Specific killing of DNA damage-response deficient cells with inhibitors of poly(ADP-ribose) glycohydrolase

Abstract

Poly(ADP-ribosylation) of proteins following DNA damage is well studied and the use of poly(ADP-ribose) polymerase (PARP) inhibitors as therapeutic agents is an exciting prospect for the treatment of many cancers. Poly(ADP-ribose) glycohydrolase (PARG) has endo- and exoglycosidase activities which can cleave glycosidic bonds, rapidly reversing the action of PARP enzymes. Like addition of poly(ADP-ribose) (PAR) by PARP, removal of PAR by PARG is also thought to be required for repair of DNA strand breaks and for continued replication at perturbed forks. Here we use siRNA to show a synthetic lethal relationship between PARG and BRCA1, BRCA2, PALB2, FAM175A (ABRAXAS) and BARD1. In addition, we demonstrate that MCF7 cells depleted of these proteins are sensitive to Gallotannin and a novel and specific PARG inhibitor PDD00017273. We confirm that PARG inhibition increases endogenous DNA damage, stalls replication forks and increases homologous recombination, and propose that it is the lack of homologous recombination (HR) proteins at PARG inhibitor-induced stalled replication forks that induces cell death. Interestingly not all genes that are synthetically lethal with PARP result in sensitivity to PARG inhibitors, suggesting that although there is overlap, the functions of PARP and PARG may not be completely identical. These data together add further evidence to the possibility that single treatment therapy with PARG inhibitors could be used for treatment of certain HR deficient tumours and provide insight into the relationship between PARP, PARG and the processes of DNA repair.

Keywords: BARD1; BRCA1; Breast cancer; FAM175A (ABRAXAS); PALB2; PARG inhibition; Synthetic lethality.

Fig. 1

Synthetic lethal screen using siRNA. (A) Relative expression of mRNA for each test gene compared to scrambled control 24 h post transfection as measured by qRT-PCR and normalised to U1 snRNA. Letters above target gene names refer to separate oligos (a–d) for each gene. (B) Protein expression of PARG and PARP 48 h post transfection with each PARG siRNA, representative western blot and mean and standard deviation of quantification of three independent repeats are shown. (C) MCF7 cell viability 5 days post transfection with each PARG siRNA measured by MTT assay, mean and standard deviation of four independent repeats are shown. (D) MCF7 cell viability as measured by MTT assay 5 days post transfection with combinations of individual siRNA oligos. For each target DDR gene siRNA + PARG siRNA survival fraction is calculated compared to DDR gene alone, mean of two independent repeats is shown.

Fig. 2

Synthetic lethal screen using PARG inhibitors. (A) Quantification of PARG activity in the presence of increasing concentrations of PDD00017273 as measured by the ability of recombinant PARG to hydrolyse biotinylated PAR polymers from histones. (Bi) Western Blot for poly(ADP-ribose) (PAR), PARP1, PARG and tubulin, in MCF7 cells control (DMSO), treated with PARG inhibitors (Gallotannin (GLTN) and PDD00017273) or PARP inhibitor (Olaparib). (Bii) Quantification represents mean intensity of PAR over three independent repeats relative to tubulin loading control. (C) Survival fraction of MCF7 cells untreated (DMSO), treated with PARG inhibitors (GLTN and PDD00017273) or PARP inhibitor (Olaparib) as measured by clonogenic survival assay, mean and standard deviation of three independent repeats are shown. Statistical significance was calculated using the Student’s T-test, compared to DMSO control. (D) Survival fraction of pooled target DDR gene siRNA transfected MCF7 cells treated with PARG inhibitors Gallotannin (GLTN) (E) PDD00017273 compared to corresponding siRNA transfected DMSO treated cells. Survival was measured by clonogenic survival assay; mean and standard deviation of three independent repeats are shown. Statistical significance was calculated using the Student’s T-test, comparing DDR gene + inhibitor to scrambled control + inhibitor where * = p < 0.05, ** = p < 0.01.

Fig. 3

γH2AX formation screen with siRNA. Fold increase in cells with greater than five γH2AX foci/cell in MCF7 cells 24 h post transfection with combinations of pools of target DDR gene siRNA plus pooled PARG siRNA compared to corresponding target DDR gene alone. 100 cells were counted on three separate occasions; mean and standard deviation are shown. Statistical significance was calculated using the Student’s T-test, compared to scrambled siRNA + PARG siRNA control, where * = p < 0.05, ** = p < 0.01. Representative images are shown in Supplementary data S3.

Fig. 4

Confirmation of synthetic lethal relationships by clonogenic survival assay. (A) Survival fraction of MCF7 cells as measured by clonogenic survival assay 14 days post transfection with siRNA. Combinations of two different siRNA targeting PARG (PARG1 or PARG3) are combined with two different siRNA targeting the test DDR gene (indicated by a letter after the test gene name). For each DDR gene siRNA + scrambled/PARG siRNA survival fraction is calculated compared to corresponding scrambled/PARG siRNA alone. Statistical significance was calculated using the Student’s T-test, comparing DDR gene + PARG gene siRNAs to DDR gene + scrambled control siRNAs. (B) Survival fraction of wildtype (BXPC3) and BRCA2 deficient (CAPAN1) cells treated with DMSO, PARP inhibitor (olaparib), or the PARG inhibitors (gallotannin (GLTN) and PDD00017273), as measured by clonogenic survival assay. Statistical significance was calculated using the Student’s T-test, comparing BXPC3 to CAPAN1 for each treatment. (C) Survival fraction of MCF7 cells as measured by clonogenic survival assay 14 days post transfection with DDR gene siRNA with addition of the PARG inhibitors Gallotannin (GLTN) or PDD00017273. For each DDR gene siRNA + DMSO/inhibitor survival fraction is calculated compared to corresponding DMSO/inhibitor + scrambled siRNA. Statistical significance was calculated using the Student’s T-test, comparing PARG inhibitor treated cells to respective DMSO control. In each case mean and standard deviation of three independent repeats are shown. * = p < 0.05, ** = p < 0.01.

Fig. 5

PARG depletion/inhibition disrupts replication forks and induces accumulation of γH2AX and RAD51 foci. (A) Percentage of cells with greater than five γH2AX foci/cell in MCF7 cells 24 h post treatment with control (DMSO), PARP inhibitor (Olaparib (OLA)), the PARG inhibitors (gallotannin (GLTN) and PDD00017273), scrambled siRNA and PARG siRNA (a pool of PARG1 + PARG3 siRNA). Example images are shown. Statistical significance was calculated using the Student’s T-test, compared to DMSO or scrambled control, where * = p < 0.05, ** = p < 0.01, *** = p < 0.001. (B) DNA fibre analysis of replication fork stalling in MCF7 cells treated with PARP and PARG inhibitors. Cells were incubated in inhibitor and then pulse labeled with CldU for 20 min, and then labeled switched to IdU for 20 min. Fork stalling was calculated as a percentage of CIdU only labeled tracts (red) from continuous forks (CIdU (red) and IdU (green) labeled tracts). Example images of replication forks are shown. At least 100 forks were counted on each of three separate occasions. Data bars present the mean and standard deviation of three independent experiments. Statistical significance was calculated using the Student’s T-test compared to DMSO control. (C) Percentage of cells with greater than 10 RAD51 foci/cell in MCF7 cells 24 h post treatment with control (DMSO), PARP inhibitor (Olaparib (OLA)), the PARG inhibitors (Gallotannin (GLTN) and PDD00017273), scrambled siRNA and PARG siRNA (a pool of PARG1 + PARG3 siRNA). Example images are shown. Statistical significance was calculated using the Student’s T-test, compared to DMSO or scrambled control.

Fig. 6

Homologous recombination after PARG depletion. (A) Percentage of cells with greater than 10 RAD51 foci/cell in MCF7 cells 24 h post treatment with scrambled or target DDR gene siRNA. (B) Percentage of cells with greater than 10 RAD51 foci/cell in MCF7 cells 24 h post treatment with scrambled, PARG siRNA (a pool of PARG1 + PARG3 siRNA) and PARG + target DDR gene siRNA. For each 100 cells were counted on three separate occasions and mean and standard deviation shown. Statistical significance was calculated using the Student’s T-test, compared to PARG siRNA alone control, where * = p < 0.05, ** = p < 0.01, *** = p < 0.001.