The Pennsylvania State University The Graduate School College of Medicine THE NOVEL MECHANISTIC ROLE OF PIGN IN LEUKEMIA PROGRESSION A Dissertation in Biomedical Sciences by Emmanuel Kwame Teye 2017 Emmanuel Kwame Teye Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2017 The dissertation of Emmanuel Kwame Teye was reviewed and approved* by the following: Jeffrey J Pu Assistant Professor of Medicine Dissertation Advisor Chair of Committee Jong K Yun Associate Professor of Pharmacology Director, Translational Therapeutics Option, Biomedical Sciences Douglas B Stairs Assistant Professor of Pathology and Pharmacology Gregory S Yochum Associate Professor of Biochemistry and Molecular Biology Hong-Gang Wang Professor of Pediatrics and Pharmacology Ralph L Keil Associate Professor of Biochemistry and Molecular Biology Chair, Biomedical Sciences Graduate Program *Signatures are on file in the Graduate School iii ABSTRACT Genomic instability plays a pivotal role in the leukemia progression of myelodysplastic syndromes (MDS). However, the precise genetic cause and the underlying mechanisms of MDS leukemia progression or transformation to acute myeloid leukemia (AML) remain elusive. Moreover, the current approach to MDS progression risk-stratification using the International Prognostic Scoring System (IPSS) or WHO Prognostic Scoring System (WPSS) is limited and fails to address the dynamic nature of the disease. This presents a need for the identification of novel prognostic markers and a better understanding of the mechanisms involved. In this study, phosphatidylinositol glycan anchor biosynthesis, class N (PIGN), a gene encoding an enzyme participating in the final steps of the glycophosphatidylinositol-anchored protein (GPI-AP) biosynthesis pathway and a cancer chromosomal instability (CIN) suppressor, was highly ranked as a predictor of the risk of MDS leukemia progression. We also observed the progressive loss of PIGN during MDS progression to AML. Moreover, PIGN gene expression aberrations (i.e. increased gene expression but diminished to no protein production) were observed in a subset of high-risk MDS and AML patients with myelodysplasia-related changes. PIGN gene expression aberrations were associated with increased frequency of GPI-AP deficiency in leukemic cells and correlated with the elevation of genomic instability that was independent of the TP53 regulatory pathway. PIGN gene expression aberrations were attributed to novel partial intron retentions between exons 14 and 15 resulting in frameshifts and premature termination. Interestingly, in an MDS leukemia progression model, this mutation was identified in the leukemia stage (i.e. MDS-L) cells but was absent in the MDS stage (i.e. MDS92) cells. Transient suppression or ablation of PIGN induced DNA damage response which was rescued following PIGN restoration. We also observed an increase in the frequency of CIN with PIGN loss. Moreover, PIGN physically interacted with and/or regulated the spindle assembly iv checkpoint via MAD1, MAD2, MPS1, and BUBR1. Thus, PIGN is crucial in the regulation of mitotic integrity for the maintenance of chromosomal stability and ultimately prevents leukemic transformation/progression. This study for the first time identified PIGN as a prognostic marker of MDS transformation and revealed the link between PIGN gene expression aberration, genomic instability, and MDS progression/leukemia transformation. PIGN gene expression aberration is associated with genomic instability and leukemogenesis and could serve as a basis for improved risk-stratification of MDS patients. v TABLE OF CONTENTS List of Figures .......................................................................................................................... vii List of Tables ........................................................................................................................... viii Abbreviations ........................................................................................................................... ix Preface ... ................................................................................................................................. xii Acknowledgements .................................................................................................................. xiii LITERATURE REVIEW ........................................................................................ 1 Myelodysplastic Syndromes (MDS) ................................................................................ 2 Current MDS therapies ............................................................................................. 4 MDS risk-stratification and current challenges ........................................................ 6 Molecular markers of MDS progression .................................................................. 9 Chromosomal instability .......................................................................................... 15 Role of CIN in MDS/AML progression ................................................................... 20 CIN as a marker of MDS leukemic transformation.................................................. 22 Therapeutic targeting of CIN ................................................................................... 24 The spindle assembly checkpoint ............................................................................. 25 Cross-talk between DDR and CIN induction ........................................................... 27 SAC/mitotic checkpoint dysregulation in MDS/AML progression ......................... 29 Phosphatidylinositol Glycan Class N (PIGN) .................................................................. 30 PIGN and GPI-AP biosynthesis ............................................................................... 32 Role of PIGN in CIN and leukemic progression ...................................................... 36 PIGN gene expression aberration is associated with genomic instability and leukemic progression in acute myeloid leukemia with myelodysplasia-related changes ............................................................................................................................. 37 Abstract ............................................................................................................................ 38 Introduction ...................................................................................................................... 39 Materials and Methods ..................................................................................................... 41 Results .............................................................................................................................. 49 Discussion ........................................................................................................................ 73 Conclusions ...................................................................................................................... 78 PIGN spatiotemporally regulates the spindle assembly checkpoint ........................ 79 Abstract ............................................................................................................................ 80 Introduction ...................................................................................................................... 81 Materials and Methods ..................................................................................................... 83 Results .............................................................................................................................. 88 Discussion ........................................................................................................................ 101 Conclusions ...................................................................................................................... 107 vi SUMMARY & FUTURE DIRECTIONS ............................................................... 108 Translational Application ................................................................................................. 118 Study Limitations ............................................................................................................. 119 CONCLUSION ................................................................................................................ 122 Appendix A Permissions .................................................................................................. 123 Appendix B Supplemental Tables .................................................................................... 128 Appendix C Primer Sequences......................................................................................... 129 Appendix D Cell cycle frequency post-PIGN knockout .................................................. 130 Appendix E RT-qPCR profile post-PIGN knockout ........................................................ 131 Appendix F Co-localization analyses of mutant PIGN .................................................... 132 REFERENCES................................................................................................................. 133 vii LIST OF FIGURES Figure 1-1 Representative Wright-Giemsa stained film of a high-risk MDS patient with refractory anemia with excess blasts. ............................................................................... 3 Figure 1-2 Mutational overlap of frequently mutated