UCSF UC San Francisco Electronic Theses and Dissertations Title Genomic exploration of the peripheral nervous system: Identification of candidate genes for neuroblastoma, hearing loss, and other aspects of neuron biology and tumorigenesis Permalink https://escholarship.org/uc/item/1cx4w7m3 Author Hackett, Christopher Sultan Publication Date 2010 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California Genomic exploration of the peripheral nervous system: Identification of candidate genes neuroblastoma, hearing loss, and other aspects of neuron biology and tumorigenesis by Christopher S. Hackett DISSERTATION Submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biomedical Sciences in the GRADUATE DIVISION of the UNIVERSITY OF CALIFORNIA, SAN FRANCISCO Copyright (2010) By Christopher S. Hackett ii This work is dedicated to my parents, who have always fostered the spirit of exploration. iii Acknowledgements I gratefully acknowledge all of the people who have supported me during graduate school and who have contributed to the work in this thesis. First, I would like to thank members of the Weiss Lab, past and present. In addition to providing helpful advice, the lab members created a wonderful working atmosphere that fostered a sprit for scientific exploration and a springboard for a career in science. In particular, I would like to thank QiWen Fan for providing molecular biology mentorship and amusing conversations over the several years we sat together in the lab. Nadia Milshteyn, David Goldenberg, Slava Yakovenko, and Kim Nguyen maintained our mouse colony and provided general lab management and maintenance, without which this project would not have been possible. Christine Cheng provided some very skilled technical assistance for some of the biochemical aspects of the project, and Yvan Chanthery contributed some important in vivo techniques. Justin Chen assisted with some of the informatics involved, and provided some useful genomics advice. Anders Persson and Fredrik Swartling provided interesting insight into neuron biology. I am grateful to all lab members for providing a wonderful social atmosphere in the lab. I am also particularly indebted to Clay Gustafson and Theo Nicolaides for their helpful guidance regarding the interface of science and clinical practice. I would also like to thank our collaborators at UCSF. Pui-Yan Kwok and his lab, in particular Denise Lind and Ludmila Pawlikowska, facilitated all of the SNP genotyping described. Saunak Sen assisted with the interpretation of our genotyping results. Allan Balmain, Jin-Hua Mao, and David Quigley provided direction for our modifier screen, and assisted with the data analysis. David Quigley, in particular, enthusiastically contributed a cutting-edge analysis approach to our data that yielded exciting results. The members of Zena Werb’s lab, in particular Pengfei Lu and Aditi Sharma, taught me critical techniques for the breast cancer iv aspects of this project. Nigel Killeen and his lab, in particular Steve Chmura and DongJi Zhang, assisted with the generation of the two lines of genetically engineered mice described. Graeme Hodgson, Sue Hariono, Greg Hamilton, and Taku Tokuyasu helped to train me in all aspects of array CGH analysis. I am also grateful to the neuropathology tissue bank and Joanna Phillips for fulfilling all of our pathology needs. I am also grateful to our collaborators outside of UCSF. In particular, David Largaespada has generously assisted our lab in adopting the Sleeping Beauty system he developed. Aron Geurts, Adam Dupuy, Lara Collier, Timothy Starry, and Vincent Keng also provided technical assistance, reagents, and mice for the project. At the NCI, Javed Khan and his lab, in particular Young Song, taught me how to use and analyze expression arrays, and graciously welcomed me into their lab for extended periods of time. Terry Van Dyke very generously provided arrays and advice that were both critical for the project. I would also like to thank my thesis committee: Zena Werb, Nigel Killeen, Kevin Shannon, and the chair, Allan Balmain, who provided encouragement, helpful advice and keen insight throughout the project, and welcomed me into their own labs to learn many of the necessary skills. I am tremendously grateful to my thesis advisor, Bill Weiss, who has provided a limitless amount of support, encouragement, inspiration, and insight with regards to science, medicine, careers, and life while I was in his lab. Bill provided me with the opportunity to explore everything I was interested in scientifically and I was blessed to have such a wonderful mentor. Finally, I would like to thank my family. My parents provided support and encouragement that got me though rough patches from grade school to graduate school and v beyond, in addition to serving as occasional scientific collaborators. I would also like to thank my fiancé, Nan Chen, who among many other things gave me constant encouragement, reinforced our mutual love for science, and made the long hours we both spent at UCSF enjoyable. This work contains published material from the following publications: Chapter 5: Geurts AM*, Hackett CS*, Bell JB, Bergemann TL, Collier LS, Carlson CM, Largaespada DA, Hackett PB. Structure-based prediction of insertion-site preferences of transposons into chromosomes. Nucleic Acids Res. 2006 May 22;34(9):2803-11. PMID: 16489096 Chapter 6: Hackett, CS, Geurts, AM, Hackett, PB. Predicting preferential DNA vector insertion sites: implications for functional genomics and gene therapy. Genome Biol. 2007;8 Suppl 1:S12. PMID: 18047689 Chapter 7: Collier LS, Adams DJ, Hackett CS, Bendzick LE, Akagi K, Davies MN, Diers MD, Rodriguez FJ, Bender AM, Tieu C, Matise I, Dupuy AJ, Copeland NG, Jenkins NA, Hodgson JG, Weiss WA, Jenkins RB, Largaespada DA. Whole-body sleeping beauty mutagenesis can cause penetrant leukemia/lymphoma and rare high-grade glioma without associated embryonic lethality. Cancer Res. 2009 Nov 1;69(21):8429-37. PMID: 19843846 Note on figure and section order: For chapters derived from published manuscripts and manuscripts in preparation, figure order has been preserved, with supplementary figures re- vi numbered and appended to the normal figures. Thus, the text may reference figures seemingly out of order, but this was done to retain the original order of the main figures in the published manuscripts. Additionally, the order of the sections (Introduction, Results, Discussion, and Methods, Figure Legends, References) has been preserved for the published manuscripts and may thus be inconsistent between chapters. Unpublished work follows the order above. vii Abstract Neuroblastoma is a deadly tumor derived from neuronal tissue for which the molecular drivers remain a mystery. Here we have applied classical genetics, analysis of expression quantitative trait loci (eQTL), and forward insertional mutagenesis to uncover novel pathways in the disease. We showed that liver arginase is a candidate susceptibility gene and interacts with component of the GABA pathway both genetically and biochemically to influence tumor susceptibility, and both of these pathways represent potential therapeutic targets. We then constructed a gene coexpression network in tumors and in sympathetic ganglia to explore novel genetic/functional interactions in both neuroblastoma and normal neurons. In particular, we used the coexpression network to identify novel candidate genes for several hereditary hearing loss loci. In a separate project, we focused on forward genetics utilizing the Sleeping Beauty insertional mutagenesis system. We developed a novel algorithm to predict local insertion site preferences of the vector, and show that the transposon system does not cause widespread genomic instability. We then generated a novel transgenic line, TH-SB11, to drive tumors in the peripheral sympathetic nervous system. Finally, we explored methods to drive tumors in the mammary gland, and generated a novel knock-in line capable of driving high-level conditional transposase expression in any tissue. This work illustrates the genetic complexity of neuroblastoma, and has identified novel functional pathways in the disease and a novel therapeutic target. In addition, this work lays the foundation for further gene discovery in neuroblastoma and other tumor types. viii Table of contents: Abstract viii List of Tables x List of Figures xi Chapter 1 1 Chapter 2 9 Chapter 3 59 Chapter 4 91 Chapter 5 101 Chapter 6 147 Chapter 7 192 Chapter 8 235 Chapter 9 253 Chapter 10 271 Chapter 11 287 Library Release 291 ix List of Tables Table 2.1 29 Table 2.2 30 Table 3.1 75 Table 3.2 77 Table 5.1 125 Table 5.2 126 Table 6.1 179 Table 7.1 214 Table 7.2 215 x List of Figures Figure 2.1 44 Figure 2.2 46 Figure 2.3 48 Figure 2.4 50 Figure 2.5 52 Figure 2.6 54 Figure 2.7 56 Figure 2.8 58 Figure 3.1 80 Figure 3.2 82 Figure 3.3 84 Figure 3.4 86 Figure 3.5 88 Figure 3.6 90 Figure 5.1 128 Figure 5.2 130 Figure 5.3 132 Figure 5.4 134 Figure 5.5 136 Figure 5.6 138 Figure 5.7 140 Figure 5.8 142 Figure 5.9 144 Figure 5.10 146 xi Figure 6.1 181 Figure 6.2 183 Figure 6.3 185 Figure 6.4 187 Figure 6.5 189 Figure 6.6 191 Figure 7.1 220 Figure 7.2 222 Figure 7.3 224 Figure 7.4 226 Figure 7.5 228 Figure 7.6 230 Figure 7.7 232 Figure 7.8 234 Figure 8.1 248 Figure 8.2 250 Figure 8.3 252 Figure 9.1 268 Figure 9.2 270 Figure 10.1 284 Figure 10.2 286 xii Chapter 1: Introduction Source: The following contains background on neuroblastoma and genetic screening technology relevant to several of the following chapters. Contributions: This is an original review of the literature that has been aided by conversations with several individuals.