No abstract

Oncogenic BRAF mutations initiate tumor formation by unleashing the autoinhibited kinase conformation and promoting RAS-decoupled proliferative RAF-MEK-ERK signaling. We have engineered luciferase-based biosensors to systematically track full-length BRAF conformations and interactions affected by tumorigenic kinase mutations and GTP loading of RAS. Binding of structurally diverse αC-helix-OUT BRAF inhibitors (BRAFi) showed differences in specificity and efficacy by shifting patient mutation–containing BRAF reporters from the definitive opened to more closed conformations. Unexpectedly, BRAFi engagement with the catalytic pocket of V600E-mutated BRAF stabilized an intermediate and inactive kinase conformation that enhanced binary RAS:RAF interactions, also independently of RAF dimerization in melanoma cells. We present evidence that the interference with RAS interactions and nanoclustering antagonizes the sequential formation of drug-induced RAS:RAF tetramers. This suggests a previously unappreciated allosteric effect of anticancer drug-driven intramolecular communication between the kinase and RAS-binding domains of mutated BRAF, which may further promote paradoxical kinase activation and drug resistance mechanisms.

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.

Compartmentalization of diverse types of signaling molecules contributes to the precise coordination of signal propagation. The primary cilium fulfills this function by acting as a spatiotemporally confined sensory signaling platform. For the integrity of ciliary signaling, it is mandatory that the ciliary signaling pathways are constantly attuned by alterations in both oscillating small molecules and the presence or absence of their sensor/effector proteins. In this context, ciliary G protein-coupled receptor (GPCR) pathways participate in coordinating the mobilization of the diffusible second messenger molecule 3′,5′-cyclic adenosine monophosphate (cAMP). cAMP fluxes in the cilium are primarily sensed by protein kinase A (PKA) complexes, which are essential for the basal repression of Hedgehog (Hh) signaling. Here, we describe the dynamic properties of underlying signaling circuits, as well as strategies for second messenger compartmentalization. As an example, we summarize how receptor-guided cAMP-effector pathways control the off state of Hh signaling. We discuss the evidence that a macromolecular, ciliary-localized signaling complex, composed of the orphan GPCR Gpr161 and type I PKA holoenzymes, is involved in antagonizing Hh functions. Finally, we outline how ciliary cAMP-linked receptor pathways and cAMP-sensing signalosomes may become targets for more efficient combinatory therapy approaches to counteract dysregulation of Hh signaling.