Development and Application of High-Throughput Chemical Genomic Screens for Functional Studies of Cancer Therapeutics by Kahlin Cheung-Ong A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Molecular Genetics University of Toronto © Copyright by Kahlin Cheung-Ong 2013 Development and Application of High-Throughput Chemical Genomic Screens for Functional Studies of Cancer Therapeutics Kahlin Cheung-Ong Doctor of Philosophy Department of Molecular Genetics University of Toronto 2013 Abstract Chemotherapeutic agents act by targeting rapidly dividing cancer cells. The full extent of their cellular mechanisms, which is essential to balance efficacy and toxicity, is often unclear. In addition, the use of many anticancer drugs is limited by dose-limiting toxicities as well as the development of drug resistance. The work presented in this thesis aims to address the basic biology that underlies these issues through the development and application of chemical genomic tools to probe mechanisms of current and novel anticancer compounds. Chemical genomic screens in the yeast Saccharomyces cerevisiae have been used to successfully identify targets and pathways related to a compound‟s mode of action. I applied these screens to examine the mode of action of potential anticancer drugs: a class of platinum-acridine compounds and the apoptosis-inducing compound elesclomol. By analogy to the yeast screens, I developed an RNAi-mediated chemical genomic screen in human cells which has the potential to reveal novel targets and drug mechanisms. This screen was applied to further understand doxorubicin‟s mode of action. In parallel with the loss-of-function assays, our lab developed a human ORF overexpression screen in human cells. I applied this gain-of-function screen to identify those genes that, when overexpressed, are toxic to cells. Characterization of such genes that cause toxicity can provide insight into human diseases where gene amplification is prevalent. ii Acknowledgements First and foremost, I would like to thank my supervisors, Corey Nislow and Guri Giaever, for their guidance and support throughout my Ph.D. Thank for you all your encouragement, wise words, and contagious enthusiasm for science. I could not have asked for better mentors. I would also like to thank my committee members Jason Moffat and Dan Durocher for excellent advice and guidance on my research. Thank you to everyone in the lab for your support and friendship over the years. To Elke Ericson, thanks for taking me under your wing during the early years of my graduate career. To Anthony Arnoldo and Jing Kittanakom, thanks for your friendship, support, and magic protocols. To Ron Ammar, Simon Alfred, and Nikko Torres, thanks for the helpful discussions and constant laughs. Thanks to Anna Lee, Daniel Shabtai, Kevin Song, and Larry Heisler working with me on the platinum project. To Marinella Gebbia, Andrew Smith, Malene Urbanus, and Elena Lissina, thanks for answering all my questions about yeast. Also thanks to the Moffat, Brown, and Stagljar labs for sharing advice, protocols, and reagents. I would like to thank my friends for putting up with me and helping me keep my sanity. Finally, I would like to thank my family for their unconditional love and support during these years, even though you never really understood what I was doing. iii Table of Contents Acknowledgements ........................................................................................................................ iii Table of Contents ........................................................................................................................... iv List of Tables ................................................................................................................................. ix List of Figures ................................................................................................................................. x List of Abbreviations .................................................................................................................... xii Chapter 1 - Introduction .................................................................................................................. 1 1.1 Cancer chemotherapy .......................................................................................................... 1 1.1.1 DNA-damaging agents as cancer chemotherapeutics ............................................. 1 1.1.1.1 DNA-reactive agents ................................................................................ 3 1.1.1.2 Antimetabolites ......................................................................................... 5 1.1.1.3 Topoisomerase poisons ............................................................................ 5 1.1.2 Mitochondria as a target for cancer chemotherapy ................................................. 7 1.1.3 Limitations of current chemotherapeutics .............................................................. 8 1.1.4 New designs for chemotherapeutics ..................................................................... 10 1.2 Chemical genomics in model organisms .......................................................................... 11 1.2.1 Importance of screening to understand drug mechanisms of action ..................... 11 1.2.2 Chemical genomic screens in yeast ...................................................................... 13 1.2.2.1 Haploinsufficiency profiling .................................................................. 15 1.2.2.2 Homozygous profiling ............................................................................ 16 1.2.2.3 Multicopy suppression profiling ............................................................. 16 1.3 Functional genomics in mammalian cells ......................................................................... 17 1.3.1 Loss-of-function screens ....................................................................................... 18 1.3.1.1 RNA interference .................................................................................... 18 1.3.1.2 Using RNAi for genome-scale loss-of-function screens ........................ 20 iv 1.3.2 Gain-of-function screens ....................................................................................... 22 1.3.2.1 Examples of systematic gain-of-function mammalian screens .............. 22 1.3.2.2 Downstream applications of overexpression studies .............................. 24 1.4 Project rationale ................................................................................................................ 24 Chapter 2 - Comparative chemogenomics to examine the mechanism of action of DNA- targeted platinum-acridine anticancer agents ........................................................................... 26 2.1 Introduction ....................................................................................................................... 26 2.2 Results ............................................................................................................................... 29 2.2.1 Global analysis of fitness profiles ......................................................................... 29 2.2.2 Specific genes involved in DNA-damage response to these novel platinum compounds ............................................................................................................ 31 2.2.3 Genome-wide profiling of DNA-damaging platinum-acridines in S. pombe ....... 39 2.2.4 Effect of platinum-acridines on DNA-replication ................................................ 39 2.2.5 Effect of platinum-acridines on mitochondria ...................................................... 40 2.3 Discussion ......................................................................................................................... 45 2.4 Methods ............................................................................................................................. 47 2.4.1 Reagents ................................................................................................................ 47 2.4.2 Yeast strains and media ........................................................................................ 47 2.4.3 Deletion pool growth and chip experiments ......................................................... 48 2.4.4 Data analysis ......................................................................................................... 48 2.4.5 Comparing genome-wide profiles ......................................................................... 48 2.4.6 Gene set enrichment analysis (GSEA) .................................................................. 49 2.4.7 DNA content analysis ........................................................................................... 50 2.4.8 Molecular combing ............................................................................................... 50 2.4.9 Microscopy ........................................................................................................... 51 Chapter 3 - Mitochondrial electron transport is the cellular target of the oncology drug elesclomol ................................................................................................................................ 53 v 3.1 Introduction ....................................................................................................................... 53 3.2 Results ..............................................................................................................................