An unbiased in vitro screen for activating epidermal growth factor receptor mutations |
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Author: | Chakroborty, Deepankar1,2,3; Kurppa, Kari J.1,4; Paatero, Ilkka3; |
Organizations: |
1Institute of Biomedicine and Medicity Research Laboratories, University of Turku, Turku 20520, Finland 2Turku Doctoral Programme of Molecular Medicine, University of Turku, Turku 20520, Finland 3Turku Center for Biotechnology, University of Turku and Åbo Akademi University, Turku 20520, Finland
4Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215
5Structural Bioinformatics Laboratory, Biochemistry, Faculty of Science and Engineering, Åbo Akademi University, 20500 Turku, Finland 6Department of Oncology and Radiotherapy, Oulu University Hospital and MRC Oulu, Oulu 90220, Finland 7Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215 8urku Center for Biotechnology, University of Turku and Åbo Akademi University, Turku 20520, Finland 9Department of Oncology, Turku University Hospital, Turku 20521, Finland |
Format: | article |
Version: | published version |
Access: | open |
Online Access: | PDF Full Text (PDF, 4 MB) |
Persistent link: | http://urn.fi/urn:nbn:fi-fe202003097621 |
Language: | English |
Published: |
American Society for Biochemistry and Molecular Biology,
2019
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Publish Date: | 2020-04-05 |
Description: |
AbstractCancer 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 dependence 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. see all
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Series: |
Journal of biological chemistry |
ISSN: | 0021-9258 |
ISSN-E: | 1083-351X |
ISSN-L: | 0021-9258 |
Volume: | 294 |
Issue: | 24 |
Pages: | 9377 - 9389 |
DOI: | 10.1074/jbc.RA118.006336 |
OADOI: | https://oadoi.org/10.1074/jbc.RA118.006336 |
Type of Publication: |
A1 Journal article – refereed |
Field of Science: |
3122 Cancers 1182 Biochemistry, cell and molecular biology |
Subjects: | |
Funding: |
Supported by the University of Turku and University of Turku Graduate School (Turku Doctoral Program in Molecular Medicine). Supported by the University of Turku and the Finnish Cultural Foundation. Supported by the Graduate School of Åbo Akademi University (Informational and Structural Biology Doctoral Network). Supported by the Joe, Pentti, and Tor Borg Memorial Fund and Sigrid Jusélius Foundation. Supported by the European Research Council, Academy of Finland, and Sigrid Jusélius Foundation. P. A. Jänne has received consulting fees from AstraZeneca, Boehringer Ingelheim, Pfizer, Merrimack Pharmaceuticals, Roche/Genentech, Chugai Pharmaceuticals, ACEA Biosciences, and Ariad Pharmaceuticals and sponsored research funding from Astellas Pharmaceuticals, AstraZeneca, Daiichi Sankyo, and PUMA and receives post-marketing royalties on DFCI-owned intellectual property on EGFR mutations licensed to Lab Corp. K. Elenius has a research agreement with Boehringer Ingelheim and ownership interest in Abomics, Orion, and Roche. |
Copyright information: |
© 2019 Chakroborty et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc. |