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Updated: Feb 26, 2026
Identification of Kinase-substrate Pairs Using High Throughput Screening
Published on: August 29, 2015
Identification and Validation of Driver Kinases from Next-Generation Sequencing Data
Andri Leonidou1,2, Barrie Peck1,2, Rachael Natrajan1,2
1Division of Breast Cancer, The Institute of Cancer Research, 123 Old Brompton Road, London, SW7 3RP, UK.
Abstract:
It is well appreciated that activating mutations in kinase genes result in kinome reprogramming that leads to altered downstream signaling networks that drive tumor progression. Indeed small-molecule inhibition of activated kinases has heralded the wave of precision medicine in the past decade. The advent of next-generation sequencing has identified a plethora of potentially activating mutations and fusion genes in previously unreported kinase genes that can potentially be developed as targeted therapies. However, the bottleneck in the translation of these alterations into clinically useful therapies lies in their functional validation. Here we describe a set of in vitro functional assays we have optimized to assess whether mutations in kinases are activating. Through overexpression of wild-type and mutant kinase cDNA constructs, we described growth assays in two and three dimensions to ascribe functionality using breast cancer as a model system.
Insights
This study presents new in vitro assays to functionally validate activating mutations in kinase genes. These methods help translate genetic discoveries into targeted cancer therapies.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Activating mutations in kinase genes reprogram cellular signaling, driving tumor progression.
- Targeted therapies, like small-molecule kinase inhibitors, are crucial in precision oncology.
- Next-generation sequencing identifies numerous novel kinase mutations and fusions with therapeutic potential.
Purpose of the Study:
- To develop and optimize in vitro functional assays for validating kinase mutations.
- To assess the activating potential of identified kinase alterations.
- To facilitate the translation of genetic findings into targeted cancer treatments.
Main Methods:
- Overexpression of wild-type and mutant kinase cDNA constructs.
- Development of 2D and 3D growth assays.
- Utilizing breast cancer as a model system for functional validation.
Main Results:
- Established a set of optimized in vitro assays to determine if kinase mutations are activating.
- Demonstrated the utility of these assays in assessing kinase functionality.
- Provided a framework for functional validation of kinase alterations in cancer.
Conclusions:
- Optimized in vitro assays are essential for validating kinase mutations.
- Functional assessment is a critical bottleneck in developing targeted kinase therapies.
- These assays can accelerate the development of precision medicines for cancer treatment.
