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INPP4B) Overexpression on Autophagy in Acute Myeloid Leukemia (AML)

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INPP4B) Overexpression on Autophagy in Acute Myeloid Leukemia (AML) Investigating the Role of Inositol Polyphosphate 4-Phosphatase Type II (INPP4B) Overexpression on Autophagy in Acute Myeloid Leukemia (AML) By Mark Hani Sharobim A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Pharmacology and Toxicology University of Toronto © Copyright by Mark Sharobim 2016 Investigating the Role of Inositol Polyphosphate 4-Phosphatase Type II (INPP4B) Overexpression on Autophagy in Acute Myeloid Leukemia (AML) Mark H. Sharobim Master of Science Department of Pharmacology and Toxicology University of Toronto 2016 ABSTRACT BACKGROUND: Inositol polyphosphate-4-phosphatase type-II (INPP4B) is a lipid phosphatase that dephosphorylates PI(3,4)P2 into PI(3)P. The depletion of PI(3,4)P2 prevents aberrant AKT signalling. Therefore, INPP4B is classically known as a tumour suppressor. Recent studies however demonstrate INPP4B overexpression may promote cancer generation and progression. We investigated effects of INPP4B overexpression in acute myeloid leukemia (AML). METHODS: AML cell lines that overexpress wild-type (INPP4Bwt) and mutant (INPP4Bmut) INPP4B were used to determine what phenotypes are governed by its phosphatase activity. RESULTS: Overexpression of wild-type INPP4B conferred advantages in growth, chemoresistance and colony forming assays, suggesting a role for INPP4B opposite to that of a tumour suppressor. These phenotypes were either absent or greatly diminished in INPP4Bmut cells. INPP4Bwt cells also had increased accumulation of autophagosomes with or without autophagy inhibitors when compared to control. CONCLUSION: INPP4B-mediated phenotypes in AML are phosphatase-dependent and this is subsequently associated with increased potential to undergo autophagy. Word Count: 150/150 ii ACKNOWLEDGEMENTS It would be remiss of me to acknowledge anyone prior to my Lord Jesus Christ and His help throughout my entire graduate degree. I couldn’t have done it without Him and His constant love. My family is a rock in my life that has kept me steady throughout the tough times and pushed me through obstacles I felt I could not overcome. It is with their support, guidance and love that I am where I am today. My dad is an example of integrity and virtue and not only do I learn from his actions everyday, the lessons I have learned from him I will keep close to my heart forever. My mother is the definition of a role-model and an exemplar for how I wish to carry myself in my personal and professional life throughout my years. My sisters are the greatest gift I could ever ask for and the relationship I have with them is something I will cherish forever. Their company keeps me cheerful throughout the tough times. My supervisor Dr. Lenny Salmena, PhD is by far and large the greatest inspiration I have in science and as a mentor. Though I was his first student and have technically graduated, I will consider him my mentor and teacher forever. His passion as well as dedication to his work and craft are attributes I aspire to attain one day. His love and enthusiasm for science is something I wish to pass on to those intending to pursue any sort of scientific career. Without him, without a doubt I would not be where I am today. His constant mentoring is something that cannot be understated. He has literally shaped the way I think and tackle science as a whole. Thank you so much Lenny. My lab mates, Martino Gabra, Emily Mangialardi, Anthony To, Meong-Hi Son, Lydia To, Thais Fontanezi-Maciel, Shayne Greenberg, Ayesha Rashid, Erik Dzneladze and John Woolley are a great family that has positively shaped my future forever. The mentoring I received from Dr. Michael Jain, Dr. Rob Laister, Dr. Vuk Stambolic and Ayesha Rashid are little gifts I cherish and I hope someone could be as lucky as me to have been able to pick their brains the past 3 years. I want to thank all those in the lab that taught me that science is an exercise in thinking. It is an open garden of creativity and is conducive to forming bright ideas, innovations and concepts. It is this I am most thankful that I learned while pursuing my degree, and a skill that could not have been cultivated elsewhere. iii CONTRIBUTIONS Martino Gabra treated all the cells with daunorubicin and counted their viability (Figure 11). Emily Mangialardi created the immortalized Inpp4b knockout and wild type MEF cell lines (Figure 16). Anthony To helped with and performed western blots (Figures 15-17). Dr. Meong Hi Son did the colony forming cell experiment (Figure 10). Dr. John Woolley did the low serum viability counts (Figure 9B). iv Table of Contents Abstract ........................................................................................................................................... ii Acknowledgements ........................................................................................................................ iii Contributions.................................................................................................................................. iv Table of Contents ............................................................................................................................ v List of Abbreviations .................................................................................................................... vii List of Figures ................................................................................................................................. x List of Tables ................................................................................................................................. xi List of Appendices ........................................................................................................................ xii 1. Introduction ............................................................................................................................. 1 1.1 Inositol Phospholipids ......................................................................................................... 1 1.2 Phosphoinositide Signalling Overview ............................................................................... 2 1.3 Phosphoinositide Signalling in Cancer ............................................................................... 5 1.3.1 The PI3K Pathway .................................................................................................... 5 1.3.2 Antagonizing PI3K signalling................................................................................... 7 1.4 Inositol Polyphosphate 4-Phosphatase, Type II (INPP4B) ................................................. 9 1.4.1 Discovery .................................................................................................................. 9 1.4.2 Structure .................................................................................................................. 10 1.4.3 Functions ................................................................................................................. 11 1.5 INPP4B and Cancer .......................................................................................................... 14 1.5.1 INPP4B is a Tumour Suppressor ............................................................................ 14 1.5.2 INPP4B Overexpression in Cancer ......................................................................... 16 1.6 Acute Myeloid Leukemia ................................................................................................. 24 1.6.1 Overview ................................................................................................................. 24 1.6.2 The PI3K pathway in AML .................................................................................... 25 1.7 Autophagy ......................................................................................................................... 28 1.7.1 Overview ................................................................................................................. 28 1.7.2 Macroautophagy ..................................................................................................... 29 1.7.3 The PI3K-mTOR Pathway and Autophagy ............................................................ 33 1.7.4 Autophagy and Cancer: a Double Edged Sword? ................................................... 34 2. Rationale, Aims and Hypothesis ........................................................................................... 39 2.1 Rationale and Aims ........................................................................................................... 39 v 2.1.1 Investigating mechanisms of INPP4B-mediated phenotypes in AML cells ........... 39 2.1.2 INPP4B Overexpression and PI(3)P signalling: Implications for Autophagy........ 40 2.2 Hypothesis......................................................................................................................... 41 3. Materials and Methods .......................................................................................................... 42 3.1 Cell Culture ....................................................................................................................... 42 3.2 Lentivirus production ........................................................................................................ 42 3.3 DNA plasmids ................................................................................................................... 43 3.4 Methylcellulose Colony Formation
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