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Ung)-Initiated Dna Base Excision Repair in The ELUCIDATING A ROLE FOR URACIL DNA GLCYOSYLASE (UNG)-INITIATED DNA BASE EXCISION REPAIR IN THE CELLULAR SENSITIVITY TO THE ANTIFOLATE, PEMETREXED by LACHELLE DAWN WEEKS Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Advisor: Dr. Stanton L. Gerson Department of Pathology CASE WESTERN RESERVE UNIVERSITY January 2014 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of ___________Lachelle Dawn Weeks__________ candidate for the Doctor of Philosophy degree*. (signed) ______Shigemi Matsuyama__________ (chair of the committee) _______Alexandru Almasan__________ _________Ruth A. Keri______________ _________George R. Stark__________ __________Stanton L. Gerson________ (date) _____August 23, 2013____________ * We also certify that written approval has been obtained for any proprietary material contrained therein. i Dedication To Clarissa Williams and Wesley Weeks. ii Table of Contents Dedication……………….........................................................................................ii Table of Contents……………………………………………………………………….iii List of Tables…………………………………………………………….………………vi List of Figures..…………………………………………………………………………vii Acknowledgements……………………………………………………………………..xi List of Abbreviations…………………………………………………………………...xiii Abstract…………………………………………………………………………………..1 Chapter 1: Introduction: Uracil misincorporation and thymine-less death – old and new hypotheses on the mechanism of action of antifolates and TS inhibitors ……………………………..3 1.1 Antifolates in cancer therapy………………………………………….......5 1.2 DNA repair in human cancer development and therapy………………12 1.3 Base excision repair of uracil-DNA………………………………………13 1.4 Proposed consequences of antifolate mediated- dUTP incorporation ...………………………………..…………………...22 1.5 Proposed clinical value for studying the role of UNG/BER in cancer chemotherapy……………………………………..34 1.6 Summary …………………………………………………………………..46 Chapter 2: Statement of objectives…………………………………………………..49 Chapter 3: Uracil DNA glycosylase determines human lung cancer cell sensitivity to pemetrexed………………………………………..54 3.1 Introduction…………………………………………………………………56 3.2 Materials and Methods……………………………………………………58 3.3 Results……………………………………………………………………...66 3.3.1 A spectrum of UNG expression exists in human lung cancer…………………………………………………………..66 3.3.2 Loss of UNG expression increases lung cancer sensitivity to pemetrexed…………………………………………..70 iii 3.3.3 Limited uracil removal is associated with increased DNA damage in UNG deficient cells……………..…………….73 3.3.4 UNG is induced in response to acute and chronic pemetrexed exposure………………………………………………79 3.3.5 Transcriptional regulation of UNG induction in pemetrexed treated cells………………………………………………….83 3.3.6 BER inhibition overrides pemetrexed resistance due to chronic exposure……………………………………………………..86 3.4 Discussion …………………………………………………………………88 Chapter 4: Uracil DNA glycosylase (UNG) loss enhances DNA double strand break formation in human cancer cells exposed to pemetrexed…………………………………...…………………………………….97 4.1 Introduction…………………………………………………………………99 4.2 Materials and Methods…………………………………………………..101 4.3 Results…………………………………………………………………….108 4.3.1 Loss of UNG expression hypersensitizes human cancer cells to pemetrexed……………………………………….108 4.3.2 Increased DNA double strand break formation in UNG-/-cells during pemetrexed exposure………………………………111 4.3.3 Delayed recovery from S-phase arrest in pemetrexed treated UNG-/- cells…………………………………………………………118 4.4 Discussion ………………………………………………………………..122 Chapter 5: Enhanced hematopoietic sensitivity in UNG-/- mice during antifolate response…………………………………………………...133 5.1 Introduction.…...………………………………………………………….135 5.2 Materials and Methods…………………………………………………..137 5.3 Results…………………………………………………………………….142 5.3.1 Increased in vitro sensitivity of UNG-/- MEFs and primary bone marrow cells pemetrexed…..…………………………..….142 5.3.2 Increased in vivo sensitivity of UNG-/- mice to iv pemetrexed…………………………………………………………………..145 5.3.3 Increased sensitivity to pemetrexed in UNG mutant human lymphoblastoid cells…...…………………………………….……150 5.4 Discussion ………………………………………………………………..153 Chapter 6: Towards the development of a novel small molecule inhibitor of uracil DNA glycosylase (UNG)……………………………157 6.1 Introduction……………………………………………………………….158 6.2 Materials and Methods…………………………………………………..159 6.3 Results…………………………………………………………………….161 6.3.1 Optimization and validation of HTS assay methodology…………………………………………………………………161 6.3.2 Determination of UNGi-2 IC50 using gel- based cutting assay………………………………………………………...164 6.3.3 Intracellular activity of UNGi-2 in combination with pemetrexed……………………………………………..169 6.4 Summary …………………………………………………………………170 Chapter 7: Summary and Conclusions…………………………………………….172 7.1 Summary of major findings……………………………………………..172 7.2 Limitations of this research……………………………………………..173 7.3 Conclusions………………………………………………………………175 Chapter 8: Future Directions………………………………………………………..178 References Cited……………………………………………………………………..183 v List of Tables Table 1-1 Important antifolates and TS inhibitors………………..…………..……..8 Table 1-2 Known specificities of human uracil DNAglycosylases.……………….19 Table 1-3 Base excision repair inhibitor strategies in various stages of development……………………………………………………………………………35 Table 3-1 Description of cell lines used in this study ……………………………...65 Table 3-2 Correlation of linear regression (RQ value vs. IC50)……………………68 Table 3-3 Multivariable regression statistics for ʻUNG+Geneʼ Pairs……………..69 Table 3-4 Cross-sensitivity of H1975 cells to other DNA damaging agents ……71 Table 4-1 Characteristics of γ-H2AX-enriched sequences………………………117 Table 4-2 Possible pathways to DSB formation and cell death in pemetrexed treated cells…………………………………………………………………………...132 Table 5-1 Pemetrexed-induced changes in animal body weight and peripheral blood count……………………………………………………………………………146 Table 5-2 Pemetrexed-induced changes in bone marrow cell number and CFU potential………………………………………………………………………………..149 Table 6-1 Properties of selected compounds from UNG inhibitor drug screen efforts…………………………………………………………………………………..167 vi List of Figures Figure 1-1 Important antifolates and TS inhibitors…………………………………..7 Figure 1-2 Mechanism of antifolate inhibition of nucleotide metabolism………….9 Figure 1-3 Schematic representation of DNA base excision repair of misincorporated uracil…………………………………………………………………16 Figure 1-4. Comparison of gene expression for significant BER genes in cancer cells vs. normal cells…………………………………………………………………..17 Figure 1-5 Futile cycle hypothesis……………………………………………………23 Figure 1-6 Deoxyribonucleotide biosynthesis ..…………………………………….26 Figure 1-7 Proposed model of cell death in UNG deficient cells………………….32 Figure 1-8 BER inhibition strategies to improve antifolate therapy in UNG proficient cells…………………………………………………………………………..36 Figure 3-1. UNG expression and pemetrexed sensitivity in human lung cancer………………………………………………………………………………...67 Figure 3-2 Loss of UNG expression sensitizes lung cancer cells to pemetrexed……………………………………………………………………………..72 Figure 3-3 Reduced uracil excision in UNG deficient cells………………………..74 Figure 3-4 Increased DNA damage in UNG deficient cells………………………..77 Figure 3-5 AP site detection in H1975 and H1975shUNG cells…………………..79 vii Figure 3-6 Induction of UNG in response to acute pemetrexed exposure………80 Figure 3-7 Induction of UNG in response to chronic pemetrexed exposure…….82 Figure 3-8 Cross-resistance of pemetrexed resistant subline to other DNA damaging agents………………………………………………………………………83 Figure 3-9 Transcription factor binding to UNG nuclear promoter in A549 cells during pemetrexed exposure…………………………………………………………85 Figure 3-10 Coordination of c-Myc expression with UNG expression and induction in lung cancer……………………………………………………………….86 Figure 3-11 Coordination of UNG and TOPOIIα expression in human lung cancer………………………………………………………………….88 Figure 4-1 Loss of UNG enhances pemetrexed sensitivity in DLD1 human colon cancer cells……………………………………………………………………………110 Figure 4-2 Increased DNA DSB formation in UNG-/- cells treated with pemetrexed……………………………………………………………………..113 Figure 4-3 Comparison of DNA DSB repair capacity in UNG+/+ and UNG-/- cells………………………………………………………………………114 Figure 4-4 γ-H2AX ChIP sequencing in UNG+/+ and UNG-/- cells……………….116 Figure 4-5 Delayed recovery from cell cycle arrest in pemetrexed treated UNG-/- cells…………………………………………………………………………………….119 Figure 4-6 Uracil base substitutions attenuate polymerase extension efficiency in vitro and correspond to intracellular pemetrexed-induced viii replication instability ………………………………………………………………...121 Figure 4-7 UNG and H2AX are over expressed in many human cancers……..130 Figure 4-8 UNG and H2AX are co-expressed in human cancer………………..131 Figure 5-1 Increased in vitro sensitivity of UNG-/- MEFs and primary bone marrow cells to pemetrexed…………………………………………………………………..144 Figure 5-2 Increased transient hematologic toxicity in pemetrexed-treated UNG-/- mice……………………………………………………………………………………147 Figure 5-3 Increased uracil in splenocytes and bone marrow cells of pemetrexed treated mice…………………………………………………………………………...150 Figure 5-4 Increased sensitivity to pemetrexed in human lymphoblastoid cells possessing inactivating mutations in UNG……………………………………..….152 Figure 6-1 Principle of molecular beacon substrate for HTS screening of compounds for UNG inhibitor activity………………………………………………162 Figure 6-2 Optimization and validation of HTS assay……………………………163 Figure 6-3 Results of HTS screening of 100 compounds
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