The Pennsylvania State University The Graduate School College of Medicine DIFFERENTIAL P53 SIGNALING IN RESPONSE TO 5-FU AND ETOPOSIDE IN MODULATING TOXICITY VIA DPYD AND CHK2 IN CANCER THERAPY A Dissertation in Molecular Medicine by Prashanth R Gokare Ó 2017 Prashanth R Gokare Submitted in Partial Fulfillment of the Requirements for the Degree, of Doctor of Philosophy December 2017 The dissertation of Prashanth R Gokare to be reviewed and approved* by the following: Wafik S. El-Deiry, Professor, Department Hematology/Oncology William Wikoff Smith Chair in Cancer Research Deputy Cancer Center Director for Translational Research Co-Leader Molecular Therapeutics Fox Chase Cancer Center Dissertation Adviser Committee Co-Chair Charles H. Lang, Distinguished Professor of Cellular and Molecular Physiology Director of the Molecular Medicine Graduate Program Committee Co-Chair Rosalyn Irby, Associate Professor, Department of Medicine Jin-Ming Yang, Professor of Pharmacology, Department of Medicine, Nelson S. Yee, (Special Member), Assistant Professor, Department of Hematology Department of Medicine, *Signatures are required on file for the Graduate School ii ABSTRACT Tp53 is a major transcription factor that controls a multitude of processes involved in the response to cellular stress. It plays a critical role in cell cycle arrest and/or apoptosis after DNA damage. p53 downstream effects can impact on cellular metabolism and these are increasingly becoming unraveled. The studies here in highlights previously unappreciated signaling by Tp53 that is relevant to toxicity and efficacy of chemotherapeutic agents. Nucleotide metabolism can influence malignant behavior and intrinsic resistance to cancer therapy. Alterations in pyrimidine metabolism are key to the mechanism of action of the chemotherapeutic antimetabolite 5-fluorouracil (5-FU). The initial study describes a novel role of p53 in controlling the rate-limiting enzyme in the pyrimidine catabolic pathway, dihydropyrimidine dehydrogenase (DPYD) and its effect on pharmacokinetics of and response to 5-FU. P53 binds to a p53 DNA-binding site (p53BS) downstream of the DPYD gene and reduces its expression at both the mRNA and protein level. The reduced expression of DPYD follows the inhibition of thymidylate synthase (TS) and is dependent on DNA-dependent protein kinase (DNA-PK) and Ataxia telangiectasia mutated (ATM) signaling. Overall this study highlights the regulation of DPYD and its implications on toxicity and efficacy of 5-FU. (Chk2) is a serine/threonine kinase that transduces DNA damage response (DDR) signals from the kinases ATM and to some extent also Ataxia Telangiectasia and Rad3-Related Protein (ATR). It plays a critical role in inducing cell death following radiation in a p53- dependent manner. However, the role of Chk2 in toxicity of chemotherapeutics is less iii well studied with regard to the involvement of the ChK2-ATM-p53 pathway. Our experiments addressed the role of Chk2 in Dose Limiting toxicity (DLT) of Topisomearase II (TOP2) inhibitors. We found Chk2 mediates toxicity from TOP2 inhibitors but not with other classes of chemotherapeutics, both in-vitro and in-vivo. Functional screens identified NSC105171 as a novel Chk2 inhibitor. NSC105171 protected from DLT following Etoposide treatment. This study has implications for a potentially effective strategy to preventing DLT. Thus, we have identified a role for p53 in controlling nucleotide metabolism through repression of DPYD following DNA damage through the involvement of TS inhibition. The study highlights different responses of chemotherapeutic agents that signal through p53 activation. In the second study, we described targeting the ATM-Chk2 p53 pathway in the context of Topoisomerase 2 inhibition to preferentially limit toxicity. Depending on the nature and type of damage there appears to be a differential response mediated by p53. Taken together, this research explores ways in which p53 signaling and biology can be used to enhance efficacy and limit toxicity following specific chemotherapy treatments. iv TABLE OF CONTENTS LIST OF FIGURES ........................................................................................................ viii LIST OF TABLES ............................................................................................................ x ABBREVIATIONS ........................................................................................................... xi ACKNOWLEDGEMENTS ............................................................................................. xiii CHAPTER 1: LITERATURE REVIEW ............................................................................. 1 P53 and cancer ........................................................................................................... 1 P53 and tumor suppression ...................................................................................... 2 WT P53 induces cell cycle arrest after cellular stress or DNA damage .................... 3 P53 and apoptosis ..................................................................................................... 4 Is role of p53 in cell cycle arrest and apoptosis important for tumor suppression? ... 6 P53 and DNA damage repair .................................................................................... 8 P53 and metabolism ................................................................................................... 9 P53 regulation of glycolysis and the Pentose Phosphate Pathway (PPP) .............. 10 P53 control of mitochondrial metabolism and ROS ................................................. 12 P53 and nucleotide metabolism .............................................................................. 13 Exploiting p53 status and signaling pathway for therapy .................................... 15 Can p53 act as transcriptional repressor? ............................................................. 17 Indirect repression by p53 ....................................................................................... 17 Recruitment to chromatin modifiers ......................................................................... 18 Interference and competition with transcription factors ........................................... 18 Non-coding RNA and p53 mediated repression ...................................................... 19 v Direct repression by p53 ......................................................................................... 19 Chemotherapeutics that trigger p53 responses .................................................... 20 5-Fluorouracil (5-FU) ............................................................................................... 20 Mechanism of action ............................................................................................ 21 Modes of action and mechanisms of resistance to 5-FU ..................................... 22 Thymidylate synthase (TS) inhibition by 5-FU ................................................. 22 DNA damage, TS, and 5-FU ............................................................................ 23 RNA damage by 5-FU ...................................................................................... 24 Dihydropyrimidine dehydrogenase (DPYD) and 5-FU sensitivity .................... 25 Tumor suppressor p53 status and 5-FU response .......................................... 25 Topoisomerase poisons, inhibitors and p53 pathway .......................................... 26 Mechanism and modes of action ......................................................................... 26 Mechanisms of resistance and toxicity of TOP drugs .......................................... 27 Dihydropyrimidine Dehydrogenase (DPYD) gene regulation ............................... 29 Characterization of the gene and the protein .......................................................... 29 Regulation of the DPYD gene ................................................................................. 29 Clinical Pharmacogenetics of DPYD ....................................................................... 31 Check-point kinase 2 (Chk2) regulation of p53 ...................................................... 34 Chk2 and DNA Damage Repair (DDR) ............................................................... 36 Chk2 and p53-dependent cell cycle arrest .......................................................... 37 Chk2 and p53-dependent apoptosis .................................................................... 38 Role of Chk2 in mitosis ........................................................................................ 39 SCOPE OF THE THESIS ............................................................................................... 41 CHAPTER 2 ................................................................................................................... 43 P53 represses pyrimidine catabolic gene dihydropyrimidine dehydrogenase (DPYD) expression in response to thymidylate synthase (TS) targeting. .............. 43 vi Introduction ............................................................................................................... 43 Material and Methods ............................................................................................... 45 Results ....................................................................................................................... 52 Discussion ................................................................................................................