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Development of a Phosphoprotein Enrichment Method to Identify and Characterize Phosphoproteins Within Leukemia Following Treatment with the PP2A Activator, FTY720

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Development of a Phosphoprotein Enrichment Method to Identify and Characterize Phosphoproteins Within Leukemia Following Treatment with the PP2A Activator, FTY720 Development of a phosphoprotein enrichment method to identify and characterize phosphoproteins within leukemia following treatment with the PP2A activator, FTY720 THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Justin Charles Staubli Graduate Program in Pathology The Ohio State University 2012 Master's Examination Committee: James W. Waldman, PhD, Advisor Michael F. Freitas, PhD Copyright by Justin Charles Staubli 2012 Abstract . Chronic lymphocytic leukemia (CLL) is the most common adult leukemia and is characterized by the accumulation of CD5+ B lymphocytes in the blood and lymph organs. CLL B lymphocytes have shown a number of abnormal cellular disruptions resulting in the inhibition of several key tumor suppressors and subsequent reliance on pro-survival pathways, a term known as ‘oncogenic addiction’. This reliance on a single robust survival pathway offers a powerful new tool in cancer therapy with the development of specific molecular targets to be exploited within these diseased cells. Given the therapeutic potential of imatinib in chronic myelogenous leukemia (CML), additional malignancies have kinases and phosphatases that are disproportionately activated or deactivated that are ripe for targeting. Most notably, the serine/threonine phosphatase PP2A is a known tumor suppressor that has been implicated in several blood-borne malignancies including CML and CLL. Treatment with the sphingolipid derivative and S1P agonist FTY720 results in an increase in enzymatic activity of PP2A, which results in the apoptosis of CLL cells. However, the mechanism by which FTY720 up regulates PP2A remains elusive. To fully understand the pathways affected by FTY720 in leukemia cells, an integrated, global phosphoprotein study is necessary. ii Numerous methods exist for phosphoprotein investigation, including immunoaffinity and metabolic labeling. While these methods have formed the backbone of phosphoproteomic studies, unfortunately, their reliance on known cellular targets and limited scope in target identifications limits our ability to fully comprehend a phosphatase network; especially one as pervasive and intricate as PP2A. The continued development of ever-increasing sensitivity in both the hardware of mass spectrometry and the protein and post-translational modification identification software has given life to the prospect of a true ‘global phosphoproteome fingerprint’ of a phosphorylation-manipulating therapy, such as FTY720. Mass spectrometry (MS)-based approaches have several advantages over immunoaffinity and metabolic labeling methods: MS investigation requires no prior knowledge of potential targets and can directly identify the site of phosphorylation. Despite these advantages, MS-based phosphoproteomics techniques are time and labor intensive and often times must be tailored to individuals’ needs. Thus, the development of unique phosphoprotein enrichment methods and MS techniques are crucial. The work I have done in this area, specifically looking at total phosphoprotein enrichment from global lysates have yielded excellent reproducibility in the global phosphoproteome fingerprint of the Burkitt lymphoma cell line Ramos, a model system for PP2A manipulation. Utilizing a modified technique with the TALON PMAC Phosphoprotein enrichment kit from Clontech along with a modified TCA sample cleanup prior to in- solution protein digest, a highly reproducible phosphoprotein identification method has iii been developed. With total Ramos cell lysate, technical replicates of phosphoprotein identification was as high as 93% (n=5), and biological replicates reproducibility was 88.5% (n=5). While further validation is necessary to determine the reproducibility of this method within primary leukemia cells, my work gives the first tantalizing steps toward reliable identification of a cancer ‘phosphoprotein fingerprint’ that could aid in the elucidation of phosphorylation pathways in cancer and subsequent molecular targeted therapies. iv Dedication This document is dedicated to my family, The Family, and you (all). v Acknowledgments I would like to acknowledge the professional, intellectual and technical assistance provided by the members of the Freitas and Byrd laboratories. Your assistance was instrumental in the completion of this project. vi Vita May 2002 .......................................................Cambridge High School May 2007 .......................................................B.S. Biology, UW – Stevens Point June 2007 to present.......................................Graduate Research Associate, Department of Pathology, The Ohio State University Publications 1. Ghebranious N, Blank RD, Raggio CL, Staubli J, McPherson E, Ivacic L, Rasmussen K, Jacobsen FS, Faciszewski T, Burmester JK, Pauli RM, Boachie- Adjei O, Glurich I, Giampietro PF. A missense T (Brachyury) mutation contributes to vertebral malformations. J Bone Miner Res. 2008 Oct;23(10):1576- 83. 2. Lapalombella R, Wang L, Ramanunni A, Yeh Y, Rafiq S, Jha S, Staubli J, Lucas DM, Herman SEM, Johnson AJ, Lozanski A, Andritsos L, Jones J, Flynn J, Thompson P, Algate P, Stromatt S, Jarjoura D, Mo X, Wang D, Chen C, Lozanski G, Tridandapani S, Freitas MA, Muthusamy N, Byrd JC. Tetraspanin CD37 Directly Mediates Transduction of Survival and Apoptotic Signals. In press at Cancer Cell. vii Fields of Study Major Field: Pathology Emphasis: Cancer Immunology viii Table of Contents Abstract ............................................................................................................................... ii Dedication ........................................................................................................................... v Acknowledgments.............................................................................................................. vi Vita .................................................................................................................................... vii Table of Contents ............................................................................................................... ix List of Tables ................................................................................................................... xiii List of Figures .................................................................................................................. xiv CHAPTER 1 INTRODUCTION ....................................................................................... 1 1.1 ONCOGENIC ADDICTION AND TARGETED MOLECULAR THERAPY .. 1 1.2 SIGNALING CASCADES AND CHRONIC LYMPHOCYTIC LEUKEMIA .. 3 1.3 THE SERINE/THREONINE PROTEIN PHOSPHATASE 2A (PP2A) ............. 6 1.4 THE PP2A-ACTIVATOR FTY720 ..................................................................... 8 1.5 CONCERTED EFFORT TO STUDY GLOBAL PHOSPHOPROTEOMICS ... 9 1.6 SUMMARY OF RESEARCH ........................................................................... 13 ix CHAPTER 2 PHOSPHOPROTEOMIC INDENTIFICATIONS IN RAMOS POINT TO A POTENTIAL ROLE OF THE PP2A INHIBITOR SET IN THE MODE OF FTY720 ACTION. .......................................................................................................................... 15 2.1 INTRODUCTION .............................................................................................. 15 2.2 EXPERIMENTAL ............................................................................................. 17 2.2.1 Materials ..................................................................................................... 17 2.2.2 Cell drugging and lysate preparation .......................................................... 17 2.2.3 Phos-Tag Phosphoprotein Enrichment ..................................................... 199 2.2.4 SDS-PAGE and Pro-Q staining .................................................................. 19 2.2.5 Band excision and in-gel digestion ............................................................. 20 2.2.6 Capillary ESI-HPLC-MS/MS ..................................................................... 21 2.2.7 Data analysis ............................................................................................... 22 2.2.8 Western blotting and immunoprecipitation ...................................................... 23 2.3 RESULTS AND DISCUSSION ........................................................................ 24 2.3.1 Phosphoprotein enrichment of Ramos cells by Phos-Tag Zn2+ .................. 24 2.3.2 LC-MS/MS Identification of differential phosphoproteins between FTY720 treated and untreated samples. .................................................................... 25 2.3.3 Protein expression of the PP2A-inhibitor SET in Ramos and primary CLL B cells.......................................................................................................... 35 x 2.3.4 Potential inhibition of the Hsp90 pathway by FTY720. ............................. 37 2.4 CONCLUSION .................................................................................................. 43 UTILIZATION OF THE PHOSPHOPROTEIN ENRICHMENT KIT TALON PMAC OFFERS A ROBUST AND REPRODUCIBLE METHOD FOR EXPLORING THE PHOSPHOPROTEOME OF LEUKEMIA. .................................. 45 3.1 INTRODUCTION ................................................................................................... 45 3.2 EXPERIMENTAL .................................................................................................
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