CHARACTERIZATION OF URACIL DNA GLYCOSYLASE AS A THERAPEUTIC TARGET FOR SENSITIZATION OF FLOXURIDINE IN CANCER WITH P53 MUTATION OR DEFICIENCY by YAN YAN Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Thesis Advisor: Dr. Stanton L. Gerson, M.D. Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY August, 2017 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Yan Yan candidate for the degree of Doctor of Philosophy *. Committee Chair Youwei Zhang Committee Member Stanton Gerson Committee Member Maria Hatzoglou Committee Member Derek Taylor Committee Member Alexandru Almasan Date of defense April 24th, 2017 *We also certify that written approval has been obtained for any proprietary material contained therein. Page | ii TABLE OF CONTENTS Table of Contents…………………………….…………………………………………...iii List of Tables……………………………………….………………….…………………vii List of Figures…………………….……………………………….…….………………viii Acknowledgements………………………………………………………….……………xi List of Abbreviations…………………………………………………….……….………xii Abstract……………………………………………………………………………………1 Chapter 1. Introduction……………………………………………………………….……4 1.1 TS inhibitors and the directed effects………………………………………………4 1.1.1 TS inhibition by fluoropyrimidines………………………...…………….….6 1.1.2 TS inhibition by antifolates…………………………………………...……...8 1.1.3 Thymineless death…………………………………………………………...9 1.2 Impact of BER repair pathway on TS inhibitors……………...…….…………….10 1.2.1 Uracil and 5-FU excision by DNA glycosylases……………………………13 1.2.2 Downstream components of BER………………………………….……….16 1.3 Molecular determinants of response to TS inhibitors…………………………….19 1.3.1 Thymidylate synthase………………………………………………………19 1.3.2 p53………………………………………………………………………….20 1.3.3 dUTPase……………………………………………………………………22 1.3.4 Other DNA repair pathways…….……………………….…….…….….….24 Page | iii 1.4 Tumor suppressor p53 and its regulation…………………………………………27 1.5 The role of p53 in apoptosis…………………………………………….………...29 1.6 p53 germline and somatic mutations…………….………………….…………...30 1.7 Mutant p53 gain of functions and their link to chemoresistance………....……….31 1.8 Statement of objectives…………………………………………………………...35 Chapter 2. Inhibition of uracil DNA glycosylase (UDG) sensitizes cancer cells to 5- fluorodeoxyuridine through replication fork collapse-induced DNA damage……........…38 2.1 Abstract…………………………………….……………...………………….….39 2.2 Introduction……………………………………………….……………………...40 2.3 Materials and methods………………….………………………………………...42 2.4 Results……………………………………………………………………………48 2.4.1 UDG removes uracil and 5-FU incorporated into DNA following 5-FdU treatment…………………………………………………………………………48 2.4.2 Loss of UDG enhances cytotoxicity of 5-FdU in cancer cells......................50 2.4.3 Thymidine treatment after 5-FdU exposure cannot fully rescue the enhanced cytotoxicity in UDG depleted cells due to the retention of genomic uracil and 5- FU.……………………………………………………………………………….51 2.4.4 UDG depletion leads to cell cycle arrest at late G1 and early S phase by 5- FdU…....................................................................................................................52 Page | iv 2.4.5 Loss of UDG inhibits DNA replication progression in response to 5-FdU treatment…………………………………………………………………………53 2.4.6 DNA damage persists in UDG depleted cells and is not due to apoptosis by 5-FdU treatment………………………………………………………………….54 2.5 Discussion………………………………….…………………………………….56 Chapter 3. Knockdown of uracil DNA glycosylase selectively re-sensitizes p53 mutant and deficient human cancer cells to 5-fluorodeoxyuridine………………………….…....72 3.1 Abstract…………………………………….……………...………………….….73 3.2 Introduction………………………………………………………………………74 3.3 Materials and methods………………….………………………………………...77 3.4 Results……………………………………………………………………………80 3.4.1 p53 mutation or deficiency affords 5-FdU resistance among different types of cancer cells……………………………………………………………….……80 3.4.2 UDG depletion sensitizes cancer cells with p53 mutation or deficiency to 5- FdU exposure…………………………………………………………………….81 3.4.3 5-FdU resistance in p53 knockout (KO) or knockdown (KD) cells is reversed by UDG depletion………………………………………………………….…….82 3.4.4 UDG depletion selectively sensitizes p53 KO cancer cells to pemetrexed and 5-FU.......................................................................................................................83 Page | v 3.4.5 5-FdU activates cell death in p53 KO cancer cells with depleted UDG………….………….……………………………………………………….84 3.5 Discussion……………………………….……………………………………….85 Chapter 4. Conclusions and future directions…………………………………………...101 4.1 Conclusions…………………….……………………………………………….101 4.2 Future directions………………………………………………………………...103 4.2.1 Evaluation of the interplay between p53 and thymidylate synthase (TS) on cytotoxicity of TS inhibitors…………………………………………………….103 4.2.2 Characterization of the effect of other glycosylases (SMUG1, TDG, MBD4) in processing genomic uracil and 5-FU induced by 5-fluorodeoxyuridine...…....106 4.2.3 Exploitation of DNA damage response signaling as a target to achieve ‘synthetic sickness or lethality’ with UDG depletion in p53 mutant cancer cells after DNA damage……….……………………………………………………...114 4.2.4 Exploration of the potential gain of functions for p53 mutations that are responsible for 5-FdU resistance……………….……………………………….118 4.2.5 Identification of small molecule inhibitors of UDG via high-throughput screening.……………………………………………………………………….120 Appendix…………………………………………………………………………….….122 Reference……………………………………………………………………………….128 Page | vi LIST OF TABLES Table 1-1. Summary of uracil and 5-FU lesions repaired by mammalian DNA glycosylases…………………………….……………………………………………….14 Table 1-2. Role of downstream BER proteins in response to TS inhibitors………………18 Table 1-3. Select list of mutant p53 upregulated genes and chemoresistance…………….34 Table 3-1. Cell lines and strains used in this work…………………………….….………88 Page | vii LIST OF FIGURES Figure 1-1. Thymidylate synthesis and two classes of TS inhibitors……….…...………….5 Figure 1-2. Metabolism of fluoropyrimidines…………………….……………………….7 Figure 1-3. Base excision repair (BER) of mis-incorporated uracil (U) and 5-FU……...12 Figure 1-4. dUTPase prevents dUTP/FdUTP from incorporation into DNA during TS inhibition…………………………………………………………………………………23 Figure 1-5. Mismatch repair (MMR) of mis-incorporated 5-FU………………………….26 Figure 2-1. UDG depletion causes incorporation of uracil and 5-FU into genomic DNA by 5-FdU…………………………………………………………………………………….60 Figure 2-2. UDG depletion enhances 5-FdU sensitivity in cancer cells..............................62 Figure 2.3. Thymidine treatment after 5-FdU exposure cannot fully rescue increased cytotoxicity in UDG depleted cells……………………………………………………….64 Figure 2-4. Loss of UDG induces cell cycle arrest at late G1 and early S phase by 5-FdU exposure………………………………………………………………………………….66 Figure 2-5. UDG depletion inhibits replication fork progression following 5-FdU treatment............................................................................................................................68 Figure 2-6. DNA damage accumulates in UDG depleted cells in a caspase independent manner. …………………………………………………………….…………………….70 Page | viii Figure 3-1. 5-FdU resistance in different types of cancer cells with p53 mutation or deficiency………………………………………………………………………………...89 Figure 3-2. UDG depletion selectively sensitizes cells with p53 mutation or deficiency to 5-FdU…………………………………………………………………………………….91 Figure 3-3. 5-FdU resistance due to loss of p53 is reversed by UDG depletion…………...93 Figure 3-4. p53 knockdown re-sensitizes cancer cells with UDG depletion to 5-FdU……95 Figure 3-5. UDG depletion selectively sensitizes p53 KO cells to pemetrexed and 5- FU......................................................................................................................................97 Figure 3-6. UDG depletion induces cell death caused by 5-FdU in p53 KO cancer cells…99 Figure 4-1. Evaluation of uracil and 5-FU levels in TDG treated genomic DNA extracted following 5-FdU exposure via AP site detection assay………………………………….109 Figure 4-2: Evaluation of UDG and TDG activities via glycosylase activity assay...…...111 Figure 4-3. Synthetic sickness or lethality between DNA damage response signaling and p53………………………………………………………………………………….….117 Page | ix Appendix 1. Retention of uracil and 5-FU in HEC1A UDG depleted cells during thymidine recovery following 5-FdU exposure…………………………………………………….123 Appendix 2. Loss of UDG induces HEC1A cell cycle arrest at late G1 and early S phase following 5-FdU exposure………………………………………………………………124 Appendix 3. DNA damage accumulates in HEC1A UDG depleted cells in a caspase independent manner…………………………………………………………………….125 Appendix 4. Effect of irradiation on p21 induction in various cancer cells with different p53 status………………………………….…………………………………………….127 Page | x ACKNOWLEDGEMENTS First and foremost, it has been a privilege to have Dr. Stanton Gerson as my mentor. I would like to express my deepest gratitude for his patient guidance, immense knowledge, and gracious support throughout the course of my project and my career as a scientist. I would also like to express my sincere appreciation to my committee members, Dr. Youwei Zhang, Dr. Maria Hatzoglou, Dr. Derek Taylor, and Dr. Alexandru Almasan, for their insightful comments, hard questions, and tremendous help alongside my Ph.D. pursuit. For their encouragement, kindness, and collaboration, I would like to thank Xiangzi Han, Shuming Yang, Shashank Gorityala, Yan Xu, and the