Selective Loss of PARG Restores PARylation and Counteracts PARP Inhibitor-Mediated Synthetic Lethality

Gogola, Ewa; Duarte, Alexandra A.; Ruiter, Julian R. de; Wiegant, Wouter W.; Schmid, Jonas; Bruijn, Roebi de; James, Dominic I.; Llobet, Sergi Guerrero; Vis, Daniël J.; Annunziato, Stefano; Broek, Bram van den; Barazas, Marco; Kersbergen, Ariena; Ven, Marieke van de; Tarsounas, Madalena; Ogilvie, Donald; Vugt, Marcel A.T.M. van; Wessels, Lodewyk F.A.; Bártková, Jiřina; Gromova, Irina; Andújar-Sánchez, Miguel; Bártek, Jiří; Lopes, Massimo; Attikum, Haico van; Borst, Piet; Jonkers, Jos; Rottenberg, Sven

doi:10.1016/j.ccell.2019.05.012

Cited by 87 publications

(111 citation statements)

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“…5a ), we observed that BRCA1 scored as the 18th ranked gene for synthetic lethality with PARP1 (Z-score -3.0), and BRCA2 ranked 54th (Z score -2.4). Conversely, PARG, which catabolizes poly(ADP-ribose), scored as a top buffering gene (rank 2, Z-score 2.8), as has been observed previously 48 . To broadly assess these genetic and small molecule screens, we used several benchmark gene sets ( Supplementary Data 4 ): a curated set of genes involved in homologous recombination provided by the Wood laboratory (n = 21) 49 ; the Reactome "DNA Repair" gene set (n = 106) 50 ; known protein-protein interactors with PARP1 curated by BioGrid (n = 289) 51 ; and a high-confidence set of hits identified by Zimmermann and colleagues via CRISPR screens for olaparib sensitivity (n = 73) 52 .…”

Section: Anchor Screens With Parp1 Knockout and Parp Inhibitorssupporting

confidence: 77%

Genetic screens in isogenic mammalian cell lines without single cell cloning

DeWeirdt

Sanson

Hanna

et al. 2019

Preprint

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Isogenic pairs of cell lines, which differ by a single genetic modification, are powerful tools for understanding gene function. Generating such pairs for mammalian cells, however, is labor-intensive, time-consuming, and impossible in some cell types. Here we present an approach to create isogenic pairs of cells and screen them with genome-wide CRISPR-Cas9 libraries to generate genetic interaction maps. We queried the anti-apoptotic genes BCL2L1 and MCL1, and the DNA damage repair gene PARP1, via 25 genome-wide screens across 4 cell lines. For all three genes, we identify a rich set of both expected and novel buffering and synthetic lethal interactions. Further, we compare the interactions observed in genetic space to those found when targeting these genes with small molecules and identify hits that may inform the clinical uses for these inhibitors. We anticipate that this methodology will be broadly useful to comprehensively study genes of interest across many cell types.Genetic interaction networks can suggest functional roles of uncharacterized genes and capture subtle biological interactions, which may prove critical for interpreting genetic signal from genome-wide association studies of common disease states. Crosses of yeast knockout strains have yielded rich networks of genetic interactions, and have further shown that the shape of the network will change based on growth conditions 1-5 . In mammalian cells, the construction of such networks is orders of magnitude more complicated, due to increased genome size, the diversity of cell types, and numerous technical factors. One approach is to use either RNAi 6 or CRISPR technology 7-11 to screen a library of all possible combinatorial perturbations within a focused gene list. This approach has been used to generate genetic interaction maps for up to hundreds of genes 12 ; however, screening all combinations of protein coding genes in the human genome would require, at bare minimum, approximately 400 million perturbations and 200 billion cells, which is equivalent to 5,000 concurrent genome-wide screens with typical guide libraries and currently exceeds the practical limits of tissue culture. This scale is exacerbated by the diversity of cell types in which to study such interactions.A second, complementary approach to query genetic interactions leverages isogenic pairs of human cells, akin to mutant strains of model organisms, to enable the delineation of a given gene's contribution to phenotypes of interest. Initial gene targeting approaches in human cell lines to create even a single knockout have yielded valuable insights but were quite laborious to generate [13][14][15][16][17][18][19] . Today, CRISPR technology has made cell line engineering possible for a broad range of researchers, but that is distinct from making it easy. Creating the site-specific nuclease for Cas9 is as simple as ordering a short nucleic acid, in contrast to the more expensive and time-consuming task of assembling a customized pair of zinc finger nucleases or TALENs 20 . After design...

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Section: Anchor Screens With Parp1 Knockout and Parp Inhibitorssupporting

confidence: 77%

Genetic screens in isogenic mammalian cell lines without single cell cloning

DeWeirdt

Sanson

Hanna

et al. 2019

Preprint

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“…While these studies that not all PARPi responders with PCa harbor HR-defective tumors, and not all PCa tumors that exhibit aberrant DNA repair are PARPi responsive, there is clinical evidence that PARPi resistance is associated with restored HR function in multiple tumor types (Edwards et al, 2008;Barber et al, 2013;Christie et al, 2017;Kondrashova et al, 2017;Pishvaian et al, 2017;Weigelt et al, 2017), including PCa (Goodall et al, 2017;Quigley et al, 2017). Additionally, PARPi resistance has been associated with differential DNA damage response (DDR) network functioning (Jaspers et al, 2013;Johnson et al, 2013;Gogola et al, 2018). These mechanisms of resistance to PARPi indicate that for these tumors, DDR defects likely led to PARPi responses.…”

Section: Introductionmentioning

confidence: 99%

PARP‐1 regulates DNA repair factor availability

et al. 2018

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PARP‐1 holds major functions on chromatin, DNA damage repair and transcriptional regulation, both of which are relevant in the context of cancer. Here, unbiased transcriptional profiling revealed the downstream transcriptional profile of PARP‐1 enzymatic activity. Further investigation of the PARP‐1‐regulated transcriptome and secondary strategies for assessing PARP‐1 activity in patient tissues revealed that PARP‐1 activity was unexpectedly enriched as a function of disease progression and was associated with poor outcome independent of DNA double‐strand breaks, suggesting that enhanced PARP‐1 activity may promote aggressive phenotypes. Mechanistic investigation revealed that active PARP‐1 served to enhance E2F1 transcription factor activity, and specifically promoted E2F1‐mediated induction of DNA repair factors involved in homologous recombination (HR). Conversely, PARP‐1 inhibition reduced HR factor availability and thus acted to induce or enhance “BRCA‐ness”. These observations bring new understanding of PARP‐1 function in cancer and have significant ramifications on predicting PARP‐1 inhibitor function in the clinical setting.

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“…or in the RNASEH2 complex(Zimmermann et al, 2018); secondly, when PARPi sensitivity occurs via mechanisms that preserve RAD51 foci formation, e.g., alterations in the MRN complex, RAD51AP1, polymerase eta, or ERCC1(Kawamoto et al, 2005;Wiese et al, 2007;Oplustilova et al, 2012;Postel-Vinay et al, 2013); thirdly, when HRR-deficient tumors have acquired PARPi resistance via RAD51independent mechanisms such as loss of PARG, mutations in PARP1, or those that involve replication fork stabilization(Guillemette et al, 2015;Chaudhuri et al, 2016;Kais et al, 2016; Yazinski et al, 2017;Gogola et al, 2018;Michelena et al, 2018;Pettitt et al, 2018);…”

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confidence: 99%

A RAD 51 assay feasible in routine tumor samples calls PARP inhibitor response beyond BRCA mutation

Castroviejo-Bermejo¹,

et al. 2018

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Poly(ADP‐ribose) polymerase (PARP) inhibitors (PARPi) are effective in cancers with defective homologous recombination DNA repair (HRR), including BRCA1/2‐related cancers. A test to identify additional HRR‐deficient tumors will help to extend their use in new indications. We evaluated the activity of the PARPi olaparib in patient‐derived tumor xenografts (PDXs) from breast cancer (BC) patients and investigated mechanisms of sensitivity through exome sequencing, BRCA1 promoter methylation analysis, and immunostaining of HRR proteins, including RAD51 nuclear foci. In an independent BC PDX panel, the predictive capacity of the RAD51 score and the homologous recombination deficiency (HRD) score were compared. To examine the clinical feasibility of the RAD51 assay, we scored archival breast tumor samples, including PALB2‐related hereditary cancers. The RAD51 score was highly discriminative of PARPi sensitivity versus PARPi resistance in BC PDXs and outperformed the genomic test. In clinical samples, all PALB2‐related tumors were classified as HRR‐deficient by the RAD51 score. The functional biomarker RAD51 enables the identification of PARPi‐sensitive BC and broadens the population who may benefit from this therapy beyond BRCA1/2‐related cancers.

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Selective Loss of PARG Restores PARylation and Counteracts PARP Inhibitor-Mediated Synthetic Lethality

Cited by 87 publications

References 0 publications

Genetic screens in isogenic mammalian cell lines without single cell cloning

Genetic screens in isogenic mammalian cell lines without single cell cloning

PARP‐1 regulates DNA repair factor availability

A RAD 51 assay feasible in routine tumor samples calls PARP inhibitor response beyond BRCA mutation

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