Identifying Novel Mechanisms of Sensitivity and Resistance to Poly-ADP-Ribose Polymerase (PARP) Inhibitors through Genome-wide CRISPR Knockout and Activation Screens
Open Access
- Author:
- Clements, Kristen Eloie
- Graduate Program:
- Biomedical Sciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- January 30, 2020
- Committee Members:
- George Lucian Moldovan, Dissertation Advisor/Co-Advisor
George Lucian Moldovan, Committee Chair/Co-Chair
James Riley Broach, Committee Member
Kristin Ann Eckert, Committee Member
Raymond J Hohl, Outside Member
Zhonghua Gao, Committee Member
Ralph Lauren Keil, Program Head/Chair - Keywords:
- PARP inhibitor
CRISPR screen
DNA repair
drug resistance
olaparib - Abstract:
- Inhibitors of poly-ADP-ribose polymerase 1 (PARPi) are highly effective in killing cells deficient in the homologous recombination (HR) DNA repair pathway, such as those lacking BRCA1 or BRCA2. In light of this, PARPi have been utilized in recent years to treat BRCA2-mutant tumors, with many patients deriving impressive clinical benefit. However, positive response to PARPi is not universal, even among patients with HR-deficient tumors. In this dissertation, I present the results of three genome-wide CRISPR knockout and activation screens which provide an unbiased look at genetic determinants of PARPi response in wildtype or BRCA2-knockout cells. Additionally, I reveal the novel mechanisms through which depletion of two hits from the screens—E2F7 and TIP60—lead to resistance to PARPi in BRCA2-deficient cells. Strikingly, I reveal that depletion of the transcription factor E2F7, a top hit from the screens, robustly reverses the PARPi sensitivity caused by BRCA2 deficiency. Moreover, I show that the mechanism underlying this activity involves increased expression of RAD51, a target for E2F7-mediated transcriptional repression, which enhances both HR DNA repair and replication fork stability in BRCA2-deficient cells. Notably, restoration of homologous recombination independent of a reversion mutation has not previously been associated with PARPi resistance in BRCA2-deficient cells. In addition, I demonstrate that loss of the histone acetyltransferase TIP60, a second hit from the screen, also abolishes the sensitivity of BRCA2-deficient cells to PARPi. Mechanistically, I reveal that TIP60 depletion rewires double strand break repair in BRCA2-deficient cells by promoting 53BP1 binding to double strand breaks to suppress end resection. My work provides a comprehensive set of putative biomarkers that serve to better understand and predict PARPi response, and identifies novel pathways of PARPi resistance in BRCA2-deficient cells.