Regulation of Ras-Dependent Signal Transduction Pathways

Division Of Basic Sciences - Nci

Investigators

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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 Ras signaling can promote human disease states, with somatic mutations in the RAS genes (HRAS, KRAS and NRAS) being prominent drivers of tumorigenesis and Ras germline mutations contributing to a group of related developmental disorders known collectively as the RASopathies. Elucidating the molecular mechanisms that regulate RAS pathway signaling and identifying strategies to disrupt signal transmission in human disease states is a major health priority and 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 live 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 still occur, events that can strongly influence protein binding as well as signal progression. During this review period, we have utilized the BRET assay to investigate the RAS/RAF interaction and our studies have 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, whereas BRAF demonstrates a unique selectivity for KRAS. By generating different RAS and RAF chimeric proteins, our work revealed that the KRAS selectivity of BRAF is determined by the acidic N-terminal segment of BRAF, which interacts with polybasic residues in the hypervariable region of KRAS, thus identifying a new RAS-binding epitope that uniquely contributes to the KRAS/BRAF interaction. 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. Thus, these findings have revealed a previously unappreciated role for CRAF in potentiating BRAF function. Working in collaboration with the NCI-Molecular Targets Program and utilizing the NCI's large and diverse collection of natural product extracts, our lab has gone on to utilize the BRET assay in a high-throughput screen to identify 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). Importantly, the screen has also resulted in the identification of numerous promising compounds that inhibit RAS/RAF binding (Kim et al., 2020) and may lead to the development of new therapeutic agents to counteract aberrant RAS signaling in human disease states. 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 two studies evaluating a number of the most prevalent RASopathy-associated CRAF and BRAF mutants (Kota et al., 2019). 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.