IDENTIFYING, CHARACTERIZING AND INHIBITING THE TELOMERASE REGULATORY NETWORK APPROVED BY SUPERVISORY COMMITTEE Jerry Shay, PhD Woodring Wright, MD, PhD David Corey, PhD Rolf Brekken, PhD Melanie Cobb, PhD DEDICATION Dedicated to my parents, Mark and Tracy Holohan, as well as my siblings, Kelly Nudleman and Kyle Holohan for their support and encouragement. IDENTIFYING, CHARACTERIZING AND INHIBITING THE TELOMERASE REGULATORY NETWORK by BRODY CHRISTOPHER HOLOHAN DISSERTATION / THESIS Presented to the Faculty of the Graduate School of Biomedical Sciences The University of Texas Southwestern Medical Center at Dallas In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY The University of Texas Southwestern Medical Center at Dallas Dallas, Texas May, 2015 Copyright by Brody Christopher Holohan, 2015 All Rights Reserved ACKNOWLEDGEMENTS I would like the thank my mentors, Jerry Shay and Woodring Wright for their continuous support, advice and encouragement, as well as allowing me time and resources to pursue my ideas. They are a fantastic team, and I could not have asked for better mentors. I would also like to thank my lab-mates, particularly Dr. Andrew Ludlow, Dr. Yong Zhou, Dr. Tsung-Po Lai, Dr. Oliver Delgado, Dr. Wanil Kim, Dr. Guido Stadler, Kimberly Batten, Ryan Laranger and Neal Jones. I would also like to thank Kevin Kennon for administrative support. Without my collaborators much of this would have been impossible, so I would like to thank Dr. Tim De Meyer, Dr. Abraham Aviv, Dr. Fowzan Aklayura, Dr. Steve Hunt, Dr. Tim Spector, Dr. Massimo Mangino, Dr. Daphne Friedman and Dr. Daniel Eisenberg for their help, intellectual discussions and willingness to pursue challenenging ideas. I thank the University of Texas Southwestern Medical Center and the Department of Cell Biology for the opportunity to earn a PhD, as well as the Cancer Prevention and Research Institute of Texas and National Institute of Health for funding. Further, I thank my fellow students that made these years enjoyable and acted as my support network in tough times. v IDENTIFYING, CHARACTERIZING AND INHIBITING THE TELOMERASE REGULATORY NETWORK Brody Christopher Holohan, PhD The University of Texas Southwestern Medical Center at Dallas, 2015 Supervising Professor: Jerry W. Shay, PhD Telomeres, which are structures that cap the ends of linear chromosomes are maintained by telomerase, a reverse transcriptase. Telomere length limits the self-renewal capacity for telomerase negative cells, and nearly all tumors circumvent this limitation through telomerase expression; as such, telomerase is an attractive target for cancer therapy. In order to identify new targets for anti-telomerase therapy, I demonstrate that a number of candidate genes are required for telomere maintenance in vitro through shRNA-mediated knockdown and telomere length analysis. Further, I show that Perifosine, a drug identified upstream of a number of the candidates can act as a telomerase inhibitor in a majority of cell lines evaluated in vitro as well as induce shortening of the shortest telomeres in tumors from vi human patients treated with Perifosine in a phase II clinical trial. Additionally, I identify a trans-generational trend in telomere length at birth in human populations that may bias estimates of telomere shortening rate that has public health implications. Lastly, using data from a large twin study, I have identified a network of genes that regulate the rate of telomere shortening in humans that may be used to clarify the association between telomere length, aging and age-related disease. vii TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................................ v ABSTRACT ......................................................................................................................... vi PRIOR PUBLICATIONS .................................................................................................. xii LIST OF FIGURES .......................................................................................................... xiii LIST OF TABLES ........................................................................................................... xvii LIST OF APPENDICES .................................................................................................. xix LIST OF DEFINITIONS .................................................................................................. xx CHAPTER 1: INTRODUCTION ........................................................................................ 1 Historical overview of telomere biology ......................................................................... 1 Modern overview of telomeres and telomerase ............................................................... 5 Molecular Biology of the telomere .................................................................................. 7 Molecular Biology of telomerase ..................................................................................... 9 Heritability of telomere length ....................................................................................... 12 Telomeres and cancer .................................................................................................... 13 Telomeropathies ............................................................................................................. 15 Summary and hypothesis ............................................................................................... 21 CHAPTER 2: VALIDATION OF AN IN VITRO SCREEN FOR POSITIVE REGULATORS OF TELOMERASE .............................................................................. 22 Introduction .................................................................................................................... 22 Knockdown of the hits chosen for validation leads to telomere shortening over time .. 26 viii Reduction in telomerase activity, telomere shortening and altered telomerase mRNA splicing upon knockdown of larp7 ........................................................................................ 30 Humans with a loss-of-function mutation in larp7 suffer a classical telomeropathy .... 34 Discussion ...................................................................................................................... 37 Methods .......................................................................................................................... 46 CHAPTER 3: CHARACTERIZATION OF PERIFOSINE AS A POSSIBLE TELOMERASE INHIBITOR .......................................................................................... 51 Introduction .................................................................................................................... 51 AKT inhibitors induce telomere shortening in some cell lines in vivo ......................... 52 Long-term treatment with perifosine inhibits colony formation in soft agar ................. 56 The HCC38 breast cancer cell line responds most robustly to perifosine of the cell lines tested .................................................................................................................................. 58 Perifosine may act as a telomerase inhibitor in a xenograft model, but does not reduce metastatic tumor burden ....................................................................................................... 60 Perifosine induces shortening of the shortest telomeres and reduces telomerase enzymatic activity in CLL samples from a phase II clinical trial ......................................................... 63 Perifosine’s effects on cell growth, telomerase activity and telomere length are uncorrelated with one another .............................................................................................. 68 Expression data indicates stress response networks may alter Perifosine sensitivity .... 72 Perifosine activates p38 activity in some but not all of the sensitive cell lines ............. 81 Perifosine treatment alters TRF2 expression in some cell lines .................................... 83 Discussion ...................................................................................................................... 85 ix Methods .......................................................................................................................... 95 CHAPTER 4: IDENTIFICATION OF A TIME-DEPENDENT TREND IN TELOMERE LENGTH .................................................................................................. 100 Introduction .................................................................................................................. 100 Results: Cross-sectional estimates of telomere shortening rate are consistently lower than longitudinal estimates ........................................................................................................ 108 PBY predicts telomere length better than FAB and models of cross-sectional data which do not assume fixed initial telomere length are consistent with longitudinal studies ........ 109 Collinearity between Age, FAB and PBY ................................................................... 113 Mediation analysis reveals heterogeneity in the PBY effect and a larger FAB effect than previously reported ............................................................................................................ 115 The age-associated telomere shortening rate from mediation analysis is more consistent