Acsvl3 Is a Novel Therapeutic Target in Glioblastoma: Drug Discovery and Role in Protein Lipidation

ACSVL3 IS A NOVEL THERAPEUTIC TARGET IN GLIOBLASTOMA: DRUG DISCOVERY AND ROLE IN PROTEIN LIPIDATION by Emily M. Clay A dissertation submitted to The Johns Hopkins University in conformity with the requirements of the degree of Doctor of Philosophy Baltimore, Maryland October 2017 © Emily M. Clay 2017 All rights reserved Abstract Glioblastoma is an aggressive form of brain cancer with a five-year survival rate after diagnosis of less than 6%. More efficacious treatments are needed to improve patient prognosis. Very long-chain acyl-CoA synthetase (ACSVL3) is not detected in healthy glial cells, but is highly expressed in gliomas. Knockout of ACSVL3 in glioblastoma cells in culture decreases tumorigenicity and disrupts growth-factor induced cell signaling. However, the mechanism through which ACSVL3 promotes malignancy is not known. To address this question, lipid-based post-translational modifications were studied in wild-type and ACSVL3-knockout glioblastoma lines in cell culture. No significant differences were found in levels of protein palmitoylation or GPI anchors between wild-type and knockout cells. However, a global decrease in protein stearoylation was observed with ACSVL3 knockout, and mass spectrometry revealed several individual proteins that exhibited increased or decreased stearoylation with ACSVL3 knockout. From this we conclude that ACSVL3 is partially responsible for mediating stearoylation in glioblastoma cells. Next, we searched for inhibitors of ACSVL3. Inhibitors are desired both as tools to study the mechanism of ACSVL3- promoted malignancy and as potential therapeutic agents in glioblastoma. Two methods were established to screen for inhibitors of ACSVL3. The first was a small screen of known inhibitors of the homologous enzyme ACSVL1. This screen determined whether these drugs and compounds inhibited the activation of the 18-carbon fatty acid stearate by glioblastoma cells. The second was development of a screen to evaluate the ability of an ACSVL3/ACSVL1 hybrid enzyme to uptake fluorescent fatty-acid analogs. In the first ii screen, the small molecule CB5 was found to greatly inhibit activation of stearate. When glioblastoma cells in culture were treated with CB5, the compound slowed the rate of cell proliferation, induced differentiation, and decreased β-oxidation of long-chain fatty acids. Growth of subcutaneous xenografts produced from U87, but not Mayo-22, glioblastoma cells implanted subcutaneously in mice was significantly slowed by CB5 treatment. We conclude that the small molecule CB5 inhibits ACSVL3 activity in vitro and in vivo, and ACSVL3 is a promising novel therapeutic target in glioblastoma. The second screen shows high potential for conducting a high-throughput screen for additional inhibitors. Thesis Advisor/Reader: Paul A. Watkins, M.D., Ph.D. Thesis Reader: Daniel M. Raben, Ph.D. iii Acknowledgments I would like to acknowledge my committee members, who provided very valuable advice and direction over the course of my thesis research: Drs. Jun Liu, John Laterra, Jin Zhang, Paul Englund, and my committee chair Daniel Raben. I would also like to thank all of the current and former members of the Watkins lab for all their help around the lab, advice, friendship, and camaraderie, especially Yanqiu Liu, Dr. Xiaohai Shi, and Dr. Liz Kolar. They made coming to work every day enjoyable, even when it was frustrating. I was incredibly fortunate to work with the best advisor imaginable, Dr. Paul Watkins. Paul was unwaveringly supportive and always available to troubleshoot or just talk science. I thank him for instilling in me a love of lipids. Finally, I thank my family, friends, and Lance for their encouragement throughout my time as a graduate student. iv Table of Contents Abstract ............................................................................................................................... ii Acknowledgments.............................................................................................................. iv List of Tables ..................................................................................................................... vi List of Figures ................................................................................................................... vii Background ..........................................................................................................................1 Chapter 1: Identification and characterization of inhibitors of ACSVL3 Introduction ............................................................................................................14 Materials and methods ...........................................................................................19 Results ....................................................................................................................33 Discussion ..............................................................................................................53 Chapter 2: GPI-anchored proteins in WT and ACSVL3-KO U87 cells Introduction ............................................................................................................59 Materials and methods ...........................................................................................62 Results ....................................................................................................................68 Discussion ..............................................................................................................77 Chapter 3: Protein acylation in WT and ACSVL3-KO U87 cells Introduction ............................................................................................................80 Materials and methods ...........................................................................................82 Results ....................................................................................................................90 Discussion ............................................................................................................101 References ........................................................................................................................105 Curriculum Vitae .............................................................................................................122 v List of Tables Table 1: Systematic names of drugs and compounds included in assays .........................20 Table 2: Primer sequences used to detect transcripts encoding for common GPI-anchored proteins ......................................................................................................64 Table 3: Stearoylated proteins more abundant in WT U87 cells vs. ACSVL3-KO U87 cells ....................................................................................................................................99 Table 4: Stearoylated proteins more abundant in ACSVL3-KO U87 cells vs. WT U87 cells ..................................................................................................................................100 vi List of Figures Figure 1: Phylogenetic tree of human ACS proteins ...........................................................3 Figure 2: Sequence alignment of ACSVL3 and ACSVL1 ................................................16 Figure 3: Expression of ACSVL3 in WT and ACSVL3-KO U87 cells ............................33 Figure 4: Total activation of long- and very-long chain fatty acids in WT and ACSVL3- KO U87 cells......................................................................................................................34 Figure 5: Inhibition of C18:0 activation by ACSVL1 inhibitors in WT and ACSVL3-KO U87 cells ...........................................................................................................................37 Figure 6: Characterization of the ACSVL1/3 hybrid enzyme ...........................................39 Figure 7: Inhibition of ACSVL1/3 by ACSVL1 inhibitors ...............................................41 Figure 8: Inhibition of U87 proliferation by CB5 ..............................................................43 Figure 9: Morphology of U87 cells treated with CB5 .......................................................44 Figure 10: Reversion of growth rate in U87 treated with CB5 after drug removal ..........45 Figure 11: Treatment of human fibroblasts with CB5 .......................................................46 Figure 12: Scratch-wound assay of CB5-treated U87 cells ...............................................47 Figure 13: β-oxidation of long- and very long-chain fatty acids in U87 cells ...................48 Figure 14: Expression of GFAP in U87 cells ....................................................................49 Figure 15: Treatment of mice bearing glioblastoma xenograft tumors with CB5 .............52 Figure 16: Diagram of a human GPI-anchored protein .....................................................60 Figure 17: Expression of GPI-anchored proteins in WT and ACSVL3-KO U87 cells .....69 Figure 18: Expression and purification of PI-PLC from E. coli ........................................71 vii Figure 19: Assessment of purified PI-PLC activity using the Ellman assay .....................74 Figure 20: Detection of GPI-anchored proteins in WT and ACSVL3-KO U87 cells .......76 Figure 21: Detection of acylated proteins in WT and ACSVL3-KO U87 cells ................92 Figure 22: Activation of Akt by EGF in U87 cells ............................................................93

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