Regulation of RAF family kinases: new insights from recent structural and biochemical studies.

The RAF kinases are required for signal transduction through the RAS-RAF-MEK-ERK pathway, and their activity is frequently up-regulated in human cancer and the RASopathy developmental syndromes. Due to their complex activation process, developing drugs that effectively target RAF function has been a challenging endeavor, highlighting the need for a more detailed understanding of RAF regulation. This review will focus on recent structural and biochemical studies that have provided 'snapshots' into the RAF regulatory cycle, revealing structures of the autoinhibited BRAF monomer, active BRAF and CRAF homodimers, as well as HSP90/CDC37 chaperone complexes containing CRAF or BRAFV600E. In addition, we will describe the insights obtained regarding how BRAF transitions between its regulatory states and examine the roles that various BRAF domains and 14-3-3 dimers play in both maintaining BRAF as an autoinhibited monomer and in facilitating its transition to an active dimer. We will also address the function of the HSP90/CDC37 chaperone complex in stabilizing the protein levels of CRAF and certain oncogenic BRAF mutants, and in serving as a platform for RAF dephosphorylation mediated by the PP5 protein phosphatase. Finally, we will discuss the regulatory differences observed between BRAF and CRAF and how these differences impact the function of BRAF and CRAF as drivers of human disease.

Bioluminescence Resonance Energy Transfer (BRET)-based Assay for Measuring Interactions of CRAF with 14-3-3 Proteins in Live Cells.

CRAF is a primary effector of RAS GTPases and plays a critical role in the tumorigenesis of several KRAS-driven cancers. In addition, CRAF is a hotspot for germline mutations, which are shown to cause the developmental RASopathy, Noonan syndrome. All RAF kinases contain multiple phosphorylation-dependent binding sites for 14-3-3 regulatory proteins. The differential binding of 14-3-3 to these sites plays essential roles in the formation of active RAF dimers at the plasma membrane under signaling conditions and in maintaining RAF autoinhibition under quiescent conditions. Understanding how these interactions are regulated and how they can be modulated is critical for identifying new therapeutic approaches that target RAF function. Here, I describe a bioluminescence resonance energy transfer (BRET)-based assay for measuring the interactions of CRAF with 14-3-3 proteins in live cells. Specifically, this assay measures the interactions of CRAF fused to a Nano luciferase donor and 14-3-3 fused to a Halo tag acceptor, where the interaction of RAF and 14-3-3 results in donor-to-acceptor energy transfer and the generation of the BRET signal. The protocol further shows that this signal can be disrupted by mutations shown to prevent 14-3-3 binding to each of its high-affinity RAF docking sites. This protocol describes the procedures for seeding, transfecting, and replating the cells, along with detailed instructions for reading BRET emissions, performing data analysis, and confirming protein expression levels. In addition, example assay results, along with optimization and troubleshooting steps, are provided.

Protocol for measuring BRAF autoinhibition in live cells using a proximity-based NanoBRET assay.

BRAF is frequently activated via mutation in human cancer and the RASopathy syndromes; however, for BRAF activation to occur, autoinhibitory interactions between the regulatory and catalytic domains must be relieved. Here, we present a proximity-based NanoBRET (bioluminescence resonance energy transfer) assay for real-time measurement of BRAF autoinhibition in live cells. We describe steps for seeding, transfecting, and replating cells. We then detail procedures for reading the NanoBRET emissions and confirming protein expression. For complete details on the use and execution of this protocol, please refer to Spencer-Smith et al. (2022).1.

RASopathy mutations provide functional insight into the BRAF cysteine-rich domain and reveal the importance of autoinhibition in BRAF regulation.

BRAF is frequently mutated in human cancer and the RASopathy syndromes, with RASopathy mutations often observed in the cysteine-rich domain (CRD). Although the CRD participates in phosphatidylserine (PS) binding, the RAS-RAF interaction, and RAF autoinhibition, the impact of these activities on RAF function in normal and disease states is not well characterized. Here, we analyze a panel of CRD mutations and show that they increase BRAF activity by relieving autoinhibition and/or enhancing PS binding, with relief of autoinhibition being the major factor determining mutation severity. Further, we show that CRD-mediated autoinhibition prevents the constitutive plasma membrane localization of BRAF that causes increased RAS-dependent and RAS-independent function. Comparison of the BRAF- and CRAF-CRDs also indicates that the BRAF-CRD is a stronger mediator of autoinhibition and PS binding, and given the increased catalytic activity of BRAF, our studies reveal a more critical role for CRD-mediated autoinhibition in BRAF regulation.

Identification of the nucleotide-free state as a therapeutic vulnerability for inhibition of selected oncogenic RAS mutants.

RAS guanosine triphosphatases (GTPases) are mutated in nearly 20% of human tumors, making them an attractive therapeutic target. Following our discovery that nucleotide-free RAS (apo RAS) regulates cell signaling, we selectively target this state as an approach to inhibit RAS function. Here, we describe the R15 monobody that exclusively binds the apo state of all three RAS isoforms in vitro, regardless of the mutation status, and captures RAS in the apo state in cells. R15 inhibits the signaling and transforming activity of a subset of RAS mutants with elevated intrinsic nucleotide exchange rates (i.e., fast exchange mutants). Intracellular expression of R15 reduces the tumor-forming capacity of cancer cell lines driven by select RAS mutants and KRAS(G12D)-mutant patient-derived xenografts (PDXs). Thus, our approach establishes an opportunity to selectively inhibit a subset of RAS mutants by targeting the apo state with drug-like molecules.

Distinct Binding Preferences between Ras and Raf Family Members and the Impact on Oncogenic Ras Signaling.

The Ras GTPases are frequently mutated in human cancer, and, although the Raf kinases are essential effectors of Ras signaling, the tumorigenic properties of specific Ras-Raf complexes are not well characterized. Here, we examine the ability of individual Ras and Raf proteins to interact in live cells using bioluminescence resonance energy transfer (BRET) technology. We find that C-Raf binds all mutant Ras proteins with high affinity, whereas B-Raf exhibits a striking preference for mutant K-Ras. This selectivity is mediated by the acidic, N-terminal segment of B-Raf and requires the K-Ras polybasic region for high-affinity binding. In addition, we find that C-Raf is critical for mutant H-Ras-driven signaling and that events stabilizing B-Raf/C-Raf dimerization, such as Raf inhibitor treatment or certain B-Raf mutations, can allow mutant H-Ras to engage B-Raf with increased affinity to promote tumorigenesis, thus revealing a previously unappreciated role for C-Raf in potentiating B-Raf function.

Correction: Targeting the α4-α5 dimerization interface of K-RAS inhibits tumor formation in vivo.

Additionally, a further error has been corrected on page five in the sentence: Subsequent improvements to the chemistry of these lead compounds have resulted in the most recent interaction, with ARS-1620, which demonstrates selective inhibition of K-RAS(G12C) mutant tumor models in vivo [23]. The word 'with' has been removed from this sentence to ensure the correct meaning is communicated, such that the sentence now is: Subsequent improvements to the chemistry of these lead compounds have resulted in the most recent interaction, ARS-1620, which demonstrates selective inhibition of K-RAS(G12C) mutant tumor models in vivo [23].

Direct inhibition of RAS: Quest for the Holy Grail?

RAS GTPases (H-, K-, and N-RAS) are the most frequently mutated oncoprotein family in human cancer. However, the relatively smooth surface architecture of RAS and its picomolar affinity for nucleotide have given rise to the assumption that RAS is an "undruggable" target. Recent advancements in drug screening, molecular modeling, and a greater understanding of RAS function have led to a resurgence in efforts to pharmacologically target this challenging foe. This review focuses on the state of the art of RAS inhibition, the approaches taken to achieve this goal, and the challenges of translating these discoveries into viable therapeutics.

Targeting the α4-α5 interface of RAS results in multiple levels of inhibition.

Generation of RAS-targeted therapeutics has long been considered a "holy grail" in cancer research. However, a lack of binding pockets on the surface of RAS and its picomolar affinity for guanine nucleotides have made isolation of inhibitors particularly challenging. We recently described a monobody, termed NS1, that blocks RAS signaling and oncogenic transformation. NS1 binds to the α4-ß6-α5 interface of H-RAS and K-RAS thus preventing RAS dimerization and nanoclustering, which in turn prevents RAS-stimulated dimerization and activation of RAF. Interestingly, NS1 reduces interaction of oncogenic K-RAS, but not H-RAS, with RAF and reduces K-RAS plasma membrane localization. Here, we show that these isoform specific effects of NS1 on RAS:RAF are due to the distinct hypervariable regions of RAS isoforms. NS1 inhibited wild type RAS function by reducing RAS GTP levels. These findings reveal that NS1 disrupts RAS signaling through a mechanism that is more complex than simply inhibiting RAS dimerization and nanoclustering.

Targeting the α4-α5 dimerization interface of K-RAS inhibits tumor formation in vivo.

RAS genes are the most commonly mutated oncogenes in human cancers. Despite tremendous efforts over the past several decades, however, RAS-specific inhibitors remain elusive. Thus, targeting RAS remains a highly sought-after goal of cancer research. Previously, we have reported a new approach to inhibit RAS-dependent signaling and transformation in vitro by targeting the α4-α5 dimerization interface with a novel RAS-specific monobody termed NS1. Expression of NS1 inhibits oncogenic K-RAS and H-RAS signaling and transformation in vitro. Here, we evaluated the efficacy of targeting RAS dimerization as an approach to inhibit tumor formation in vivo. Using a doxycycline (DOX)-regulated NS1 expression system, we demonstrate that DOX-induced NS1 inhibited oncogenic K-RAS-driven tumor growth in vivo. Furthermore, we observed context-specific effects of NS1 on RAS-mediated signaling in 2D vs 3D growth conditions. Finally, our results highlight the potential therapeutic efficacy of targeting the α4-α5 dimerization interface as an approach to inhibit RAS-driven tumors in vivo.

Investigating Potential Chromosomal Rearrangements during Laboratory Culture of Neisseria gonorrhoeae.

Comparisons of genome sequence data between different strains and isolates of Neisseria spp., such as Neisseria gonorrhoeae, reveal that over the evolutionary history of these organisms, large scale chromosomal rearrangements have occurred. Factors within the genomes, such as repetitive sequences and prophage, are believed to have contributed to these observations. However, the timescale in which rearrangements occur is not clear, nor whether it might be expected for them to happen in the laboratory. In this study, N. gonorrhoeae was repeatedly passaged in the laboratory and assessed for large scale chromosomal rearrangements. Using gonococcal strain NCCP11945, for which there is a complete genome sequence, cultures were passaged for eight weeks in the laboratory. The resulting genomic DNA was assessed using Pulsed Field Gel Electrophoresis, comparing the results to the predicted results from the genome sequence data. Three cultures generated Pulsed Field Gel Electrophoresis patterns that varied from the genomic data and were further investigated for potential chromosomal rearrangements.

Inversion of Correia repeat enclosed elements in Neisseria gonorrhoeae.

Neisseria gonorrhoeae is capable of causing gonorrhoea and more complex diseases in the human host. Within the gonococcal genome are over 100 copies of the insertion sequence-like Correia repeat enclosed element (CREE), which has been predicted to be mobile within the neisserial genomes. Although there is evidence of ancestral movement of these elements, no previous study has provided evidence for current mobilization. CREE has the ability to alter gene expression and regulation in many ways: by insertional mutagenesis, by introducing promoter elements, by generating mRNA processing sites and by association with non-coding RNAs. Previous studies have compared the genomic locations of CREEs in the Neisseria spp., demonstrating that otherwise identical regions have either the element or the target TA insertion site. In this study, we report for the first time, to our knowledge, movement of CREEs, through inversion of the element at its chromosomal location. Analysis of Ion Torrent generated genome sequence data from N. gonorrhoeae strain NCCP11945 passaged for 8 weeks in the laboratory under standard conditions and stress conditions revealed a total of 37 inversions: 24 were exclusively seen in the stressed sample, 7 were seen in the control sample and the remaining 3 were seen in both samples. These inversions have the capability to alter gene expression in N. gonorrhoeae through the previously determined activities of the sequence features of these elements, potentially resulting in reversible phase-variable gene expression.

Inhibition of RAS function through targeting an allosteric regulatory site.

RAS GTPases are important mediators of oncogenesis in humans. However, pharmacological inhibition of RAS has proved challenging. Here we describe a functionally critical region, located outside the effector lobe of RAS, that can be targeted for inhibition. We developed NS1, a synthetic binding protein (monobody) that bound with high affinity to both GTP- and GDP-bound states of H-RAS and K-RAS but not N-RAS. NS1 potently inhibited growth factor signaling and oncogenic H-RAS- and K-RAS-mediated signaling and transformation but did not block oncogenic N-RAS, BRAF or MEK1. NS1 bound the α4-ß6-α5 region of RAS, which disrupted RAS dimerization and nanoclustering and led to blocking of CRAF-BRAF heterodimerization and activation. These results establish the importance of the α4-ß6-α5 interface in RAS-mediated signaling and define a previously unrecognized site in RAS for inhibiting RAS function.

Correia Repeat Enclosed Elements and Non-Coding RNAs in the Neisseria Species.

Neisseria gonorrhoeae is capable of causing gonorrhoea and more complex diseases in the human host. Neisseria meningitidis is a closely related pathogen that shares many of the same genomic features and virulence factors, but causes the life threatening diseases meningococcal meningitis and septicaemia. The importance of non-coding RNAs in gene regulation has become increasingly evident having been demonstrated to be involved in regulons responsible for iron acquisition, antigenic variation, and virulence. Neisseria spp. contain an IS-like element, the Correia Repeat Enclosed Element, which has been predicted to be mobile within the genomes or to have been in the past. This repeat, present in over 100 copies in the genome, has the ability to alter gene expression and regulation in several ways. We reveal here that Correia Repeat Enclosed Elements tend to be near non-coding RNAs in the Neisseria spp., especially N. gonorrhoeae. These results suggest that Correia Repeat Enclosed Elements may have disrupted ancestral regulatory networks not just through their influence on regulatory proteins but also for non-coding RNAs.

DNA uptake sequences in Neisseria gonorrhoeae as intrinsic transcriptional terminators and markers of horizontal gene transfer.

DNA uptake sequences are widespread throughout the Neisseria gonorrhoeae genome. These short, conserved sequences facilitate the exchange of endogenous DNA between members of the genus Neisseria. Often the DNA uptake sequences are present as inverted repeats that are able to form hairpin structures. It has been suggested previously that DNA uptake sequence inverted repeats present 3' of genes play a role in rho-independent termination and attenuation. However, there is conflicting experimental evidence to support this role. The aim of this study was to determine the role of DNA uptake sequences in transcriptional termination. Both bioinformatics predictions, conducted using TransTermHP, and experimental evidence, from RNA-seq data, were used to determine which inverted repeat DNA uptake sequences are transcriptional terminators and in which direction. Here we show that DNA uptake sequences in the inverted repeat configuration occur in N. gonorrhoeae both where the DNA uptake sequence precedes the inverted version of the sequence and also, albeit less frequently, in reverse order. Due to their symmetrical configuration, inverted repeat DNA uptake sequences can potentially act as bi-directional terminators, therefore affecting transcription on both DNA strands. This work also provides evidence that gaps in DNA uptake sequence density in the gonococcal genome coincide with areas of DNA that are foreign in origin, such as prophage. This study differentiates for the first time, to our knowledge, between DNA uptake sequences that form intrinsic transcriptional terminators and those that do not, providing characteristic features within the flanking inverted repeat that can be identified.

Phase variable DNA repeats in Neisseria gonorrhoeae influence transcription, translation, and protein sequence variation.

There are many types of repeated DNA sequences in the genomes of the species of the genus Neisseria, from homopolymeric tracts to tandem repeats of hundreds of bases. Some of these have roles in the phase-variable expression of genes. When a repeat mediates phase variation, reversible switching between tract lengths occurs, which in the species of the genus Neisseria most often causes the gene to switch between on and off states through frame shifting of the open reading frame. Changes in repeat tract lengths may also influence the strength of transcription from a promoter. For phenotypes that can be readily observed, such as expression of the surface-expressed Opa proteins or pili, verification that repeats are mediating phase variation is relatively straightforward. For other genes, particularly those where the function has not been identified, gathering evidence of repeat tract changes can be more difficult. Here we present analysis of the repetitive sequences that could mediate phase variation in the Neisseria gonorrhoeae strain NCCP11945 genome sequence and compare these results with other gonococcal genome sequences. Evidence is presented for an updated phase-variable gene repertoire in this species, including a class of phase variation that causes amino acid changes at the C-terminus of the protein, not previously described in N. gonorrhoeae.

Sequence features contributing to chromosomal rearrangements in Neisseria gonorrhoeae.

Through whole genome sequence alignments, breakpoints in chromosomal synteny can be identified and the sequence features associated with these determined. Alignments of the genome sequences of Neisseria gonorrhoeae strain FA1090, N.gonorrhoeae strain NCCP11945, and N. gonorrhoeae strain TCDC-NG08107 reveal chromosomal rearrangements that have occurred. Based on these alignments and dot plot pair-wise comparisons, the overall chromosomal arrangement of strain NCCP11945 and TCDC-NG08107 are very similar, with no large inversions or translocations. The insertion of the Gonococcal Genetic Island in strain NCCP11945 is the most prominent distinguishing feature differentiating these strains. When strain NCCP11945 is compared to strain FA1090, however, 14 breakpoints in chromosomal synteny are identified between these gonococcal strains. The majority of these, 11 of 14, are associated with a prophage, IS elements, or IS-like repeat enclosed elements which appear to have played a role in the rearrangements observed. Additional rearrangements of small regions of the genome are associated with pilin genes. Evidence presented here suggests that the rearrangements of blocks of sequence are mediated by activation of prophage and associated IS elements and reintegration elsewhere in the genome or by homologous recombination between IS-like elements that have generated inversions.