Epigenetic silencing of BEND4, a novel DNA damage repair gene, is a synthetic lethal marker for ATM inhibitor in pancreatic cancer

Abstract

Synthetic lethality is a novel model for cancer therapy. To understand the function and mechanism of BEN domain-containing protein 4 (BEND4) in pancreatic cancer, eight cell lines and a total of 492 cases of pancreatic neoplasia samples were included in this study. Methylation-specific polymerase chain reaction, CRISPR/Cas9, immunoprecipitation assay, comet assay, and xenograft mouse model were used. BEND4 is a new member of the BEN domain family. The expression of BEND4 is regulated by promoter region methylation. It is methylated in 58.1% (176/303) of pancreatic ductal adenocarcinoma (PDAC), 33.3% (14/42) of intraductal papillary mucinous neoplasm, 31.0% (13/42) of pancreatic neuroendocrine tumor, 14.3% (3/21) of mucinous cystic neoplasm, 4.3% (2/47) of solid pseudopapillary neoplasm, and 2.7% (1/37) of serous cystic neoplasm. BEND4 methylation is significantly associated with late-onset PDAC (> 50 years, P < 0.01) and tumor differentiation (P < 0.0001), and methylation of BEND4 is an independent poor prognostic marker (P < 0.01) in PDAC. Furthermore, BEND4 plays tumor-suppressive roles in vitro and in vivo. Mechanistically, BEND4 involves non-homologous end joining signaling by interacting with Ku80 and promotes DNA damage repair. Loss of BEND4 increased the sensitivity of PDAC cells to ATM inhibitor. Collectively, the present study revealed an uncharacterized tumor suppressor BEND4 and indicated that methylation of BEND4 may serve as a potential synthetic lethal marker for ATM inhibitor in PDAC treatment.

Keywords

BEND4 / DNA methylation / synthetic lethality / NHEJ pathway

1 Introduction

Pancreatic cancer is predicted to be the second cause of cancer-related mortality by 2030 [1]. At the time of diagnosis, around 50% of patients exhibited distant metastases, and systemic chemotherapy remains the primary treatment [2,3]. Actionable mutational targets occur at low prevalence, and mutational analysis of tumors has largely been exhausted as a strategy to discover druggable new targets [4,5].

Poly(ADP-ribose) polymerase (PARP) inhibitor targeting BRCA1/2 deficient breast cancer cell is a classical synthetic lethality model [6–8]. Tumors that share molecular features of BRCA mutant tumors are defined as “BRCAness”, and it is reasonable to apply the same treatment regimens to target DNA repair deficient tumors [9].

Epigenetics is associated with developmental, genomic, and environmental factors [10]. Epigenetic silencing of gene expression may cause tumor suppressor inactivation or “loss of function” according to Knudson’s two hit theory [11]. Aberrant DNA methylation may be involved in many signaling pathways, including Wnt, TGF-β, NF-κB, cell cycle, and DNA repair [12].

The BEN (BANP, E5R, and NAC1) domain was identified using bioinformatics and later found to be a conserved DNA-binding domain [13]. BEN domain-containing proteins are involved in various biological processes, including DNA damage response, development, transcriptional repression, and differentiation [14–19]. Recently, it was reported that BEND4 synergizes with BEND5 to bind to the chromatin boundary and promote epiblast-like cell induction in reporter mouse embryonic stem cells [20]. However, the regulation of expression and the function of BEND4 in cancer remains to be elucidated. In the present study, the function and underlying mechanism of BEND4 in PDAC were explored for the first time, and the synthetic lethal therapeutic value of BEND4 was revealed.

2 Materials and methods

2.1 Human pancreatic cancer cell lines and tissue samples

Eight PDAC cell lines, including PANC03.11, PANC-1, SW1990, MIA PaCa-2, AsPC1, PANC05.04, PANC10.05, and CFPAC1, were obtained from American Type Culture Collection. PDAC cells were authenticated by short tandem repeat profiling carried out by BIOWING (Shanghai, China). A total of 492 cases with different types of pancreatic tumor samples were included in this study, including 303 cases of pancreatic ductal adenocarcinoma (PDAC), 42 cases of intraductal papillary mucinous neoplasms (IPMN), 42 cases of pancreatic neuroendocrine tumors (PNET), 21 cases of mucinous cystic neoplasms (MCN), 47 cases of solid pseudopapillary neoplasms (SPN), 37 cases of serous cystic neoplasms (SCN), and 15 cases of noncancerous tissue samples were used to exclude tissue specific methylation. All samples were collected from the Chinese PLA General Hospital, and no chemo-radiotherapy was performed before surgery. Tumors were classified according to TNM staging. The procedure was approved by the Institutional Review Board of the Chinese PLA General Hospital.

2.2 RNA and DNA preparation, 5-aza treatment, RT-PCR, methylation-specific PCR, bisulfite sequencing, and immunohistochemistry

All procedures were performed as described previously [21]. RT-PCR, methylation-specific PCR (MSP), and bisulfite sequencing (BSSQ) primer sequences are listed in Table S1. Antibodies used for immunohistochemistry (IHC) staining are listed in Table S2.

2.3 Plasmid construction and screening for BEND4 expressing monoclonal cells

pCDH-CMV-MCS-EF1-Puro and Lipofectamine 3000 (Invitrogen, USA) were used to construct BEND4 stably expressed cells. The coding sequence region of human BEND4 (GenBank accession number: NM_207406) was cloned using the primers 5′-CTAGTCTAGAATGGAGGAAGAGATGCAGCCG-3′ (F) and 5′-CTAGCTAGCCTAATCCCCAGATCCATCCTGGGAA-3′ (R). BEND4 expressing cells were selected by puromycin treatment at a concentration of 6 μg/mL (SW1990) and 2 μg/mL (PANC10.05) for 3 days. Single-cell clones were screened out by limited dilution in 96-well plates and validated using Western blotting. The pcDNA3.1(+) plasmid was used for transient expression.

2.4 CRISPR/Cas9 knockout and siRNA knockdown

CRISPR/Cas9 knockout was used to establish BEND4 null cells. Single guide RNA (sgRNA) sequences were designed by Guide Design Resources. siRNA (JTS Scientific, Beijing, China) transfection was performed using RNAiMax (Invitrogen, USA) following the manufacturer’s instructions. sgRNA and siRNA sequences are listed in Table S1.

2.5 MTT, colony formation, cell cycle, apoptosis, and transwell assay

The procedures were performed as previously described [21]. In MTT assay (KeyGEN Biotech, Jiangsu, China), cells were seeded at 2 × 10³ (PANC10.05), 1.5 × 10³ (SW1990), and 2 × 10³ (CFPAC1) per well in 96-well plates. In colony formation assay, PANC10.05 and SW1990 cells were seeded for 300 cells/well in 6-well plates growing for 14 days. CFPAC1 cells were seeded for 500 cells/well growing for 12 days. For evaluating the efficacy of cisplatin (Selleck, USA) and AZD0156 (an ATM inhibitor, Selleck, USA), colony formation was performed using 1000 cells/well. Cells were treated with cisplatin (0.1 μmol/L) and AZD0156 (0.05 μmol/L) for 48 h, and the medium was changed and grown for 10 days. For migration and invasion analysis, 4 × 10⁴ (PANC10.05), 6 × 10⁴ (SW1990), and 6 × 10⁴ (CFPAC1) cells were grown for 36 h and 48 h, respectively.

2.6 Evaluating the sensitivity of PDAC cells to AZD0156 and VE-822

The cells were seeded at 3 × 10³ (PANC10.05), 2 × 10³ (SW1990), and 2 × 10³ (CFPAC1) per well in 96-well plates. The inhibitory concentration 50% (IC₅₀) was determined 48 h after AZD0156 or VE-822 (an ATR inhibitor, Selleck, USA) treatment.

2.7 Western blotting and coimmunoprecipitation (Co-IP) analysis

All procedures were performed in accordance with a previous study [22]. The antibodies are listed in Table S2.

2.8 Homologous recombination and non-homologous end joining assay

Homologous recombination (HR) and non-homologous end joining (NHEJ) assays were performed as previously reported [23,24]. In brief, siBEND4 and scramble control were transfected into U2OS cells, which were integrated with direct repeat GFP (DR-GFP) or end-joining 5 GFP (EJ5-GFP) reporters. Then, the cells were infected by I-Scel and control lentivirus after being grown for 24 h. Forty-eight hours after siRNA transfection, GFP-positive cells were analyzed to evaluate the HR and NHEJ efficiency using flow cytometry.

2.9 Comet assay

Alkaline comet electrophoresis was performed as previously described [25]. 0.5 μmol/L cisplatin was used to induce exogenous DNA. The cell/agarose mixture (80 μL) was pipetted and gently spread on a coated comet slide. After immersing in a pre-chilled lysing solution for 1 h at 4 °C in the dark, the slides were then incubated in alkali unwinding solution at 4 °C for 15 min. Electrophoresis was performed at 25 V for 30 min. Imaging was performed by epifluorescence microscopy after staining with propidium iodide. The quantitation of the tail moments (n = 100 cells in each group) was analyzed using CometScore.

2.10 Chromatin fraction isolation

The chromatin fraction was isolated in accordance with a previously published study [26]. In brief, PDAC cells were exposed to 2 μmol/L cisplatin for 0.5 h and 4 h. Subsequently, the cells were lysed using CSK-100 buffer (300 mmol/L sucrose, 100 mmol/L NaCl, 3 mmol/L MgCl₂, 1 mmol/L EGTA, 10 mmol/L PIPES, pH 6.8, 0.2% TritonX-100) at 4 °C for 20 min. Then, chromatin-associated proteins were released from the cell pellets by treating them with lysis buffer (50 mmol/L HEPES pH 7.5, 0.05% SDS, 50 mmol/L NaCl, 10% glycerol, 2 mmol/L MgCl₂, 10 units of benzonase nuclease, 0.1% TritonX-100) at 4 °C overnight.

2.11 PDAC cell xenograft model and synthetic lethal therapeutic study

The animal experiments were performed in accordance with the procedures approved by the Animal Ethics Committee of the Chinese PLA General Hospital. Four-week-old BALB/c nude mice were grouped randomly. To evaluate the role of BEND4 in tumor xenograft growth, a total of 3 × 10⁶ BEND4 unexpressed and stably re-expressed SW1990 cells were injected subcutaneously into the flanks of BABL/c nude mice (n = 6) [21]. For synthetic lethal therapeutic study, a total of 2 × 10⁶ BEND4 unexpressed and stably re-expressed SW1990 cells were injected, and then the mice were randomly selected for treatment with control (n = 6), cisplatin (n = 6, 2 mg/kg), AZD0156 (n = 6, 30 mg/kg), and cisplatin combined with AZD0156 (n = 6, 2 mg/kg cisplatin, and 30 mg/kg AZD0156). Cisplatin was injected intraperitoneally, and AZD0156 was administered by oral gavage twice a week for 2 weeks.

2.12 Statistical analysis

Statistical analyses were performed using SPSS and GraphPad Prism. Kaplan–Meier plots with a log-rank test was used to evaluate overall survival (OS). The association between risk factors and OS was assessed using univariate and multivariate Cox proportional hazards regression models. Student’s t-test was used to test the difference between the two groups, and the results are presented as mean ± SD. A Chi-square test was used to compare the categorical variables. P < 0.05 was regarded as statistically significant.

3 Results

3.1 BEND4 is frequently methylated, and its expression is regulated by promoter region methylation in human pancreatic neoplasms

To evaluate the association of BEND4 expression with its promoter region methylation, mRNA expression and methylation data of 178 cases of PDAC were extracted from The Cancer Genome Atlas database. As shown in Fig.1 and Fig. S1, the reduced expression of BEND4 was significantly associated with CpG methylation (all P < 0.0001), indicating that the expression of BEND4 is potentially regulated by promoter region methylation.

To analyze the epigenetic regulation of BEND4, the expression level and methylation status of BEND4 were detected by semiquantitative RT-PCR and MSP. The results showed that PDAC cells without BEND4 expression were completely methylated, and PDAC cells with reduced expression were partially methylated (Fig.1 and 1C), demonstrating that the loss or reduced expression of BEND4 is correlated to the promoter region methylation status. A DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5-aza), was used to validate the epigenetic regulation of BEND4 expression. The expression of BEND4 was restored in BEND4 deficient cells after 5-aza treatment (Fig.1). To validate the efficiency of MSP primers and explore the methylation density in the promoter region of BEND4, BSSQ was performed in completely methylated PANC10.05 and SW1990 cells and partially methylated CFPAC1 cells. As shown in Fig.1, dense methylation was observed in PANC10.05 and SW1990 cells, and partial methylation was found in CFPAC1 cells. The above results indicate that the expression of BEND4 is regulated by promoter region methylation.

No methylation was detected in 15 noncancerous tissue samples, eliminating tissue-specific methylation in the pancreas (Fig.1). BEND4 was methylated in 58.1% (176/303) of PDAC, 33.3% (14/42) of IPMN, 31.0% (13/42) of PNET, 14.3% (3/21) of MCN, 4.3% (2/47) of SPN, and 2.7% (1/37) of SCN, indicating that BEND4 is frequently methylated in pancreatic neoplasms (Fig.1). As shown in Tab.1, BEND4 methylation was significantly associated with late-onset PDAC (> 50 years old, P < 0.01) and poor tumor differentiation (P < 0.0001). IHC was used to evaluate the protein level of BEND4 in 64 pairs of cancerous and noncancerous tissue samples, and its level was higher in noncancerous tissue samples than in cancerous tissues (Fig.1 and 1G, P < 0.0001). In addition, BEND4 methylation was found in 87.1% (27/31) of cancer samples with reduced BEND4 expression, whereas only 18.2% (6/33) of cancer samples with BEND4 normal expression were methylated (Fig.1, P < 0.0001). The above mentioned data indicate that the expression of BEND4 is regulated by promoter region methylation in PDAC.

3.2 BEND4 methylation is an independently poor prognostic marker

Kaplan–Meier and Cox proportional hazards models were used to explore the prognostic value of BEND4 methylation in PDAC. Among 207 PDAC patients with available follow-up data, BEND4 was methylated in 118 patients (57.0%). The median OS was 28 months (95% CI 19–36 months) in the BEND4 unmethylated group and 22 months (95% CI 20–24 months) in the BEND4 methylated group. OS was longer in BEND4 unmethylated patients (log-rank, P < 0.001; Fig.1). Multivariate analysis indicated that BEND4 methylation is an independent marker of poor prognosis (Tab.2, P < 0.01).

3.3 BEND4 suppresses cell growth, inhibits migration and invasion, and induces G1/S arrest and apoptosis in PDAC cells

BEND4 was overexpressed in PANC10.05 and SW1990 cells as well as knocked out (KO) in CFPAC1 cells to explore its function in PDAC. MTT assay and colony formation were used to investigate the role of BEND4 in cell proliferation. As shown in Fig.2, cell proliferation was attenuated significantly in BEND4 overexpressing PANC10.05 and SW1990 cells compared with empty vector controls, whereas it was enhanced in BEND4-KO CFPAC1 cells compared with wild-type (WT) controls. Consistent with these findings, the clone numbers were reduced significantly by restoring BEND4 and increased by knocking out BEND4 (Fig.2). The above mentioned results demonstrated that BEND4 suppressed PDAC cell proliferation.

Flow cytometry was performed to evaluate whether BEND4 modulates cell cycle and apoptosis. The percentage of G0/G1-phase cells increased significantly, and S-phase cells decreased in BEND4 overexpressing PANC10.05 and SW1990 cells compared with control groups. By contrast, G0/G1-phase cells were reduced, and S-phase cells increased by knocking out BEND4 in CFPAC1 cells (Fig.2). Western blotting was performed to confirm the results of cell distribution, indicating that BEND4 decreased the protein levels of cyclinA2, cyclinD1, cyclinE1, and CDK2 in PDAC cells (Fig.2). Moreover, the percentage of apoptotic cells increased significantly in BEND4 overexpressing PANC10.05 and SW1990 cells compared with control cells, whereas it decreased in BEND4 knockdown CFPAC1 cells compared with scramble control cells (Fig.2 and S2). Furthermore, BEND4 increased the protein levels of cleaved-caspase 3 and BAX and decreased those of Bcl-2 (Fig.2). These results indicated that BEND4 induced G1/S checkpoint arrest and apoptosis in PDAC cells.

Subsequently, transwell assay was used to investigate the effects of BEND4 on migration and invasion. As shown in Fig.2, the migratory and invasive abilities of PANC10.05 and SW1990 cells were attenuated significantly after BEND4 overexpression. By contrast, BEND4 knockout promoted migration and invasion in CFPAC1 cells (Fig.2). Western blotting further showed that BEND4 decreased the protein levels of MMP2, MMP7, and MMP9 (Fig.2).

3.4 BEND4 suppresses PDAC cell growth in vivo

To explore the effect of BEND4 on pancreatic cancer cell growth in vivo, BEND4 unexpressed and re-expressed SW1990 cell xenograft model was used. The tumor volume and weight were reduced significantly by BEND4 re-expression (Fig.2–2K, P < 0.0001), indicating the suppressing role of BEND4 in tumor growth in vivo. Collectively, these data indicated that BEND4 exerts tumor-suppressive functions in PDAC in vitro and in vivo.

3.5 BEND4 promotes DNA damage repair in PDAC

Co-IP and silver staining were used to further understand the underlying mechanism of BEND4 in PDAC. As shown in Fig.3, a distinct band was excised from BEND4 re-expressed SW1990 cells for mass spectrometry, and Ku80 was determined as an interesting candidate. The interaction between BEND4 and Ku80 was validated in SW1990 cells by reciprocal Co-IP (Fig.3).

HR and NHEJ are two major pathways for DNA double-strand break (DSB) repair. Ku80 forms a heterodimer with Ku70 and plays a central role in NHEJ. To explore the function of BEND4 in DSB repair, comet assay, a sensitive method for monitoring DSB, was used. Reduced comet tail moment was observed in BEND4 overexpressing PANC10.05 and SW1990 cells compared with control cells after cisplatin treatment. Conversely, BEND4-KO lead to increased tail moment in CFPAC1 cells (Fig.3). Above results indicated that BEND4 was involved in DSB repair.

Subsequently, the key molecules in NHEJ signaling were examined by Western blotting. BEND4 expression did not affect the protein level of Ku80 (Fig.3). DNA-dependent protein kinase catalytic subunit (DNAPKcs) plays an important role in NHEJ signaling. Elevated p-DNAPKcs and XRCC4 levels were observed in BEND4 re-expressed PANC10.05 and SW1990 cells under cisplatin treatment, whereas decreased p-DNAPKcs and XRCC4 levels were observed in BEND4-KO CFPAC1 cells (Fig.3). Above results indicated that BEND4 promotes DNA damage repair (DDR) in PDAC cells.

3.6 BEND4 activates NHEJ repair via Ku80

The efficacy of BEND4 on DDR was further analyzed by HR and NHEJ assays. After detecting the efficiency of three BEND4 targeting siRNAs, siBEND4#2 and siBEND4#3 were selected for further experiment. BEND4 knockdown reduced the efficiency of NHEJ, whereas no effect was observed on HR (Fig.4 and S3A). NHEJ is the major pathway for DDR in dividing and non-dividing somatic cells. Ku80 and Ku70 form a heterodimer to bind DSBs for recruiting other DNA repair factors to perform DNA repair [27]. It was validated by IP and reciprocal Co-IP, indicating that BEND4 interacted with Ku70 (Fig. S3B). DNAPKcs is the core enzyme that combined with the Ku80/Ku70 heterodimer to form the active DNAPK holoenzyme to perform DDR function [28]. Then, Ku70, Ku80, and DNAPKcs antibodies were used to analyze their interaction using co-IP. Re-expression of BEND4 enforced the interaction of DNAPKcs with Ku80/Ku70 (Fig.4). To further validate the role of BEND4 in NHEJ, chromatin fraction isolation experiment was performed. As shown in Fig.4, the levels of Ku80, Ku70, and DNAPKcs were increased in BEND4 expressed cells under cisplatin treatment, indicating that BEND4 enforced repair efficiency on damaged chromatin.

The comet assay was used to further investigate the role of BEND4 in NHEJ through Ku80. DNA comet moments induced by cisplatin was increased significantly by knocking down Ku80 in BEND4 expressed cells (Fig.4 and S3C). To confirm the results of the comet assay, Western blotting was performed to investigate the changes of key molecules in NHEJ under Ku80 knockdown. The results demonstrated that the effect of BEND4 on elevating p-DNAPKcs and XRCC4 was abrogated when Ku80 was knocked down (Fig.4). Collectively, these results demonstrate that BEND4 participates in NHEJ repair through interacting with Ku80.

3.7 Loss of BEND4 sensitizes PDAC cells to AZD0156

Synthetic lethality has emerged as a promising therapeutic strategy for cancer by targeting backup pathways. BEND4 is involved in NHEJ signaling, and its expression is silenced frequently by promoter region methylation in PDAC. Thus, we analyzed the sensitivity of PDAC cells to ATM or ATR inhibitors, with or without BEND4 expression. The results showed that the IC₅₀ of AZD0156 increased fivefold in BEND4 overexpressing PANC10.05 and tenfold in SW1990 cells compared with controls. By contrast, IC₅₀ decreased threefold in BEND4-KO CFPAC1 cells (Fig.5), indicating that the loss of BEND4 sensitizes PDAC cells to ATM inhibitor. Next, we examined the sensitivity of PDAC cells to ATR inhibitor, and no significant difference was found regarding the IC₅₀ of VE-822 in PDAC cells with or without BEND4 expression (Fig. S4), demonstrating that no synthetic lethality effect was found between ATR inhibitor and loss of BEND4 expression.

To further understand the synthetic lethality of BEND4 defects and ATM inhibitors, a colony formation assay was performed. Under cisplatin and AZD0156 treatment, the relative colony formation efficiency was 21.01% ± 3.72% versus 46.48% ± 3.02% in PANC10.05 cells and 20.18% ± 0.71% versus 32.18% ± 1.71% in BEND4 unexpressed and forcefully expressed SW1990 cells (all P < 0.001, Fig.5). In CFPAC1 cells, the normalized colony efficiency was 65.36% ± 4.46% versus 38.76% ± 6.03% before and after BEND4 knockout (P < 0.01, Fig.5), indicating the synthetic lethality of the BEND4 defect and ATM inhibitor in vitro. We further examined the ATM/CHK2 signaling and γ-H2AX using Western blotting. P-ATM and p-CHK2 were activated in BEND4 unexpressed PANC10.05 and SW1990 cells and BEND4-KO CFPAC1 cells under cisplatin treatment, indicating that ATM/CHK2 is a backup pathway in BEND4 deficient PDAC cells (Fig.5). Furthermore, γ-H2AX was elevated when AZD0156 was applied to BEND4 unexpressed PANC10.05 and SW1990 cells as well as BEND4-KO CFPAC1 cells, indicating that the synthetic effect of BEND4 defect and ATM inhibitor.

3.8 BEND4 defect and ATM inhibitor play synthetic lethal role in SW1990 cell xenograft

SW1990 cell xenografts were used to further investigate the synthetic lethal effects of BEND4 defect and AZD0156. The tumor volume and weight were reduced significantly in empty vector cells compared with BEND4 overexpressing cells under cisplatin and AZD0156 treatment, respectively (Fig.6–6C). In addition, increased level of p-DNAPKcs and XRCC4 was observed in BEND4 re-expressed cell xenografts compared with those in controls under cisplatin treatment, indicating the promoting role of BEND4 in NHEJ signaling (Fig.6). Collectively, the results indicated that BEND4 participates in the NHEJ pathway and epigenetic loss of BEND4 sensitized PDAC cells to ATM inhibitors (Fig.6).

4 Discussion

With next-generation sequencing of tumors becoming increasingly commonplace, targeted therapy has been a reality based on distinct molecular features and germline alterations. Despite success in certain cancers, targeted therapy remains unsatisfactory in PDAC. Several large sequencing studies have revealed that PDAC genome landscape was characterized as four mutational “mountains” (KRAS, CDKN2A, TP53, and SMAD4) as well as a larger number of less frequently mutated “hills” [29–37]. However, targeting KRAS and tumor suppressors (CDKN2A, TP53, and SMAD4) remains a challenge clinically, and other targeted therapies fail to improve the survival of PDAC because of genomic heterogeneity [38,39].

DNA damage is a common event that requires immediate repair to ensure the exact transfer of genetic information during cell division. A recent report found that Olaparib for germline BRCA-mutated metastatic pancreatic cancer significantly prolonged progression-free survival [40]. Human genome encodes approximately 450 genes that are involved in DDR, including BRCA1, BRCA2, PALB2, and ATM, whereas the mutation prevalence is relatively low [41–43]. The discovery of new genes related to DDR is critical for the development of novel therapeutic strategies. BEND members are transcriptional factors that participate in development. BEND4 is a new member of this family that is involved in cell fate. Methylation of BEND4 was found frequently in human pancreatic tumors in this study, and its expression is regulated by promoter region methylation. BEND4 methylation is associated with poor tumor differentiation and is a late-onset marker of PDAC. Moreover, BEND4 methylation is an independent poor prognostic factor for PDAC. BEND4 inhibits cell growth, induces apoptosis in PDAC cells, and suppresses PDAC cell xenograft growth in mice, indicating its function as a tumor suppressor. IP, mass spectrometry, and other techniques were used to understand the mechanism of BEND4 in pancreatic cancer. BEND4 is involved in NHEJ by binding to Ku80. Comet and NHEJ assays were performed to validate the role of BEND4 in NHEJ repair. With cisplatin treatment, increased p-DNAPKcs and XRCC4 levels were observed by re-expression of BEND4 in PDAC cells, which further suggesting the effect of BEND4 on NHEJ signaling. Synthetic lethality is a new strategy for cancer therapy, which targets the backup DNA repair pathway; however, limited known DDR gene mutations hinder its extensive application. Epigenetic silencing of tumor suppressor expression functionally joins Knudson’s two-hit event. Our previous studies found that epigenetic silencing of TMEM176A and NRN1 serve as an ATM or ATR inhibitor-sensitive marker [21,44]. Epigenetic silencing of BEND4 occurs frequently in pancreatic tumors, and BEND4 promotes NHEJ signaling. Therefore, we analyzed the synthetic lethal effect of an ATM inhibitor in BEND4 defect PDAC cells. Loss of BEND4 expression increased the sensitivity of PDAC cells to AZD0156 both in vitro and in vivo, suggesting that BEND4 methylation is an ATM inhibitor-sensitive marker (Fig.6). A plethora of studies are trying to use demethylating agents for cancer therapy without locus-specific selection [45,46]. Recent research has demonstrated that active DNA demethylation may disturb cell fate and DNA damage response [47,48]. It may be a better choice to target backup pathways based on aberrant changes in major components of cell fate determination and DDR signaling. In conclusion, BEND4 methylation is a late-onset and independent marker of poor prognosis in PDAC. BEND4 serves as a tumor suppressor that suppresses PDAC cell growth both in vitro and in vivo. BEND4 plays an important role in NHEJ signaling, and methylation of BEND4 is a synthetic lethal marker for ATM inhibitors.

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