Analyses of the oncogenic BRAFD594G variant reveal a kinase-independent function of BRAF in activating MAPK signaling

Abstract

Class 3 mutations in B-Raf proto-oncogene, Ser/Thr kinase (BRAF), that result in kinase-impaired or kinase-dead BRAF have the highest mutation frequency in BRAF gene in lung adenocarcinoma. Several studies have reported that kinase-dead BRAF variants amplify mitogen-activated protein kinase (MAPK) signaling by dimerizing with and activating WT C-Raf proto-oncogene, Ser/Thr kinase (CRAF). However, the structural and functional principles underlying their activation remain elusive. Herein, using cell biology and various biochemical approaches, we established that variant BRAFD594G, a kinase-dead representative of class 3 mutation-derived BRAF variants, has a higher dimerization potential as compared with WT BRAF. Molecular dynamics simulations uncovered that the D594G substitution orients the αC-helix toward the IN position and extends the activation loop within the kinase domain, shifting the equilibrium toward the active, dimeric conformation, thus priming BRAFD594G as an effective allosteric activator of CRAF. We found that B/CRAF heterodimers are the most thermodynamically stable RAF dimers, suggesting that RAF heterodimers, and not homodimers, are the major players in determining the amplitude of MAPK signaling in cells. Additionally, we show that BRAFD594G:CRAF heterodimers bypass autoinhibitory P-loop phosphorylation, which might contribute to longer duration of MAPK pathway signaling in cancer cells. Last, we propose that the dimer interface of the BRAFD594G:CRAF heterodimer may represent a promising target in the design of novel anticancer therapeutics.

Drosophila RASopathy models identify disease subtype differences and biomarkers of drug efficacy

“…Of note, some variants act as inactivating mutations but still give rise to overlapping patient outcomes ( Figure S1 ); this is consistent with previous studies that suggest these alleles can direct elevated pathway activity in a context-dependent fashion (e.g. ( Oishi et al., 2006 ; Pandit et al., 2007 ; Machado et al., 2017 ; Li et al., 2018 ; Cope et al., 2020 ; Lorca et al., 2020 ; Lu et al., 2020 )). Using standard transgenic technology ( Venken and Bellen, 2012 ), each transgene was inserted into the same genomic site to reduce expression differences due to insertion site.…”

Retrospective Case Series Analysis of RAF Family Alterations in Pancreatic Cancer: Real-World Outcomes From Targeted and Standard Therapies

“…One notable exception to these subgroup definitions is for the class 3 kinase-dead BRAF D594G variant, which does not confer similar actionability as the class 1 BRAF V600E despite its position nearby within Exon 15. 39 , 40 This distinction is important because these BRAF variants are considered RAS -dependent and enriched for co-occurrence with KRAS mutations. In the Clinical Cohort, all but two of the BRAF short variants identified in protein-coding regions outside of Exons 11 and 15 were found alongside a confounding driver (Fig 1 A).…”

Recent Developments in Targeting RAS Downstream Effectors for RAS-Driven Cancer Therapy

“…[81]. In a subsequent study, Raf dimer breaker has been shown to be active against oncogenic B-Raf D594G :C-Raf dimers [82], thus providing further evidence that allosteric type IV inhibitors targeting the Raf dimer interface have potential to be developed as an anticancer drug.…”