Establishing Ubiquitylation Pattern In Tissues And Cells Efficient Method To Monitor Cancer Drug Function And Biomarkers

The cell regulates its function by altering ubiquitylation status of many key proteins, especially oncoproteins. As a result, these ubiquitin proteasome system (UPS) substrates and the relevant UPS enzymes have been identified as attractive anticancer targets. However, to date, the only effective way to measure these effects has been by immunoprecipitation coupled with immunoblot analysis (IP/IB) or by mass spectrometry methods, which are either insensitive, low throughput, or highly intensive. Here, we describe two Tandem Ubiquitin Binding Entity (TUBE)-based assays, UbiQuant S and UbiTest, that provide a platform to efficiently detect ubiquitylation of anticancer targets. UbiQuant S is a high-throughput assay to quantify ubiquitylation status in a cell or tissue. Ubiquitylated proteins and the nature poly-ubiquitin are identified by energy transfer between tagged TUBE and antibody against the target protein that is poly-ubiquitylated. Interaction between TUBE and antibody is registered in a label-free manner in a solution. In case the level of ubiquitylated protein is extremely low or cannot be detected by antibody, ubiquitylated proteins can be affinity purified by TUBEs attached to agarose or magnetic beads and FRET or alphaLISA experiment is repeated. Furthermore, we have extended the technology to identify K63- and K48-linked poly-ubiquitin proteins by poly-ubiquitin chain selective TUBEs. UbiTest is an immunoblot-based assay, which can be used to verify the result of UbiQuant S. Compared to traditional ubiquitin IP/IB, this assay avoids epitope masking of antibodies caused by ubiquitin modification. Several important therapeutic targets have been used to verify these assays, including Cbl-b and USP7 immune oncology drug targets. The high efficiency and sensitivity of these assays have wide applications in drug discovery targeting ubiquitylation-dependent signaling pathways. Examples from DUBs and ubiquitin ligase inhibitors will illustrate the power of this system. These novel technologies will equip researchers with versatile tools to better understand the role of ubiquitylation in cancer treatment and monitoring biomarkers in response to drug action. Citation Format: Xiaolong Lu, Rajesh Singh, Chengcheng Song, Peter Foote. Establishing ubiquitylation pattern in tissues and cells efficient method to monitor cancer drug function and biomarkers [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A038.