Date of Award
Fall 11-1-2023
Embargo Period
11-1-2025
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Cell and Molecular Pharmacology and Experimental Therapeutics
College
College of Graduate Studies
First Advisor
John P. O'Bryan
Second Advisor
Denis Guttridge
Third Advisor
Michael Ostrowski
Fourth Advisor
Nathan Dolloff
Fifth Advisor
G. Aaron Hobbs
Abstract
The RAS family of small GTPases, consisting of HRAS, KRAS and NRAS, represent the most frequently mutated oncogenes in human cancers. As such, targeting RAS has remained at the forefront of efforts for cancer therapeutic development. For decades, these efforts were largely unsuccessful. However, recent seminal discoveries led to the development of the first clinically-approved inhibitors of a common oncogenic RAS mutant. While a major milestone, most oncogenic RAS mutants remain without inhibitors. One emerging theme over the past several years has been the notion that RAS multimerization plays a critical role in RAS function and that this process may represent a therapeutic vulnerability. Our lab has previously developed a synthetic binding protein, the NS1 monobody, that inhibits RAS through disruption of RAS–RAS interactions. NS1 binds to the α4-α5 allosteric lobe of HRAS and KRAS, which was previously proposed to be the interface for RAS dimerization. However, several groups reported conflicting evidence on the effect of mutating residues in the α4-α5 region on RAS function, igniting debate on the nature of these interactions and whether bona fide RAS dimerization is a prerequisite for RAS activity. We thus sought to determine the role of individual amino acids in the α4-α5 allosteric lobe on RAS activity through stringent cell signaling, biological transformation, and protein–protein interaction assays (Chapter 2). In addition, we developed a new monobody that binds and inhibits NRAS in a similar manner as NS1, which to our knowledge, is the first NRAS-specific inhibitory reagent (Chapter 3). In summary, we demonstrate that residues in the α4-α5 allosteric lobe of RAS do not mediate RAS dimerization. Instead, RAS–RAS interactions necessary for viii function are likely in the form of transient clustering at the cytoplasmic face of the cell membrane, and these interactions are likely driven by the membrane anchors (hypervariable regions) of RAS, not the G-domain.
Recommended Citation
Whaby, Michael, "Utilizing Monobody Technology to Investigate RAS Signaling Mechanisms and Potential Therapeutic Vulnerabilities" (2023). MUSC Theses and Dissertations. 823.
https://medica-musc.researchcommons.org/theses/823
Rights
Copyright is held by the author. All rights reserved.