Abstract A39: Understanding poly-ADP-ribose polymerase (PARP) inhibitor resistance in BRCA2-deficient cells through dual CRISPR knockout and activation screens

Cells deficient in the DNA repair pathway homologous recombination (HR) show particular sensitivity to inhibitors of poly-ADP-ribose polymerases (PARPi). Based on this observation, several PARPi, such as olaparib, have been FDA approved for the treatment of BRCA2-mutated ovarian cancer. Indeed, clinical trials have demonstrated that use of these agents significantly improved progression-free survival (PFS), for example, from 5.5 months to 19 months in one cohort of ovarian cancer patients harboring BRCA1/2 mutations (SOLO2 trial). However, even in this trial, only 65% of patients who were predicted to be genetically susceptible to this treatment attained 12 months PFS. This indicates that sensitivity to PARPi is mediated by more than simply BRCA2 status. Moreover, investigations into mechanisms governing sensitivity and resistance to PARPi continue to further our understanding of basic DNA repair and replication pathways. To this end, we conducted dual CRISPR screens in BRCA2-deficient HeLa cells as an unbiased approach for identifying proteins whose loss or overexpression confers resistance to PARPi. First, over 19,000 genes were individually knocked out in BRCA2-deficient cells. Then, these cells were treated or not with olaparib. Sequencing and computational analysis were used to identify which genes were lost most often in cells surviving olaparib treatment, yielding hundreds of potential hits. Hits were tested in multiple cell lines using cellular viability, apoptosis, and clonogenic survival assays. Several of these hits were successfully validated, including the histone acetyltransferase TIP60 (KAT5) and the ubiquitin ligase HUWE1. Mechanistically, we show that the rescue of PARPi sensitivity caused by TIP60 depletion is dependent on the downstream proteins 53BP1 and REV7, proteins known to inhibit end resection, thus promoting NHEJ; this indicates that the role of TIP60 as a modulator of double-strand break repair pathway choice underlies this resistance. The finding that increased 53BP1 binding contributes to PARPi resistance in BRCA2-deficient cells is particularly interesting given that loss of 53BP1 has been shown to cause resistance to PARPi in BRCA1-deficient cells. This indicates that pathways that govern sensitivity to PARPi in BRCA1-deficient cells may have opposite effects on this phenotype in BRCA2-deficient cells. To further understand the networks governing PARPi resistance in BRCA2-deficient cells, we also completed a CRISPR activation screen using the same experimental design as the knockout screen. By combining the knockout and activation screens for the first time in this context, we are able to better construct overarching networks regulating PARPi sensitivity in BRCA2-deficient cells. We have identified many potential mediators of PARPi resistance in BRCA2-deficient cells by combining CRISPR-knockout and CRISPR-activation screens. Our work identifies TIP60 as a novel potential biomarker for PARPi response in BRCA2-deficient cells. Citation Format: Kristen E. Clements, Anastasia Hale, Nathanial J. Tolman, George-Lucian Moldovan. Understanding poly-ADP-ribose polymerase (PARP) inhibitor resistance in BRCA2-deficient cells through dual CRISPR knockout and activation screens [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A39.