Thesis: Targeting β-catenin in MPNSTs. Jed Kendall August 8th, 2016 Previous Degree: B.S. from Brigham Young University Degree to be conferred: Doctorate of Philosophy Department: Molecular and Developmental Biology Committee Chair: Nancy Ratner Abstract MPNSTs are a highly invasive soft tissue sarcoma that arises from aberrantly regulated Schwann cells/Schwann cell precursors. Fifty percent of MPNSTs originate in patients with Neurofibromatosis type 1, an autosomal dominant disease that affects 1:3500 people, and the other fifty percent are sporadic in origin. Curative drug treatments are lacking due to MPNSTs high resistance to chemotherapy. The combination of MPNST’s invasive nature and ability to resist chemotherapy results in a dismal 5-year survival rate Malignant peripheral nerve sheath tumors (MPNSTs) are soft tissue sarcomas that are a major cause of mortality of Neurofibromatosis type 1 (NF1) patients. MPNST patients have few therapeutic options available and only complete surgical resection can be curative. MPNST formation and survival are dependent on activated β- catenin signaling. The goal of my work is to identify novel genes important in MPSNT progression and survival and also to screen any developed anti cancer agents against MPNST cell lines. With this approach we can identify readily available therapeutics and also identify potential therapeutics of the future. CK2 is a known oncogenic kinase in diverse types of cancer through maintaining. Often times CK2 maintains oncogenic phenotypes through stabilizing β-catenin, which is a critical factor for MPNST survival. We found that CK2α is over expressed in MPNSTs and is critical for maintaining cell survival, as the CK2 inhibitor, CX-4945 (Silmitasertib), and shRNA targeting CK2α in vitro each significantly reduced MPNST cell viability. These effects were preceded by loss of critical signaling 2 pathways in MPNSTs, including destabilization of β-catenin and TCF-8. CX-4945 administration in vivo slowed tumor growth and extended survival time. We conclude that CK2 inhibition is a promising approach to blocking β-catenin in MPNST cells, although combinatorial therapies may be required for maximal efficacy. To find potential therapeutic targets we identified genes critical for MPNST cell survival. We used microarray analysis, stringent selection criteria, and shRNA knock down. Through these methods we identified PITX2 as a critical survival gene specific to MPNST cells. Knocking down PITX2 leads to apoptosis in MPNST cells. We discovered a novel relationship between PITX2 expression and β-Catenin protein stability. Through microarray analysis of PITX2 shRNA treated MPNST cells as compared to controls we have identified NLK, FZD6, and GAS2 as genes differentially regulated by PITX2 expression, containing bicoid response elements within the promoter, and implicate β-Catenin signaling. Furthermore, we identified GAS2 as a mechanism used by MPNSTs to stabilize β-catenin. 3 4 Preface The work contained in this thesis is published or is in preparation for publication. 1. Kendall JJ, Katherine CE, Patel AV, Rizvi TA, Largaespada DA, Ratner N. CK2 blockade causes MPNST cell apoptosis and promotes degradation of β- catenin. Oncotarget. 2014 July 2. Kendall JJ, Patel AV, Choi K, Ratner N. PITX2 over-expression is critical for MPNST survival and promotes β-catenin protein stability. Manuscript in preparation. 5 Acknowledgments Through the course of my dissertation studies I have been graced with assistance and friendship of many colleagues. I would first like to thank Nancy Ratner for building a successful and well-funded lab. I have been the benefactor of her tremendous success. Nancy has imparted an abundance of wisdom and guidance to many, including me, through the years. I will always be grateful to Nancy for allowing me to pursue topics about which I am most passionate. The topics in retrospect are not so important; rather the refinement of thought from my own trial and error is by far and away the greatest skill that I will take away from my years as a PhD student. Outside of scientific accolades and above all else Nancy is a good person. I would like to thank my thesis committee, Dr. Aaron Zorn, Dr. Brian Gebellein, Dr. Lionel Chow, and Dr. David Largaespada. Each member of my committee has given valuable feedback for the betterment of my projects. Additionally, my committee has challenged me as a scientist and encouraged me to think outside of the box that often traps me. Additionally, I would like to thank the Molecular and Developmental and MSTP program for their support. Through the good and bad times I have had the support and friendship of many Ratner lab members. Since Ami Patel had to endure the brunt of my naïve ideas during my early years she definitely deserves the most credit. Ami, Preeti, and Tilat all have become my good friends and I will miss each of you. Kwangmin, I greatly appreciate your willingness to run a thousand different bioinformatics analyses that have been integral in forming working hypotheses. I would like to 6 thank Lindsey Aschlbacher-Smith, Katie Chaney, Jianqiang Wu and Josh Pressler for creating a fun and productive work environment. I would also like to thank Meghan Brundage for taking me under her wing when I was a new arrival. Finally, I would like to thank all of my family members. To my mom, dad and grandparents thank you for a wonderful life and magical childhood. Each of you have made tremendous sacrifices on my behalf. Any achievements that I happen to stumble upon are to your credit. Most of all, I would like to thank my lovely wife. She has been the source of my greatest happiness. From our move to Cincinnati where we purchased our first home, to raising our two beautiful children, thank you for all your love and support. 7 Table of Contents ABSTRACT ................................................................................................................................................................... 2 CHAPTER 1: ETIOLOGY, PATHWAYS, AND THERAPEUTICS OF MPNSTS ........................... 12 1.1 THE ETIOLOGY OF MPNSTS ......................................................................................................................... 12 1.2 FROM BENIGN TO MALIGNANT .................................................................................................................... 14 1.3 MPNST OUTCOMES AND TREATMENT ...................................................................................................... 15 1.4 THERAPEUTIC ADVANCEMENTS IN MPNSTS PAST AND PRESENT. ....................................................... 16 1.5 Β-CATENIN AS A POTENTIAL THERAPEUTIC TARGET IN MPNSTS ........................................................ 19 1.5.1. Canonical WNT signaling cascade ................................................................................................. 19 1.5.2 Pathogenesis of β-catenin signaling in MPNSTS ....................................................................... 21 1.5.3 WNT signaling therapeutics .............................................................................................................. 23 CHAPTER 2: CK2 AS AN ONCOTARGET ......................................................................................... 24 2.1 INTRODUCTION TO CK2 ................................................................................................................................. 24 2.2 CK2 AND DNA DAMAGE REPAIR ................................................................................................................. 26 2.3 CK2 AND CELL CYCLE PROGRESSION ........................................................................................................... 28 2.4 CK2 AND CELL DEATH/APOPTOSIS ............................................................................................................ 29 2.5 CK2 AND CANCER ........................................................................................................................................... 30 2.6 CROSS TALK BETWEEN CK2 AND PRO-SURVIVAL/PROLIFERATIVE PATHWAYS. ................................ 32 2.6.1 NF-κB and CK2 ......................................................................................................................................... 32 2.6.2 PI3K/AKT and CK2 ................................................................................................................................. 32 2.6.3 CK2 and β-catenin .................................................................................................................................. 34 2.8 RESULTS ............................................................................................................................................................ 38 2.8.1 CK2 is overexpressed in MPNSTs ...................................................................................................... 38 2.8.2 CK2 inhibition induces cell death and cell cycle arrest in MPNSTs in vitro. ................. 38 2.8.3 CK2 regulates β-catenin protein stability in MPNSTs in vitro. ........................................... 39 2.8.4 CK2 regulates TCF8 and other survival pathways in MPNSTs in vitro. .......................... 40 2.8.5 CX-4945 slows tumor growth in vivo ............................................................................................. 41 2.8.6 MPNST treatment with CX-4945 in combination with PD0325901 ................................. 42 2.9 DISCUSSION, IMPLICATIONS, AND LIMITATIONS ......................................................................................