Eight ESCC cells were used in this study, including KYSE30, KYSE140, KYSE150, KYSE180, KYSE450, KYSE510, KYSE520, and colo680n. These cells were derived from primary ESCC and cultured in RPMI-1640 medium (Gibco, No. 31800089) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. HEK293 cell line was used for lentivirus production and was maintained in DMEM (Gibco, No. 12100061).

Esophageal tissue samples, including 142 esophageal dysplasia (ED) and 1007 primary ESCC tissue samples, were collected from the Chinese PLA General Hospital. These samples were not subjected to chemo-radiotherapy prior to surgical resection. Sample collection adhered to the guidelines authorized by the Chinese PLA General Hospital Institutional Review Board (IRB number: 20090701-015). Tumor classification was performed using the TNM staging system (AJCC 8^th).

For cell treatment, 5-aza-2’-deoxycytidine (5-aza, Sigma-Aldrich, No. A3656) was added to RPMI-1640 medium at a concentration of 2 μmol/L. RNA isolation and semi-quantitative RT-PCR procedures were conducted in accordance with our previously described methods[28]. RT-PCR primers for SLFN11 were designed as follows: 5′-AACGCCCGATAACCTTCACA-3′ (forward) and 5′-CTAAGGGGAGGCCCACTAGA-3′ (reverse). To evaluate the quality of cDNA, GAPDH was amplified for 25 cycles[28].

The methods for DNA extraction, bisulfite modification, and methylation-specific PCR (MSP) were described as previously[28]. The primer sequences for MSP targeting SLFN11 were designed as below: 5′-TTTGGAAGGTGGGATCGTAGGTATC-3′ (MF) and 5′-ACCCAAACAACTATCGACTCCTACG-3′ (MR); 5′-TATTTGGAAGGTGGGATTGTAGGTATT-3′ (UF) and 5′-AAACCCAAACAACTATCAACTCCTACA-3′ (UR).

The PCDH-CMV-MCS-puro vector was employed to construct a full-length cDNA (GenBank accession number: NM_91607) expression vector for human SLFN11. The lentiviral supernatant was obtained after transfection into HEK293 cells by lipofectamine 3000 growing for 48 h and 72 h following the instructions of the manufacturer (Invitrogen, No. L3000008). Subsequently, the lentiviral supernatant was added into the culture medium. Puromycin (MCE, No. HY-15695) was then used to screen for SLFN11 expressing KYSE30 (2.5 μg/mL) and KYSE450 (1.0 μg/mL) cells 48 h after lentiviral transfection, with a duration of 3 d for the screening process. The monoclonal SLFN11 expression cells were obtained through limited dilution in 96 well plates, and further validated via Western blot.

The CRISPR/Cas9 technique was employed to generate SLFN11 deleted KYSE510 cells. Single guide RNA (sgRNA) was designed using Guide Design Resources from http://crispr.mit.edu. The LentiCRISPRv2 vector was utilized to construct the CRISPR/Cas9 knockout system with sgRNA1 5′-CACCGCAGCCTGACAACCGAGAAAT-3′ and sgRNA2 5′-AAACATTTCTCGGTTGTCAGGCTGC-3′. SLFN11 knockout KYSE510 cell clones were selected following above procedure with puromycin (0.5 μg/mL), and validated by DNA sequencing and Western blot.

Anti-SLFN11 (No. 34858), anti-p-ATM Ser1981 (No. 13050S), anti-ATR (No. 2790S), anti-p-ATR Ser428 (No. 2853S), and anti- γ-H2AX Ser139 (No. 9718) antibodies were ordered in the Cell Signaling Technology. Anti-DNA-PKcs (No. 200618-6D1), anti-p-DNA-PKcs Ser2056 (No. 380800), anti-CHK2 (No. R23921), anti-p-CHK2 Thr68 (No. 240766) and anti-CHK1 (No. 380200) antibodies were ordered from ZENBIO. Anti-ATM (No. HX12561) and anti-XRCC4 (No. HX19688) antibodies were obtained from Huaxingbio. Anti-β-actin (No. 66009-1-Ig) and anti-p-CHK1 Ser345 (No. GTX100065) antibodies were from Proteintech and Genetex, respectively. The procedures were performed as described previously[28].

For assessment of the sensitivity of ESCC cells to different reagents, MTT assay was employed. Cells were grown in 96-well plates with 2000 cells for each well, and treatment was performed after seeding for 24 h. The IC₅₀ was evaluated by treatment with gradient dilution of cisplatin (Selleck, No. S1166) for 48 h. The sensitivity of three different cell lines to AZD0156 (MCE, No. HY-100016), an ATM inhibitor, was tested using DNA damaging cell models induced by low dose cisplatin with gradient dilution of AZD0156 for 48 h. GraphPad Prism software was employed for data analysis.

Colony formation assays were performed using 35 mm dishes. For chemosensitivity detection, KYSE30, KYSE450, and KYSE510 cells were inoculated with the density of 3 × 10³ cells each well. They were cultured in medium supplemented with 1 μmol/L and 2 μmol/L of cisplatin for 24 h. Then the medium was changed for growing 14 d. To assess the impact of SLFN11 on DDR, ESCC cell models induced with 0.05 μmol/L cisplatin were treated with 0.20 μmol/L AZD0156. The medium was changed after 24 h of growth for a total of 14 d. The cell colonies were then fixed with 75.0% ethanol and stained with 0.2% crystal violet (Solarbio, No. G1063) for 30 min. The relative efficiency of colony formation was determined by normalizing the colony areas to the control. This process was repeated in three independent experiments.

Four-week-old BALB/c nude mice weighing around 20 g were procured from SPF Company (Beijing, China) and housed under conditions that met standard pathogen-free requirements. SLFN11 silenced and re-expressed KYSE30 cells (6 × 10⁶ cells in 0.15 mL sterilized PBS) were injected subcutaneously into the mice. A caliper was used for tumor size measuring. The volume was calculated as the formula: V = length × width²/2. Once the average tumor volume reached 50 mm³, both SLFN11 unexpressed and re-expressed xenograft mice were randomly divided into four groups (six mice per group). Mice were administrated with 0.9% saline, cisplatin (2 mg/kg), AZD0156 (30 mg/kg) or combined cisplatin and AZD0156 for every three days in different groups. Cisplatin was administered via intraperitoneal injection, and AZD0156 was administered orally. The Animal Ethics Committee of the Chinese PLA General Hospital (approval number: 2022-X18-72) approved the animal experimental procedures, with strict adherence to protocols aimed at minimizing the discomfort of mice.

SPSS 21.0 (NY, United States) and GraphPad Prism 8.0 (CA, United States) were used for statistical analysis. P < 0.05 was regarded significantly difference. The association between methylation status and clinical-pathological factors was examined with the χ² test. Quantitative data were described as mean ± SD and analyzed using the student’s two-tailed t test.

To assess the relation between the expression of SLFN11 and its methylation status in the promoter region, 171 cases of ESCC data were extracted from The Cancer Genome Atlas (TCGA) database (http://xena.ucsc.edu/). The reverse association between SLFN11 mRNA levels and CpG sites (cg13341380, cg18108623, cg05224998, cg18608369, cg01348733, cg14380270, cg26573518, and cg05504685, all P < 0.05) methylation around the transcript start sites was observed, as depicted in Figure 1A.

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Figure 1 The expression and methylation status of Schlafen-11 in esophageal squamous cell carcinoma. A: Correlation analysis between Schlafen-11 (SLFN11) mRNA expression and methylation levels of 8 CpG sites around transcription start site retrieved from TCGA datasets (n = 171). Scatter plots shown inverse relevance of SLFN11 expression and methylation status in representative CpG sites (cg18108623, cg13341380, and cg26573518); B: Semi-quantitative RT-PCR showing the expression of SLFN11 in esophageal squamous cell carcinoma (ESCC) cell lines before and after treatment with 5-aza-2’-deoxycytidine (5-aza); C: Detection of the methylation status of SLFN11 by methylation-specific polymerase chain reaction (MSP) in ESCC cells; D: Representative MSP results of SLFN11 in esophageal tissue samples. TSS: Transcription start site; KYSE30, KYSE140, KYSE150, KYSE180, KYSE450, KYSE510, KYSE520, and colo680n are ESCC cells; 5-aza: 5-aza-2’-deoxycytidine; GAPDH: Internal control of RT-PCR; H₂O: Double distilled water; (-): Absence of 5-aza; (+): Administration of 5-aza; U: Unmethylated alleles; M: Methylated alleles; IVD: In vitro methylated DNA as methylation control; NL: Normal peripheral lymphocytes DNA as unmethylation control; ED: Esophageal dysplasia; EC: Esophageal squamous cell carcinoma.

The expression and promoter region methylation were detected by semi-quantitative RT-PCR and MSP. SLFN11 was highly expressed in KYSE510 and colon680n cells whereas SLFN11 was silenced in KYSE520, KYSE450, KYSE180, KYSE150, KYSE140, and KYSE30 cell lines (Figure 1B). SLFN11 was unmethylated in KYSE510 and colo680n cells, and completely methylated in KYSE520, KYSE450, KYSE180, KYSE150, KYSE140, and KYSE30 cell lines (Figure 1C), indicating the correlation between loss of expression and promoter region hypermethylation. To further validate the regulatory role of DNA methylation in SLFN11 expression, 5-aza, an inhibitor of DNA methyltransferase, was employed. The induction of SLFN11 expression by 5-aza was observed in methylated ESCC cells (Figure 1B), suggesting that DNA methylation regulates the expression of SLFN11.

To explore the epigenetic changes of SLFN11 during esophageal carcinogenesis, methylation status was detected in 142 cases of ED and 1007 cases of primary ESCC tissues. Methylation of SLFN11 was found in 9.15% (13/142) of ED and 25.62% (258/1007) of ESCC samples, indicating a progressive tendency during carcinogenesis (Figure 1D). Furthermore, SLFN11 methylation was significantly associated with poor tumor differentiation and tumor size (both P < 0.05, Table 1). However, no significant association was observed between methylation and age, gender, smoking, alcohol consumption, TNM stage or lymph node metastasis (Table 1).

To evaluate the impact of SLFN11 on cisplatin sensitivity, we utilized SLFN11 epigenetic silenced and knockout ESCC cell models. As shown in Figure 2A, the IC₅₀ value of cisplatin was 12.45 μmol/L ± 1.16 μmol/L vs 7.03 μmol/L ± 0.69 μmol/L in KYSE30 cells (P < 0.01) and 8.57 μmol/L ± 0.87 μmol/L vs 4.30 μmol/L ± 0.71 μmol/L in KYSE450 cells (P < 0.01) before and after re-expressing SLFN11, indicating that SLFN11 increased the sensitivity of ESCC cells to cisplatin. In SLFN11 highly expressed KYSE510 cells, the IC₅₀ value was 0.79 μmol/L ± 0.12 μmol/L vs 1.93 μmol/L ± 0.09 μmol/L (P < 0.001) before and after deletion of SLFN11, demonstrating that deletion of SLFN11 reduced sensitivity to cisplatin.

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Figure 2 Schlafen-11 was correlated with chemoresistance to cisplatin in esophageal squamous cell carcinoma cells. A: MTT assay showing the sensitivity to cisplatin in KYSE30 and KYSE450 cells before and after re-expression of Schlafen-11 (SLFN11), and in KYSE510 cells before and after knockout of SLFN11, data are representative of three independent experiments; B: Representative colony formation assay under the treatment of 1 μmol/L and 2 μmol/L cisplatin for 14 d in esophageal squamous cell carcinoma cells. Each experiment was repeated in triplicate. The average normalized colony efficiency was indicated by a bar diagram. Statistical significance was analyzed by t test (^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001). KO: Knockout; SLFN11: Schlafen-11.

To further investigate the impact of SLFN11 on cisplatin sensitivity, colony formation assay was performed. Before and after the restoration of SLFN11 expression in KYSE30 cells, the normalized colony efficiency was 73.59% ± 12.51% vs 55.73% ± 12.18% (1 μmol/L cisplatin), and 38.52% ± 2.13% vs 9.49% ± 2.25% (2 μmol/L cisplatin), respectively (both P < 0.05, Figure 2B). The normalized colony efficiency was 77.86% ± 8.26% vs 20.79% ± 1.54% (1 μmol/L cisplatin) and 38.15% ± 9.95% vs 9.16% ± 1.79% (2 μmol/L cisplatin) in KYSE450 cells before and after the expression of SLFN11, respectively (both P < 0.05, Figure 2B). In KYSE510 cells, the normalized colony efficiency was 25.30% ± 5.40% vs 76.24% ± 4.34% (1 μmol/L cisplatin) and 5.15% ± 1.55% vs 17.92% ± 3.05% (2 μmol/L cisplatin) before and after knockout of SLFN11, respectively (both P < 0.01, Figure 2B). Above results validated the chemo-sensitive role of SLFN11 in human ESCC.

To elucidate the mechanism of SLFN11 in ESCC, low dose cisplatin induced DNA damage cell models were employed. The majority of chemotherapeutic reagents primarily induce DNA double strand breaks (DSBs), which are the most harmful DNA lesions. DSBs are repaired through two major signaling pathways, namely homologous recombination repair (HR) and non-homologous end-joining (NHEJ). Subsequently, the expression levels of ATM, ATR, p-CHK1, p-CHK2, DNAPKcs, and XRCC4 were examined in SLFN11 expressed and unexpressed ESCC cells. Under the treatment of low dose cisplatin, the expression of p-DNAPKcs and XRCC4 was elevated in KYSE30 and KYSE450 cells by re-expressing SLFN11, indicating that the NHEJ pathway was activated by SLFN11 (Figure 3A). The involvement of SLFN11 in NHEJ signaling pathway was then further demonstrated through SLFN11 knockout in KYSE510 cell lines (Figure 3A). As shown in Figure 3B and C, the expression of p-ATR and p-CHK1 was elevated, while the expression of p-ATM and p-CHK2 was suppressed after re-expression of SLFN11 in KYSE30 and KYSE450 cell lines, suggesting that SLFN11 activates ATR/CHK1 signaling and inhibits ATM/CHK2 signaling in ESCC cells. These results were further validated by deletion of SLFN11 in KYSE510 cell lines.

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Figure 3 The role of Schlafen-11 on DNA damage repair network in esophageal squamous cell carcinoma cells. A: The levels of Schlafen-11 (SLFN11), p-DNAPKcs, DNAPKcs, and XRCC4 under the treatment of 1 μmol/L cisplatin for 12 h and 24 h in KYSE30 and KYSE450 cells before and after SLFN11 re-expression and in KYSE510 before and after SLFN11 knockout; B: The effects on ATR/CHK1 signaling in KYSE30 and KYSE450 cells after SLFN11 re-expression and in KYSE510 cells after SLFN11 knockout under the treatment of 1 μmol/L cisplatin for 12 h and 24 h; C: The effects on ATM/CHK2 signaling in KYSE30 and KYSE450 cells after the restoration of SLFN11 and in KYSE510 cells after SLFN11 knockout under the treatment of 1 μmol/L cisplatin for 12 h and 24 h. β-actin: Internal control.

SLFN11 is regulated by promoter region methylation, and its expression suppresses ATM signaling. Then, the effects of AZD0156 were assessed in SLFN11 deficient ESCC cells. As shown in Figure 4A, the IC₅₀ value was 5.91 μmol/L ± 1.35 μmol/L vs 13.04 μmol/L ± 2.54 μmol/L (P < 0.05) in KYSE30 cells and 5.79 μmol/L ± 0.83 μmol/L vs 10.77 μmol/L ± 1.08 μmol/L (P < 0.01) in KYSE450 cells before and after re-expressing SLFN11, respectively, demonstrating that epigenetic silencing of SLFN11 sensitizes ESCC cells to AZD0156. The IC₅₀ value was 3.52 μmol/L ± 0.48 μmol/L vs 1.62 μmol/L ± 0.29 μmol/L (P < 0.01) in SLFN11 highly expressed and deleted KYSE510 cells, respectively (Figure 4A), further supporting the notion that SLFN11 deficiency sensitizes ESCC cells to AZD0156.

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Figure 4 Schlafen-11 silencing sensitized esophageal squamous cell carcinoma cells to AZD0156. A: MTT assay to evaluate the sensitivity of esophageal squamous cell carcinoma (ESCC) cells to AZD0156 under the treatment of low dose cisplatin, data are representative of three independent experiments; B: Representative colony formation assay under the treatment of low dose cisplatin, AZD0156 and combined mini-dose cisplatin with AZD0156 for 14 d in ESCC cells, each experiment was repeated in triplicate, the average normalized colony efficiency was indicated by a bar diagram, statistical significance was analyzed by t test (^bP < 0.01, ^cP < 0.001, ^dP < 0.0001); C: The protein levels of ATM/CHK2 signaling and γ-H2AX in ESCC cells under the treatment of low dose cisplatin, AZD0156 and combined cisplatin with AZD0156 in ESCC cells. KO: Knockout; β-actin: Internal control.

To further evaluate the sensitivity of SLFN11 to AZD0156, colony formation assay was applied. Under the treatment of low dose cisplatin and AZD0156, the normalized colony efficiency was 8.67% ± 1.93% vs 30.36% ± 5.01% (P < 0.001) in KYSE30 cells and 10.72% ± 1.78% vs 27.94% ± 7.76% (P < 0.01) in KYSE450 cells before and after re-expressing SLFN11, hinting that the relative colony formation efficiency was inhibited by SLFN11 (Figure 4B). The relative colony formation efficiency was 30.53% ± 8.56% vs 12.18% ± 1.42% (P < 0.01) in KYSE510 cells before and after deleting SLFN11, respectively, providing further evidence for the role of SLFN11 in sensitizing AZD0156. The levels of γ-H2AX, a DNA damage marker, were detected in these cells with or without SLFN11 expression (Figure 4C). The levels of γ-H2AX were elevated by administration of AZD0156 in ESCC cells without SLFN11, further validating above results.

To further investigate the impact of SLFN11 on ATM inhibitor in vivo, KYSE30 cell xenograft models were employed. The tumor volume and weight in control groups, which did not receive cisplatin or AZD0156 treatment, were normalized as 100%. When subjected to low dose cisplatin, AZD0156 or a combination of both, the relative normalized tumor volumes were 84.33% ± 8.87% vs 84.83% ± 5.33% (P > 0.05), 26.07% ± 6.00% vs 57.90 ± 9.92% (P < 0.0001), and 12.43% ± 3.81% vs 31.95% ± 4.17% (P < 0.0001) in SLFN11 silenced and re-expressed xenografts, respectively (Figure 5C and D).

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Figure 5 Invivo efficacy of sensitivity of Schlafen-11 deficient cells to AZD0156. A: Schematic diagram of xenograft mouse models generation and medication; B: Tumors derived from Schlafen-11 (SLFN11) unexpressed and re-expressed KYSE30 cell xenografts in mice treated as indicated; C: Growth curves of xenograft tumors treated with 2 mg/kg cisplatin, 30 mg/kg AZD0156 and combination of 2 mg/kg cisplatin plus 30 mg/kg AZD0156; D and E: Normalized tumor volume and weight in KYSE30 unexpressed and re-expressed xenografts under different modes of treatment, statistical significance was analyzed by t test (^dP < 0.0001); F: Schematic model of synthetic lethality of SLFN11 with ATM inhibitor in esophageal squamous cell carcinoma cells.

The results showed that in the cisplatin, AZD0156, and combined cisplatin with AZD0156 treatment groups, the normalized tumor weight was 92.62% ± 2.36% vs 90.25% ± 1.65% (P > 0.05), 37.98% ± 2.13% vs 64.93% ± 4.20% (P < 0.001), and 17.67% ± 1.97% vs 40.92% ± 1.56% (P < 0.0001) in SLFN11 silenced and re-expressed xenografts, respectively (Figure 5E). The combined cisplatin and AZD0156 treatment led to a significant reduction in tumor volume and weight in SLFN11 silenced KYSE30 cell xenografts, demonstrating that the loss of SLFN11 expression increased the sensitivity of ESCC cells to AZD0156 in vivo.