Mutation-oriented profiling of autoinhibitory kinase conformations predicts RAF inhibitor efficacies
Related Experiment Video
Updated: Nov 29, 2025
Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods
Published on: July 17, 2019
Johanna E Mayrhofer1, Florian Enzler1,2, Andreas Feichtner1
1Institute of Biochemistry and Center for Molecular Biosciences, University of Innsbruck, 6020 Innsbruck, Austria.
Abstract:
Kinase-targeted therapies have the potential to improve the survival of patients with cancer. However, the cancer-specific spectrum of kinase alterations exhibits distinct functional properties and requires mutation-oriented drug treatments. Besides post-translational modifications and diverse intermolecular interactions of kinases, it is the distinct disease mutation which reshapes full-length kinase conformations, affecting their activity. Oncokinase mutation profiles differ between cancer types, as it was shown for BRAF in melanoma and non-small-cell lung cancers. Here, we present the target-oriented application of a kinase conformation (KinCon) reporter platform for live-cell measurements of autoinhibitory kinase activity states. The bioluminescence-based KinCon biosensor allows the tracking of conformation dynamics of full-length kinases in intact cells and real time. We show that the most frequent BRAF cancer mutations affect kinase conformations and thus the engagement and efficacy of V600E-specific BRAF inhibitors (BRAFi). We illustrate that the patient mutation harboring KinCon reporters display differences in the effectiveness of the three clinically approved BRAFi vemurafenib, encorafenib, and dabrafenib and the preclinical paradox breaker PLX8394. We confirmed KinCon-based drug efficacy predictions for BRAF mutations other than V600E in proliferation assays using patient-derived lung cancer cell lines and by analyzing downstream kinase signaling. The systematic implementation of such conformation reporters will allow to accelerate the decision process for the mutation-oriented RAF-kinase cancer therapy. Moreover, we illustrate that the presented kinase reporter concept can be extended to other kinases which harbor patient mutations. Overall, KinCon profiling provides additional mechanistic insights into full-length kinase functions by reporting protein-protein interaction (PPI)-dependent, mutation-specific, and drug-driven changes of kinase activity conformations.
Insights
A new KinCon biosensor tracks kinase activity in real-time, revealing how specific mutations impact cancer drug effectiveness. This technology aids in selecting personalized, mutation-oriented cancer therapies for improved patient outcomes.
Area of Science:
- Oncology
- Molecular Biology
- Pharmacology
Background:
- Kinase-targeted therapies offer promise for cancer treatment but require mutation-specific drug selection due to distinct kinase alterations.
- Cancer-driving mutations reshape kinase conformations, influencing their activity and response to inhibitors.
- Understanding these mutation-specific conformational changes is crucial for effective, personalized cancer therapy.
Purpose of the Study:
- To introduce and validate the kinase conformation (KinCon) reporter platform for real-time, live-cell measurement of kinase activity states.
- To investigate the impact of common BRAF cancer mutations on kinase conformations and their response to BRAF inhibitors (BRAFi).
- To demonstrate the utility of KinCon reporters in predicting drug efficacy for various BRAF mutations.
Main Methods:
- Development of a bioluminescence-based KinCon biosensor for tracking full-length kinase conformation dynamics in intact cells.
- Application of KinCon reporters to measure the effects of patient-derived BRAF mutations on kinase activity and drug engagement.
- Validation of KinCon-based drug efficacy predictions using proliferation assays and downstream signaling analysis in patient-derived lung cancer cell lines.
Main Results:
- KinCon reporters successfully tracked real-time kinase conformation dynamics, revealing mutation-specific alterations in kinase activity.
- The efficacy of clinically approved BRAFi (vemurafenib, encorafenib, dabrafenib) and a preclinical compound (PLX8394) varied significantly based on patient BRAF mutations.
- KinCon-based predictions of drug efficacy for non-V600E BRAF mutations were confirmed in proliferation assays and signaling analyses.
Conclusions:
- The KinCon reporter platform enables live-cell, real-time assessment of kinase conformation states and their response to targeted therapies.
- KinCon profiling provides mechanistic insights into mutation-specific kinase behavior and drug interactions, facilitating personalized cancer treatment decisions.
- The KinCon reporter concept is extendable to other kinases, offering a versatile tool for advancing mutation-oriented cancer therapy.
Related Concept Videos
01:07MAPK Signaling Cascades
02:34Inhibition of Cdk Activity
01:22PI3K/mTOR/AKT Signaling Pathway