Identifying Novel Mechanisms of Sensitivity and Resistance to Poly-ADP-Ribose Polymerase (PARP) Inhibitors through Genome-wide CRISPR Knockout and Activation Screens
- Restrictions on Access:
- Restricted (Penn State Only).
- 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 screensE2F7 and TIP60lead 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.
- Dissertation Note:
- Ph.D. Pennsylvania State University 2020.
- Technical Details:
- The full text of the dissertation is available as an Adobe Acrobat .pdf file ; Adobe Acrobat Reader required to view the file.
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