Ponatinib Inhibits STAT3 Activity and Reduces Colorectal Cancer Growth

Ponatinib Inhibits STAT3 Activity and Reduces Colorectal Cancer Growth Fiona Hue Yee Tan Submitted to The University of Melbourne in total fulfilment of the requirements of the degree of Doctor of Philosophy November 2017 The Royal Melbourne Hospital The Department of Surgery The University of Melbourne ABSTRACT Colorectal cancer is the 4th most common cancer globally and the 2nd most common cancer in Australia. Constitutive activation of Signal Transducer and Activator of Transcription 3 (STAT3) has been observed in over 50% of human colorectal carcinomas and its role in tumour progression has been confirmed in numerous mouse models and clinically in human samples. Previous data suggests the hyper-activation of upstream molecules notably, Epidermal Growth Factor Receptor (EGFR) and the Interleukin (IL)-6-gp130 family of cytokines, contributes to enhanced STAT3 activation and tumourigenesis [1]. With the aim of overcoming STAT3-driven tumourigenicity, we evaluated a panel of 1167 FDA approved agents for their ability to inhibit STAT3 activity. In this initial drug screen, human colorectal cancer cell lines were assessed by the adenoviral STAT3 luciferase reporter assay. In the presence of inhibitors, cells were also stimulated with EGF and IL-6 allowing enhanced STAT3 activity at 10µM. We identified 51 FDA approved agents to have reduced STAT3 activity by ≥50%. Our secondary drug screen further evaluated inhibitors by EGF and IL-6 mediated STAT3 activity by western blot and resulted with 9 inhibitors to have shown successive reduction at 1µM. It has been recently shown that IL-11, a closely related IL-6 family member has a more prominent role than IL-6 during the progression of gastrointestinal cancers, including colorectal tumours [5], and therefore in our tertiary screen we further evaluated agents that could inhibit IL-11, IL-6 and EGF mediated STAT3 phosphorylation by western blot analyses. As a result, Ponatinib (AP24534) markedly reduced EGF, IL-6 and IL-11 driven STAT3 activation. Ponatinib is a multi-targeted tyrosine kinase inhibitor and is currently approved for the treatment of chronic myeloid leukaemia and Philadelphia chromosome-positive acute lymphoblastic leukaemia. With further in-vitro and in-vivo analyses performed, Ponatinib also reduced transcriptional gene expression of STAT3 regulated genes (SOCS3), cell viability, migration and tumour growth. In addition, Ponatinib was also observed to reduce LIF (another member of the IL-6 family of cytokines) driven STAT3 activity. This study demonstrated Ponatinib’s preferentially targets the IL-11 driven STAT3 pathway in CRC cell lines when compared to IL-11 driven AKT and ERK1/2 signalling pathways. We therefore, further explored the possibility of Ponatinib to directly target IL- ii 11R/gp130 and speculate possible interaction however, further analysis is required to determine this theory. While the effectiveness of Ponatinib requires additional investigation, our findings both in-vitro and in-vivo offer proof-of-principle evidence for the potential use of Ponatinib for the treatment of STAT3 driven colorectal cancers. iii DECLARATION This declaration is to certify that: i. The thesis comprises only their original work towards the Ph.D. except where indicated in the preface. ii. Due acknowledgement has been made in the text to all other material used. iii. The thesis is fewer than 100,000 words in length, exclusive of tables, maps, bibliographies and appendices. Fiona Hue Yee Tan The Royal Melbourne Hospital The Department of Surgery The University of Melbourne iv PREFACE The experimental data described in this thesis comprises only my original work, except for the following results, obtained in collaboration: • The Ad-APRE-luc promoter was developed by the Zhu laboratory from the Department of Surgery at the University of Melbourne, The Royal Melbourne Hospital. • The L-gp130 construct was obtained from the Putoczki laboratory at the Walter and Elisa Hall Institute of Medical Research. • The plasmid preparation of IL-11R, L-gp130 and STAT3C was performed by our Research Assistant, Lucy Paradiso. v ACKNOWLEDGEMENTS I would first like to thank my primary supervisor, Dr. Rodney Luwor for all his commitment, time and patience throughout my Ph.D. candidature. I remember in the earlier years, I would knock on your door at least twice a day and I thank you for having great patience with all my endless questions. Over the past 3.5 years, I have grown into a more confident scientist and I cannot thank you enough for your continual support and guidance. I am truly grateful to have a supervisor who is always available when I’m in a pickle or if I need clarification. I would not have been able to complete my Ph.D. without all your help and wisdom. Thank you! I would also like to thank my co-supervisor Dr. Stanley Stylli for your joyful nature and your willingness to help me throughout these years. I am sincerely grateful for all the discussions we’ve had and for your endless encouragement throughout this journey of mine. I sincerely value the time you have sacrificed out of your day to see how I am going with experiments and thesis writing. Thank you for spreading joy in the lab with your ‘dad’ jokes and endless humour, never change. To my other co- supervisor, Dr. Tracy Putoczki, I sincerely thank you for all your knowledge and on- going support throughout the years. Your drive and passion is evident in the several studies and manuscripts published and I am truly grateful to have you as my co- supervisor. I am also thankful for my Ph.D. committee members, Dr. Hong-Jian Zhu and Dr. Robert O’Donoghue for all their advice and support throughout my Ph.D. journey. Thank you for taking the time to suggest possible experiments that could help with my project. Your knowledge and encouragement has truly been invaluable. I would like to thank my fellow lab members, past and present, for all their added support and numerous enjoyable conversations. In particularly, Lucy Paradiso for all her endless help with the ins and outs of the lab and for teaching me how to perform RNA extractions and for assisting with plasmid preparations. My gratitude also extends out to the members of the Zhu lab and Hovens lab. It has been a pleasure coming in everyday to have lovely corridor conversations with you all! I acknowledge the various facilities involved with my project, especially to the members of the BRF facility for taking care of my mice and for all their assistance with vi animal training. Additionally, I would like to also thank the MIPS facility in particularly, Dr. Cameron Nowell and Sandy Fung for all their assistance with FACS sorting. I would also like to thank my family: my parents, my brother and sister for their endless support and encouragement throughout this journey of mine. Especially to my parents and their restaurant, Double Happiness for funding me and my belly throughout my Ph.D. life. I would also like to personally thank my friends, Jessica Ventura and Sonia Aithal who have been of great support. Lastly, I would like to thank my partner, for his endless support, patience and endless Gong Chas during my Ph.D. Your constant encouragement has been so important in allowing myself to always push through and to never give up – thank you! vii TABLE OF CONTENTS ABSTRACT ................................................................................................................................ ii DECLARATION ....................................................................................................................... iv PREFACE ................................................................................................................................... v ACKNOWLEDGEMENTS ...................................................................................................... vi TABLE OF CONTENTS .......................................................................................................... viii LIST OF ABBREVIATIONS ................................................................................................... xii LIST OF TABLES ..................................................................................................................... xiv LIST OF FIGURES ................................................................................................................... xvi 1. CHAPTER 1 - INTRODUCTION .................................................................................... 2 1.1 Introduction to Colorectal Cancer ....................................................................................... 2 Colorectal Cancer Epidemiology ........................................................................................ 2 Polyp to cancer (Pathogenesis) ........................................................................................... 5 Associated gene mutations .................................................................................................. 6 Current treatments for Colorectal Cancer ........................................................................... 8 Current targeted therapies and prognosis ............................................................................ 9 Resistance against current treatments ................................................................................. 11 1.2 Signal transduction, STAT3 and CRC ................................................................................ 13 Signal transduction and oncogenesis .................................................................................

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