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

Author Manuscript Published OnlineFirst on May 29, 2019; DOI: 10.1158/0008-5472.CAN-18-1037 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Title Dysfunction of poly(ADP-ribose) glycohydrolase induces a synthetic lethal effect in dual specificity phosphatase 22-deficient lung cancer cells.

Authors and affiliations Yuka Sasaki1,2*, Hiroaki Fujimori1,2*, Miyuki Hozumi2,3, Takae Onodera1,2, Tadashige Nozaki1,4, Yasufumi Murakami3, Kazuto Ashizawa5, Kengo Inoue6, Fumiaki Koizumi7, Mitsuko Masutani1,2 1Department of Frontier Life Sciences, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8588, Japan 2Division of Chemotherapy and Clinical Research, National Cancer Center Research Institute, 5-1-1, Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan 3Department of Biological Science and Technology, Faculty of Industrial Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo, 125-8585, Japan 4Department of Pharmacology, Faculty of Dentistry, Osaka Dental University, 8-1, Kuzuhahanazono-cho, Hirakata, Osaka, 573-1121, Japan 5Department of Clinical Oncology, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8588, Japan 6Pharma Valley Center, 1007 Shimonagakubo, Nagaizumi-cho, Shunto-gun, Shizuoka, 411-8777, Japan 7Department of Laboratory Medicine, Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, 3-18-22, Honkomagome, Bunkyo-ku, Tokyo, 113-8677, Japan *Equally contributed.

Running title Dysfunction of PARG and DUSP22 induces synthetic lethality

Keywords PARG, poly(ADP-ribose), synthetic lethality, DUSP22, cancer therapy

A conflict of interest disclosure statement We have no financial relationships to disclose.

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 29, 2019; DOI: 10.1158/0008-5472.CAN-18-1037 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Financial supports This research is partially supported by The Practical Research for Innovative Cancer Control from Japan Agency for Medical Research and Development, AMED (15Ack0106021, 17ck0106286), and Grant-in-Aid for Scientific Research (KibanB 22300343, H23-Jitsuyoka(Gan)-004) to M.M.

Corresponding author Mitsuko Masutani Department of Frontier Life Sciences, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8588, Japan Phone and Fax: +81-95-819-8502 E-mail address: [email protected]

227 words of abstract 6124 words of text 1 table and 6 figures

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

Statement of significance This study identified DUSP22 as a novel synthetic lethal gene under the condition of PARG dysfunction and elucidated the mechanism of synthetic lethality in lung cancer cells.

Introduction Poly(ADP-ribosylation) is a post-translational modification by which some PARP family proteins catalyze the transfer of ADP-ribose to target proteins in a nicotinamide adenine dinucleotide (NAD+)-dependent manner (1,2). This reaction is involved in various biological processes, including cell death, chromatin regulation, and DNA repair of single strand breaks (SSBs) and double strand breaks (DSBs) (1,3). PARP inhibitors were recently developed as a novel anticancer agent based on the concept of synthetic lethality (4,5). PARP inhibitors selectively induce cell death in homologous recombination repair (HRR)-deficient cancers such as those associated with mutations in BRCA1/2 (6,7), RAD51 (8), and PTEN (9). The development of novel anticancer agents based on the concept of synthetic lethality is a valuable cancer chemotherapy strategy because these drugs show increased tumor selectivity with reduced adverse effects on normal cells (4). Poly(ADP-ribose) (PAR) synthesized by PARP is rapidly degraded to ADP-ribose by poly(ADP-ribose) glycohydrolase (PARG) (10) and ADP-ribosyl hydrolase (ARH3) (11). PARG is the main enzyme catabolizing PAR to ADP-ribose through its endo- and exo-glycohydrolase activities (12). As previously reported, PARG is required for the efficient repair of DSBs and SSBs (13). PARG deficiency induces PAR accumulation and a delay of DNA repair (14,15). And, PAR accumulation induces cell death (parthanatos) accompanied by the translocation of apoptosis inducing factor from mitochondria to nuclei, leading to fragmentation of large-sized DNA in neuronal cells and cancer cells such as HeLa cells (16,17). As previously reported, certain human cancer cell lines with PARG knockdown synergistically show higher sensitivity to alkylating agents (14,18) and cisplatin treatment (18). PARG hypomorphic mouse ES cells with residual 10% PARG activity did not exhibit growth defect but showed higher sensitivity to alkylating agents, cisplatin, photon and particle beam irradiation compared to wild-type ES cells (14,19,20). In addition, BRCA2 (21) and Bruton’s tyrosine kinase (BTK) (22) defects increase PARG inhibition-induced cytotoxicity (21-23). These findings led us to hypothesize that PARG could serve as a novel therapeutic target for anticancer agents in both monotherapy and combination therapy with radiotherapy or other DNA targeting chemotherapeutic agents for particular types of cancers. Recently, PARG inhibitors such as phenolic hydrazide hydrazones (24),

rhodanine-based PARG inhibitors (RBPIs) (25), xanthene compounds (26),

ADP-HPD (27), and PDD00017273 (28), which has an IC50 in the sub-microM range, have been developed. However, specific and potent PARG inhibitors for clinical applications remain to be developed. Here, we screened genes whose deficiency enhances sensitivity in a synthetic lethal manner to develop a novel anticancer agent targeting PARG. We identified dual specificity phosphatase 22 (DUSP22) as such a novel gene. Synthetic lethality induced by PARG and DUSP22 dysfunction in lung cancer cells led to TP63-dependent apoptosis by upregulating p53 upregulated modulator of apoptosis (PUMA). Double knockdown of PARG and DUSP22 inhibited tumor growth in a mouse xenograft model. These results indicated that alterations in DUSP22 expression levels may serve as a predictive biomarker for PARG inhibitors.

Materials and methods Cell culture and reagents The TRHmPARG#8 cell line is a tetracycline-inducible PARG knockdown strain derived from the human T-REx HeLa cell line described previously (29). TRHmPARG#8 and PC14 were cultured in Minimum Essential Medium and Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific), respectively. A549 and SBC5 cells were grown in RPMI1640 (Thermo Fisher Scientific). Media were supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin (Invitrogen) as needed. Cells were

maintained in a humidified atmosphere with 5% CO2 at 37°C. The cell line A549 was obtained from the ATCC. The cell line PC-14 was obtained from Dr. Hayata, Tokyo Medical College. The cell line SBC-5 was obtained from Okayama University in 1994. Cell line authentication of all cell lines was performed by STR (short tandem repeat)-PCR (Promega, August 2018). Mycoplasma testing was carried out using e-Myco plus Mycoplasma PCR Detection kit (iNtRON Biotechnology) for all cell lines used in this study and all cell lines were mycoplasma free. All cell lines were passaged less than 15 times prior to use.

Negative screening using a lentivirus shRNA library Negative screening was performed using the Decode RNAi Pooled Lentiviral shRNA Screening Libraries: Annotated Genome Negative Selection Kit (Thermo Fisher Scientific). TRHmPARG#8 cells were infected with a lentiviral siRNA expression library (Thermo Scientific) using the TransDux reagent (System Biosciences). Green fluorescent protein-positive cells were selected using puromycin for 3 days and divided into two populations. Cells were cultured for 6 days in the presence or absence of 40 ng/ml tetracycline, and genomic DNA was purified from two populations using a DNA purification kit (Dojindo). Amplification of barcode sequences in genomic DNA and purification of DNA were performed using the Decode shRNA Negative Selection Kit (Thermo Fisher Scientific) and Gene JET PCR Purification Kit (Thermo Fisher Scientific), respectively, as recommended by the manufacturer. Then, the genomic DNA was labeled using a Genomic DNA Enzymatic Labeling Kit (Agilent Technologies) and purified using Amicon Ultra-0.5 ml Centrifugal Filters (Millipore). The labeled barcode

sequences were hybridized to microarray slides for 17 h, and the slides were washed according to the Agilent CpG microarray protocol.

siRNA transfection Cells were seeded onto 6-well plates or 24-well plates. Transfection with siRNA was performed using Lipofectamine RNAi MAX (Life Technologies) according to the manufacturer’s protocol. Individual siRNAs were used at final concentration of 10 nM in Opti-MEM. siRNAs (PARG#2, DUSP22#2, PUMA, TP63) targeting DUSP22, PARG, PUMA, and TP63 were purchased from Integrated DNA Technologies. The siRNA sequence of DUSP22#1 is based on shRNA sequence of oligo ID:V2LHS_225030 in the Decode RNAi Pooled Lentiviral shRNA Screening Libraries and it was constructed from Integrated DNA Technologies. PARG#1 siRNA was obtained as described previously (14). DS NC1 siRNA (Integrated DNA Technologies) and scrambled siRNA (Ambion/Applied Biosystems) were used as negative controls.

Quantitative RT-PCR (qRT-PCR) RNA was prepared from each individual cell line and reverse transcribed using a High Capacity Reverse Transcription Kit (Thermo Fisher Scientific). The quantitative RT-PCR (qRT-PCR) analysis was performed using SYBR Green with the CFX96 Real-Time System (Bio-Rad). The mRNA levels were normalized to GUSB mRNA. The sequences of primer pairs are listed in Supplementary Table S1.

Cell proliferation assay Cell viability was measured using the Cell Counting Kit-8 (Dojindo Laboratories) according to the manufacturer’s instructions. Cells were seeded onto 96-well plates and cultured for 1 week. Cell proliferation rate was determined using the Cell Counting Kit-8 containing water soluble tetrazolium dys (WST-8). Plates were analyzed using a microtiter plate reader at 450 nm with a reference of 600 nm.

Western blot analysis Western blotting was performed as described previously (29). Cell extracts were prepared with Laemmli’s buffer. Proteins were separated by

SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membranes. The following antibodies were used for immunoblotting: anti-PARG (Millipore), anti-p-p38 (Cell Signaling), anti--Actin (Sigma-Aldrich), anti-DUSP22 (Gene Tex), anti-AKT (pan) (C67E7) (Cell Signaling), anti-p-AKT (Ser473) (Cell Signaling), anti-p-mTOR (Ser2448) (Cell Signaling), anti-p-mTOR (Ser2481) (Cell Signaling), anti-mTOR (7C10) (Cell Signaling), anti-PTEN (138G6) (Cell Signaling), anti-p-PTEN (Ser380) (Cell Signaling). Immune complexes were visualized using a horseradish peroxidase-linked secondary antibody and enhanced chemiluminescence (Millipore). Image quantification was performed with the Image J software (NIH).

Colony formation assay Cells were transfected with siRNA against the target gene and cultured in 6-well plates for 9 days. Colonies were fixed with 4% neutralized formalin and stained with 0.02% crystal violet for counting.

Cell cycle analysis Cell cycle distribution was analyzed by flow cytometry as previously described (30). Cells were fixed with 70% ethanol. Fixed cells were incubated with PBS containing 50 μg/ml propidium iodide (Sigma-Aldrich) and 20 μg/ml RNase A (Sigma-Aldrich) for 2 h and analyzed using the FACSCalibur system (Becton-Dickinson).

Antibody array A549 cells were transfected with siRNA against PARG and/or DUSP22 and cultured for 3 days. Cell lysates were analyzed with PathScan Stress and Apoptosis Signaling Antibody Array (Cell Signaling, #12856). This array was performed according to the manufacturer’s instructions.

Animal experiments A549 cells were transfected with siRNA against PARG and/or DUSP22 or control siRNA. On the next day, cells (2.9 × 105) were mixed with Growth Factor Reduced Matrigel (BD Biosciences) and injected subcutaneously into both legs of 11-week-old Balb/c-nu/nu nude mice. Tumor diameters were measured every 3 days with micrometer calipers, and tumor volume was

calculated using the following formula: (smallest diameter) × (largest diameter) × (height) / 2. All animal studies were approved by the Animal Experimental Committee of the National Cancer Center and performed following the Guidelines for Animal Experiments of the National Cancer Center, which meet the ethical guidelines for experimental animals in Japan.

Statistical analysis Data were expressed as meant ± S.E.. Statistical significance was indicated when p value was less than 0.05. In this study, data were analyzed using Tukey’s test or Mann-Whitney U-test. Synergistic effects were analyzed by 2-way ANOVA.

Results Identification of synthetic lethal genes related to PARG dysfunction A comprehensive analysis was performed to identify synthetic lethal genes related to PARG dysfunction and understand the function of PARG in cancer cells. For this purpose, a siRNA library was screened using a negative screening strategy. Inducible PARG knockdown T-REx HeLa cells (TRHmPARG#8) were established in which PARG knockdown was induced in the presence of tetracycline (Tc) (29). The cells were infected with lentiviral shRNA pools targeting approximately 10,000 genes. The relative abundance of individual shRNAs after PARG knockdown was determined by microarray analysis (Fig. 1A). Seventeen candidate siRNAs that suppressed the growth of PARG knockdown cells were identified at a 4-fold or higher signal rate (Fig. 1B, Table 1). The targeted genes were classified into functional categories, including metabolism, signal transduction, and posttranslational modification (Fig. 1B, Table 1). Among these genes, we focused on DUSP22, because DUSP22 mutations are reported frequently in lung, colon, and other tumors in the CanSAR database (Fig. 1C), and its expression is downregulated in certain cancers (31,32). To determine whether dysfunction of PARG and DUSP22 exerts a synthetic lethal effect in cancer cells, siRNA against DUSP22 was introduced into the inducible T-REx HeLa cells. As shown in Fig. 1Da, PARG expression was decreased to approximately 50% of the control in the presence of Tc and transfection of cells with siRNA targeting DUSP22 decreased DUSP22 mRNA to approximately 10% of control levels (Fig. 1Db). DUSP22 protein levels decreased to 29% in DUSP22 knockdown cells (Fig. 1Dc). DUSP22 belongs to the DUSP family of proteins, which function in the dephosphorylation of JNK, p38, and ERK (33). Inducible PARG knockdown T-REx HeLa cells were transfected with siRNA against DUSP22 or control siRNA, and the phosphorylation level of p38 was determined using a phospho-p38 antibody. The results showed that p38 phosphorylation levels were 2.3-fold higher in DUSP22 knockdown cells than in control cells (Fig. 1Dc), suggesting that DUSP22 was necessary for the dephosphorylation of p38. To determine whether dysfunction of PARG and DUSP22 induces synthetic lethality, the survival 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 PARG knockdown cells to

approximately 40% of that of single knockdown and control cells. This result suggested that DUSP22 deficiency induces synthetic lethality under conditions of PARG inhibition.

Dysfunction of PARG and DUSP22 efficiently suppressed the growth of lung cancer cells Because the frequency of DUSP22 mutation is higher in lung cancer than in other types of tumors (Fig. 1C), we examined whether DUSP22 and PARG dysfunction induced synthetic lethality in the lung cancer cell lines A549, PC14, and SBC5. To exclude the possibility of off-target effects of PARG and DUSP22 knockdown, the effect of two different siRNA sets (#1 and #2) against PARG and DUSP22 was tested (Fig. 2Aa-b, 2Ba-b) using a clonogenic survival assay (Fig. 2Ca-c). Double knockdown of PARG and DUSP22 in A549 showed synergistic growth inhibition in comparison with single knockdown and N.C. cells (Fig. 2Ca). In PC14 and SBC5 cells, one of each siRNA set showed a synergistic effect but the other showed an additive effect, respectively (Fig. 2Cb, c). These results suggested that dysfunction of PARG and DUSP22 efficiently induced synthetic lethality in particular lung cancer cell lines.

PARG and DUSP22 double knockdown promoted apoptosis by upregulating PUMA To examine the mechanism underlying lethality in DUSP22 and PARG double knockdown cells, the expression levels of apoptosis- and cell cycle-related genes were analyzed by qRT-PCR. As shown in Fig. 3Aa-c, PUMA mRNA levels were higher in double knockdown cells than in single knockdown cells in these lung cancer cell lines. In PC14 cells, DUSP22 single knockdown and DUSP22 and PARG double knockdown upregulated the expression of NOXA, a gene involved in apoptosis induction, compared with the levels in control cells (Fig. 3Ab). The expression level of CDKN2A, a cell cycle negative regulator and cell senescence-related factor, was also elevated in response to DUSP22 single knockdown and double knockdown conditions in SBC5 cells (Fig. 3Ac). To determine whether apoptosis induction was involved in the decreased cell viability caused by PARG and DUSP22 double knockdown in lung cancer cell lines, we performed cell cycle distribution analysis. In all cell lines tested, PARG and DUSP22 double knockdown

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

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

cultured in the presence or absence of the p38 MAPK-specific inhibitor SB203580, and cell growth was analyzed by the cell proliferation assay. As shown in Supplementary Fig. S2, SB203580 treatment restored cell growth in DUSP22 and PARG knockdown A549 cells. These results suggested that the upregulation of phosphorylated p38 level under DUSP22 dysfunction is involved in promoting PUMA-mediated apoptosis in double knockdown A549 cells. Additionally, we examined whether PARG and DUSP22 knockdown suppressed the protein expression of cell proliferation-related genes in A549 cells. PTEN, phospho-PTEN (Ser380), and phospho-mTOR (Ser2448) levels were decreased in double knockdown A549 but not in single knockdown cells (Fig. 5). Phospho-AKT (Thr308 and Ser473) and phospho-mTOR (Ser2481) were downregulated in both double knockdown and 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 Figure 6Ba and Bb, double knockdown of DUSP22 and PARG in A549 cells suppressed tumor growth compared with that of tumors transfected with control siRNA (Figure 6Bb, 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 synthetic lethal genes using inducible PARG knockdown cells and a shRNA library. Among the candidate genes identified, we focused on DUSP22, because it is frequently mutated in various types of cancer (Fig. 1C). DUSP22 is a member of the DUSP subfamily of protein tyrosine phosphatases. Its dephosphorylation substrate remains unclear, whereas other DUSP family proteins function in the dephosphorylation of JNK, p38, and/or ERK (33). Here, we showed that DUSP22 directly or indirectly dephosphorylated p38 by silencing DUSP22 in inducible PARG knockdown T-REx HeLa cells (Fig. 1Dc). The p38 MAPK pathway is activated by phosphorylation of p38 and is involved in the induction of cell death through apoptosis (36). Colony formation assays showed that double knockdown of DUSP22 and PARG in the lung cancer cell lines A549, PC14, and SBC5 efficiently induced cell death compared with the effect of single knockdown. Cell cycle analysis showed that double knockdown increased the sub-G1 population, and treatment with the p38 MAPK inhibitor SB203580 restored cell survival in double knockdown A549 cells. Taken together, these results suggest that the synthetic lethal effect of dysfunction of DUSP22 and PARG was mediated in part by the induction of apoptosis via the p38 MAPK pathway in A549 cells. Under oxidative stress conditions, PARP1 promotes the phosphorylation of p38 in association with the downregulation of MAPK

phosphatase-1, which is involved in the dephosphorylation of JNK and p38 MAP kinases, resulting in increased cell death (37). In this study, we demonstrated that increased p38 phosphorylation induced by DUSP22 knockdown and PAR accumulation induced by PARG knockdown (Supplementary Fig. S3) exerted a synthetic lethal effect by promoting apoptotic cell death in lung cancer cells. In the apoptosis pathway, the tumor suppressor p53 activates apoptosis-related factors, and p53 missense mutations are present in various types of cancer including lung cancer (38). On the other hand, the p53 homolog TP63 is rarely mutated (39). TP63 has common transcription targets with p53 and promotes the expression of PUMA to induce p53-independent apoptosis (40). In this study, we showed that knockdown of DUSP22 and PARG in A549 cells upregulated TP63 (Fig. 4C) and PUMA expression. In addition, despite the fact that PC14 and SBC5 cells bear p53 mutations (41,42), double knockdown of DUSP22 and PARG in these cells induced apoptosis by upregulating PUMA (Fig. 3Aa-c). These results indicated that the synthetic lethal effect of DUSP22 and PARG dysfunction was mediated by the induction of apoptosis through the TP63 pathway. The induction of apoptosis through a p53-independent pathway has important implications because many cancers have p53 pathway mutations. The present results suggested that dysfunction of PARG and DUSP22 in A549 cells affects the PI3K/AKT/mTOR pathway, which is frequently involved in cancer cell proliferation (43). As shown in Fig. 5, the level of phospho-mTOR (Ser-2448) was lower in double knockdown cells than in control cells despite the downregulation of PTEN and phospho-PTEN, a negative regulator of the PI3K/AKT pathway, in double knockdown cells. The phosphorylation level of AKT (Thr308 and Ser473) and mTOR (Ser2481) was reduced in both double knockdown A549 cells and single knockdown cells. Overall, these results indicated that the promotion of TP53-independent apoptosis and suppression of PI3K/AKT/mTOR pathway activity induced synthetic lethality in DUSP22 and PARG double knockdown A549 cells. As shown in Fig. 1C, various cancers including lung and colon cancers occasionally bear DUSP22 gene mutations. The complete genome sequence analysis from a lung cancer patient showed that DUSP22 was inactivated by loss of heterozygosity and point mutations (31). DUSP22 expression is also downregulated in breast cancer and anaplastic lymphoma

kinase-negative anaplastic large cell lymphoma (32). In this study, the double deficiency of DUSP22 and PARG suppressed lung tumor growth in a xenograft model (Fig. 6), suggesting that DUSP22 could be useful as a biomarker for monitoring the synthetic lethal effect of PARG inhibitors. PARG deficiency sensitizes cancer cells to alkylating agents (14,18), cisplatin (18) and -irradiation (18,19). Cancers with low expression levels of DUSP22 may increase sensitivity to combination therapy with these drugs and PARG inhibitors. The development of PARG inhibitors for clinical application is awaited. Both inhibition of BTK and BRCA2 in combination with PARG inhibition has a moderate synthetic lethal effect (21,22). The present study suggested that PARG-specific inhibitors could be useful for cancer therapy by exerting a synthetic lethal effect on cancers with DUSP22 deficiency.

Acknowledgements We thank for kind supports by Dr. Toshio Imai of Central Animal Division, National Cancer Center and technical assistance by Hiromi Harada.

References

1. Miwa M, Masutani M. PolyADP-ribosylation and cancer. Cancer science 2007;98:1528-35 2. Sugimura T. Poly(adenosine diphosphate ribose). Prog Nucleic Acid Res Mol Biol 1973;13:127-51 3. Rodriguez MI, Majuelos-Melguizo J, Marti Martin-Consuegra JM, Ruiz de Almodovar M, Lopez-Rivas A, Javier Oliver F. Deciphering the insights of poly(ADP-ribosylation) in tumor progression. Med Res Rev 2015;35:678-97 4. Chan DA, Giaccia AJ. Harnessing synthetic lethal interactions in anticancer drug discovery. Nature reviews Drug discovery 2011;10:351-64 5. Brown JS, Kaye SB, Yap TA. PARP inhibitors: the race is on. Br J Cancer 2016;114:713-5 6. Bryant HE, Schultz N, Thomas HD, Parker KM, Flower D, Lopez E, et al. Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature 2005;434:913-7 7. Farmer H, McCabe N, Lord CJ, Tutt AN, Johnson DA, Richardson TB, et al. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 2005;434:917-21 8. McCabe N, Turner NC, Lord CJ, Kluzek K, Bialkowska A, Swift S, et al. Deficiency in the repair of DNA damage by homologous recombination and sensitivity to poly(ADP-ribose) polymerase inhibition. Cancer research 2006;66:8109-15 9. Mendes-Pereira AM, Martin SA, Brough R, McCarthy A, Taylor JR, Kim JS, et al. Synthetic lethal targeting of PTEN mutant cells with PARP inhibitors. EMBO molecular medicine 2009;1:315-22 10. Miwa M, Tanaka M, Matsushima T, Sugimura T. Purification and properties of glycohydrolase from calf thymus splitting ribose-ribose linkages of poly(adenosine diphosphate ribose). The Journal of biological chemistry 1974;249:3475-82 11. Oka S, Kato J, Moss J. Identification and characterization of a mammalian 39-kDa poly(ADP-ribose) glycohydrolase. The Journal of biological chemistry 2006;281:705-13 12. Brochu G, Duchaine C, Thibeault L, Lagueux J, Shah GM, Poirier GG. Mode of action of poly(ADP-ribose) glycohydrolase. Biochimica et biophysica acta 1994;1219:342-50

13. Mortusewicz O, Fouquerel E, Ame JC, Leonhardt H, Schreiber V. PARG is recruited to DNA damage sites through poly(ADP-ribose)- and PCNA-dependent mechanisms. Nucleic acids research 2011;39:5045-56 14. Shirai H, Poetsch AR, Gunji A, Maeda D, Fujimori H, Fujihara H, et al. PARG dysfunction enhances DNA double strand break formation in S-phase after alkylation DNA damage and augments different cell death pathways. Cell Death Dis 2013;4:e656 15. Wei L, Nakajima S, Hsieh CL, Kanno S, Masutani M, Levine AS, et al. Damage response of XRCC1 at sites of DNA single strand breaks is regulated by phosphorylation and ubiquitylation after degradation of poly(ADP-ribose). Journal of cell science 2013;126:4414-23 16. Andrabi SA, Kim NS, Yu SW, Wang H, Koh DW, Sasaki M, et al. Poly(ADP-ribose) (PAR) polymer is a death signal. Proc Natl Acad Sci U S A 2006;103:18308-13 17. Fatokun AA, Dawson VL, Dawson TM. Parthanatos: mitochondrial-linked mechanisms and therapeutic opportunities. Br J Pharmacol 2014;171:2000-16 18. Fujihara H, Ogino H, Maeda D, Shirai H, Nozaki T, Kamada N, et al. Poly(ADP-ribose) Glycohydrolase deficiency sensitizes mouse ES cells to DNA damaging agents. Current cancer drug targets 2009;9:953-62 19. Shirai H, Fujimori H, Gunji A, Maeda D, Hirai T, Poetsch AR, et al. Parg deficiency confers radio-sensitization through enhanced cell death in mouse ES cells exposed to various forms of ionizing radiation. Biochemical and biophysical research communications 2013;435:100-6 20. Nakadate Y, Kodera Y, Kitamura Y, Tachibana T, Tamura T, Koizumi F. Silencing of poly(ADP-ribose) glycohydrolase sensitizes lung cancer cells to radiation through the abrogation of DNA damage checkpoint. Biochemical and biophysical research communications 2013;441:793-8 21. Fathers C, Drayton RM, Solovieva S, Bryant HE. Inhibition of poly(ADP-ribose) glycohydrolase (PARG) specifically kills BRCA2-deficient tumor cells. Cell Cycle 2012;11:990-7 22. Rotin LE, Gronda M, MacLean N, Hurren R, Wang X, Lin FH, et al. Ibrutinib synergizes with poly(ADP-ribose) glycohydrolase inhibitors to induce cell death in AML cells via a BTK-independent mechanism. Oncotarget 2016;7:2765-79 23. Gravells P, Grant E, Smith KM, James DI, Bryant HE. Specific killing of DNA damage-response deficient cells with inhibitors of poly(ADP-ribose) glycohydrolase. DNA repair 2017;52:81-91 24. Islam R, Koizumi F, Kodera Y, Inoue K, Okawara T, Masutani M. Design and synthesis of phenolic hydrazide hydrazones as potent poly(ADP-ribose) glycohydrolase (PARG)

inhibitors. Bioorganic & medicinal chemistry letters 2014;24:3802-6 25. Finch KE, Knezevic CE, Nottbohm AC, Partlow KC, Hergenrother PJ. Selective small molecule inhibition of poly(ADP-ribose) glycohydrolase (PARG). ACS chemical biology 2012;7:563-70 26. Okita N, Ashizawa D, Ohta R, Abe H, Tanuma S. Discovery of novel poly(ADP-ribose) glycohydrolase inhibitors by a quantitative assay system using dot-blot with anti-poly(ADP-ribose). Biochemical and biophysical research communications 2010;392:485-9 27. Slama JT, Aboul-Ela N, Goli DM, Cheesman BV, Simmons AM, Jacobson MK. Specific inhibition of poly(ADP-ribose) glycohydrolase by adenosine diphosphate (hydroxymethyl)pyrrolidinediol. Journal of medicinal chemistry 1995;38:389-93 28. James DI, Smith KM, Jordan AM, Fairweather EE, Griffiths LA, Hamilton NS, et al. First-in-Class Chemical Probes against Poly(ADP-ribose) Glycohydrolase (PARG) Inhibit DNA Repair with Differential Pharmacology to Olaparib. ACS chemical biology 2016;11:3179-90 29. Sasaki Y, Hozumi M, Fujimori H, Murakami Y, Koizumi F, Inoue K, et al. PARG Inhibitors and Functional PARG Inhibition Models. Current protein & peptide science 2016;17:641-53 30. Fujimori H, Sato A, Kikuhara S, Wang J, Hirai T, Sasaki Y, et al. A comprehensive analysis of radiosensitization targets; functional inhibition of DNA methyltransferase 3B radiosensitizes by disrupting DNA damage regulation. Scientific reports 2015;5:18231 31. Lee W, Jiang Z, Liu J, Haverty PM, Guan Y, Stinson J, et al. The mutation spectrum revealed by paired genome sequences from a lung cancer patient. Nature 2010;465:473-7 32. Feldman AL, Dogan A, Smith DI, Law ME, Ansell SM, Johnson SH, et al. Discovery of recurrent t(6;7)(p25.3;q32.3) translocations in ALK-negative anaplastic large cell lymphomas by massively parallel genomic sequencing. Blood 2011;117:915-9 33. Patterson KI, Brummer T, O'Brien PM, Daly RJ. Dual-specificity phosphatases: critical regulators with diverse cellular targets. The Biochemical journal 2009;418:475-89 34. Flores ER, Tsai KY, Crowley D, Sengupta S, Yang A, McKeon F, et al. p63 and p73 are required for p53-dependent apoptosis in response to DNA damage. Nature 2002;416:560-4 35. Kaufman B, Shapira-Frommer R, Schmutzler RK, Audeh MW, Friedlander M, Balmana J, et al. Olaparib monotherapy in patients with advanced cancer and a germline BRCA1/2

mutation. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2015;33:244-50 36. Zarubin T, Han J. Activation and signaling of the p38 MAP kinase pathway. Cell research 2005;15:11-8 37. Racz B, Hanto K, Tapodi A, Solti I, Kalman N, Jakus P, et al. Regulation of MKP-1 expression and MAPK activation by PARP-1 in oxidative stress: a new mechanism for the cytoplasmic effect of PARP-1 activation. Free radical biology & medicine 2010;49:1978-88 38. Murai Y, Hayashi S, Takahashi H, Tsuneyama K, Takano Y. Correlation between DNA alterations and p53 and p16 protein expression in cancer cell lines. Pathology, research and practice 2005;201:109-15 39. Moll UM, Slade N. p63 and p73: roles in development and tumor formation. Molecular cancer research : MCR 2004;2:371-86 40. Bergamaschi D, Samuels Y, Jin B, Duraisingham S, Crook T, Lu X. ASPP1 and ASPP2: common activators of p53 family members. Molecular and cellular biology 2004;24:1341-50 41. Kashii T, Mizushima Y, Monno S, Nakagawa K, Kobayashi M. Gene analysis of K-, H-ras, p53, and retinoblastoma susceptibility genes in human lung cancer cell lines by the polymerase chain reaction/single-strand conformation polymorphism method. Journal of cancer research and clinical oncology 1994;120:143-8 42. Fujita T, Kiyama M, Tomizawa Y, Kohno T, Yokota J. Comprehensive analysis of p53 gene mutation characteristics in lung carcinoma with special reference to histological subtypes. International journal of oncology 1999;15:927-34 43. Polivka J, Jr., Janku F. Molecular targets for cancer therapy in the PI3K/AKT/mTOR pathway. Pharmacology & therapeutics 2014;142:164-75

Table 1 Candidate genes found in the synthetic lethality screening in PARG K.D. condition. # Gene Functional classification 1 RANBP6 Protein transport 2 STK25 Signal transduction 3 RAC1 Metabolic process 4 ARL6 Signal transduction 5 RPL4 Metabolic process 6 MSH6 DNA damage response 7 IL6ST Signal transduction 8 DUSP22 Signal transduction 9 KIF9 Protein transport 10 GTSE1 DNA damage response 11 ATXN1L Signal transduction 12 ST6GALNAC2 Posttranslational modification 13 PLA2G15 Metabolic process 14 WIPF1 Cell structure/cytoskeleton 15 PARP15 Posttranslational modification 16 KRTAP10-10 Cell structure/cytoskeleton 17 TCFL5 Metabolic process

Figure legends 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, Tc; tetracycline. 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 / 6750 patients) in CanSAR database (https://cansar.icr.ac.uk/) (Tym, J. E. et al., Nucleic Acids Res. (2016) 44, D938-D943.). D. Knockdown level of PARG (a) and DUSP22 (b, 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’s test).

Figure 2 Dysfunction of PARG/DUSP22 reduced cellular survival ratio in lung cancer cell lines. A and B. siRNA targeting DUSP22 and PARG successfully knockdown 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 3 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<0.0001, **p<0.001 and ***p<0.05 (Tukey’s test). Error bars represent standard error of the mean.

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 4-5 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<0.0001 and **p<0.001 versus N.C. and single knockdown cells (Tukey’s test). B. Flow cytometry analysis. A549, PC14 and SBC5 cells were harvested at 2 days and 6 days after transfection, respectively and analyzed by flow cytometry. *p<0.0001, **p<0.001 and ***p<0.05 versus N.C. or single knockdown cells (Tukey’s test). a, A549; b, PC14;c, SBC5.

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 and PUMA) and cell viability was measured by colony formation assay (Tukey’s test, *p<0.005). C. PARG/DUSP22 knockdown induced TP63 (a) /TP73 (b) expression in A549 (Tukey’s test, *p<0.0001, **p<0.001, ***p<0.05). 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’s test, *p<0.001, **p<0.001, ***p<0.05).

Figure 5 PI3K/AKT/mTOR pathway was down-regulated by DUSP22 and PARG double knockdown in A549 cells. A549 cells were transfected with DUSP22 and/or PARG siRNA. After 3 days, cell lysates were analyzed by western blotting using antibodies for phosphorylated form of PTEN, AKT and mTOR. Relative expression levels normalized to -actin level was shown under each panel.

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 Fig. 6B. *p<0.05 relative to N.C. (Kruskal-Wallis test).

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 Published OnlineFirst May 29, 2019.

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