Mutation-oriented profiling of autoinhibitory kinase conformations predicts RAF inhibitor efficacies.

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.

• Authors: Johanna E Mayrhofer, Florian Enzler, Andreas Feichtner, Ruth Röck, Jakob Fleischmann, Andrea Raffeiner, Philipp Tschaikner, Egon Ogris, Roland G Huber, Markus Hartl, Rainer Schneider, Jakob Troppmair, Omar Torres-Quesada, Eduard Stefan
• Journal: Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci U S A) Vol. 117 Issue 49 Pg. 31105-31113 (12 08 2020) ISSN: 1091-6490 [Electronic] United States
• PMID: 33229534 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2020 the Author(s). Published by PNAS.

Chemical References

• Carbamates
• Heterocyclic Compounds, 2-Ring
• Imidazoles
• Oximes
• PLX8394
• Protein Kinase Inhibitors
• Sulfonamides
• Vemurafenib
• encorafenib
• Phosphotransferases
• BRAF protein, human
• Proto-Oncogene Proteins B-raf
• dabrafenib

Topics

• A549 Cells
• Carbamates (chemistry, pharmacology)
• Heterocyclic Compounds, 2-Ring (pharmacology)
• Humans
• Imidazoles (chemistry, pharmacology)
• Lung Neoplasms (drug therapy, genetics, pathology)
• Mutation (drug effects)
• Oximes (chemistry, pharmacology)
• Phosphotransferases (antagonists & inhibitors, ultrastructure)
• Protein Conformation (drug effects)
• Protein Kinase Inhibitors (chemistry, pharmacology)
• Protein Processing, Post-Translational (drug effects, genetics)
• Proto-Oncogene Proteins B-raf (antagonists & inhibitors, genetics, ultrastructure)
• Sulfonamides (chemistry, pharmacology)
• Vemurafenib (chemistry, pharmacology)