Identification of regulators of poly-ADP-ribose polymerase (PARP) inhibitor response through complementary CRISPR knockout and activation screens

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 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 HRdeficient tumors. Here, we 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. Strikingly, we reveal that depletion of the histone acetyltransferase TIP60, a top hit from our screens, robustly reverses the PARPi sensitivity caused by BRCA2 deficiency. Mechanistically, we show that TIP60 depletion rewires double strand break repair in BRCA2-deficient cells by promoting 53BP1 binding to double strand breaks to suppress end resection. Our work provides a comprehensive set of putative biomarkers that serve to better understand and predict PARPi response, and identifies a novel pathway of PARPi resistance in BRCA2-deficient cells.

PARP Inhibitors: Clinical Relevance, Mechanisms of Action and Tumor Resistance

“…Inhibition of REV7 via shRNA has been shown to inhibit NHEJ and consequently, promote HR. This shRNA mediated inhibition of REV7 induces PARPi resistance and rescue cells from Olaparib-induced cytotoxicity ( Clements et al, 2019 ). In support of this theory, elevated expression of TRIP13 ATPase has been identified in a large cohort of PARPi resistant BRCA1 mutated carcinomas.…”

Solenoid architecture of HUWE1 contributes to ligase activity and substrate recognition

“…A growing list of HUWE1 substrates has since been reported ( Kao et al, 2018 ), including the stress-responsive regulator of mTORC1 signaling, DDIT4 ( Brugarolas et al, 2004 ; Thompson et al, 2014 ), and many DNA damage response factors, such as the BRCA1 tumor suppressor, TopBP1, Cdc6, and CHEK1 ( Cassidy et al, 2020 ; Hall et al, 2007 ; Herold et al, 2008 ; Wang et al, 2014 ). Loss of HUWE1 sensitizes cells not only to DNA damage but also to a variety of other stressors, including both oxidative and hypoxic stress ( Amici et al, 2019 ; Bosshard et al, 2017 ; Clements et al, 2019 ; Kao et al, 2018 ; Olivieri et al, 2020 ). In addition, HUWE1 has been shown to mediate the destruction of unassembled constituents of multi-protein complexes and free histones ( Liu et al, 2005 ; Singh et al, 2009a , 2009b ; Xu et al, 2016 ), contributing to protein quality control ( Sung et al, 2016 ; Xu et al, 2016 ) and cell cycle checkpoint decisions.…”

Knowledge Graph-based Recommendation Framework Identifies Novel Drivers of Resistance in EGFR mutant Non-small Cell Lung Cancer

“…The primary data for pre-clinical drug resistance to EGFRi was generated from pooled, genome-wide CRISPR loss-of-function (LoF) and gain-of-function (GoF) studies, analogous to [59]. In the screens every gene in the genome was knocked out or over expressed and the impact on osimertinib resistance was assessed based on a treatment-control comparison of guide RNA prevalence.…”