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MEK Inhibition in Pancreas Cancer Francesca Vena

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MEK Inhibition in Pancreas Cancer Francesca Vena MEK Inhibition In Pancreas Cancer Francesca Vena A thesis submitted to the University College London for the degree of Doctor of Philosophy December 2015 CRUK Drug-DNA Interactions Research Group Department of Oncology Cancer Institute University College London 72 Huntley Street, London, WC1E 6BT, UK 1 DECLARATION I, Francesca Vena confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in this thesis. 2 ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis and is resistant to chemotherapy. Gemcitabine, a nucleoside analogue, is an important component of treatment for locally advanced and metastatic PDAC, but provides only modest survival benefit. The enzyme Ribonucleotide Reductase (RR) catalyses the conversion of ribonucleotides into deoxyribonucleotides, required for DNA synthesis and repair. Several studies have demonstrated that RR large subunit-1 (RRM1) is associated with gemcitabine resistance in PDAC. The RAS/RAF/MEK/ERK signalling pathways regulates cellular proliferation, differentiation and survival. Targeting downstream effectors of the RAS gene by direct inhibition of MEK (MAPKK) proteins is a rational therapeutic strategy, since aberrant activation of this pathway occurs frequently in PDAC. In this study, the ability of pimasertib (AS703026), a highly selective and allosteric MEK1/2 inhibitor, to enhance gemcitabine efficacy was tested and the molecular mechanism of their interaction was investigated. Synergistic antiproliferative effects and increased apoptosis were observed when pimasertib was combined sequentially with gemcitabine in human pancreatic cancer cells. Importantly, pimasertib reduced the expression of RRM1 protein and this was associated with sensitivity to gemcitabine. Pre-treatment with the proteasome inhibitor MG132 impaired RRM1 downregulation induced by pimasertib, suggesting RRM1 is degraded through the ubiquitin-proteasome system. Immunoprecipitation experiments indicated enhanced MDM2-mediated polyubiquitination of RRM1 through lys48-mediated linkage following pimasertib treatment, an effect in part mediated by AKT. Finally, the combination treatment of pimasertib with gemcitabine caused significant tumor growth delay in an orthotopic pancreatic cancer model, with RRM1 downregulation in pimasertib-treated mice. These results indicate MEK as a potential target to sensitize gemcitabine therapy through modulation of RRM1 protein stability and underline the importance of drug scheduling in achieving efficacious antitumor activity. This study has provided mechanistic insights to the synergistic interaction between gemcitabine and pimasertib that could be further investigated in design of future clinical trials. 3 ACKNOWLEDGEMENTS First I would like to thank my supervisors Professor Daniel Hochhauser and Professor John Hartley for giving me the opportunity to carry out a PhD under their guidance. Dear Daniel, thank you very much for your constant support, for keeping up with my mood swings for the entire four years. Thank you for reminding me always to be positive during the hard times, for teaching me how to be a tough and critical scientist and for just being a great PI! I would also like to thank Dr. Samantha Goodstal for supervising the project from overseas and the MERCK Serono team in Billerica (MA) for funding my PhD. I would like to thank Dr. Manuel Rodriguez Justo for performing immunohistochemistry quantification and for critical reading of the manuscript. An important thank you goes to Eleonora Li Causi, my collaborator at the Barts Cancer Institute (London, UK), who helped me with the animal work. Thank you to all the members of the DRUG/DNA interactions lab: Rouchen, Luke, Helen, Sylwia, Fei, Juanjo, Miguel and Kostas. And to the former members, Raisa and Giammy for mentoring me from the beginning of this tough journey and for our amazing coffee breaks gossiping about the cancer institute life. A particular thank you to the amazing Trio: Hanna, Michael and Valenti, for keeping up with my pointless dramas about the paper revision, the thesis writing and for all the great Friday nights spent together at the Jeremy Bentham pub after work. Un grande grazie goes to my ex colleague and friend Valeria. I would not have made it without your support and friendship. I miss you a lot Vale! Thank you to Lucia, Anto and Sebastian for our fun spinning classes at Fitness First together and to Nieves for helping me with my PowerPoint presentations. To my PhD buddy Jocelyn and our nights out in Mayfair that helped us to distract from our lab life. Thank you to my London partners in crime: Letizia, Pascale, Maddalena and Matilde. Even though they had no idea what a MEK inhibitor is, they have always been interested in hearing about my experiments’ progress. Thank you to all my Italian friends for your long distance support. In particular, to my dear friend Michela and to all my Bolognese friends: Caterina, Agnese, Angela, 4 Barbara, Simona and Cate. Grazie alla mia Fede, for our millions nights out together, for our trips and for our chats on the phone. You have truly always been there for me and always will. Thank you to Paul for introducing me to the PhD student’s world and taught me how to swim without sinking. You are a great example of how hard work and dedication can lead to great success. Thank you to LIGC! I am so glad to be part of this amazing gospel choir and to have found such great friends. Now, a very special OBRIGADO goes to Ema for supporting me throughout the final stage of my PhD…the writing! Thank you for being very patient with me, for always understanding me and for standing by my side. You are an amazing person and I am so glad to have you in my life. The biggest thank you goes to my big fat Italian family who has always believed in me. You are the most precious gift life could ever give me. To my mother, my biggest inspiration: no one could ever do a better job than you! To my great father who always allowed me to follow my dreams. All that I am I owe it to you two. To my aunt Stefania who followed me everywhere I went: Buffalo, Seattle or London. No matter what part of the world I was moving to she has always come along to make sure I was fine! There is no one quite as special as my older sister Vale who has taught me what the real values in life are.To my grandfather Mario who has watched over me during these four years and to zia Giovanna who transmitted me the passion for medical science.To my cousins Luca, Matteo, Fausto and Martina who represent the most fun part of my life. Marty: you are like sister to me and I am sure you will shine bright like a diamond one day!! To my amazing grandparents: Nonna Luciana (I love you so much) e Nonno Betto, for their unconditional love, tenderness, and words of wisdom. My curiosity for science began many years ago when as a little child I used to play in my auntie’s pharmacy store, wondering why and how these pills would work to cure human diseases. Therefore my last thank you goes to my auntie Dina, who walks beside us every day, to whom this thesis is dedicated Thank you all for having taken part of this amazing journey. 5 “It’s the possibility of having a dream come true that makes life interesting” (Paulo Coelho) 6 To my auntie Dina 7 COMMUNICATIONS PRESENTATIONS Poster Presentation at 10th NCRI cancer conference. 2-5th November 2014, Liverpool (UK). MEK inhibition enhances gemcitabine efficacy by increasing MDM2-mediated ubiquitination and degradation of RRM1. Vena F, Li Causi E, Hartley J.A, Hagemann T, Goodstal S, Hochhauser D. Poster Presentation at EORTC-NCI-AACR 17th-21st November 2014 Barcelona. (Spain). MEK inhibition enhances gemcitabine efficacy by increasing MDM2- mediated ubiquitination and degradation of RRM1. Vena F, Li Causi E, Hartley J.A, Hagemann T, Goodstal S, Hochhauser D. Poster Presentation at UCL Cancer Institute/Colin Woolf Memorial Fund. 18th March 2015, London (UK). MEK inhibition enhances gemcitabine efficacy by increasing MDM2-mediated ubiquitination and degradation of RRM1. Vena F, Li Causi E, Hartley J.A, Hagemann T, Goodstal S, Hochhauser D. Poster Presentation at AACR/Croucher Summer Course in Cancer Biology. 10th- 14th August 2015, Hong Kong (CH). The combination of MEK1/2 inhibitor pimasertib with gemcitabine induces schedule-dependent antitumor activity in pancreatic cancer models by reducing RRM1 protein. Vena F, Li Causi E, Justo- Rodriguez M, Goodstal S, Hartley J.A, Hochhauser D. PUBLICATIONS Vena F, Li Causi E, Rodriguez J.M, Goodstal S, Hagemann T, Hartley J.A, Hochhauser D. (2015). ”The MEK1/2 Inhibitor Pimasertib Enhances Gemcitabine Efficacy In Pancreatic Cancer Models By Altering Ribonucleotide Reductase Subunit-1 (RRM1)“. Clin Cancer Res. 8 TABLE OF CONTENTS DECLARATION .......................................................................................................... 2 ABSTRACT ................................................................................................................ 3 ACKNOWLEDGEMENTS ............................................................................................ 4 COMMUNICATIONS ................................................................................................. 8 TABLE OF CONTENTS ................................................................................................ 9 LIST OF FIGURES..................................................................................................... 14 LIST OF TABLES
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