator (yellow) and receiver (blue; αC-helix in red) kinase domains and the A702V site highlighted in spheres. The inset shows the proximity of A702V to hydrophobic residues (Ile-941 of activator and Ala-767 from αC-helix of the receiver) enabling more favorable binding of the two kinase domains. C, the A702V mutation is modeled in the crystal structure of a symmetric, inactive EGFR kinase dimer (Protein Data Bank code 3GT8). The two kinase domains are shown in blue and yellow, and the inset shows how the A702V mutation may introduce steric clashes with the C-terminal helix of the opposing kinase domain that weaken the inactive dimer.