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Modulation of P53 Signalling and Response to MDM2-P53 Binding Antagonists

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Modulation of P53 Signalling and Response to MDM2-P53 Binding Antagonists Modulation of p53 signalling and response to MDM2-p53 binding antagonists Thesis submitted in partial fulfilment of the requirements of the degree of Doctor of Philosophy Arman Esfandiari University of Newcastle upon Tyne Faculty of Medical Sciences Molecular Oncology Research Group Northern Institute for Cancer Research 09/2015 Abstract Mutational inactivation of the p53 tumour suppressor protein, encoded by the TP53 gene, occurs in ≈50% of malignancies overall. Non-genotoxic activation of p53 signalling in the remaining TP53 wild-type malignancies is a promising therapeutic strategy. MDM2 inhibitors, such as Nutlin-3 and RG7388, can activate p53 in a non- genotoxic manner, mobilising p53-dependent signalling; however, sensitivity to these compounds varies widely among TP53 wild-type cell lines. In this study p53 signalling network components involved in the response to DNA damage and p53 homeostasis are investigated for their role as determinants of cellular sensitivity to MDM2 inhibitors. Deciphering determinants of sensitivity to this group of compounds will enable optimisation of their therapeutic potential. Chemical inhibition of kinases, ATM and DNA-PKcs, which are critical for DNA double strand break repair and activation of p53 signalling in response to DNA damage, did not affect the cellular sensitivity to Nutlin-3 in the absence of DNA damage. However, inhibition of these kinases enhanced the cellular sensitivity of TP53 wild-type cells to the combined effect of Nutlin-3 and DNA damage induced by ionising radiation, in a cell type dependent manner. In a neuroblastoma derived TP53 wild-type and mutant, otherwise isogenic, cell line pair, ionising radiation increased the growth inhibitory effect of Nutlin-3 in a p53-dependent manner and this was enhanced significantly in the presence of the DNA-PKcs inhibitor NU7441. In contrast, in the osteosarcoma derived TP53 wild-type and mutant, otherwise isogenic, cell line pair, exposure to ionising radiation decreased the growth inhibitory effect of Nutlin-3 in a p53-dependent manner and DNA-PKcs inhibition did not influence this protective effect. Given that ATM and DNA-PKcs activate p53 through phosphorylation of key residues, inhibition of the WIP1 phosphatase (encoded by the PPM1D gene) that dephosphorylates one such residue, was tested for the effect on cellular sensitivity to MDM2 inhibitors. Cellular growth/proliferation was assessed in TP53 wild-type and matched mutant/null cell line pairs, differing in their PPM1D genetic status, when treated with MDM2 inhibitors Nutlin-3/RG7388 + a highly selective WIP1 inhibitor GSK2830371 or transient siRNA knockdown of WIP1 expression. The effects of GSK2830371 and transient WIP1 siRNA knockdown on MDM2 inhibitor induced p53Ser15 phosphorylation, p53-mediated global transcriptional activity and apoptosis I were also investigated. WIP1 transient siRNA knockdown increased p53Ser15 phosphorylation and sensitivity to MDM2 inhibitors in TP53 wild-type parental cell lines. TP53 wild-type and mutant cell line pairs were relatively insensitive to single agent GSK2830371 regardless of their PPM1D status. However, a non-growth inhibitory dose of GSK2830371 markedly potentiated the response to MDM2 inhibitors in TP53 wild-type cell lines, most notably in those harbouring PPM1D activating mutations or copy number gain (up to 5.8-fold decrease in GI50). Potentiation also concurred with significant increase in MDM2 inhibitor induced cell death endpoints which were preceded by a marked increase in phosphorylated p53Ser15, a WIP1 negatively regulated substrate, and known to increase p53 transcriptional activity. Microarray-based gene expression profiling showed that the combination treatment increases the subset of early RG7388 induced p53-transcriptional target genes involved in growth inhibition and apoptosis. Increased mRNA and protein expression of WIP1 has been associated with poor clinical outcome in various malignancies in which MDM2 inhibitors are being considered as a potential therapeutic strategy. For neuroblastoma mining the Amsterdam microarray databank showed WIP1 mRNA expression to correlate with worse survival. Therefore, WIP1 protein expression was assessed by immunohistochemical (IHC) staining of neuroblastoma tissue microarrays. A wide range of WIP1 IHC staining was found, however there was no significant association between high WIP1 staining and clinical outcome. Overall these findings show that manipulating p53 post-translational modification following its activation by MDM2 inhibitors or DNA damaging agents can increase cellular sensitivity to this class of compounds. Furthermore, these observations provide evidence to support the inhibition of WIP1 phosphatase activity as a strategy for enhancing the efficacy of MDM2 inhibitors, particularly in TP53 wild-type, PPM1D overexpressing/overactive malignancies. II Declaration I certify that this thesis is my own work, except where stated, and has not been previously submitted for a degree or any qualification at this or any other university. III Acknowledgments I would like to thank my supervisor Professor John Lunec for his infinite patience, persistent support and guidance throughout my MRes/PhD degrees. My gratitude also goes to the other members of the molecular oncology research group for technical support and stimulating discussions. I would also like to thank the following colleagues for providing tissue, cell lines, reagents and/or technical support: Professors Herbie Newell, Nicola J Curtin, Olaf Heidenreich and Craig N Robson, Dr Ximena Montano, Dr Sari Alhasan, Dr Ahmad Mahdi, Dr Laura Woodhouse, Dr Chiao-En Wu, Dr Ashleigh Herriot, Dr Gesa Junge, Dr Lindi Chen, Dr Clair Hutton, Dr Jennifer Jackson, Dr Steven Darby, Dr Olivier Binda, Dr Richard Heath and Mr Ricky Tirtakusuma. I thank the Undergraduate and MRes students whom I supervised over the last 3 years, for their hard work and dedication to advancement of their projects. I must also thank my parents Iraj Esfandiari and Fahimeh Alipour, and my siblings Ramin Esfandiari, Fardin Esfandiari and Elham Toussi (sibling-in-law), for their unrelenting support of my academic endeavours. Finally, I would also like to acknowledge and thank all cancer patients and their families for donating valuable tissue samples to potentially lifesaving research. IV Table of Contents Abstract .......................................................................................................................... I Declaration ................................................................................................................... III Acknowledgments ........................................................................................................ IV List of Figures .............................................................................................................. XII List of tables .............................................................................................................. XXV List of abbreviations ................................................................................................ XXVII Chapter 1 Introduction ...................................................................................................... 1 1.2 Genomic integrity .................................................................................................... 2 1.3 Cancer ..................................................................................................................... 2 1.4 Hallmarks of cancer ................................................................................................ 3 1.4.1 Proto-oncogenes ............................................................................................... 5 1.4.2 Tumour suppressor genes ................................................................................. 5 1.5 A brief history of p53 .............................................................................................. 6 1.5.1 Discovery ......................................................................................................... 6 1.5.2 Oncogenic tendencies....................................................................................... 6 1.5.3 A classic tumour suppressor ............................................................................ 7 1.5.4 Trp53 knockout transgenic mice .................................................................... 10 1.5.5 Human germline mutations in TP53 .............................................................. 11 1.5.6 TP53 gene and its isoforms ............................................................................ 11 1.5.7 TP53 mutations: Gain-of-function or dominant-negative? ............................ 12 1.6 p53 structure and function ..................................................................................... 14 1.6.1 Transactivation domain .................................................................................. 14 1.6.2 Proline rich region .......................................................................................... 16 1.6.3 DNA binding domain ..................................................................................... 17 1.6.4 C-terminus ...................................................................................................... 21 1.7 Cell cycle checkpoints ........................................................................................... 25 1.7.1 Regulation of cell cycle progression .............................................................
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