The O'Bryan Laboratory

Research Description

The ability of cells to respond to extracellular signals is critical for the normal homeostasis of an organism. Disruption of these normal cell signaling pathways underlies numerous pathological conditions including cancer. The research in the O’Bryan laboratory centers on understanding the regulation of these cellular signaling pathways and how alterations to these pathways contribute to cancer. Current projects in the lab are focused on two major areas:

Regulation of RAS GTPases

RAS functions as a critical molecular switch to regulate cellular signaling pathways involved in growth, proliferation, development, differentiation and survival (Fig. 1). Indeed, RAS is the most frequently mutated oncogene in human cancers with 27% of all human tumors possessing mutations in one of the three RAS genes (KRAS, HRAS, or NRAS) and some cancers, such as pancreatic ductal adenocarcinoma (PDACs), exhibiting RAS mutants in >90% of tumors. Importantly, RAS serves as a driver of tumor development, with many cancers “addicted” to the continued presence of oncogenic RAS. Thus, much effort has focused on development of pharmacological inhibitors of RAS for use as therapeutic agents in treatment RAS-mutant cancers. Despite many years of research, there remain no FDA-approved anti-RAS therapeutics in the clinic. Thus, there remains a great unmet need to develop effective strategies for inhibiting RAS. While recent work has resulted in the development of direct RAS inhibitors, these compounds selectively target a very specific mutant form of RAS, namely RAS(G12C), which is present in lung tumors (14%) and colorectal tumors (5%). However, given the overall low frequency of G12C mutations across human tumors (16%), more broadly efficacious RAS inhibitors are needed.

Figure 1. RAS Proteins. A) GTPase cycle. Normally, RAS proteins reside in the GDP-bound or inactive state. Following mitogenic stimulation by growth factors, GEFs are recruited to the plasma membrane. Bind of GEFs to RAS results in destabilization in nucleotide binding leading to the release of GDP from RAS and creation of a transient nucleotide free state. Given the high concentration of GTP in cells relative to GDP, RAS proteins load with GTP resulting in the switch to the active state. RAS-GTP recruits and activates it downstream targets such as RAF and PI3K. Termination of RAS signaling occurs through hydrolysis of GTP to GDP. Although RAS possesses intrinsic GTPase activity, it is a poor enzyme. This inactivation step is aided by GTPase accelerating/activating proteins which enhance the GTPase activity of RAS by nearly 100-fold, returning RAS to the inactive, GDP-bound state. B) RAS family members. KRAS4A and KRAS4B are derived from alternative splicing of the same gene resulting in different C-termini. Grey shading highlights residues that are identical in all four RAS proteins. SW1, switch 1 region (aa 30-40); SW2, switch 2 region (aa 60-76); HVR, hypervariable region. Proteins were aligned with Clustal multiple alignment. C) Mutation frequency in RAS alleles. Data were compiled from the Catalogue of Somatic Mutations (COSMIC), v86. Figure taken from: O’Bryan, J.P. Pharmacological targeting of RAS: Recent success with direct inhibitors (2019) Pharmacological Research, Jan;139:503-511. doi: 10.1016/j.phrs.2018.10.021

We have taken an unbiased approached to discover novel modalities to inhibit RAS using Monobody technology, developed by our collaborator Dr. Shohei Koide. Using this approach, we have isolated a Monobody, termed NS1, that inhibits RAS by binding the allosteric lobe and preventing self-association/clustering (Fig. 2). As a result, RAS no longer promotes the dimerization and activation of the RAF Ser/Thr kinase, which is the major effector of RAS. NS1 inhibits RAS signaling and oncogenic transformation both in vitro and in animal models for tumorigenesis. These results highlight a new approach to potentially therapeutically inhibit RAS, namely by disrupting RAS self-association. In our continued collaboration with Dr. Koide, we have isolated additional Monobodies that inhibit RAS and are currently working to define their mechanisms of action and biological activity. These studies will help to define new vulnerabilities in RAS that can be exploited in the development of small molecule inhibitors that target this critical oncoprotein.

Figure 2. NS1 inhibits RAS. A) Crystal structure of NS1:H-RAS-GDP. NS1 (shown in ribbon) binds to the a4-a5 interface (shaded brown) in the allosteric lobe of RAS (shown in space filling model). B) In quiescent or unstimulated cells, RAS predominantly GDP-bound. However, growth factor stimulation or oncogenic mutation, shifts RAS to the active, GTP-bound state resulting in dimerization of RAS which in turns results in the recruitment, dimerization, and activation of RAF, shown here as the formation of a heterodimer of BRAF and CRAF. NS1 inhibits RAS signaling and oncogenic transformation by blocking RAS self-association. These figures were taken from Spencer-Smith, R. et al. Inhibition of RAS function through targeting an allosteric regulatory site. (2017) Nature Chemical Biology, 13(1):62-68. doi: 10.1038/nchembio.2231. PMCID: PMC5193369.

Intersectin scaffold protein

Intersectin is a multi-domain scaffold protein first implicated in the regulation of clathrin-dependent endocytosis. Indeed, intersectin functions to recruit a number of endocytic accessory proteins to the sites of clathrin-coated pit formation. However, we discovered that intersectin also interacts with and activates a number cellular signaling proteins, including several RAS family GTPases (Fig. 3). Thus, we have focused on defining the cellular signaling pathways regulated by intersectin and determining the importance of these intersectin-regulated pathways in normal and pathological conditions. Indeed, we and others have shown that intersectin is overexpressed in a number of neurodegenerative conditions such as Alzheimer Disease and Down Syndrome and that intersectin may contribute to a number of polyglutamine-expansion diseases such as Huntington’s Disease and Kennedy’s Disease. More recently, we have uncovered a role for intersectin in a pediatric cancer called neuroblastoma. Intersectin is prominently expressed in neuroblastoma tumors and is critical to the tumorigenic properties of neuroblastoma cells. We are currently working to define the mechanism(s) underlying intersectin’s role in neuroblastoma tumorigenesis as well as explore the potential role of this multi-domain scaffold in additional cancers.

Figure 3. Intersectin (ITSN) acts as an integrator of biochemical pathways. A) ITSN interacts with and regulates a number of RAS family GTPases including RAS. B) ITSN regulates a variety of kinases including receptor tyrosine kinases (e.g., EGFR and EphB2), intracellular protein kinases (e.g., JNK and WNK) and lipid kinases (e.g. PIK3C2B). C) ITSN activates the Cbl E3 ubiquitin ligase to regulate EGFR ubiquitylation and trafficking. However, ITSN may also interact with a number of additional E3 ligases as well.

Laboratory Staff
Imran Khan, Ph.D. Postdoc	Vinodh Rajagopalan, Ph.D., DVM Postdoc
Matt Rhett, Ph.D. Staff Scientist	Mariyam Zuberi, Ph.D. Postdoc