Abstract 3539: Establishing ubiquitylation patterns in cells: Efficient monitoring of oncogenic ubiquitylation activity

Abstract

Abstract Cells regulate their internal functions by the ubiquitylation of many key proteins, especially onco-proteins and tumor suppressors. As a result, ubiquitin proteasome system (UPS) substrates and the relevant UPS enzymes have been identified as attractive anti-cancer targets. However, traditional methods for monitoring ubiquitylation patterns, including by immunoprecipitation coupled with immunoblot analysis (IP/IB) or by mass spectrometry methods, are either insensitive, low throughput, or highly intensive. Here, we describe three Tandem Ubiquitin Binding Entity (TUBE)-based assays, UbiTest, HT-UbiTest, and UbiQuant S, which provide a platform to efficiently detect ubiquitylation of anti-cancer targets. UbiTest is an immunoblot-based assay, which can be used to easily identify ubiquitlyated proteins. Compared to traditional ubiquitin IP/IB, this assay avoids epitope masking of antibodies caused by ubiquitin modification. HT-UbiTest extends this concept using a TUBE pulldown followed by two-antibody detection in a 96-well plate, and allows high-throughput absolute quantification of a target of interest. UbiQuant S is a high-throughput assay to quantify ubiquitylation status in cells or tissue. Ubiquitylated proteins are identified by energy transfer between AlphaLISA bead-labeled TUBE and antibody against the endogenous protein of interest in a homogenous plate-based assay. Furthermore, we have extended these technologies to identify K63- and K48-linked poly-ubiquitylated proteins using poly-ubiquitin linkage-selective TUBEs. Several prominent therapeutic targets in oncology and immuno-oncology have been used to verify these assays, including Cbl-b and its substrates, p53, and USP7. The efficiency and sensitivity of these assays have wide applications in drug discovery targeting ubiquitylation-dependent signaling pathways. Examples using DUB and ubiquitin ligase inhibitors illustrate the power of this system. These novel assays are versatile tools for understanding the role of ubiquitylation in cancer treatment, and provide platforms to monitor disease biomarkers in response to drug action. Citation Format: Peter K. Foote, Xiaolong Lu, Rajesh Singh. Establishing ubiquitylation patterns in cells: Efficient monitoring of oncogenic ubiquitylation activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3539.