Dysfunction of Poly

Published OnlineFirst May 29, 2019; DOI: 10.1158/0008-5472.CAN-18-1037

Cancer Molecular Cell Biology Research

Dysfunction of Poly (ADP-Ribose) Glycohydrolase Induces a Synthetic Lethal Effect in Dual Speciﬁcity Phosphatase 22-Deﬁcient Lung Cancer Cells Yuka Sasaki1,2, Hiroaki Fujimori1,2, Miyuki Hozumi2,3,Takae Onodera1,2,Tadashige Nozaki1,4, Yasufumi Murakami3, Kazuto Ashizawa5, Kengo Inoue6, Fumiaki Koizumi7, and Mitsuko Masutani1,2

Abstract

Poly (ADP-ribose) glycohydrolase (PARG) is the main cancer A549, PC14, and SBC5 cells, and inhibited the enzyme responsible for catabolism of poly (ADP-ribose) PI3K/AKT/mTOR pathway in A549 cells, suggesting that (PAR), synthesized by PARP. PARG dysfunction sensitizes dual depletion of PARG and DUSP22 induced apoptosis by certain cancer cells to alkylating agents and cisplatin by upregulating PUMA and suppressing the PI3K/AKT/mTOR perturbing the DNA damage response. The gene mutations pathway. Consistently, the growth of tumors derived from that sensitize cancer cells to PARG dysfunction-induced death double knockdown A549 cells was slower compared with remain to be identified. Here, we performed a comprehensive those derived from control siRNA-transfected cells. Taken analysis of synthetic lethal genes using inducible PARG together, these results indicate that DUSP22 deficiency knockdown cells and identified dual specificity phosphatase exerts a synthetic lethal effect when combined with PARG 22 (DUSP22) as a novel synthetic lethal gene related to PARG dysfunction, suggesting that DUSP22 dysfunction could dysfunction. DUSP22 is considered a tumor suppressor and its be a useful biomarker for cancer therapy using PARG mutation has been frequently reported in lung, colon, and inhibitors. other tumors. In the absence of DNA damage, dual depletion of PARG and DUSP22 in HeLa and lung cancer A549 cells Significance: This study identified DUSP22 as a novel reduced survival compared with single-knockdown counter- synthetic lethal gene under the condition of PARG dysfunction parts. Dual depletion of PARG and DUSP22 increased the and elucidated the mechanism of synthetic lethality in lung apoptotic sub-G1 fraction and upregulated PUMA in lung cancer cells.

ADP-ribose to target proteins in a nicotinamide adenine dinu- Introduction þ cleotide (NAD )-dependent manner (1, 2). This reaction is Poly (ADP-ribosylation) is a posttranslational modification involved in various biological processes, including cell death, by which some PARP family proteins catalyze the transfer of chromatin regulation, and DNA repair of single-strand breaks (SSB) and double-strand breaks (DSB; refs. 1, 3). PARP inhibitors 1Department of Frontier Life Sciences, Nagasaki University Graduate School of were recently developed as a novel anticancer agent based on the Biomedical Sciences, Nagasaki, Japan. 2Division of Chemotherapy and Clinical concept of synthetic lethality (4, 5). PARP inhibitors selectively Research, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan. induce cell death in homologous recombination repair (HRR)– 3Department of Biological Science and Technology, Faculty of Industrial Science deficient cancers such as those associated with mutations in and Technology, Tokyo University of Science, Katsushika-ku, Tokyo, Japan. BRCA1/2 (6, 7), RAD51 (8), and PTEN (9). The development of 4 Department of Pharmacology, Faculty of Dentistry, Osaka Dental University, novel anticancer agents based on the concept of synthetic lethality Hirakata, Osaka, Japan. 5Department of Clinical Oncology, Nagasaki University Graduate School of Biomedical Sciences, Sakamoto, Nagasaki, Japan. 6Pharma is a valuable cancer chemotherapy strategy because these drugs Valley Center, Nagaizumi-cho, Shunto-gun, Shizuoka, Japan. 7Department of show increased tumor selectivity with reduced adverse effects on Laboratory Medicine, Tokyo Metropolitan Cancer and Infectious Diseases Center normal cells (4). Komagome Hospital, Bunkyo-ku, Tokyo, Japan. Poly (ADP-ribose) (PAR) synthesized by PARP is rapidly Note: Supplementary data for this article are available at Cancer Research degraded to ADP-ribose by poly (ADP-ribose) glycohydrolase Online (http://cancerres.aacrjournals.org/). (PARG; ref. 10) and ADP-ribosyl hydrolase (ARH3; ref. 11). PARG Y. Sasaki and H. Fujimori contributed equally to this article. is the main enzyme catabolizing PAR to ADP-ribose through its endo- and exo-glycohydrolase activities (12). As reported previ- Corresponding Author: Mitsuko Masutani, Nagasaki University, Sakamoto 1-7-1, ously, PARG is required for the efficient repair of DSBs and Nagasaki 852-8588, Japan. Phone: 81-95-819-8502; Fax: 81-95-819-8502; E-mail: fi [email protected] SSBs (13). PARG de ciency induces PAR accumulation and a delay of DNA repair (14, 15). PAR accumulation induces cell Cancer Res 2019;79:3851–61 death (parthanatos) accompanied by the translocation of apo- doi: 10.1158/0008-5472.CAN-18-1037 ptosis inducing factor from mitochondria to nuclei, leading to 2019 American Association for Cancer Research. fragmentation of large-sized DNA in neuronal cells and cancer

www.aacrjournals.org 3851

Sasaki et al.

cells such as HeLa cells (16, 17). As previously reported, certain 3 days and divided into 2 populations. Cells were cultured for human cancer cell lines with PARG knockdown synergistically 6 days in the presence or absence of 40 ng/mL Tc, and genomic show higher sensitivity to alkylating agents (14, 18) and cisplatin DNA was purified from 2 populations using a DNA Purification treatment (18). PARG hypomorphic mouse embryonic stem cells Kit (Dojindo). Amplification of barcode sequences in genomic with residual 10% PARG activity did not exhibit growth defect but DNA and purification of DNA were performed using the showed higher sensitivity to alkylating agents, cisplatin, photon, Decode shRNA Negative Selection Kit (Thermo Fisher Scien- and particle beam irradiation compared with wild-type embry- tific) and Gene JET PCR Purification Kit (Thermo Fisher onic stem cells (14, 19, 20). In addition, BRCA2 (21) and Bruton Scientific), respectively, as recommended by the manufacturer. tyrosine kinase (BTK; ref. 22) defects increase PARG inhibition- Then, the genomic DNA was labeled using a Genomic DNA induced cytotoxicity (21–23). These findings led us to hypothe- Enzymatic Labeling Kit (Agilent Technologies) and purified size that PARG could serve as a novel therapeutic target for using Amicon Ultra-0.5 mL Centrifugal Filters (Millipore). The anticancer agents in both monotherapy and combination therapy labeledbarcodesequenceswerehybridizedtomicroarrayslides with radiotherapy or other DNA targeting chemotherapeutic for 17 hours, and the slides were washed according to the agents for particular types of cancers. Recently, PARG inhibitors Agilent CpG microarray protocol. such as phenolic hydrazide hydrazones (24), rhodanine-based PARG inhibitors (RBPI; ref. 25), xanthene compounds (26), siRNA transfection ADP-HPD (27), and PDD00017273 (28), which has an IC50 in Cells were seeded onto 6-well plates or 24-well plates. Trans- the sub-microM range, have been developed. However, specific fection with siRNA was performed using Lipofectamine RNAi and potent PARG inhibitors for clinical applications remain to MAX (Life Technologies) according to the manufacturer's proto- be developed. col. Individual siRNAs were used at final concentration of Here, we screened genes whose deficiency enhances sensi- 10 nmol/L in Opti-MEM. siRNAs (PARG#2, DUSP22#2, PUMA, tivity in a synthetic lethal manner to develop a novel anticancer TP63) targeting DUSP22, PARG, PUMA, and TP63 were purchased agent targeting PARG. We identified dual specificity phospha- from Integrated DNA Technologies. The siRNA sequence of tase 22 (DUSP22) as such a novel gene. Synthetic lethality DUSP22#1 is based on shRNA sequence of oligo ID: induced by PARG and DUSP22 dysfunctioninlungcancercells V2LHS_225030 in the Decode RNAi Pooled Lentiviral shRNA led to TP63-dependent apoptosis by upregulating p53 upregu- Screening Libraries and it was constructed from Integrated DNA lated modulator of apoptosis (PUMA). Double knockdown of Technologies. PARG#1 siRNA was obtained as described previ- PARG and DUSP22 inhibited tumor growth in a mouse xeno- ously (14). DS NC1 siRNA (Integrated DNA Technologies) and graft model. These results indicated that alterations in DUSP22 scrambled siRNA (Ambion/Applied Biosystems) were used as expression levels may serve as a predictive biomarker for PARG negative controls (N.C.). inhibitors. qRT-PCR RNA was prepared from each individual cell line and reverse Materials and Methods transcribed using a High Capacity Reverse Transcription Kit Cell culture and reagents (Thermo Fisher Scientific). The qRT-PCR analysis was per- The TRHmPARG#8 cell line is a tetracycline (Tc)-inducible formed using SYBR Green with the CFX96 Real-Time System PARG knockdown strain derived from the human T-REx HeLa (Bio-Rad). The mRNA levels were normalized to GUSB mRNA. cell line described previously (29). TRHmPARG#8 and PC14 were The sequences of primer pairs are listed in Supplementary cultured in Minimum Essential Medium and DMEM (Thermo Table S1. Fisher Scientific), respectively. A549 and SBC5 cells were grown in RPMI1640 (Thermo Fisher Scientific). Media were supplemented Cell proliferation assay with 10% FBS (Gibco) and 1% penicillin–streptomycin Cell viability was measured using the Cell Counting Kit-8 (Invitrogen) as needed. Cells were maintained in a humidified (Dojindo Laboratories) according to the manufacturer's instruc- atmosphere with 5% CO2 at 37 C. The cell line A549 was tions. Cells were seeded onto 96-well plates and cultured for obtained from the ATCC. The cell line PC-14 was obtained from 1 week. Cell proliferation rate was determined using the Cell Dr. Hayata, Tokyo Medical College (Tokyo Japan). The cell line Counting Kit-8 containing water soluble tetrazolium dys SBC-5 was obtained from Okayama University in 1994. Cell line (WST-8). Plates were analyzed using a microtiter plate reader at authentication of all cell lines was performed by short tandem 450 nm with a reference of 600 nm. repeat (STR)-PCR (Promega, August 2018). Mycoplasma testing was carried out using e-Myco plus Mycoplasma PCR Detection Kit Western blot analysis (iNtRON Biotechnology) for all cell lines used in this study and all Western blotting was performed as described previously (29). cell lines were mycoplasma free. All cell lines were passaged less Cell extracts were prepared with Laemmli's buffer. Proteins were than 15 times prior to use. separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. The following antibodies were Negative screening using a lentivirus shRNA library used for immunoblotting: anti-PARG (Millipore), anti-p-p38 Negative screening was performed using the Decode RNAi (Cell Signaling Technology), anti-b-actin (Sigma-Aldrich), Pooled Lentiviral shRNA Screening Libraries: Annotated Genome anti-DUSP22 (Gene Tex), anti-AKT (pan; C67E7; Cell Signaling Negative Selection Kit (Thermo Fisher Scientific). TRHmPARG#8 Technology), anti-p-AKT (Ser473; Cell Signaling Technology), cells were infected with a lentiviral siRNA expression library anti-p-mTOR (Ser2448; Cell Signaling Technology), anti- (Thermo Fisher Scientific) using the TransDux reagent (System p-mTOR (Ser2481; Cell Signaling Technology), anti-mTOR Biosciences). GFP-positive cells were selected using puromycin for (7C10; Cell Signaling Technology), anti-PTEN (138G6; Cell

3852 Cancer Res; 79(15) August 1, 2019 Cancer Research

Dysfunction of PARG and DUSP22 Induces Synthetic Lethality

Signaling Technology), anti-p-PTEN (Ser380; Cell Signaling Cell-cycle analysis Technology). Immune complexes were visualized using a horse- Cell-cycle distribution was analyzed by flow cytometry as radish peroxidase-linked secondary antibody and enhanced previously described (30). Cells were fixed with 70% ethanol. chemiluminescence (Millipore). Image quantification was per- Fixed cells were incubated with PBS containing 50 mg/mL formed with the ImageJ software (NIH). propidium iodide (Sigma-Aldrich) and 20 mg/mL RNase A (Sigma-Aldrich) for 2 hours and analyzed using the FACSCalibur Colony formation assay system (Becton–Dickinson). Cells were transfected with siRNA against the target gene and cultured in 6-well plates for 9 days. Colonies were fixed with 4% Antibody array neutralized formalin and stained with 0.02% crystal violet for A549 cells were transfected with siRNA against PARG and/or counting. DUSP22 and cultured for 3 days. Cell lysates were analyzed with

Figure 1. Comprehensive analysis to identify synthetic lethal genes in the condition of PARG dysfunction identified DUSP22 gene as the synthetic lethal gene. A, Experimental procedure of negative screening. Puro., puromycin. B, Classification of identified candidates. Seventeen genes showed the decreased signal rates (Cy5/Cy3) more than 4-fold. C, The percentage of nonsynonymous substitution mutation of DUSP22 in each tumor (total 191 mutations of tumor samples/6,750 patients) in CanSAR database (https://cansar.icr.ac.uk; ref. 44). D, Knockdown level of PARG (a)andDUSP22 (b and c) and resulting phosphorylation of p38 (c) in inducible PARG knockdown T-REx HeLa cells. E, Relative cell growth under PARG and DUSP22 knockdown. , P < 0.05 (Tukey test).

www.aacrjournals.org Cancer Res; 79(15) August 1, 2019 3853

Sasaki et al.

PathScan Stress and Apoptosis Signaling Antibody Array (Fig. 1B; Table 1). Among these genes, we focused on DUSP22, (Cell Signaling Technology, #12856). This array was performed because DUSP22 mutations are reported frequently in lung, according to the manufacturer's instructions. colon, and other tumors in the CanSAR database (Fig. 1C), and its expression is downregulated in certain cancers (31, 32). To Animal experiments determine whether dysfunction of PARG and DUSP22 exerts a A549 cells were transfected with siRNA against PARG and/or synthetic lethal effect in cancer cells, siRNA against DUSP22 was DUSP22 or control siRNA. On the next day, cells (2.9 105) were introduced into the inducible T-REx HeLa cells. As shown mixed with Growth Factor Reduced Matrigel (BD Biosciences) in Fig. 1D, a, PARG expression was decreased to approximately and injected subcutaneously into both legs of 11-week-old Balb/c- 50% of the control in the presence of Tc and transfection of cells nu/nu nude mice. Tumor diameters were measured every 3 days with siRNA-targeting DUSP22 decreased DUSP22 mRNA to with micrometer calipers, and tumor volume was calculated using approximately 10% of control levels (Fig. 1D,b). DUSP22 protein the following formula: (smallest diameter) (largest diameter) levels decreased to 29% in DUSP22 knockdown cells (Fig. 1D, c). (height)/2. All animal studies were approved by the Animal DUSP22 belongs to the DUSP family of proteins, which function Experimental Committee of the National Cancer Center and in the dephosphorylation of JNK, p38, and ERK (33). Inducible performed following the Guidelines for Animal Experiments of PARG knockdown T-REx HeLa cells were transfected with siRNA the National Cancer Center, which meet the ethical guidelines for against DUSP22 or control siRNA, and the phosphorylation experimental animals in Japan. level of p38 was determined using a phospho-p38 antibody. The results showed that p38 phosphorylation levels were Statistical analysis 2.3-fold higher in DUSP22 knockdown cells than in control cells Data were expressed as meant SE. Statistical significance was (Fig. 1D, c), suggesting that DUSP22 was necessary for the indicated when P value was less than 0.05. In this study, data were dephosphorylation of p38. To determine whether dysfunction analyzed using Tukey test or Mann–Whitney U test. Synergistic of PARG and DUSP22 induces synthetic lethality, the survival effects were analyzed by 2-way ANOVA. rates of PARG and/or DUSP22 knockdown cells were analyzed using a clonogenic survival assay (Fig. 1E). DUSP22 and PARG double knockdown suppressed the viability of inducible Results PARG knockdown cells to approximately 40% of that of single Identification of synthetic lethal genes related to PARG knockdown and control cells. This result suggested that DUSP22 dysfunction deficiency induces synthetic lethality under conditions of PARG A comprehensive analysis was performed to identify synthetic inhibition. lethal genes related to PARG dysfunction and understand the function of PARG in cancer cells. For this purpose, a siRNA library Dysfunction of PARG and DUSP22 efficiently suppressed the was screened using a negative screening strategy. Inducible PARG growth of lung cancer cells knockdown T-REx HeLa cells (TRHmPARG#8) were established Because the frequency of DUSP22 mutation is higher in lung in which PARG knockdown was induced in the presence of Tc (29). cancer than in other types of tumors (Fig. 1C), we examined The cells were infected with lentiviral shRNA pools targeting whether DUSP22 and PARG dysfunction induced synthetic lethal- approximately 10,000 genes. The relative abundance of individ- ity in the lung cancer cell lines A549, PC14, and SBC5. To exclude ual shRNAs after PARG knockdown was determined by micro- the possibility of off-target effects of PARG and DUSP22 knock- array analysis (Fig. 1A). Seventeen candidate siRNAs that sup- down, the effect of 2 different siRNA sets (#1 and #2) against pressed the growth of PARG knockdown cells were identified at a PARG and DUSP22 was tested (Fig. 2A,a and b; Fig. 2B, a and b) 4-fold or higher signal rate (Fig. 1B; Table 1). The targeted genes using a clonogenic survival assay (Fig. 2C, a–c). Double knock- were classified into functional categories, including metabolism, down of PARG and DUSP22 in A549 showed synergistic growth signal transduction, and posttranslational modification inhibition in comparison with single knockdown and N.C. cells (Fig. 2C, a). In PC14 and SBC5 cells, one of each siRNA set showed Table 1. Candidate genes found in the synthetic lethality screening in PARG K.D. a synergistic effect but the other showed an additive effect, condition respectively (Fig. 2C, b and c). These results suggested that No. Gene Functional classification dysfunction of PARG and DUSP22 efficiently induced synthetic 1 RANBP6 Protein transport lethality in particular lung cancer cell lines. 2 STK25 Signal transduction RAC1 3 Metabolic process PARG and DUSP22 double knockdown promoted apoptosis by 4 ARL6 Signal transduction PUMA 5 RPL4 Metabolic process upregulating DUSP22 6 MSH6 DNA damage response To examine the mechanism underlying lethality in 7 IL6ST Signal transduction and PARG double knockdown cells, the expression levels of 8 DUSP22 Signal transduction apoptosis-related and cell-cycle–related genes were analyzed by 9 KIF9 Protein transport qRT-PCR. As shown in Fig. 3A, a–c, PUMA mRNA levels were GTSE1 10 DNA damage response higher in double knockdown cells than in single knockdown 11 ATXN1L Signal transduction DUSP22 12 ST6GALNAC2 Posttranslational modification cells in these lung cancer cell lines. In PC14 cells, single DUSP22 PARG 13 PLA2G15 Metabolic process knockdown and and double knockdown upregu- 14 WIPF1 Cell structure/cytoskeleton lated the expression of NOXA, a gene involved in apoptosis 15 PARP15 Posttranslational modification induction, compared with the levels in control cells (Fig. 3A, 16 KRTAP10-10 Cell structure/cytoskeleton b). The expression level of CDKN2A, a cell-cycle negative regulator TCFL5 17 Metabolic process and cell senescence-related factor, was also elevated in response to

3854 Cancer Res; 79(15) August 1, 2019 Cancer Research

Dysfunction of PARG and DUSP22 Induces Synthetic Lethality

Figure 2. Dysfunction of PARG/DUSP22 reduced cellular survival ratio in lung cancer cell lines. A and B, siRNA-targeting DUSP22 and PARG successfully knocked down their targets in indicated cell lines. We used two different siRNAs for both DUSP22 and PARG (#1 and #2). C, In two independent PARG/DUSP22 siRNA sets, survival rates were compared with N.C., the single knockdown groups in three lung cancer cell lines by colony formation assay. In C, dotted lines and values marked with red show the expected survival levels with additive effects to compare with the survival levels of the combined siRNA knockdown groups. , P < 0.05; , P < 0.001; , P < 0.0001 (Tukey test). Error bars, SE of the mean.

www.aacrjournals.org Cancer Res; 79(15) August 1, 2019 3855

Sasaki et al.

Figure 3. PARG and DUSP22 knockdown in lung cancer cells caused synthetic lethality by apoptosis. A, Expression level of p53-dependent apoptosis and cell-cycle– related factors after 4 to 5 days of transfection. Target gene expression levels are normalized to GUSB level. PUMA expression level synergistically increased in double K.D. cells as compared with single K.D. cells (2-way ANOVA). , P < 0.05; , P < 0.0001 versus N.C. and single knockdown cells (Tukey test). B, Flow cytometry analysis. A549, PC14, and SBC5 cells were harvested at 2 and 6 days after transfection, respectively, and analyzed by ﬂow cytometry. , P < 0.05; , P < 0.001; , P < 0.0001 versus N.C. or single knockdown cells (Tukey test). a, A549; b, PC14; c, SBC5.

DUSP22 single knockdown and double knockdown conditions in down moderately recovered cell viability of PARG/DUSP22 dou- SBC5 cells (Fig. 3A, c). To determine whether apoptosis induction ble knockdown condition at 9 days after transfection. To identify was involved in the decreased cell viability caused by PARG and the cell death pathway involved in synthetic lethality in A549 DUSP22 double knockdown in lung cancer cell lines, we per- cells, a protein array analysis was performed using PathScan Stress formed cell-cycle distribution analysis. In all cell lines tested, and an Apoptosis Signaling Antibody Array Kit (Cell Signaling PARG and DUSP22 double knockdown increased the sub-G1 Technology). As shown in Supplementary Fig. S1, cleaved caspase population compared with that in PARG or DUSP22 single 3 and cleaved PARP1 were upregulated at 3 days after transfection knockdown cells (Fig. 3B, a–c). To determine whether PUMA in response to DUSP22 and PARG double knockdown compared induction was responsible for the suppression of cell viability, with their expression in the single knockdown condition in A549 siRNA-targeting PUMA was introduced into PARG and DUSP22 cells. Taken together, these data suggested that synthetic lethality double knockdown A549 cells (Fig. 4A). As shown in Fig. 4B, was induced by the promotion of apoptosis through the upregu- PARG/DUSP22 double knockdown reduced cell viability com- lation of PUMA under conditions of PARG and DUSP22 double pared with N.C. (P < 0.05), whereas the additional PUMA knock- knockdown in lung cancer cells.

3856 Cancer Res; 79(15) August 1, 2019 Cancer Research

Dysfunction of PARG and DUSP22 Induces Synthetic Lethality

Figure 4. PARG and DUSP22 double knockdown induced the PUMA-mediated apoptosis through TP63 pathway activation. A, A549 cells were transfected with siRNA- targeting PUMA and PUMA expression level was analyzed by qRT-PCR. B, A549 cells were treated with DsiRNA (PARG, DUSP22,andPUMA) and cell viability was measured by colony formation assay (Tukey test, , P < 0.01). C, PARG/DUSP22 knockdown induced TP63 (a) /TP73 (b) expression in A549. D, siRNA for TP63 successfully knocked down their target in A549 cells. E, TP63 knockdown rescued the synthetic lethality induced by double knockdown of PARG and DUSP22 in A549. Cell growth rate was measured by cell proliferation assay (Tukey test, , P < 0.05; , P < 0.001; , P < 0.0001).

PARG and DUSP22 dysfunction induced synthetic lethality DUSP22 knockdown cells but did not differ between double through the TP63 pathway knockdown and PARG single knockdown cells. Next, we exam- TP63 and TP73 are both involved in apoptosis induction (34). ined whether TP63 dysfunction affected cell viability in DUSP22 To determine the mechanism underlying DUSP22 and PARG and PARG knockdown A549 cells. The results showed that TP63 double knockdown-induced synthetic lethality in A549 cells, the knockdown rescued cell growth in double knockdown cells mRNA levels of TP63 and TP73 were analyzed by qRT-PCR. As (Fig. 4D and E). These suggest that TP63 expression is necessary shown in Fig. 4C, a and b, TP63 levels were synergistically for apoptosis induction in the double knockdown condition. To increased in double knockdown A549 cells than in PARG and determine whether DUSP22 and PARG knockdown-induced apo- DUSP22 single knockdown cells at 4 days after transfection, ptosis in A549 cells is dependent on reduced dephosphorylation whereas TP73 levels showed an increase compared with N.C. and of p38 MAPK (Fig. 1D, c), double knockdown cells were cultured

www.aacrjournals.org Cancer Res; 79(15) August 1, 2019 3857

Sasaki et al.

PARG or DUSP22 single knockdown cells (Fig. 5). These results suggested that the decreased survival rate of DUSP22 and PARG double knockdown cells was induced in part by the inhibition of the PI3K/AKT/mTOR pathway.

Dysfunction of PARG and DUSP22 suppresses xenograft growth of lung tumor in a mouse model Based on the in vitro data showing the induction of apoptosis in DUSP22 and PARG double knockdown cells, we examined whether PARG and DUSP22 knockdown exerted a synthetic lethal effect in A549-derived xenograft tumors. Mice were injected with A549 cells transfected with PARG siRNA and/or DUSP22 siRNA, and effect on tumor growth was observed (Fig. 6A). As shown in Fig. 6B, a and B, b, double knockdown of DUSP22 and PARG in A549 cells suppressed tumor growth compared with that of tumors transfected with control siRNA (Fig. 6B, b, P < 0.05). Although statistical differences between single and double knockdown groups were not observed, a tendency of decreased tumor volume in double knockdown condition was observed. This observation thus suggested that PARG and DUSP22 double knockdown suppressed tumor xenograft growth, possibly in a synergistic manner.

Discussion PARP inhibitors were recently shown to induce synthetic lethality in HRR-deficient cancer cells (5). Olaparib, a PARP inhibitor, was approved for the treatment of ovarian cancer harboring BRCA1/2 mutations, and this novel type of anticancer agent is effective as monotherapy against BRCA1/2-mutated cancers (5, 35). These drugs are expected to provide an effective cure for cancer with few adverse effects on normal cells. By contrast, little is known about synthetic lethal targets of PARG inhibition. PARG inhibition by gallotannin and siRNA-mediated silencing of PARG induce a weak synthetic lethal effect in BRCA2-mutated breast cancer cells (21). In addition, ibrutinib, an inhibitor of BTK, enhances the lethal effects of PARG inhibition by ethacridine (22). Despite these findings, the potential of PARG as a therapeutic target for anticancer drugs based on synthetic lethality remains unclear. In this study, we searched for novel synthetic lethal targets of PARG inhibition by performing a comprehensive analysis of Figure 5. PARG DUSP22 PARG synthetic lethal genes using inducible knockdown cells and PI3K/AKT/mTOR pathway was downregulated by and double fi knockdown in A549 cells. A549 cells were transfected with DUSP22 and/or a shRNA library. Among the candidate genes identi ed, we PARG siRNA. After 3 days, cell lysates were analyzed by Western blotting focused on DUSP22, because it is frequently mutated in various using antibodies for phosphorylated form of PTEN, AKT, and mTOR. Relative types of cancer (Fig. 1C). expression levels normalized to b-actin level are shown under each panel. DUSP22 is a member of the DUSP subfamily of protein tyrosine phosphatases. Its dephosphorylation substrate remains in the presence or absence of the p38 MAPK-specific inhibitor unclear, whereas other DUSP family proteins function in the SB203580, and cell growth was analyzed by the cell proliferation dephosphorylation of JNK, p38, and/or ERK (33). Here, we assay. As shown in Supplementary Fig. S2, SB203580 treatment showed that DUSP22 directly or indirectly dephosphorylated restored cell growth in DUSP22 and PARG knockdown A549 cells. p38 by silencing DUSP22 in inducible PARG knockdown T-REx These results suggested that the upregulation of phosphorylated HeLa cells (Fig. 1D, c). The p38 MAPK pathway is activated by p38 level under DUSP22 dysfunction is involved in promoting phosphorylation of p38 and is involved in the induction of cell PUMA-mediated apoptosis in double knockdown A549 cells. death through apoptosis (36). Colony formation assays showed In addition, we examined whether PARG and DUSP22 knock- that double knockdown of DUSP22 and PARG in the lung cancer down suppressed the protein expression of cell proliferation- cell lines A549, PC14, and SBC5 efficiently induced cell death related genes in A549 cells. PTEN, phospho-PTEN (Ser380), and compared with the effect of single knockdown. Cell-cycle analysis phospho-mTOR (Ser2448) levels were decreased in double showed that double knockdown increased the sub-G1 popula- knockdown A549 but not in single knockdown cells (Fig. 5). tion, and treatment with the p38 MAPK inhibitor SB203580 Phospho-AKT (Thr308 and Ser473) and phospho-mTOR restored cell survival in double knockdown A549 cells. Taken (Ser2481) were downregulated in both double knockdown and together, these results suggest that the synthetic lethal effect of

3858 Cancer Res; 79(15) August 1, 2019 Cancer Research

Dysfunction of PARG and DUSP22 Induces Synthetic Lethality

Figure 6. PARG and DUSP22 double knockdown suppressed tumor xenograft growth, possibly in a synergistic manner. A, Experimental procedure. B, Double knockdown of PARG and DUSP22 synthetically suppressed the increase of tumor volume (a) and tumor weight (b) in A549 xenograft model. Statistical significant difference was observed between N.C. and double knockdown groups, but not between single and double knockdown groups in B. , P < 0.05 relative to N.C. (Kruskal–Wallis test). dysfunction of DUSP22 and PARG was mediated in part by the which is frequently involved in cancer cell proliferation (43). As induction of apoptosis via the p38 MAPK pathway in A549 cells. shown in Fig. 5, the level of phospho-mTOR (Ser-2448) was lower Under oxidative stress conditions, PARP1 promotes the phos- in double knockdown cells than in control cells despite the phorylation of p38 in association with the downregulation downregulation of PTEN and phospho-PTEN, a negative regula- of MAPK phosphatase-1, which is involved in the dephosphor- tor of the PI3K/AKT pathway, in double knockdown cells. The ylation of JNK and p38 MAP kinases, resulting in increased cell phosphorylation level of AKT (Thr308 and Ser473) and mTOR death (37). In this study, we demonstrated that increased p38 (Ser2481) was reduced in both double knockdown A549 cells and phosphorylation induced by DUSP22 knockdown and PAR accu- single knockdown cells. Overall, these results indicated that the mulation induced by PARG knockdown (Supplementary Fig. S3) promotion of TP53-independent apoptosis and suppression of exerted a synthetic lethal effect by promoting apoptotic cell death PI3K/AKT/mTOR pathway activity induced synthetic lethality in in lung cancer cells. DUSP22 and PARG double knockdown A549 cells. In the apoptosis pathway, the tumor suppressor p53 activates As shown in Fig. 1C, various cancers including lung and colon apoptosis-related factors, and p53 missense mutations are present cancers occasionally bear DUSP22 gene mutations. The complete in various types of cancer including lung cancer (38). However, genome sequence analysis from a patient with lung cancer the p53 homolog TP63 is rarely mutated (39). TP63 has common showed that DUSP22 was inactivated by loss of heterozygosity transcription targets with p53 and promotes the expression of and point mutations (31). DUSP22 expression is also down- PUMA to induce p53-independent apoptosis (40). In this study, regulated in breast cancer and anaplastic lymphoma kinase- we showed that knockdown of DUSP22 and PARG in A549 cells negative anaplastic large cell lymphoma (32). In this study, the upregulated TP63 (Fig. 4C) and PUMA expression. In addition, double deficiency of DUSP22 and PARG suppressed lung tumor despite the fact that PC14 and SBC5 cells bear p53 muta- growth in a xenograft model (Fig. 6), suggesting that DUSP22 tions (41, 42), double knockdown of DUSP22 and PARG in these could be useful as a biomarker for monitoring the synthetic cells induced apoptosis by upregulating PUMA (Fig. 3A, a–c). lethal effect of PARG inhibitors. PARG deficiency sensitizes These results indicated that the synthetic lethal effect of DUSP22 cancer cells to alkylating agents (14, 18), cisplatin (18) and and PARG dysfunction was mediated by the induction of apo- g-irradiation (18, 19). Cancers with low expression levels of ptosis through the TP63 pathway. The induction of apoptosis DUSP22 may increase sensitivity to combination therapy with through a p53-independent pathway has important implications these drugs and PARG inhibitors. The development of PARG because many cancers have p53 pathway mutations. inhibitors for clinical application is awaited. Both inhibition of The present results suggested that dysfunction of PARG and BTK and BRCA2 in combination with PARG inhibition has a DUSP22 in A549 cells affects the PI3K/AKT/mTOR pathway, moderate synthetic lethal effect (21, 22). This study suggested

www.aacrjournals.org Cancer Res; 79(15) August 1, 2019 3859

Sasaki et al.

that PARG-specific inhibitors could be useful for cancer therapy by Administrative, technical, or material support (i.e., reporting or organizing exerting a synthetic lethal effect on cancers with DUSP22 data, constructing databases): H. Fujimori, T. Nozaki, M. Masutani deficiency. Study supervision: H. Fujimori, K. Inoue, M. Masutani Acknowledgments Disclosure of Potential Conflicts of Interest We are thankful for kind support by Dr. Toshio Imai of Central Animal fl No potential con icts of interest were disclosed. Division, National Cancer Center and technical assistance by Hiromi Harada. This research is partially supported by the Practical Research for Innovative Authors' Contributions Cancer Control from Japan Agency for Medical Research and Development, Conception and design: Y. Sasaki, H. Fujimori, M. Hozumi, T. Nozaki, K. Inoue, AMED (15Ack0106021, 17ck0106286), and Grant-in-Aid for Scientific F. Koizumi, M. Masutani Research [KibanB 22300343, H23-Jitsuyoka(Gan)-004] to M. Masutani. Development of methodology: H. Fujimori, M. Masutani Acquisition of data (provided animals, acquired and managed patients, The costs of publication of this article were defrayed in part by the provided facilities, etc.): Y. Sasaki, H. Fujimori, M. Hozumi, M. Masutani payment of page charges. This article must therefore be hereby marked Analysis and interpretation of data (e.g., statistical analysis, biostatistics, advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate computational analysis): Y. Sasaki, H. Fujimori, M. Hozumi, T. Onodera, this fact. K. Ashizawa, F. Koizumi, M. Masutani Writing, review, and/or revision of the manuscript: Y. Sasaki, H. Fujimori, Received April 5, 2018; revised January 23, 2019; accepted May 20, 2019; T. Onodera, T. Nozaki, Y. Murakami, K. Ashizawa, K. Inoue, M. Masutani published first May 29, 2019.

References 1. Miwa M, Masutani M. PolyADP-ribosylation and cancer. Cancer Sci 2007; 17. Fatokun AA, Dawson VL, Dawson TM. Parthanatos: mitochondrial-linked 98:1528–35. mechanisms and therapeutic opportunities. Br J Pharmacol 2014;171: 2. Sugimura T. Poly(adenosine diphosphate ribose). Prog Nucleic Acid Res 2000–16. Mol Biol 1973;13:127–51. 18. Fujihara H, Ogino H, Maeda D, Shirai H, Nozaki T, Kamada N, 3. Rodriguez MI, Majuelos-Melguizo J, Marti Martin-Consuegra JM, Ruiz de et al. Poly(ADP-ribose) Glycohydrolase deficiency sensitizes mouse Almodovar M, Lopez-Rivas A, Javier Oliver F. Deciphering the insights of ES cells to DNA damaging agents. Curr Cancer Drug Targets 2009;9: poly(ADP-ribosylation) in tumor progression. Med Res Rev 2015;35: 953–62. 678–97. 19. Shirai H, Fujimori H, Gunji A, Maeda D, Hirai T, Poetsch AR, et al. Parg 4. Chan DA, Giaccia AJ. Harnessing synthetic lethal interactions in anticancer deficiency confers radio-sensitization through enhanced cell death in drug discovery. Nat Rev Drug Discovery 2011;10:351–64. mouse ES cells exposed to various forms of ionizing radiation. 5. Brown JS, Kaye SB, Yap TA. PARP inhibitors: the race is on. Br J Cancer 2016; Biochem Biophys Res Commun 2013;435:100–6. 114:713–5. 20. Nakadate Y, Kodera Y, Kitamura Y, Tachibana T, Tamura T, Koizumi F. 6. Bryant HE, Schultz N, Thomas HD, Parker KM, Flower D, Lopez E, et al. Silencing of poly(ADP-ribose) glycohydrolase sensitizes lung cancer cells Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP- to radiation through the abrogation of DNA damage checkpoint. ribose) polymerase. Nature 2005;434:913–7. Biochem Biophys Res Commun 2013;441:793–8. 7. Farmer H, McCabe N, Lord CJ, Tutt AN, Johnson DA, Richardson TB, et al. 21. Fathers C, Drayton RM, Solovieva S, Bryant HE. Inhibition of poly(ADP- Targeting the DNA repair defect in BRCA mutant cells as a therapeutic ribose) glycohydrolase (PARG) specifically kills BRCA2-deficient tumor strategy. Nature 2005;434:917–21. cells. Cell Cycle 2012;11:990–7. 8. McCabe N, Turner NC, Lord CJ, Kluzek K, Bialkowska A, Swift S, et al. 22. Rotin LE, Gronda M, MacLean N, Hurren R, Wang X, Lin FH, et al. Ibrutinib Deficiency in the repair of DNA damage by homologous recombination synergizes with poly(ADP-ribose) glycohydrolase inhibitors to induce cell and sensitivity to poly(ADP-ribose) polymerase inhibition. Cancer Res death in AML cells via a BTK-independent mechanism. Oncotarget 2016;7: 2006;66:8109–15. 2765–79. 9. Mendes-Pereira AM, Martin SA, Brough R, McCarthy A, Taylor JR, Kim JS, 23. Gravells P, Grant E, Smith KM, James DI, Bryant HE. Specific killing of et al. Synthetic lethal targeting of PTEN mutant cells with PARP inhibitors. DNA damage-response deficient cells with inhibitors of poly(ADP-ribose) EMBO Mole Med 2009;1:315–22. glycohydrolase. DNA Repair (Amst) 2017;52:81–91. 10. Miwa M, Tanaka M, Matsushima T, Sugimura T. Purification and properties 24. Islam R, Koizumi F, Kodera Y, Inoue K, Okawara T, Masutani M. Design and of glycohydrolase from calf thymus splitting ribose-ribose linkages of poly synthesis of phenolic hydrazide hydrazones as potent poly(ADP-ribose) (adenosine diphosphate ribose). J Biol Chem 1974;249:3475–82. glycohydrolase (PARG) inhibitors. Bioorg Med Chemist Lett 2014;24: 11. Oka S, Kato J, Moss J. Identification and characterization of a mammalian 3802–6. 39-kDa poly(ADP-ribose) glycohydrolase. J Biol Chem 2006;281:705–13. 25. Finch KE, Knezevic CE, Nottbohm AC, Partlow KC, Hergenrother PJ. 12. Brochu G, Duchaine C, Thibeault L, Lagueux J, Shah GM, Poirier GG. Mode Selective small molecule inhibition of poly(ADP-ribose) glycohydrolase of action of poly(ADP-ribose) glycohydrolase. Biochim Biophys Acta (PARG). ACS Chem Biol 2012;7:563–70. 1994;1219:342–50. 26. Okita N, Ashizawa D, Ohta R, Abe H, Tanuma S. Discovery of novel poly 13. Mortusewicz O, Fouquerel E, Ame JC, Leonhardt H, Schreiber V. PARG is (ADP-ribose) glycohydrolase inhibitors by a quantitative assay system recruited to DNA damage sites through poly(ADP-ribose)- and PCNA- using dot-blot with anti-poly(ADP-ribose). Biochem Biophys Res dependent mechanisms. Nucleic Acids Res 2011;39:5045–56. Commun 2010;392:485–9. 14. Shirai H, Poetsch AR, Gunji A, Maeda D, Fujimori H, Fujihara H, et al. 27. Slama JT, Aboul-Ela N, Goli DM, Cheesman BV, Simmons AM, Jacobson PARG dysfunction enhances DNA double-strand break formation in MK. Specific inhibition of poly(ADP-ribose) glycohydrolase by adenosine S-phase after alkylation DNA damage and augments different cell death diphosphate (hydroxymethyl)pyrrolidinediol. J Med Chem 1995;38: pathways. Cell Death Dis 2013;4:e656. 389–93. 15. Wei L, Nakajima S, Hsieh CL, Kanno S, Masutani M, Levine AS, et al. 28. James DI, Smith KM, Jordan AM, Fairweather EE, Griffiths LA, Hamilton Damage response of XRCC1 at sites of DNA single-strand breaks is NS, et al. First-in-class chemical probes against poly(ADP-ribose) glycohy- regulated by phosphorylation and ubiquitylation after degradation of drolase (PARG) inhibit DNA repair with differential pharmacology to poly(ADP-ribose). J Cell Sci 2013;126:4414–23. olaparib. ACS Chem Biol 2016;11:3179–90. 16. Andrabi SA, Kim NS, Yu SW, Wang H, Koh DW, Sasaki M, et al. Poly(ADP- 29. Sasaki Y, Hozumi M, Fujimori H, Murakami Y, Koizumi F, Inoue K, et al. ribose) (PAR) polymer is a death signal. Proc Natl Acad Sci U S A 2006;103: PARG inhibitors and functional PARG inhibition models. Curr Protein 18308–13. Peptide Sci 2016;17:641–53.

3860 Cancer Res; 79(15) August 1, 2019 Cancer Research

Dysfunction of PARG and DUSP22 Induces Synthetic Lethality

30. Fujimori H, Sato A, Kikuhara S, Wang J, Hirai T, Sasaki Y, et al. new mechanism for the cytoplasmic effect of PARP-1 activation. Free Rad A comprehensive analysis of radiosensitization targets; functional inhibi- Biol Med 2010;49:1978–88. tion of DNA methyltransferase 3B radiosensitizes by disrupting DNA 38. Murai Y, Hayashi S, Takahashi H, Tsuneyama K, Takano Y. Correlation damage regulation. Sci Rep 2015;5:18231. between DNA alterations and p53 and p16 protein expression in cancer cell 31. Lee W, Jiang Z, Liu J, Haverty PM, Guan Y, Stinson J, et al. The mutation lines. Pathol Res Pract 2005;201:109–15. spectrum revealed by paired genome sequences from a lung cancer patient. 39. Moll UM, Slade N. p63 and p73: roles in development and tumor Nature 2010;465:473–7. formation. Mol Cancer Res 2004;2:371–86. 32. Feldman AL, Dogan A, Smith DI, Law ME, Ansell SM, Johnson SH, et al. 40. Bergamaschi D, Samuels Y, Jin B, Duraisingham S, Crook T, Lu X. ASPP1 Discovery of recurrent t(6;7)(p25.3;q32.3) translocations in ALK-negative and ASPP2: common activators of p53 family members. Mol Cell Biol anaplastic large cell lymphomas by massively parallel genomic sequencing. 2004;24:1341–50. Blood 2011;117:915–9. 41. Kashii T, Mizushima Y, Monno S, Nakagawa K, Kobayashi M. Gene 33. Patterson KI, Brummer T, O'Brien PM, Daly RJ. Dual-speciﬁcity phospha- analysis of K-, H-ras, p53, and retinoblastoma susceptibility genes in tases: critical regulators with diverse cellular targets. Biochem J 2009;418: human lung cancer cell lines by the polymerase chain reaction/single- 475–89. strand conformation polymorphism method. J Cancer Res Clin Oncol 34. Flores ER, Tsai KY, Crowley D, Sengupta S, Yang A, McKeon F, et al. p63 and 1994;120:143–8. p73 are required for p53-dependent apoptosis in response to DNA dam- 42. Fujita T, Kiyama M, Tomizawa Y, Kohno T, Yokota J. Comprehensive age. Nature 2002;416:560–4. analysis of p53 gene mutation characteristics in lung carcinoma with 35. Kaufman B, Shapira-Frommer R, Schmutzler RK, Audeh MW, Friedlander special reference to histological subtypes. Int J Oncol 1999;15: M, Balmana J, et al. Olaparib monotherapy in patients with advanced 927–34. cancer and a germline BRCA1/2 mutation. J Clin Oncol 2015;33:244–50. 43. Polivka J Jr., Janku F. Molecular targets for cancer therapy in the PI3K/AKT/ 36. Zarubin T, Han J. Activation and signaling of the p38 MAP kinase pathway. mTOR pathway. Pharmacol Thera 2014;142:164–75. Cell Res 2005;15:11–8. 44. Tym JE, Mitsopoulos C, Coker EA, Razaz P, Schierz AC, Antolin AA, et al. 37. Racz B, Hanto K, Tapodi A, Solti I, Kalman N, Jakus P, et al. Regulation of CanSAR: an updated cancer research and drug discovery knowledgebase. MKP-1 expression and MAPK activation by PARP-1 in oxidative stress: a Nucleic Acids Res 2016;44:D938–43.

www.aacrjournals.org Cancer Res; 79(15) August 1, 2019 3861

Dysfunction of Poly (ADP-Ribose) Glycohydrolase Induces a Synthetic Lethal Effect in Dual Specificity Phosphatase 22-Deficient Lung Cancer Cells

Yuka Sasaki, Hiroaki Fujimori, Miyuki Hozumi, et al.

Cancer Res 2019;79:3851-3861. Published OnlineFirst May 29, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-18-1037

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2019/05/29/0008-5472.CAN-18-1037.DC1

Cited articles This article cites 44 articles, 10 of which you can access for free at: http://cancerres.aacrjournals.org/content/79/15/3851.full#ref-list-1

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at Subscriptions [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/79/15/3851. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.