Elucidating and Targeting EZH2 and HELZ in the DNA Damage Response in Small Cell Lung Cancer 公开

Abstract

Small cell lung cancer (SCLC) is a highly aggressive malignancy with poor outcomes associated with resistance to the first line treatment of cisplatin with etoposide (EP). Targeting proteins critical to the repair of EP mediated DNA damage is a promising strategy for overcoming acquired EP resistance in SCLC. In this thesis, we performed synthetic lethal siRNA screens in EP resistant SCLC cells, and identified EZH2 and HELZ as two of the most promising mediators of cisplatin and etoposide resistance, respectively. We show that EZH2 has a non-catalytic and PRC2 independent role in stabilizing DDB2 to promote nucleotide excision repair (NER) and governs cisplatin resistance in SCLC. EZH2 complexes with DDB1-DDB2 and promotes DDB2 stability by impairing its ubiquitination independent of methyltransferase activity or PRC2, thereby facilitating DDB2 localization to cyclobutane pyrimidine dimer (CPD) crosslinks to govern their repair. Furthermore, targeting EZH2 for depletion with DZNep strongly sensitizes SCLC cells and tumors to cisplatin. Our findings reveal a non-catalytic and PRC2-independent function for EZH2 in promoting NER through DDB2 stabilization, suggesting a rationale for targeting EZH2 beyond its catalytic activity for overcoming cisplatin resistance in SCLC. Further, we show that HELZ, a previously uncharacterized putative RNA helicase, is a novel mediator of etoposide and DSB resistance, and that HELZ promotes homologous recombination, genomic instability, and the resolution of DNA-RNA hybrids. HELZ localizes to DSB sites in a PARP1 dependent manner. Depletion of HELZ induces DSB hypersensitivity and impairs proper formation of RAD51 foci in HR. Lower levels of HELZ are associated with better patient outcomes in cancers. Taken together, our data define roles for EZH2 and HELZ in the DDR and implicate them as highly relevant therapeutic targets that have the potential to synergize with EP therapy and impact patient outcome in SCLC.  

Table of Contents

Chapter 1: Introduction 1

1.1: Small Cell Lung Cancer 1

Figure 1.1: Inherent and Acquired Resistance 5

Figure 1.2: Synthetic Lethality 8

1.2: The DNA Damage Response 12

1.3: Nucleotide Excision Repair 14

Figure 1.3: NER Pathway Overview 19

1.4: Double Strand Break Repair 20

1.5: Homologous Recombination 21

Figure 1.4: HR Pathway Overview 23

1.6: Genomic Instability 24

1.7: EZH2 25

Figure 1.5: Domain Map of EZH2 27

Figure 1.6: Overview of EZH2 Functions 29

1.8: EZH2 in Cancer 30

1.9: EZH2 in the DDR 31

1.10: RNA Helicases 31

1.11: RNA Helicases in the DDR 32

1.12: HELZ 33

Figure 1.7: Domains and Motifs of HELZ 35

1.13: Scope of Dissertation 36

Chapter 2: EZH2 has a Non-Catalytic and PRC2 Independent Role in Stabilizing DDB2 to Promote Nucleotide Excision Repair 37

2.1 Author Contributions and Reproducibility Statement 37

2.2 Abstract 39

2.3 Introduction 40

2.4 Materials and Methods 42

2.5 Results 56

2.6 Discussion 95

2.7 Acknowledgements 99

2.8 Funding 100

2.9 Conflict of Interest 100

Chapter 3: RNA Helicase HELZ Promotes Homologous Recombination Repair to Maintain Genomic Stability 101

3.1 Author Contributions 101

3.2 Abstract 102

3.3 Introduction 103

3.4 Materials and Methods 105

3.5 Results 115

3.6 Discussion 141

3.7 Acknowledgements 145

3.8 Funding 145

3.9 Conflict of Interest 145

Chapter 4: Discussion 146

4.1 Summary of Key Results and Remaining Questions 146

4.2 The Utility of Phenotypic Screens 149

4.3 Non-Catalytic Functions of Enzymes in the DDR 152

4.4 Linking RNA Processing to the DDR 154

4.5 Translational Applications 155

References 159

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Cancer Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Cell Biology, Molecular
关键词	EZH2 chemotherapy resistance small cell lung cancer homologous recombination dna damage response nucleotide excision repair targeted theraputics dna repair cancer therapy HELZ
Committee Chair / Thesis Advisor	David Yu, Emory University

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