University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2017 An Investigation of 53BP1 in Multiple Myeloma Simms, Justin Simms, J. (2017). An Investigation of 53BP1 in Multiple Myeloma (Unpublished master's thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/24911 http://hdl.handle.net/11023/4002 master thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY An Investigation of 53BP1 in Multiple Myeloma by Justin Andrew Simms A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE GRADUATE PROGRAM IN MEDICAL SCIENCE CALGARY, ALBERTA JULY, 2017 © Justin Andrew Simms 2017 Abstract Currently, multiple myeloma is the second most common hematological malignancy, and as of yet it remains incurable. Although many therapeutic advances have been made in the recent past, there is still room for improvement in the treatment of myeloma. Here, using CRISPR/Cas9 genome editing, we show that a therapeutic combination of proteasome inhibition in combination with PARP inhibition that is in clinical trials depends on the DNA damage response protein 53BP1. These findings contribute to the understanding of the mechanisms behind potential resistance to the combination of proteasome inhibitors with PAPR inhibitors. ii Table of Contents Abstract ............................................................................................................................... ii Table of Contents ............................................................................................................... iii List of Tables .......................................................................................................................v List of Figures and Illustrations ......................................................................................... vi List of Symbols, Abbreviations and Nomenclature ......................................................... viii CHAPTER ONE: INTRODUCTION ..............................................................................12 1.1 Multiple Myeloma .................................................................................................12 1.2 Multiple Myeloma and Genome Instability ...........................................................13 1.3 Bortezomib .............................................................................................................15 1.4 Poly-ADP-Ribose Polymerase Inhibitors ..............................................................16 1.5 Double Stranded Break Repair ..............................................................................17 1.5.1 Canonical non-homologous end joining double strand break repair ...............18 1.5.2 Alternative end joining double strand break repair..........................................19 1.5.3 Homologous recombination double strand break repair ..................................22 1.6 53BP1 recruitment and displacement ....................................................................26 1.6.1 53BP1 Recruitment ..........................................................................................26 1.6.2 53BP1 Displacement/Antagonism ...................................................................27 1.6.2.1 H4K16 acetylation and 53BP1 antagonism .......................................27 1.6.2.2 Ubiquitin and 53BP1 antagonism ......................................................28 1.6.2.3 RIF1 antagonism to facilitate pathway switch ...................................29 1.7 Multiple Myeloma, bortezomib and PARP inhibitors ...........................................30 1.7.1 Previous work from Bahlis lab: Neri et al. 2011 .............................................30 Hypothesized mechanism of action for PI and PARPi combination therapy in MM33 1.8 Experimental Aims: ...............................................................................................38 CHAPTER TWO: MATERIALS AND METHODS ......................................................39 2.1 Antibodies, Plasmids, Primers, and Cell lines: ......................................................39 2.2 Cell culture methods ..............................................................................................41 2.3 Lentiviral infection with sh53BP1 containing LV particles ..................................42 2.4 Cloning of CRISPR/Cas9 Constructs ....................................................................42 2.5 CRISPR/Cas9 packaging of lentiviral plasmids and lentiviral infection: ..............43 2.6 Immunofluorescence Assays .................................................................................44 2.7 Western Blots .........................................................................................................46 2.7.1 Cell lysis buffers ..............................................................................................47 2.7.1.1 RIPA ..................................................................................................47 2.7.1.2 NP-40 (Ines) .......................................................................................47 2.7.1.3 Hypotonic lysis buffer........................................................................47 2.8 RT-PCR and qPCR for transcript expression analysis ..........................................48 2.9 RNA-seq Analysis .................................................................................................48 2.9.1 Sequence Acquisition.......................................................................................48 2.9.2 Sequence Alignment ........................................................................................48 2.9.3 Protein family gene acquisition .......................................................................49 iii 2.10 Surveyor Mutation Detection Assay ......................................................................49 2.11 Cell viability Assays ..............................................................................................50 CHAPTER THREE: ANALYSIS OF MM CELL LINES FOR MUTATIONS IN HISTONES AND DNA REPAIR PROTEINS ........................................................51 CHAPTER FOUR: GENE EDITING USING CRISPR/CAS9.......................................58 4.1 Using shRNA to silence 53BP1 in MM .................................................................58 4.2 Using TALEN to generate 53BP1-/- MM cell lines ...............................................61 4.2.1 TALEN mechanism of action and design to target 53BP1 ..............................61 4.2.2 Detection of mutation by SURVEYOR Mutation Detection assay .................65 4.2.3 Unsuccessful mutation of 53BP1 by TALEN ..................................................66 4.3 Employing the CRISPR/Cas9 genome editing technique to knock-out 53BP1 ....70 4.3.1 CRISPR/Cas9 instead of TALEN? ..................................................................70 4.3.2 CRISPR/Cas9 mechanism of action ................................................................70 4.3.3 Using CRISPR/Cas9 to edit 53BP1 and RNF168 ...........................................73 4.3.3.1 pLENTICRISPRv2 CRISPR/Cas9 of 53BP1 ....................................73 4.3.3.2 pLENTICRISPRv2 targeting RNF168 in OPM2 ..............................85 4.4 Revisiting CRISPR/Cas9 editing of 53BP1 ...........................................................87 4.4.1 Using pSp-Cas9(BB)-2A-GFP to edit 53BP1..................................................87 4.5 Other CRISPR/Cas9 targets that were unsuccessful ..............................................98 4.6 Other CRISPR/Cas9 targets that were successful ................................................101 CHAPTER FIVE: 53BP1 AND MULTIPLE MYELOMA ..........................................103 5.1 High RNF168 expression levels does not correlate with an increase in basal levels of 53BP1 foci in MM ...............................................................................................116 5.2 RNF168 loss does not confer resistance to synergy seen with concomitant treatment of MM with ABT-888 and BTZ ..........................................................................121 5.3 53BP1 is necessary for the synergistic activity of PI and PAPRi in MM............124 CHAPTER SIX: DISCUSSION AND FUTURE DIRECTIONS .................................128 REFERENCES ................................................................................................................131 SUPPLEMENTARY MATERIALS ...................................................................................141 iv List of Tables Table 1-1: Classification of MM by genetic alterations ............................................................... 14 Table 1-2: Known DNA repair pathways ..................................................................................... 25 Table 2-1: List of antibodies used for experiments detailed herein. ............................................. 39 Table 2-2: Cloning primers made to generate CRISPR/Cas9 mediated gene editing. These primers were inserted into pLENTICRISPRv2, pLentiGuide-Puro