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.

Resistor: an algorithm for predicting resistance mutations using Pareto optimization over multistate protein design and mutational signatures

“…The full sets of predictions are included in the supplementary tables S4-S7. To validate R esistor ’s predictions, we compared its predictions with two sources of experimental data: a saturation mutagenesis variant effect assay from Wagenaar et al [61] and a cell-based kinase conformation reporter assay termed KinCon by Stefan and colleagues [62, 63]. Furthermore we carried out new KinCon experiments on a number of R esistor predictions to validate R esistor ’s predictive capabilities.…”

Allosteric Kinase Inhibitors Reshape MEK1 Kinase Activity Conformations in Cells and In Silico

“…KinCon reporter: Following PCR amplification of the human MEK1 gene (MEK1: NM_002755.3), we fused it N-terminally with -F[1] and C-terminally with -F[2] of the R luc -PCA (pcDNA3.1 backbone vector) as previously described [ 27 , 28 ]. We inserted interjacent 10-aa standard linkers as previously described.…”

“…This kinase conformation reporter system, which we have named KinCon, can be extended to a collection of full-length kinases for tracking their different kinase activity states [ 26 ]. In the proof-of-concept studies, we have shown that the engineered luciferase-based biosensors can be used to systematically track mutation and drug-driven changes of RAF kinase conformations in intact cell settings [ 26 , 27 , 28 ]. It is of interest that MEK kinases are smaller in size, when compared to RAF, but still they contain a N-terminal “negative regulatory region” which participates in stabilizing an inactive kinase conformation [ 8 ].…”

“…It is of interest that MEK kinases are smaller in size, when compared to RAF, but still they contain a N-terminal “negative regulatory region” which participates in stabilizing an inactive kinase conformation [ 8 ]. Recently we showed that very rare MEK1 patient mutations were sufficient to alter MEK1 kinase conformation states [ 27 ]. Here, we show that overexpression of active BRAF variants and the integrations of the phosphorylation-mimetic mutations S218D and S222D convert the KinCon biosensor to a more opened conformation.…”

Tracking mutation and drug-driven alterations of oncokinase conformations

“…In this context it is of interest that many kinases share a similar concept for kinase regulation. This involves so-called intramolecularly acting cis-regulatory elements (CREs) or auto-inhibitory modules (AIM) which control substrate access and kinase activity states [30][31][32][33]. The kinase OFF state is characterized by interaction and association of the CRE with the catalytic cleft of the kinase domain.…”