Regulation of Ras-Dependent Signal Transduction Pathways

Division Of Basic Sciences - Nci

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Abstract

The RAS pathway is an important route of cellular signal transduction, functioning to relay vital signals that control cell survival, proliferation, and differentiation. Consistent with its central role in cell signaling, dysregulation of the RAS pathway can promote human disease states including cancer and the RASopathy developmental disorders. Elucidating the molecular mechanisms that regulate RAS pathway signaling and identifying strategies to disrupt RAS signaling in human disease states has been the focus of our laboratory's efforts for almost 30 years. Much of our research has centered on the RAF protein kinases (ARAF, BRAF and CRAF). Members of the RAF kinase family are direct effectors of activated RAS and function as the initiating enzymes in the three-tiered ERK/MAPK cascade, comprised of the RAF, MEK and ERK protein kinases. A primary contribution of our work to the field has been our identification and characterization of key protein interactions and phosphorylation events that modulate RAF function. Early studies from our group were the first to identify a mutation in the RAF kinases that disrupts RAS binding, providing researchers with a key tool to investigate the functional significance of the RAS/RAF interaction. In addition, our work analyzing RAF phosphorylation led to the discovery of inhibitory feedback phosphorylation loops that can impact the effectiveness of certain cancer therapies and are critical for the downregulation of RAS signaling under normal growth conditions and during cellular stress. Our studies demonstrating the role of RAF dimerization have also had important implications for cancer treatment, revealing how disease progression can be altered by secondary mutations or inhibitor treatments that promote RAF dimer formation. Moreover, these studies provided the "proof-of-principle" that inhibiting RAF dimerization has therapeutic potential. Realizing the importance of studying signaling events under live cell conditions, our group has recently developed bioluminescence resonance energy transfer (BRET) methodologies for analyzing RAF regulatory interactions (RAS/RAF binding and RAF dimerization) in living cells. The advantage of the BRET system is that it allows for crucial signaling interactions to be monitored in the context of the plasma membrane environment and under conditions where post-translational modifications and lipid processing still occur, events that can strongly influence protein binding as well as signal progression. Using the BRET assay to investigate the RAS/RAF interaction, our studies revealed distinct binding preferences between the highly conserved RAS and RAF family members that directly impact cancer progression and can alter how a cancer cell responds to targeted therapies (Terrell et al., 2019). More specifically, we found that mutant KRAS, the major contributor to RAS-mediated tumorigenesis, binds with high affinity to all RAF members. In contrast, mutant HRAS and NRAS exhibit preferential binding to CRAF, with BRAF demonstrating a unique selectivity for KRAS. Moreover, through depletion studies, we found that CRAF is critical for mutant HRAS-driven signaling and that events promoting stable BRAF/CRAF dimer formation, such as certain BRAF mutations or RAF inhibitor treatments, can allow mutant HRAS to engage BRAF with increased affinity to promote tumorigenesis. During this review period, our lab has continued to use the BRET RAS/RAF interaction assay in collaborative studies to determine how specific mutations in KRAS or NRAS impact the ability of these RAS proteins to interact with the various RAF members (Johnson et al, 2022 and Murphy et al, 2022). In addition, working in collaboration with the NCI-Molecular Targets Program and utilizing the NCI's large and diverse collection of natural product extracts, our lab utilized the BRET assay to conduct a high-throughput drug screen for identifying compounds that can modulate the RAS/RAF interaction. The BRET assay has proven to be a very sensitive way of detecting kinase inhibitors and other drug therapies that have the deleterious effect of augmenting RAS/RAF binding, which in turn can promote drug resistance and/or secondary tumor formation (Durrant et al. 2021). In addition, the screen identified numerous compounds that were able to inhibit RAS/RAF binding (Kim et al., 2020 and Senadeera et al., 2022), some of which may have therapeutic potential. In this review period, our lab also completed an important collaborative project with Dr. Ping Zhang in the NCI-Structural Biology Program. This project resulted in the determination of three high-resolution cryo-electron microscopy structures of full-length BRAF complexes that were isolated from mammalian cells: autoinhibited, monomeric BRAF:14-3-32:MEK and BRAF:14-3- 32 complexes, and a RAF inhibitor-bound, dimeric BRAF2:14-3-32 complex. Notably, the RAS binding domain (RBD) of BRAF was well-resolved in both of our monomeric BRAF structures, revealing for the first time the position and orientation of this critical domain in the context of the full-length, autoinhibited BRAF monomer (Martinez-Fiesco et al., 2022). Finally, during the review period, our lab was also engaged in the kick-off of the NCI-CCR Initiative on Advancing RASopathy Therapies (ART). This initiative has both clinical and basic research components and will bring together investigators in the Center for Cancer Research (CCR), Division of Cancer Epidemiology and Genetics (DCEG), patient advocacy groups, and extramural experts working on these developmental disorders. Our group has recently completed a project evaluating a number of the most prevalent RASopathy-associated CRAF and BRAF mutants. Through this effort and in collaboration with the LCDS Zebrafish Facility, we have established a screening assay using zebrafish embryos that can monitor the gain-of-function activities of RASopathy-associated mutants. This assay will be employed to analyze any previously uncharacterized RASopathy mutants that are identified through the RASopathy Initiative. Moreover, this assay is expected to provide valuable information regarding the severity of the mutation as well as the effectiveness of various drug treatments.