An unbiased in vitro screen for activating epidermal growth factor receptor mutations

Area of Science:

• Oncology
• Molecular Biology
• Genetics

Background:

• Cancer genomes contain numerous mutations, but functional characterization of oncogenic variants, particularly in receptor tyrosine kinases (RTKs), remains a challenge.
• Identifying gain-of-function mutations is crucial for understanding cancer evolution and developing targeted therapies.

Purpose of the Study:

• To develop and validate an unbiased platform, iSCREAM (screen for activating mutations), for high-throughput functional characterization of RTK variants.
• To identify novel activating mutations in the epidermal growth factor receptor (EGFR) gene using this platform.

Main Methods:

• Developed the iSCREAM platform for unbiased, single-assay functional characterization of thousands of gene variants.
• Utilized a library of 7,216 randomly mutated EGFR single-nucleotide variants tested in Ba/F3 cells dependent on interleukin-3 (IL-3) for growth.
• Employed a somatic cancer evolution model with selection for gain-of-function EGFR mutants that confer IL-3 independence.

Main Results:

• The iSCREAM platform successfully enriched 21 activating EGFR variants from 7,216 tested mutations.
• Identified EGFR A702V as a novel activating variant that stabilizes the kinase dimer interface and is sensitive to afatinib.
• Validated the platform's efficacy by recapitulating known activating mutations, including EGFR L858R.

Conclusions:

• The iSCREAM platform provides a powerful tool for unbiased identification of activating RTK variants under selection pressure.
• This approach facilitates the discovery of novel oncogenic mutations within a model of cancer heterogeneity and evolution.
• Findings highlight the potential of iSCREAM for accelerating the functional annotation of cancer-associated genetic variants.