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Identification of Novel Regulatory Genes in Acetaminophen

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Identification of Novel Regulatory Genes in Acetaminophen IDENTIFICATION OF NOVEL REGULATORY GENES IN ACETAMINOPHEN INDUCED HEPATOCYTE TOXICITY BY A GENOME-WIDE CRISPR/CAS9 SCREEN A THESIS IN Cell Biology and Biophysics and Bioinformatics Presented to the Faculty of the University of Missouri-Kansas City in partial fulfillment of the requirements for the degree DOCTOR OF PHILOSOPHY By KATHERINE ANNE SHORTT B.S, Indiana University, Bloomington, 2011 M.S, University of Missouri, Kansas City, 2014 Kansas City, Missouri 2018 © 2018 Katherine Shortt All Rights Reserved IDENTIFICATION OF NOVEL REGULATORY GENES IN ACETAMINOPHEN INDUCED HEPATOCYTE TOXICITY BY A GENOME-WIDE CRISPR/CAS9 SCREEN Katherine Anne Shortt, Candidate for the Doctor of Philosophy degree, University of Missouri-Kansas City, 2018 ABSTRACT Acetaminophen (APAP) is a commonly used analgesic responsible for over 56,000 overdose-related emergency room visits annually. A long asymptomatic period and limited treatment options result in a high rate of liver failure, generally resulting in either organ transplant or mortality. The underlying molecular mechanisms of injury are not well understood and effective therapy is limited. Identification of previously unknown genetic risk factors would provide new mechanistic insights and new therapeutic targets for APAP induced hepatocyte toxicity or liver injury. This study used a genome-wide CRISPR/Cas9 screen to evaluate genes that are protective against or cause susceptibility to APAP-induced liver injury. HuH7 human hepatocellular carcinoma cells containing CRISPR/Cas9 gene knockouts were treated with 15mM APAP for 30 minutes to 4 days. A gene expression profile was developed based on the 1) top screening hits, 2) overlap with gene expression data of APAP overdosed human patients, and 3) biological interpretation including assessment of known and suspected iii APAP-associated genes and their therapeutic potential, predicted affected biological pathways, and functionally validated candidate genes. This screen is the first genome-wide CRISPR/Cas9 knockout screen of APAP- induced hepatocyte toxicity. The top hits from this screen included numerous genes previously not linked to liver injury. We further demonstrated the implementation of intermediate time points for the identification of early and late response genes. A negative selection screen identified genes involved in fundamental processes, including NAAA, ATG2B, and MYOZ3. A positive selection screen identified numerous genes potentially involved in pathogenic processes, including LZTR1, PGM5, and EEF1D. A top essential pathway at 24 hours of APAP treatment was Regulation of Skeletal Muscle Contraction. We additionally identified 6 genes, 3 novel and 3 known, that have drug-gene interactions favorable for re-purposing existing therapies to treat APAP-induced hepatotoxicity. Collectively, this line of research has illustrated the power of a genome-wide CRISPR/Cas9 screen to systematically identify novel genes involved in APAP induced hepatocyte toxicity and to provide potential new targets to develop novel therapeutic modalities. iv APPROVAL PAGE The faculty listed below, appointed by the Dean of the School of Graduate Studies, have examined a thesis titled “Identification of Novel Regulatory Genes in Acetaminophen Induced Hepatocyte Toxicity by a Genome-Wide CRISPR/Cas9 Screen”, presented by Katherine Anne Shortt, candidate for the Doctor of Philosophy degree, and certify that in their opinion it is worthy of acceptance. Supervisory Committee Shui Qing Ye, M.D, Ph.D., Chair Division of Cell Biology and Biophysics, School of Biological Sciences Department of Biomedical and Health Informatics, School of Medicine Mary Gerkovich, Ph.D. Department of Biomedical and Health Informatics, School of Medicine Daniel P. Heruth, Ph.D. Department of Pediatrics, School of Medicine Saul Honigberg, Ph.D. Division of Cell Biology and Biophysics, School of Biological Sciences Gerald J. Wyckoff, Ph.D. Division of Molecular Biology and Biochemistry, School of Biological Sciences v CONTENTS ABSTRACT ....................................................................................................................... iii LIST OF ILLUSTRATIONS ...............................................................................................x LIST OF TABLES ............................................................................................................. xi GLOSSARY ..................................................................................................................... xii ACKNOWLEDGMENTS .............................................................................................. xiv Chapter 1. INTRODUCTION .....................................................................................................1 2. REVIEW OF THE LITERATURE ...........................................................................4 Candidate gene approaches for biomarker discovery in APAP-induced hepatotoxicity ...........................................................................................................4 “Omics” approaches for biomarker discovery in APAP-induced hepatotoxicity ....7 Gene editing approaches for biomarker discovery in other diseases .....................10 Novel approach to APAP-induced hepatotoxicity biomarker discovery ...............11 3. RESEARCH QUESTION .........................................................................................13 4. METHODS ..............................................................................................................15 Research design .....................................................................................................15 sgRNA library amplification..................................................................................15 Cell culture .............................................................................................................16 Lentivirus production and concentration ...............................................................17 Acetaminophen kill curve ......................................................................................18 vi In vitro hepatocellular carcinoma transduction using the GeCKOv2 sgRNA library .....................................................................................................................18 CRISPR/Cas9 acetaminophen screen and sample collection ................................19 CRISPR/Cas9 screen amplicon sequencing ..........................................................19 CRISPR/Cas9 Screen deconvolution and analysis ...............................................20 Pathway analysis ....................................................................................................21 Statistical analysis of gene expression datasets from the Gene Expression Analysis of overlapping data from Gene Expression Omnibus .............................21 Human APAP induced liver injury data ....................................................21 Mouse APAP induced liver injury data ....................................................23 Analysis of acetaminophen-associates SNPs in the literature ...............................24 Drug-gene interaction analysis ..............................................................................25 Functional validations in primary mouse hepatocytes ...........................................25 Semi-quantitative PCR...........................................................................................27 Western Blotting ....................................................................................................27 Plasmids .................................................................................................................28 5. RESULTS AND DISCUSSION PART 1: CRISPR/CAS9 SCREEN ......................29 Development of screening strategy and preparation of cell lines ..........................29 CRISPR/Cas9 knock-out screen and deconvolution..............................................29 Discussion ..............................................................................................................39 6. RESULTS AND DISCUSSION PART 2: OUR SCREEN IN THE CONTEXT OF OTHER ACETAMINOPHEN DATA SETS ...........................................................42 vii Overlapping analysis of our screen top hits with other gene expression acetaminophen datasets .............................................................................................42 Analysis of RNA-sequence from mice with acetaminophen-induced acute liver injury ......................................................................................................................42 Analysis of microarray of human liver biopsies from normal and acetaminophen- induced acute liver failure patients ........................................................................43 Analysis of microarray of human blood from normal and acetaminophen-dosed participants .............................................................................................................45 Identification of candidate genes from overlapping gene sets ...............................48 Discussion ..............................................................................................................49 7. RESULTS AND DISCUSSION PART 3: ACETAMINOPHEN-ASSOCIATED SINGLE NUCLEOTIDE POLYMORPHISMS IN THE LITERATURE ...............52 Acetaminophen-associated single nucleotide polymorphisms in the literature .....52 Functional in silico analyses of SNPs associated with acetaminophen-induced
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