Regulation of Ras-Dependent Signal Transduction Pathways

Division Of Basic Sciences - Nci

Investigators

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Abstract

The RAS pathway is a primary route of cellular signal transduction, relaying signals that control vital processes such as cell survival, proliferation, and differentiation. Reflecting its importance, dysregulation of this pathway can promote human disease states, including cancer and developmental disorders collectively known as the RASopathies. For nearly three decades, our laboratory has focused on elucidating the molecular mechanisms that govern RAS pathway signaling, with the ultimate goal of identifying therapeutic strategies to disrupt aberrant RAS signaling in disease states. A major emphasis of our work has been on the RAF family of protein kinases (ARAF, BRAF, and CRAF), which are direct effectors of RAS and initiate the ERK/MAPK cascade. Our early studies were among the first to identify key mutations in RAF that impair RAS binding-tools that have since been widely used to dissect the RAS-RAF interaction. In parallel, our analyses of RAF phosphorylation led to the discovery of feedback regulatory loops, including those involving ERK and stress-activated kinases, which are crucial for downregulating RAS signaling and can influence the effectiveness of targeted therapies. We have also contributed to defining the role of RAF dimerization in signaling and therapeutic response. Our findings demonstrated that dimerization is required for RAF activation under most physiological and oncogenic conditions, and that a peptide mimetic of the RAF dimer interface can disrupt dimerization and inhibit pathway activation. These studies provided proof-of-concept that targeting RAF dimerization holds therapeutic potential and highlighted how secondary mutations or drug treatments that affect dimer formation can alter disease progression. To better capture the dynamic nature of RAF regulation in living cells, our group developed bioluminescence resonance energy transfer (BRET)-based assays to study RAS-RAF binding and RAF dimerization in real time. The BRET platform enables the monitoring of protein-protein interactions in the context of native post-translational modifications and membrane localization. Using this assay, we identified distinct binding preferences between RAS and RAF isoforms that impact tumor behavior and therapeutic response. For instance, mutant KRAS binds all RAF isoforms with high affinity, whereas mutant HRAS and NRAS preferentially bind CRAF, with BRAF demonstrating selectivity for KRAS. We further showed that CRAF is essential for HRAS-driven signaling, and that events promoting BRAF-CRAF dimerization-such as BRAF mutations or certain RAF inhibitors-enable HRAS to more effectively engage BRAF, promoting tumorigenesis. In collaboration with the NCI-Molecular Targets Program and using the NCI's natural product extract library, we used the BRET assay in a high-throughput screen to identify compounds that modulate the RAS-RAF interaction. This approach successfully detected both inhibitors and undesired enhancers of RAS-RAF binding, the latter of which may promote drug resistance or secondary tumor development. Several hits from this screen that block RAS-RAF interactions are being further evaluated for therapeutic potential. Our lab also participates in the NCI-CCR Initiative on Advancing RASopathy Therapies (ART)-a multidisciplinary effort involving the Center for Cancer Research (CCR), Division of Cancer Epidemiology and Genetics (DCEG), patient advocacy groups, and extramural experts. As part of this initiative, and in collaboration with the LCDS Zebrafish Facility, we developed in vivo zebrafish embryo assays to assess the activity of RASopathy-associated BRAF mutants. This panel of assays serves as a platform to evaluate the pathogenicity of newly identified variants and to assess drug responses. Our initial studies focused on several prevalent BRAF CRD (cysteine-rich domain) mutations (Spencer-Smith et al., 2023; Spencer-Smith and Morrison, 2024), demonstrating that these mutations disrupt autoinhibition and promote dimerization-dependent signaling. These signaling changes correlate with the severity of developmental defects observed in embryos, highlighting the potential of these assays to model disease-relevant phenotypes and to guide therapeutic evaluation. In collaboration with Dr. Ping Zhang (NCI Structural Biology Program), we determined three high-resolution cryo-EM structures of full-length BRAF complexes: (1) an autoinhibited monomeric BRAF:14-3-3:MEK complex, (2) a monomeric BRAF:14-3-3 complex, and (3) a RAF inhibitor-bound dimeric BRAF:14-3-3 complex. Notably, the RAS-binding domain (RBD) was well resolved in the monomeric structures, revealing for the first time its position and orientation within the full-length, autoinhibited BRAF protein. Building on these structural insights, we have initiated a collaborative project with the NCI RAS Initiative and Lawrence Livermore National Laboratory to develop computational tools for modeling RAF conformational changes during activation. The goals of this project are: 1)To resolve intermediate structural states between the autoinhibited monomer and the active dimer. 2) To identify cellular components required for or facilitating this transition. 3)To uncover regulatory steps amenable to therapeutic targeting. Simulations will be carried out by LLNL, while experimental validation will be performed collaboratively by our group and laboratories within the NCI-RAS Initiative. In particular, our laboratory has developed an in vitro BRAF activation assay and numerous biological cell-based assays that have proven critical in testing simulation-driven models. By tightly integrating large-scale simulations with experimental data, this project aims to produce a detailed, predictive model of RAS-driven RAF activation. These structural and dynamic insights are expected to significantly advance our mechanistic understanding of the RAS-RAF interaction and open new avenues for therapeutic intervention.