An unbiased in vitro screen for activating epidermal growth factor receptor mutations
Chakroborty D; Jänne PA; Paatero I; Elenius K; Koivu M; Tamirat MZ; Ojala VK; Johnson MS; Kurppa KJ; Elo LL; Koivunen JP
An unbiased in vitro screen for activating epidermal growth factor receptor mutations
American Society for Biochemistry and Molecular Biology
Julkaisun pysyvä osoite on:
https://urn.fi/URN:NBN:fi-fe2021042821145
https://urn.fi/URN:NBN:fi-fe2021042821145
Tiivistelmä
Cancer tissues harbor thousands of mutations, and a given oncogene may
be mutated at hundreds of sites. Yet, only a few of these mutations have
been functionally tested. Here, we describe an unbiased platform for
the functional characterization of thousands of variants of a single
receptor tyrosine kinase (RTK) gene in a single assay. Our in vitro screen for activating mutations
(iSCREAM) platform enabled rapid analysis of mutations conferring
gain-of-function RTK activity promoting clonal growth. The screening
strategy included a somatic model of cancer evolution and utilized a
library of 7,216 randomly mutated epidermal growth factor receptor
(EGFR) single-nucleotide variants, that were tested in murine lymphoid
Ba/F3 cells. These cells depend on exogenous interleukin-3 (IL-3) for
growth, but this dependency can be compensated by ectopic EGFR
overexpression, enabling selection for gain-of-function EGFR
mutants. Analysis of the enriched mutants revealed EGFR A702V, a novel
activating variant that structurally stabilized the EGFR kinase dimer
interface and conferred sensitivity to kinase inhibition by afatinib. As
proof of concept for our approach, we recapitulated clinical
observations and identified the EGFR L858R as the major enriched EGFR
variant. Altogether iSCREAM enabled robust enrichment of 21 variants
from a total of 7,216 EGFR mutations. These findings indicate the
power of this screening platform for unbiased identification of
activating RTK variants that are enriched under selection pressure in a
model of cancer heterogeneity and evolution.
be mutated at hundreds of sites. Yet, only a few of these mutations have
been functionally tested. Here, we describe an unbiased platform for
the functional characterization of thousands of variants of a single
receptor tyrosine kinase (RTK) gene in a single assay. Our in vitro screen for activating mutations
(iSCREAM) platform enabled rapid analysis of mutations conferring
gain-of-function RTK activity promoting clonal growth. The screening
strategy included a somatic model of cancer evolution and utilized a
library of 7,216 randomly mutated epidermal growth factor receptor
(EGFR) single-nucleotide variants, that were tested in murine lymphoid
Ba/F3 cells. These cells depend on exogenous interleukin-3 (IL-3) for
growth, but this dependency can be compensated by ectopic EGFR
overexpression, enabling selection for gain-of-function EGFR
mutants. Analysis of the enriched mutants revealed EGFR A702V, a novel
activating variant that structurally stabilized the EGFR kinase dimer
interface and conferred sensitivity to kinase inhibition by afatinib. As
proof of concept for our approach, we recapitulated clinical
observations and identified the EGFR L858R as the major enriched EGFR
variant. Altogether iSCREAM enabled robust enrichment of 21 variants
from a total of 7,216 EGFR mutations. These findings indicate the
power of this screening platform for unbiased identification of
activating RTK variants that are enriched under selection pressure in a
model of cancer heterogeneity and evolution.
Kokoelmat
- Rinnakkaistallenteet [19207]