MEDIATORS OF RADIATION RESISTANCE IN GLIOBLASTOMA MULTIFORME by BRITTANY AGUILA Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Advisor: Scott M Welford, Ph.D. Department of Biochemistry CASE WESTERN RESERVE UNIVERSITY May 2020 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Brittany Aguila Candidate for the degree of Doctor of Philosophy* Committee Chair Hung-Ying Kao, Ph.D. Committee Member Scott Welford, Ph.D. Committee Member Jason Mears, Ph.D. Committee Member Barbara Bedogni, Ph.D. March 6, 2020 *We certify that written approval has been obtained for any proprietary material contained therein. 2 Table of Contents List of Tables .................................................................................................. 5 List of Figures ................................................................................................ 6 Acknowledgements ....................................................................................... 8 List of Abbreviations ................................................................................... 10 Abstract ........................................................................................................ 11 Chapter 1 - Introduction and Background ................................................. 13 Glioblastoma multiforme .................................................................................................... 13 Standard of care .................................................................................................................................. 14 Prognosis ................................................................................................................................................. 15 Molecular Subtypes ........................................................................................................................... 15 Therapeutic Resistance ................................................................................................................... 17 Lentiviral mediated shRNA library negative selection and genomic hybridization functional knockdown screen ............................................................... 22 PTGFRN ..................................................................................................................................... 28 CD9 ............................................................................................................................................... 30 Tetraspanin Enriched Microdomains ............................................................................. 28 AKT/PI3K signaling pathway ............................................................................................. 32 SAT1 ............................................................................................................................................ 35 Statement of Purpose ........................................................................................................... 36 Chapter 2 - The Ig superfamily protein PTGFRN coordinates survival signaling in Glioblastoma multiforme ....................................................... 39 Abstract ...................................................................................................................................... 40 Introduction .............................................................................................................................. 41 Results ........................................................................................................................................ 43 PTGFRN is overexpressed in GBM and correlates with poorer patient outcome .................................................................................................................................................... 43 PTGFRN promotes cell proliferation and tumor growth .................................................. 44 PTGFRN inhibition sensitizes GBM cells to radiation ...................................................... 46 PTGFRN is necessary for P-AKT signaling by increasing stability of p110β ...... 48 PTGFRN promotes DNA damage sensing through p110β ........................................... 51 Discussion ................................................................................................................................ 52 Chapter 3 - Tetraspanin CD9 promotes tumorigenesis and radiation resistance in Glioblastoma multiforme ..................................................... 74 Abstract ...................................................................................................................................... 75 Introduction .............................................................................................................................. 76 Results ........................................................................................................................................ 77 CD9 is overexpressed in GBM and correlates with poorer outcome ....................... 77 CD9 promotes cell proliferation and tumor growth in GBM tumors .......................... 78 3 CD9 inhibition sensitizes GBM cells to radiation ................................................................ 79 CD9 depletion hinders DNA damage repair .......................................................................... 80 CD9 signals through AKT ............................................................................................................... 81 Discussion ................................................................................................................................ 81 Chapter 4 - A novel role for spermidine/spermine N1-acetyltransferase 1 as a gene-specific transcriptional regulator that promotes brain tumor aggressiveness ................................................................................ 94 Abstract ...................................................................................................................................... 95 Introduction .............................................................................................................................. 96 Results ........................................................................................................................................ 97 SAT1 regulates gene programs controlling cell cycle and DNA dynamics ........... 97 SAT1 target genes are enriched in high-grade gliomas ................................................. 98 SAT1 regulates MELK and EZH2 by direct interaction with chromatin .................. 99 SAT1 regulates transcription of specific targets through polyamine catabolism102 Discussion .............................................................................................................................. 103 Chapter 5 - Discussion and Future Directions ........................................ 116 The vast molecular basis for radioresistance .......................................................... 116 Other signaling possibilities for PTGFRN .................................................................. 118 PTGFRN and CD9: a possible scaffolding complex? ............................................ 124 Could SAT1 be regulating CD9? .................................................................................... 128 Concluding remarks ........................................................................................................... 133 Appendix A ................................................................................................. 134 Appendix B ................................................................................................. 139 Appendix C ................................................................................................. 143 References ................................................................................................. 148 4 List of Tables Appendix C Supplementary Method Table 1: Antibodies ...................... 145 Appendix C Supplementary Method Table 2: Primers for qPCR .......... 146 Appendix C Supplementary Method Table 3: Primers for Chromatin Immunoprecipitations ............................................................................... 147 5 List of Figures Figure 1-1: Negative Selection and Genomic Hybridization Functional Knockdown Screen……………………………………………………24 Figure 1-2: Results of negative selection and genomic hybridization functional knockdown screen…………………………………….26 Figure 2-1: Relevance of PTGFRN to GBM………………………………………56 Figure 2-2: PTGFRN expression in GBM tumors with specific mutation statuses and GBM molecular subtypes……………………………...58 Figure 2-3: PTGFRN is required for cell proliferation and tumor growth…………………………………………………………………………….........60 Figure 2-4: PTGFRN depletion extends cell cycle progression……………..62 Figure 2-5: shRNA knockdown confirmation in GBM cell lines……………..64 Figure 2-6: PTGFRN reduction sensitizes GBM cells to IR…………………..66 Figure 2-7: PTGFRN depletion decreases p110β levels………………………68 Figure 2-8: PTGFRN depletion does not affect p110α protein levels and PTGFRN co-localizes with p110β……………………………………70 Figure 2-9: PTGFRN promotes DNA damage sensing………………………...72 Figure 3-1: Relevance of CD9 to GBM……………………………………………84 Figure 3-2: CD9 promotes cell proliferation and tumor growth……………..86 Figure 3-1: CD9 inhibition sensitizes GBM cells to radiation………………..88 Figure 3-2: CD9 depletion decreases