Understanding the protein-protein interactions (PPIs) between the B7-family of immune regulatory proteins and their cognate binding partners on the T-cell have led to a new era in cancer treatment, where checkpoint blockade by monoclonal antibodies inhibiting CTLA-4 and PD-1 on the T-cell has revolutionized immunotherapy. These PPIs provide both stimulatory secondary signals to promote and sustain T cell responses, and they also contribute to negative secondary signals that downregulate T cell responses. PD-L1, PD-L2, B7-H3, B7-H4 and HHLA-2 can be expressed on non-hematopoetic cells and tumors, but the role of temporal, as well as, spatial differences in ligand expression and their contributions to pathogenic and protective immune responses have not been fully elucidated. Many B7 family members, similar to other families of cell surface signaling proteins, are known to exist as dimers, related to their signaling state. Thus, characterization of B7 family interactions and oligomerization is essential to understanding the molecular mechanisms by which they alter the cancer cell to confer an oncogenic phenotype. From a candidate gene approach, an interesting and novel target protein is B7- H3, a member of the B7-family, which is overexpressed on the surface of many solid tumors. This proposal aims to develop a bioluminescence-based, sequential resonance energy transfer (BLI-SRET) reporter of PPIs that can be used to define structural features that contribute to B7-H3 dimerization and oligomerization and determine the mechanism of how these interactions promote cancer cell survival and immune evasion.
In aim 1, I will develop a BLI-SRET system which can distinguish dimeric or multimeric protein complexes in a spatiotemporal manner. This system will allow us to understand how signals are integrated at a molecular level through protein dimerization and oligomerization, and provide a platform to aid characterizing those interactions that contribute to pathogenesis.
In aim 2, I will test the hypothesis that defined regions of B7-H3 contribute to protein clustering and that ablation of these interacting domains will disrupt protein function, both intracellularly and extracellularly, through the complemented immune synapse. As each interacting domain may serve a different downstream function, understanding the similar or different mechanisms that regulate B7-H3-driven tumorigenesis may better enable pathway-specific targeting of therapeutics and improve our understanding of B7-H3 oligomerization and how this relates to its function. The research proposed herein will support my training and development as an independent and productive cancer biologist, allowing me to gain a greater understanding of assay development, genetically encoded optical biosensors, and the application of these tools to understand complex biological processes. Using the longstanding practical expertise and development of optical imaging agents in the Piwnica- Worms group and my detailed understanding of KRAS protein clustering as it relates to cancer development, we aim to develop this system that will allow us to better understand how signals are integrated at a molecular level, and use it to aid in developing therapeutics to target the B7-family of oncogenic immune regulatory proteins.
The discovery and characterization of protein-protein interactions (PPIs) as principle regulators of biochemical signal transduction make them central to understanding cellular homeostasis, and increased interest in the B7- family of immune regulatory proteins and their interactions have led to a new era in cancer treatment. As current methods lack the ability to distinguish dimeric vs. oligomeric protein complexes in living cells, this proposal aims to develop a bioluminescence-based, sequential resonance energy transfer reporter of PPIs that can be used to define structure features that contribute to B7-H3 dimerization/oligomerization and determine whether these interactions promote cancer cell survival and immune evasion. This work will enable us to both improve our basic understanding of B7-H3 oligomerization, and allow us to understand how signals are integrated at a molecular level through protein dimerization and higher order clustering, and provide a platform to aid characterizing those interactions that contribute to pathogenesis.
