The Highly Parallel Homology Directed Repair Assay and the Analysis of BRCA1 Variants Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Muhtadi Muhammad Islam, B.E. Biomedical Sciences Graduate Program The Ohio State University 2016 Dissertation Committee: Jeffrey Parvin, MD, PhD, Advisor Michael Freitas, PhD Kun Huang, PhD Amanda Toland, PhD Copyrighted by Muhtadi Muhammad Islam 2016 Abstract DNA damage and repair are processes that have been linked to cancer, both in carcinogenesis and in the treatment of cancer. We explore two different DNA repair factors, BRCA1 and HDAC10, in two distinct studies. BRCA1 is a highly penetrant gene in both breast and ovarian cancer and with several functions. We analyze BRCA1 in a novel assay that scores the homologous recombination repair function of several thousand BRCA1 variants in parallel. Our goal is to create a structure function map of BRCA1 at the amino acid resolution to not only prove the novel highly parallel functional assay, but also to provide clinically relevant data of BRCA1 variants with respect to homologous recombination repair. HDAC10 is a histone deacetylase with only one known function, stimulating homologous recombination repair of double strand breaks. Utilizing genome databases, we found that HDAC10 is deleted in up to 10% of ovarian tumors. We also found that low HDAC10 expression correlated with platinum therapy sensitivity. We demonstrated general HDAC inhibition enhanced cisplatin therapy in primary ovarian carcinomas. Additionally, HDAC10 specific inhibition sensitized a BRCA1 deficient ovarian cancer cell line to both DNA damage and cisplatin therapy. We suggest that HDAC10 is an important agent in the mechanisms of general HDAC inhibition as well as a potential new target for BRCA1 deficient ovarian tumors. ii Dedication This document is dedicated to the people and families who are struggling with cancer. iii Acknowledgements The primary driving force who supported me throughout my research was Dr. Jeffrey Parvin. His support was unwavering throughout the years, despite the few triumphs amongst the many setbacks. My committee has provided me with invaluable feedback and regularly reinvigorated my drive through their excitement at the potential of my work. My colleagues have been a continual source of both technical and emotional support, something I will dearly miss. My parents and sisters have been remarkably patient with me during these years and always believed in my ability even when I had my doubts. Finally, to my wife, who has been my partner in all my trials and tribulations these last few years. Andria has done so much more than simply share my burdens, she transformed them time and again so they were less heavy for both of us. This is something I am grateful for beyond words. Thank you all. iv Vita May 2003 .......................................................Centerville High School April 2007 .......................................................B.E. Chemical Engineering University of Dayton August 2007-present .....................................Graduate Research Assistant Department of Biomedical Informatics The Ohio State University Publications Zhang, J., Lu, K., Xiang, Y., Islam, M., Kotian, S., Kais, Z., ... & Huang, K. (2012). Weighted frequent gene co-expression network mining to identify genes involved in genome stability. PLoS Comput Biol, 8(8), e1002656. Starita, L. M., Young, D. L., Islam, M., Kitzman, J. O., Gullingsrud, J., Hause, R. J., ... & Fields, S. (2015). Massively parallel functional analysis of BRCA1 RING domain variants. Genetics, 200(2), 413-422. Field of Study Major Field: Biomedical Sciences v Table of Contents Abstract ...............................................................................................................................ii Dedication .......................................................................................................................... iii Acknowledgements ............................................................................................................ iv Vita ...................................................................................................................................... v List of Figures ..................................................................................................................... ix List of Tables....................................................................................................................... xi Chapter 1: A Review of DNA Repair and Cancer ................................................................ 1 1.1 A Recent History of Molecular Biology ................................................................ 2 1.2 BRCA1 and Breast Cancer ..................................................................................... 4 1.3 The link between DNA damage and cancer ......................................................... 7 1.4 DNA Repair review ............................................................................................... 9 1.4a Non-homologous End Joining ......................................................................... 14 1.4b Homologous Recombination .......................................................................... 15 1.5 BRCA1 ................................................................................................................. 17 1.6 Cancer treatment ............................................................................................... 20 1.7 Summary of studies presented .......................................................................... 23 Chapter 2: Highly Parallel Homology Directed Repair ..................................................... 25 2.1 Abstract .............................................................................................................. 26 vi 2.2 Introduction........................................................................................................ 26 2.3 Materials and Methods ...................................................................................... 28 2.3a Homology Directed Repair Assay ................................................................... 28 2.3b HeLa DR-FRT generation ................................................................................. 30 2.3c HeLa BRCA1-variant cell line generation ........................................................ 34 2.3d Highly Parallel Homology Directed Repair Assay ........................................... 35 2.3e Construction of barcoded variant libraries of BRCA1 .................................... 36 2.3f Sequencing to link the BRCA1 N-terminal variants to their barcode ............. 37 2.3g Sequencing barcodes from genomic DNA ...................................................... 38 2.3h Determining a functional score for each BRCA1 variant ................................ 39 2.3i Statistics .......................................................................................................... 42 2.4 Validation Results ............................................................................................... 42 2.5 BRCA1 variants ................................................................................................... 51 2.6 Discussion ........................................................................................................... 57 Chapter 3: HDAC10 as a potential therapeutic target in ovarian cancer ........................ 61 3.1 Abstract .............................................................................................................. 62 3.2 Introduction........................................................................................................ 62 3.3 Materials and Methods ...................................................................................... 64 3.3a Cell Culture and Reagents .............................................................................. 64 3.3b Homology Directed Repair Assay (HDR) ......................................................... 65 3.3c Comet Assay ................................................................................................... 65 vii 3.3d MTT Assay ....................................................................................................... 67 3.3e Statistics .......................................................................................................... 67 3.4 Results ................................................................................................................ 68 3.5 Discussion ........................................................................................................... 80 3.6 Tables ................................................................................................................. 85 Chapter 4: Summary and Future Directions .................................................................... 86 4.1 DNA repair and cancer ....................................................................................... 87 4.2 BRCA1 ................................................................................................................. 88 4.2a Highly Parallel Homology Directed Repair ..................................................... 88 4.2b BRCA1 Future Directions ................................................................................ 89 4.3 HDAC10 .............................................................................................................