The Spencer-Smith Lab

Research Interests

The RAS-MAPK pathway is hyperactivated in 30-40% of human cancers; however, there remains a dearth of effective drugs that target this pathway. The primary objective of our lab is to uncover new vulnerabilities in the RAS-MAPK pathway through the study of rare disease-associated mutations therein. We have a particular interest in the mutations that cause RASopathy developmental syndromes (Figure 1A). The RASopathies, which include Noonan, cardiofaciocutaneous (CFC), and Costello syndrome, are caused by germline mutations throughout the RAS-MAPK pathway and collectively occur in 1:1000-2000 live births. While phenotypically distinct, RASopathies can be broadly characterized by distinct craniofacial features, congenital heart defects, short stature, autism traits and an increased risk of certain cancers. Notably, these mutations occur in many of the same genes as in human cancer, however because RASopathy mutations must be tolerated during development they are often distinct from those found in cancer, either in terms of their location within the protein or in the residue that is mutated. The characterization of these unique mutations can uncover new functional regions in pathway members and identify novel drug targets in frequent oncoproteins (Figure 1B).

 

 

Projects

 

Determining the mechanisms by which germline RAF mutations drive RAS-MAPK signaling

RAF kinases (ARAF, BRAF, and CRAF) are direct effectors of RAS GTPases and are essential for signal transduction through the RAS-MAPK pathway. Somatic driver mutations in BRAF are commonplace in melanoma, thyroid cancer and colorectal cancer, whereas oncogenic mutations in ARAF and CRAF are rare. In contrast, germline mutations are frequently reported in both BRAF and CRAF, which predominantly cause CFC and Noonan syndrome, respectively. The locations of these mutations also differ, with oncogenic mutations occurring almost exclusively in the BRAF kinase domain, whereas RASopathy mutations cluster around two conserved 14-3-3 docking sites in CRAF, and within the kinase domain and cysteine-rich domain (CRD) of BRAF (Figure 2A). The CRD had previously been shown to play roles in RAF autoinhibition, membrane binding, and the RAS:RAF interaction, however the relative importance of these roles in RAF regulation were not well understood. Through the characterization of a panel of RASopathy CRD mutations we discovered that the major function of BRAF CRD is to maintain the autoinhibitory interactions of the regulatory (REG) and catalytic (CAT) domains, and mutations which relieve these contacts increased RAS-dependent and RAS-independent BRAF activity (Figure 2B). These findings also indicated that stabilizing CRD-mediated autoinhibition would be of therapeutic benefit against the elevated BRAF activity in the many cancers and the RASopathies. In addition, when we compared the properties of CRAF CRD to those of BRAF, we found that the CRD plays distinct roles in CRAF regulation. Excitingly, our ongoing studies of the CRD, along with those of the RASopathy CRAF mutations (Figure 2A), have identified several vulnerabilities in CRAF regulation. Moving forward, we will investigate the effects of targeting these vulnerabilities on CRAF signaling and the tumorigenesis of KRAS-driven lung cancer, which was recently shown to be dependent on CRAF expression.

   

 

Dissecting the RAS-MAPK pathway using NanoBRET technology

A key aspect of our research is the development of Bioluminescence Resonance Energy Transfer (BRET)-based assays for measuring protein-protein interactions in live cells and in real-time. Of note, we recently utilized NanoBRET^TM technology to develop a live cell RAF autoinhibition assay (Figure 3A), which proved invaluable for our studies of RASopathy BRAF CRD mutations. This assay utilizes the ability of the REG domain to bind and inhibit CAT domain activity when expressed as two separate proteins in cells (Figure 3B), whereby interaction of the REG and CAT results in donor to acceptor energy transfer and the generation of the NanoBRET signal (Figures 3A and 3C). To further demonstrate the functionality of this assay we showed that autoinhibition could be disrupted internally by the oncogenic V600E mutation or externally by activated KRAS (Figure 3D). In addition, we recently developed a novel assay for measuring the interactions of RAF and 14-3-3 proteins, which along with an existing assay that measures RAF:RAF binding, forms the basis of a toolkit for dissecting the interplay of RAS-MAPK signaling under live cell conditions. Moreover, NanoBRET shows promise as a live cell drug screening platform, which may prove useful for identifying novel inhibitors of RAS-MAPK signaling.   

 

Developing novel prognostic assays and treatment strategies for RASopathy patients

RASopathies present with a wide array of developmental defects that show remarkable specificity to the causative protein and/or mutation (Figure 1A). Through a number of ongoing collaborations, this project seeks to develop biochemical assays for the prediction of RASopathy patient phenotype and severity based on the causative mutation to help establish prognostic and therapeutic guidance for RASopathy patients (Figures 1B and 2B). Indeed, our studies of the RASopathy BRAF mutants showed that relief of autoinhibition is the major factor determining mutation severity in zebrafish developmental models, which correlated closely with available patient reports. We are currently investigating whether our NanoBRET autoinhibition assay (Figure 3) can be used for predicting the severity of newly discovered BRAF mutations based on their ability to relieve autoinhibition. Another goal of these studies is to test whether currently available drugs that target the RAS-MAPK pathway can be used for treating the developmental defects seen in the RASopathies using transgenic mouse models of these syndromes (Figure 1B). The results of this project will help pave the way to new treatment options and improved therapeutic guidance for RASopathy patients. 

 

Contact Us

 

Dr. Russell Spencer-Smith, PhD.

Assistant Professor

Department of Cell & Molecular Pharmacology & Experimental Therapeutics

Hollings Cancer Center

86 Jonathan Lucas St., MSC 955

Rm HO712G

Medical University of South Carolina

Charleston, SC 29425

Phone: 843-792-5234

Email: spenceru@musc.edu

 

Selected Publications

1.    Spencer-Smith R, Terrell EM, Insinna C, Agamasu C, Wagner ME, Ritt DA, Stauffer J, Stephen AG, Morrison DK. RASopathy mutations provide functional insight into the BRAF cysteine-rich domain and reveal the importance of autoinhibition in BRAF regulation. Mol Cell. 2022 Nov 17;82(22):4262-4276.e5. doi: 10.1016/j.molcel.2022.10.016. Epub 2022 Nov 7. PubMed PMID: 36347258; PubMed Central PMCID: PMC9677513

2.    Spencer-Smith R, Morrison DK. Protocol for measuring BRAF autoinhibition in live cells using a proximity-based NanoBRET assay. Cell STAR Protoc. 2023 Aug 16;4(3):102461. doi: 10.1016/j.xpro.2023.102461. PubMed PMID: 37590148; PubMed Central PMCID: PMC10440347

 

3.    Spencer-Smith R, Koide A, Zhou Y, Eguchi RR, Sha F, Gajwani P, Santana D, Gupta A, Jacobs M, Herrero-Garcia E, Cobbert J, Lavoie H, Smith M, Rajakulendran T, Dowdell E, Okur MN, Dementieva I, Sicheri F, Therrien M, Hancock JF, Ikura M, Koide S, O'Bryan JP. Inhibition of RAS function through targeting an allosteric regulatory site. Nat Chem Biol. 2017 Jan;13(1):62-68. doi: 10.1038/nchembio.2231. Epub 2016 Nov 7. PubMed PMID: 27820802; PubMed Central PMCID: PMC5193369

4.    Khan I, Spencer-Smith R, O'Bryan JP. Targeting the α4-α5 dimerization interface of K-RAS inhibits tumor formation in vivo. Oncogene. 2019 Apr;38(16):2984-2993. doi: 10.1038/s41388-018-0636-y. Epub 2018 Dec 20. PubMed PMID: 30573767; PubMed Central PMCID: PMC6474814

 

Complete List of Published Work