Discovery of metabolic regulators of DNA topology and cellular responses to topoisomerase II inhibition

Johns Hopkins University, Baltimore MD

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Abstract

Project Summary/Abstract The goal of my research is to understand the cellular mechanisms that regulate topoisomerase II (topo II) function and respond to topo II inhibition. Type II topoisomerases, such as topo II, are essential enzymes that manage DNA superhelical structure and decatenate entangled DNA strands to facilitate critical processes such as transcription, DNA replication, and chromosome segregation1. With respect to human health, topo II is a tremendously important cancer target, and a variety of chemotherapeutics are clinically used to exploit the ability to topo II to generate DNA damage and kill replicating cells. Biochemical and structural studies have provided valuable details about the ?strand passage? mechanism of topo II and the means by which synthetic inhibitors affect this activity. However, the field lacks a clear understanding of how topo II activity is regulated in accordance with cell needs. To better understand how topo II is connected to regulatory pathways and DNA damage responses, I aim to use the S. cerevisiae model system to explore the role of cellular metabolism in regulating topo II function and perform an unbiased screen for genes involved in responding to topo II inhibition. While analyzing the evolutionary conservation of eukaryotic topo IIs, I discovered a highly conserved pocket on topo II that interacts with ICRF-187, a clinically approved, synthetic inhibitor that is used in chemotherapeutic regimens2. I later discovered that this site also engages resveratrol, a natural product found in red wine3. These observations suggest that this site may be an orphan allosteric site and inspired the hypothesis that small-molecule metabolites may allosterically regulate topo II function. Preliminary data indicate that components of yeast metabolite extracts are able to modulate topo II activity. Aim 1 outlines the natural product purification strategies and mass spectrometry-based metabolite ligand screening I will employ to identify and characterize endogenous small molecules that may have topo II-regulatory function. In addition to probing previously unexplored connections between cellular metabolism and DNA topology, I aim to uncover pathways involved in cellular responses to abnormal topo II activity. Thus, in Aim 2 I propose an unbiased pharmacogenetic screen with yeast deletion and hypomorph libraries to identify alleles that affect growth in the presence of clinically- relevant topo II inhibitors. Together, these aims have the potential to discover novel interactions between topo II and regulatory pathways. Such discoveries, in turn, could unveil new ways to allosterically modulate topo II function and new targets for combinatorial therapy in conjunction with anti-topoisomerase inhibitors to improve the efficacy and/or decrease toxicity of currently available treatments.