PARG loss sensitizes cells to PARGi.

a, Ranking of co-essential genes with PARGi treatment on the basis of a DrugZ analysis of the results of CRISPR/Cas9 screening with a DDR library in HEK293A cells. The NormZ score was used to determine a possible synthetic lethality gene under PARGi treatment. Drug-sensitive genes were marked in red; drug-resistant genes were marked in blue on the basis of the false discovery rate (FDR, 0.05 cut-off). b, HEK293A WT cells and HEK293A PARG KO cells were treated with different doses of PARGi for 72 hours. Cell viability was determined by the CellTiter-Glo assay. c, HEK293A PARG KO cells, re-constituted with either full-length PARG or catalytic domain mutation of PARG, were treated with different doses of PARGi for 72 hours. Cell viability was determined by the CellTiter-Glo assay.

PARGi treatment induces NAD+- and PARP-dependent cell death in PARG KO cells.

a, HEK293A WT, PARG KO, PARP1/2 DKO, and PARG/PARP1/2 TKO cells were treated with PARGi (1µM) for 72 hours. The total cell lysates were immunoblotted with the indicated antibodies. b, HEK293A WT, PARG KO, PARP1/2 DKO, and PARG/PARP1/2 TKO cells were treated with DMSO or 10 µM PARGi for 4 hours and then fixed and stained with anti-pADPr antibody and propidium iodide (PI). c, HEK293A WT, PARG KO, PARP1/2 DKO clls, and PARG/PARP1/2 TKO cells were treated with different doses of PARGi for 72 hours. Cell viability was determined by the CellTiter-Glo assay. d, Relative NAD+ level in HEK293A WT and PARG KO cells with the indicated treatment for 48 hours. PARGi, 10 µM; PARPi, 10 µM; NAM, 100 µM; NMN, 1 mM; FK866, 10 nM. e, PARG KO cells were treated with PARGi (10 µM) or PARGi and NAM (100 µM) or NMN (1mM) for 48 hours. The total cell lysates were immunoblotted with the indicated antibodies. f, Results of clonogenic assays conducted using HEK293A PARG KO cells treated with PARGi (500 nM) or PARGi and NAM (100 µM) or NMN (1 mM) for 7 days.

PARGi treatment induces S phase-specific pADPr signaling in PARG KO cells.

a, HEK293A WT and PARG KO cells were treated with DMSO or 10 µM PARGi for 4 hours or 0.01% MMS for 30 min and then fixed and stained with anti-pADPr antibody or anti-γH2A.X antibody and PI. b, HEK293A WT and HEK293A PARG KO cells were mock-treated or pre-treated with 2 µM emetine for 90 min and then treated with PARGi for an additional 4 hours. Cells were fixed and stained with anti-pADPr antibody and PI. c, HEK293A PARG KO cells were synchronized with double thymidine block (DTB). Cells remained with DTB or were released from DTB, treated with 10 µM PARGi for 4 hours, and then fixed and stained with anti-pADPr antibody and PI. d, Representative images and results (left) of clonogenic assays conducted using control cells and DTB synchronized or released HEK293A PARG KO cells treated with the indicated doses of PARGi for 7 days, and quantification of crystal violet staining assay (right). e, Results of clonogenic assays were conducted in PARG KO cells with indicated treatment for 7 days (PARGi, 1 µM; adarotene, 200 nM; CD437, 800 nM).

Prolonged PARGi treatment induces pADPr throughout the cell cycle and DDR in PARG KO cells.

a, Immunoblots of chromatin-bound PARP1 and PARylated proteins in HEK293A WT and PARG KO cells treated with PARGi (10 µM) for 4 hours. PARylated proteins were enriched by Af1521 beads. b, Sensitivity of HEK 293A PARG/PARP1/2 TKO and PARP1 reconstitution cells to PARGi. Cells were treated with different doses of PARGi for 72 hours, and cell viability was determined by the CellTiter-Glo assay. c, Prolonged PARGi treatment induces pADPr throughout the cell cycle in PARG KO cells. HEK293A WT and PARG KO cells were treated with PARGi (10 µM) for the indicated time and then fixed and stained with anti-pADPr antibody and violet. d, Immunoblotting of γH2A.X signals and other indicated proteins and modifications induced by prolonged PARGi+/-MMS treatment.

CRISPR screening identifies regulators of pADPr and cell viability.

a, Workflow of whole-genome CRISPR screens. For FACS-based CRISPR screening, 5 days after puromycin selection, cells were treated with PARGi (10uM) for 4 hours and then stained with anti-pADPr antibody and sorted with flow cytometry. Cells with strong signals (top 25%, TOP) and weak signals (bottom 25%, BOT) were selected. The sgRNAs from these cells were then sequenced and analyzed. For cell viability screening, cells were treated with or without PARGi for 21 days before collection. b, Scatter plot of DrugZ scores of PARG KO cells treated with or without PARGi treatment. The genes in the same pathway were marked with specific colors. A positive score indicates an enhanced pADPr signal, while a minus score indicates a decreased pADPr signal. c and d, Ranking of PARGi co-essential genes on the basis of a DrugZ analysis of the results of CRISPR/Cas9 screens performed with Toronto Knock Out Library (version 3) in HEK293A PARG KO cells and HEK239A cells. e, Analysis of biological processes of PARGi co-essential genes identified in HEK293A PARG KO cells and HEK239A cells. f, HEK293A WT cells, PARG KO, POLB KO, LIG1 KO, XRCC1 KO, and LIG3 knockdown cells were treated with different doses of PARGi for 72 hours. Cell viability was determined by the CellTiter-Glo assay. g, The cell Viability of HEK293A WT and PARG cells under POLB knockdown to PARGi. Cells were treated with different doses of PARGi for 72 hours.

PARG expression is a potential marker for PARGi sensitivity.

a, PARG mRNA level comparison between sensitive (RMGI, KURAMOCHI, and OVMANA) and resistant cells (COV362, COV318, OV56, OVISE, OVSAHO, CAOV3, and OVCAR3) from Pillay’s work on the basis of CCLE data. b, Clonogenic assay results of control and PARG knockdown HeLa cells with PARGi (2 µM) treatment for 7 days. shRNA knockdown efficiency was confirmed by an immunoblot of PARG. c, Ranked PARG expression level in ovarian cancer cell lines based on the CCLE database. The sensitive and resistant cells from Pillay’s work were labeled. d, Clonogenic assay results of OVCAR3 and RMUGS treated with or without PARGi (2 µM). e, Clonogenic assay results of RMUGS treated with PARGi (2 µM), PARPi (2 µM), or both. f, Left: Representative images of PARG IHC staining in breast and ovarian tissues and tumor samples to determine the PARG expression level. The summary is listed at the bottom. Scale bar, 200 µm. Right: The scatter plot of mean immunostaining intensity of PARG in each sample. The mean of each group was plotted.

PARG is essential for cell survival.

a, Diagram of full-length PARG was presented with the indicated gRNAs (gRNA#3 and 4) which target different regions in the C-terminal catalytic domain. The boundary of the catalytic domain was depicted based on Uniprot annotation. gRNA#1 and gRNA#2 were used previously to generate the aforementioned HEK293A- and HeLa-derived PARG KO cells, respectively; while gRNA#3 and gRNA#4 were used to generate PARG complete/conditional knockout (cKO) in the presence of olaparib in HEK293A and HeLa cells. b, Immunoblotting was conducted to confirm the loss of PARG in PARG cKO cells derived from HEK293A and HeLa cells, which were cultured in the presence of 100 nM olaparib. c, Clonogenic assay results of WT and PARG cKO cells treated with or without PARPi (100nM) for 7 days. d, Left: The immunoblots to confirm reconstitution with WT PARG or catalytic inactivation PARG in HEK293A PARG cKO cells. Right: Results of clonogenic survival assay with HEK293A PARG cKO cells reconstituted with WT or catalytic inactivation mutant of PARG for 7 days. e, Representative clonogenic results conducted in HEK293A PARG cKO cells treated with NAM (100 µM) or NMN (1 mM) for 7 days. f, HEK293A PARG cKO cells were synchronized with double thymidine block (DTB). Cells remained with DTB or were released from DTB for 4 hours, and then fixed and stained with anti-pADPr antibody and FxCycle Violet dye. g, Immunoblots of soluble and chromatin-bound PARP1 and pADPr levels in HEK293A WT and PARG cKO cells treated with DMSO or olaparib (10 µM) for 2 hours.