Dataset Information

BRAF inhibitors promote intermediate BRAF(V600E) conformations and binary interactions with activated RAS.

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 ?C-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.

SUBMITTER: Rock R

PROVIDER: S-EPMC6693913 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

BRAF inhibitors promote intermediate BRAF(V600E) conformations and binary interactions with activated RAS.

Röck Ruth R Mayrhofer Johanna E JE Torres-Quesada Omar O Enzler Florian F Raffeiner Andrea A Raffeiner Philipp P Feichtner Andreas A Huber Roland G RG Koide Shohei S Taylor Susan S SS Troppmair Jakob J Stefan Eduard E

Science advances 20190814 8

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-cont ...[more]

PMID: 31453322

Similar Datasets

Project description:BackgroundMost mutations in melanoma affect one critical amino acid on BRAF gene, resulting in the V600E substitution. Patient management is often based on the use of specific inhibitors targeting this mutation.MethodsDNA and RNA mutation status was assessed in 15 melanoma cell lines by Sanger sequencing and RNA-seq. We tested the cell lines responsiveness to BRAF inhibitors (vemurafenib and PLX4720, BRAF-specific and sorafenib, BRAF non-specific). Cell proliferation was assessed by MTT colorimetric assay. BRAF V600E RNA expression was assessed by qPCR. Expression level of phosphorylated-ERK protein was assessed by Western Blotting as marker of BRAF activation.ResultsThree cell lines were discordant in the mutation detection (BRAF V600E at DNA level/Sanger sequencing and BRAF WT on RNA-seq). We initially postulated that those cell lines may express only the WT allele at the RNA level although mutated at the DNA level. A more careful analysis showed that they express low level of BRAF RNA and the expression may be in favor of the WT allele. We tested whether the discordant cell lines responded differently to BRAF-specific inhibitors. Their proliferation rate decreased after treatment with vemurafenib and PLX4720 but was not affected by sorafenib, suggesting a BRAF V600E biological behavior. Yet, responsiveness to the BRAF specific inhibitors was lower as compared to the control. Western Blot analysis revealed a decreased expression of p-ERK protein in the BRAF V600E control cell line and in the discordant cell lines upon treatment with BRAF-specific inhibitors. The discordant cell lines showed a lower responsiveness to BRAF inhibitors when compared to the BRAF V600E control cell line. The results obtained from the inhibition experiment and molecular analyses were also confirmed in three additional cell lines.ConclusionCell lines carrying V600E mutation at the DNA level may respond differently to BRAF targeted treatment potentially due to a lower V600E RNA expression.

Dataset Information

BRAF inhibitors promote intermediate BRAF(V600E) conformations and binary interactions with activated RAS.

Publications

BRAF inhibitors promote intermediate BRAF(V600E) conformations and binary interactions with activated RAS.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets