Generation of CRISPR engineered prostate cancer cell line models to study androgen receptor signalling in advanced prostate cancer Thesis submitted in partial fulfilment of the requirement of the degree of Doctor of Philosophy Evangelia Eirini Kounatidou Northern Institute for Cancer Research Faculty of Medical Sciences Newcastle University September 2019 Abstract Prostate cancer resistance to AR targeted therapies due to the emergence of AR point mutations and AR splice variants that cannot be targeted by the currently available agents comprise a major clinical challenge. There is paucity of models that accurately reflect the mechanisms of AR regulation in advanced disease. This highlights the high demand of generating novel disease relevant models. A CRISPR pipeline was developed to generate cell line models which harbour specific point mutations in the LBD of AR as well as stop codons in AR exon 5 which resulted in AR-FL knock-out so that the remaining endogenous AR-Vs could be studied discriminately of interfering AR-FL. Using a streptavidin-tagged Cas9 in conjugation with a biotinylated donor template resulted in high donor template knock-in efficiencies and yielded (i) an ARW741L CWR22Rv1 cell line derivative and (ii) an AR-FL knock-out cell line derivative called CWR22Rv1-AR-EK (Exon Knock-out). CWR22Rv1-AR-EK cells retained all endogenous AR-Vs following AR gene editing. AR-Vs acted unhindered following AR-FL deletion to drive cell growth and expression of androgenic genes. Global transcriptomics demonstrated that AR-Vs drive expression of a cohort of cell cycle and DNA damage response genes and depletion of AR-Vs sensitised cells to ionising radiation. To date, elimination of AR-Vs by pharmacological inhibition remains challenging. However, disruption of AR pre-mRNA splicing is nowadays a highly attractive option. A CRISPR-based approach, called CRIME (Cas9-directed Rapid Immunoprecipitation Mass Spectrometry of Endogenous proteins) was developed to isolate and identify the AR-V7-specific spliceosome, in association with a nuclease deficient Cas9 at AR cryptic exon 3. Mass spec-derived hits were screened for their ability to alter AR-V7 mRNA levels in CWR22Rv1 cells. SRSF3 was identified as a potential AR-V7 splicer which promotes cryptic exon 3 skipping in normal prostate. In CRPC, SRSF3 is significantly downregulated and hence cryptic exon 3 inclusion in the mature AR-V7 mRNA transcript is permitted leading to generation of AR-V7. i Acknowledgements First, I would like to thank my supervisor Dr Luke Gaughan for the opportunity he offered me to work in his group. Nothing would be possible without his constant guidance and support at difficult times. He is a true mentor and working alongside him was an incredible experience. I would also like to thank the members of the STTD group, especially, my co-supervisor Prof. Craig Robson, Dominic Jones, Lewis Chaytor, Laura Wilson and Alex Bainbridge for providing precious advice and help when needed. Special thanks to Prof. Scott Dehm, University of Minnesota, and Dr Lakis Liloglou, University of Liverpool who have significantly assisted with my PhD project by providing valuable advice and resources to achieve my scientific goals. Nobody can manage surviving a PhD without true friends. Therefore, I am eternally grateful to my lovely friends Katerina, Eirini, Argiro and Ricky who always had the patience to babysit and encourage me at times I thought I couldn’t make it. They all stood by my side despite their busy schedules and I feel extremely lucky to have them in my life. I cannot thank enough my incredible parents. They have both been by my side during this journey despite the thousands of miles that physically separate us. For this, and much more, I am beyond thankful. '' No amount of experimentation can ever prove me right; a single experiment can prove me wrong.'' A. Einstein ii iii Contents Abstract………………………………………………………………………………………………………………………………………………………… i Acknowledgements………………………………………………………………………………………………………………………………………. ii Abbreviations……………………………………………………………………………………………………………………………………………….. 1 List of Figures…………………………………………………………………………………………………………………………………………………6 List of Tables…………………………………………………………………………………………………………………………………………………. 9 Chapter 1. Introduction……………………………………………………………………………………………………………………………….. 10 1.1. Anatomy and function of the prostate ..................................................................................... 10 1.2. Development of the prostate ................................................................................................... 10 1.3. Prostate carcinogenesis ............................................................................................................ 11 1.3.1. Risk factors ........................................................................................................................ 11 1.3.2. Genomic landscape of PC .................................................................................................. 11 1.4. Prostate cancer incidence and mortality .................................................................................. 13 1.5. The androgen receptor ............................................................................................................. 14 1.5.1. The N-terminal domain ..................................................................................................... 15 1.5.2. The DNA binding domain .................................................................................................. 16 1.5.3. The Hinge region ............................................................................................................... 17 1.5.4. The Ligand binding domain ............................................................................................... 17 1.6. AR signalling .............................................................................................................................. 18 1.7. Clinical management of PC ....................................................................................................... 19 1.8. Targeting the AR signalling pathway ......................................................................................... 20 1.8.1. Targeting androgen synthesis via the hypothalamic-pituitary-gonadal axis .................... 20 1.8.2. Targeting adrenal androgen synthesis .............................................................................. 20 1.8.3. Anti-androgen therapy ...................................................................................................... 21 1.9. Mechanisms of PC Resistance to Current AR-targeted Therapies ............................................ 23 1.9.1. AR Amplification ................................................................................................................ 25 1.9.2. Nuclear Receptor Cross-talk ............................................................................................. 25 1.9.3. AR co-regulator dysregulation .......................................................................................... 26 1.9.4. Gain-of-function mutations within the LBD of AR ............................................................ 27 1.10. AR splice variants .................................................................................................................. 30 1.10.1. Origin of AR splice variants ............................................................................................... 30 1.10.2. Generation of AR splice variants ....................................................................................... 31 1.11. CRISPR genome editing ......................................................................................................... 34 iv 1.11.1. Origin of CRISPR and its application in biomedicine ......................................................... 34 1.11.2. Mechanism of CRISPR editing ........................................................................................... 35 1.12. Expansion of the CRISPR toolbox .......................................................................................... 37 1.13. Mechanism of splicing .......................................................................................................... 39 1.14. Alternative splicing ................................................................................................................ 40 1.15. Co-transcriptional splicing .................................................................................................... 41 1.16. Alternative splicing in cancer ................................................................................................ 43 1.17. Splicing in PC ......................................................................................................................... 44 1.18. Therapeutic targeting of splicing .......................................................................................... 46 Chapter 2. Aims and Objectives……………………………………………………………………………………………………….48 Chapter 3. Materials and Methods…………………………………………………………………………………………………. 49 3.1. Mammalian cell culture - Cell maintenance and passaging ..................................................... 50 3.2. Cell lines .................................................................................................................................... 50 3.3. Compounds ..............................................................................................................................