Non-homologous end joining (NHEJ) is the major pathway responsible for the repair of ionizing radiation (IR)–induced DNA double-strand breaks (DSB), and correspondingly regulates the cellular response to IR. Identification of NHEJ inhibitors could substantially enhance the tumor radiosensitivity and improve the therapeutic efficiency of radiotherapy. In this study, we demonstrated a screening for NHEJ inhibitors using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and high-resolution melting (HRM) analysis. Because NHEJ is regarded as an error-prone mechanism, the NHEJ-mediated ligation of the site-specific DSB induced by Cas9 nuclease would eventually cause the mutation of the targeted sequence. Then, HRM analysis, a reliable and rapid assay for detecting sequence variation, was performed to evaluate the mutation efficiency of the targeted site. Validating analysis confirmed the NHEJ activities were positively correlated with the mutation frequencies. Next, an approved drug library containing 1,540 compounds was interrogated by using this screening strategy. Our results identified ouabain, a cardiotonic agent, and penfluridol, an antipsychotic agent, have the capacity to restrain NHEJ activity. Further experiments in vitro revealed the radiosensitizing effects of these compounds. Overall, we presented a cell-based screening for NHEJ inhibitors, which could promote the discovery of novel radiosensitizers. Mol Cancer Ther; 17(2); 419–31. ©2017 AACR.

See all articles in this MCT Focus section, “Developmental Therapeutics in Radiation Oncology.”

Induction of DNA double-strand breaks (DSB) is one of the fundamental mechanisms by which ionizing radiation (IR) and chemotherapeutic drugs exert their cytotoxic effects on tumor cells (1–3). DSBs could also be generated in response to a plethora of endogenous and exogenous genotoxic agents (4, 5). DSB is generally regarded as the most dangerous type of DNA lesion, because one single unrepaired DSB is sufficient to initiate programmed cell death (6). To counteract the threat of DSB and maintain genome integrity, cells have evolved an elaborate network to coordinate the lesion recognition, signal transmission, cell-cycle synchronization, and damage restoration (7). In eukaryotic cells, there are two major mechanisms of DSB repair: homologous recombination (HR) and non-homologous end joining (NHEJ; refs. 3, 8). HR is an error-free repair pathway and requires the intact sequence information from the sister chromatid; thus, its function is believed to restrict during S and G2 phases of the cell cycle when the homologous template is available. Unlike HR, NHEJ can operate throughout the cell cycle and work in a template-independent manner. NHEJ involves the direct processing and religation of the broken ends, which could result in deletion or insertion of the nucleotides. Although NHEJ is inaccurate, it is estimated to repair up to 85% of IR-induced DSBs in the mammalian cells (9–11).

The proficiency of DSB repair in tumor cells mainly, if not all, determines the sensitivity to IR-induced cytotoxicity. Therefore, targeting DSB repair pathway might hold the promise for improving the outcome of cancer radiotherapy (1–3, 12). Indeed, increasing efforts have been invested in discovering DSB repair inhibitors, particularly the small molecules that could suppress the effector proteins involved in HR and NHEJ. To name a few, NU7441 and CC-115, inhibitors of DNA-dependent protein kinase catalytic subunit (DNA-PKcs; refs. 13, 14), KU55933 and CP466722, inhibitors of ataxia-telangiectasia mutated (ATM; refs. 15, 16), olaparib and rucaparib, inhibitors of poly(ADP-riose) polymerase (17, 18), have been identified to inhibit DSB repair and substantially increase the antitumor activity of IR.

Currently, the targeted genome editing technology has brought a revolutionary surge in life science, especially the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, originally discovered as an adaptive immune system of bacteria and archaea to defend themselves against viral infection (19, 20). Cas9 nuclease monomer is able to induce a site-specific DSB by the direction of a customized single guide RNA (sgRNA); hence, desired gene knockin or knockout could be easily accomplished by DSB repair through HR or NHEJ. Here, we used the CRISPR/Cas9 system to produce a DSB in the gene loci of hypoxanthine guanine phosphoribosyltransferase (HPRT) or heparin-binding EGF-like growth factor (HBEGF), and analyze their mutation frequencies that could reflect the NHEJ activity. To rapidly and quantitatively evaluate the mutation frequencies, high-resolution melting (HRM) analysis, a simple PCR-based method for detecting DNA sequence variation, was performed following the NHEJ repair of the Cas9 nuclease-mediated DSB. HRM analysis relies on saturating dye, such as EvaGreen and LCGreen, instead of SYBR Green, and it is so sensitive that even a single-nucleotide variant could be identified (21, 22).

In this study, by taking advantage of CRISPR/Cas9 system and HRM analysis, a cost-effective screening method for NHEJ inhibitors has been developed. Validation of this novel method demonstrated the satisfactory sensitivity and reliability to quantitate the CRISPR/Cas9-induced mutation, as well as the suppressive effects of DNA-PKcs inhibitors on NHEJ activity. Next, an approved drug library was screened, and a collection of compounds were identified to impair NHEJ activity. Further experiments in vitro verified the suppressive effects on DSB repair and the radiosensitizing function of these drugs. Our results revealed ouabain, a cardiotonic agent, and penfluridol, an antipsychotic agent, might be potential candidates as radiosensitizers.

Cell culture and chemical reagents

HEK293T and HeLa cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) in September 2015. T98G cells were obtained from the ATCC in November 2015. All the cell lines were characterized by Genetic Testing Biotechnology Corporation (Suzhou, China) using short tandem repeat (STR) markers. These cells were cultured in DMEM supplemented with 10% FBS (Hyclone). NU7441 and KU-0060648 were purchased from Selleck Chemicals and dissolved in dimethyl sulfoxide (DMSO) to obtain a stock solution, stored at −20°C and diluted to the desired concentration in fresh medium immediately before use.

DNA vector preparation and transfection

Cas9/sgRNA-expressing vector construction kit was obtained from ViewSolid Biotech. The sgRNA against HPRT (sgHPRT) or HBEGF (sgHBEGF) were inserted into the prelinearized vector according to the manufacturer's instructions. The sequences of sgHPRT and sgHBEGF were shown in Fig. 1A and Supplementary Fig. S1A (23, 24). CRISPR/Cas9 plasmids were transfected into cells using Lipofectamine 2000 Reagent (Invitrogen).

Figure 1.

Quantitative detection of sgHPRT-induced mutation by HRM analysis. A, Graphical representation of the targeting of HPRT locus using CRISPR/Cas9 system. B, HEK293T cells were transfected with sgHPRT plasmid, and genomic DNA was extracted at different time points. T7E1 cleavage assay was performed to evaluate the targeted mutation. C, Melting data were collected and normalized as described in Materials and Methods (top), and a subtractive plot showed the relative fluorescence difference (ΔF) to the control curve (bottom). D, Different mutation efficiencies were induced by transfection of proportional sgHPRT and control plasmid. The data were presented in a three-dimension plot where x-axis showed the temperature, y-axis showed the ΔF, and z-axis showed the mutation efficiency. E, Correlation of the mutation efficiency with the maximum value of ΔF (ΔFmax).

Figure 1.

Quantitative detection of sgHPRT-induced mutation by HRM analysis. A, Graphical representation of the targeting of HPRT locus using CRISPR/Cas9 system. B, HEK293T cells were transfected with sgHPRT plasmid, and genomic DNA was extracted at different time points. T7E1 cleavage assay was performed to evaluate the targeted mutation. C, Melting data were collected and normalized as described in Materials and Methods (top), and a subtractive plot showed the relative fluorescence difference (ΔF) to the control curve (bottom). D, Different mutation efficiencies were induced by transfection of proportional sgHPRT and control plasmid. The data were presented in a three-dimension plot where x-axis showed the temperature, y-axis showed the ΔF, and z-axis showed the mutation efficiency. E, Correlation of the mutation efficiency with the maximum value of ΔF (ΔFmax).

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Genomic DNA extraction

For cells cultured in dishes, genomic DNA was extracted by MiniBEST Universal Genomic DNA Extraction Kit (Takara Bio). For cells cultured in 96-well plates, genomic DNA extraction was performed by using Chelex-100 ion exchange resin (Sigma). Briefly, cells in each well were lysed in 200 μL 5% (w/v) Chelex-100 suspension in deionized water containing 0.2 mg/mL proteinase K. Then the lysates were incubated at 56°C for 30 minutes, subsequently at 95°C for 10 minutes, and vigorously vortexed for 10 seconds. After centrifugation at 12, 000 × g for 5 minutes, the supernatant containing genomic DNA was collected for further analysis.

PCR and T7 endonuclease 1 (T7E1) assay

CRISPR/Cas9 plasmid–targeted site was amplified from 100 ng genomic DNA by using PCR Master Mix (Thermo Fisher Scientific). Primer sequences were: forward 5′-ATCCAATCAAATGTTTGTATCCTGT-3′; reverse 5′-CCCTTCAATGTTTACTTTGTTCTGG-3′. PCR thermal cycling conditions were 1 cycle at 95°C for 2 minutes; 35 cycles at 95°C for 30 seconds, at 53°C for 30 seconds, at 72°C for 40 seconds; and 1 cycle at 72°C for 10 minutes. Next, the PCR amplicons were denatured at 95°C for 5 minutes, and annealed to allow the heteroduplex formation by cooling down to 75°C for 20 minutes and to 15°C for 30 minutes. Finally, the heteroduplex products were digested by 5 units of T7E1 (ViewSolid Biotech) at 37°C for 30 minutes, and immediately subjected to 2% agarose gel electrophoresis.

HRM analysis

HRM analysis premix (containing hot start Taq DNA polymerase, dNTPs, EvaGreen dye, HRM PCR buffer and ROX reference dye) was obtained from Tiangen Biotech. Primer sequences for HPRT were: forward 5′-AGGTTATGACCTTGATTTATTTTGCA-3′; reverse 5′-AACAGCTGCTGATGTTTGAAATTAA-3′. Primer sequences for HBEGF were: forward 5′-TGGGCGGGTGTCTGATG-3′; reverse 5′-CAGCTGGTCCGTGGATACAGT-3′. The real-time PCR reaction mixture consisted of 20 ng genomic DNA, 200 nmol/L forward and reverse primers, and HRM analysis premix in a final volume of 20 μL. Real-time reaction was run in 96-well plates by using ViiA 7 Real-time PCR System (Applied Biosystems). The thermal cycling conditions were: 95°C for 2 minutes; 40 cycles at 95°C for 15 seconds, 60°C (HPRT locus) or 61°C (HBEGF locus) for 1 minutes. The PCR amplicons were then denatured at 95°C for 15 seconds and annealed at 60°C for 1 minute, followed by gradually temperature elevation from 60°C to 95°C by 0.02°C/second increment. The fluorescent data were continuously acquired during the slow DNA melting process.

HRM data analyses were performed as described previously (25, 26). The exponential background subtraction method was applied to normalized raw fluorescent data. Next, the melting curves were converted into a subtractive plot in which the relative fluorescence difference (ΔF) to a control curve against temperature was shown. Finally, the maximum value of ΔF (ΔFmax) was used to determine the mutation frequency of the CRISPR/Cas9 plasmid-induced NHEJ.

Approved drug library screening

Approved drug library containing 1,540 compounds was obtained from TargetMol and supplied as predissolved DMSO solution with the concentration of 10 mmol/L. HEK293T cells were transfected with CRISPR/Cas9 plasmid for 5 hours, and then the cells were trypsinized and seeded in 96-well plates. The approved drugs were added in a final concentration of 5 μmol/L. After 24 hours, the genomic DNA was extracted for HRM analysis. The quality of the screening was evaluated by Z' factor, defined as 1 – [3 (σc+ + σc-)/|μc+μc-|] (27), where σc+ and σc- are the SDs of the ΔFmax of the NU7441 and DMSO-treated samples, and μc+ and μc- are the means of the ΔFmax of the NU7441 and DMSO-treated samples.

Cell viability assay

After treated with indicated conditions, cells were incubated with 10 μL CCK-8 solution at 37°C for 2 hours. The absorbance was measured at 450 nm using a spectrophotometer.

NHEJ and HR reporter plasmid assay

NHEJ or HR activities were measured by using NHEJ or HR reporter plasmid as described previously (28, 29). Briefly, the plasmids were digested with I-SceI enzyme (New England Biolabs) and then cotransfected with pCMV-DsRed plasmids into HEK293T cells using Lipofectamine 2000 Reagent. Successful NHEJ or HR reconstitutes the functional GFP gene, and GFP-positive cells were quantified by flow cytometry (BD Biosciences). The red fluorescence served as an internal control for transfection efficiency. The ratio of GFP-positive cells to DsRed-positive cells was used to determine the NHEJ or HR activities.

Neutral comet assay

DSBs were analyzed by using the Trevigen Comet Assay Kit (Trevigen). HeLa cells were exposed to 8 Gy of X-ray (160 kV, 1.15 Gy/minute) irradiation by a biological research irradiator (Rad Source Technologies) and cultured for 12 hours. Next, 10 μL trypsinized cells were added to 100 μL molten LMA agarose, then immediately spread onto a CometSlide. The slides were placed at 4°C for 10 minutes in the dark and subsequently immersed in cold lysis solution for 30 minutes. The slides were then transferred to a horizontal electrophoresis chamber. Electrophoresis at 20 V for 25 minutes was performed. The slides were immersed in 70% ethanol for 5 minutes and stained with SYBR Gold. Images were captured by fluorescence microscope (Leica) and analyzed using CaspLab software (30).

Clonogenic survival assay

HeLa cells were plated in triplicate into 6-well plate and subjected to various doses of X-ray. Cells were further cultured for 14 days, and then fixed with methanol and stained with 1% crystal violet. Colonies consisting of more than 50 cells were counted as a single colony.

Immunofluorescence

HeLa cells were treated with 8 Gy of X-ray and cultured for 12 hours. Then, cells were fixed with 4% paraformaldehyde in the presence of 0.2% Triton X-100 for 20 minutes. Then cells were immunostained with antibody against phosphorylated histone H2A variant (γH2AX; Abcam) or p53-binding protein 1 (53BP1; Novus Biologicals).

Western blotting

Cell lysates containing equal amount of protein were subjected to SDS-PAGE, and electrically transferred to polyvinylidene difluoride membranes. Nonspecific binding was blocked with Tris-buffered saline containing 5% (w/v) BSA. The membranes were incubated with primary antibody for phosphorylated DNA-PKcs (S2056), DNA-PKcs, phosphorylated ATM (S1981), ATM, phosphorylated checkpoint kinase 2 (Chk2), Chk2 (Abcam), and β-actin (Santa Cruz Biochemicals) and then incubated with horseradish peroxidase–conjugated second antibody. β-Actin was used as a loading control. Immunoblotting signals were detected using an enhanced chemiluminescence method.

Statistical analysis

Data were expressed as mean ± SD. Analysis was performed by Student t test or one-way ANOVA following multiple comparisons using SPSS 18.0 software (SPSS Inc.). Results with P < 0.05 were considered statistically significant.

Quantification of CRISPR/Cas9–induced mutation by HRM analysis

HPRT is located on the X chromosome and has been intensively investigated as a surrogate marker of somatic mutations (31), meanwhile it is also a well-defined candidate gene for targeted genome editing (23, 32–34). Here, we first constructed a CRISPR/Cas9 plasmid containing the sgRNA sequence targeting the exon 2 of HPRT gene (Fig. 1A). After transfection of the plasmid into HEK293T cells, disruption of the HPRT gene could be validated by the overlapping peaks in the Sanger sequencing data, suggesting the error-prone NHEJ repair of the target site. To monitor the time-dependent manner of the Cas9 nuclease–induced mutation, genomic DNA was extracted at different time points after the CRISPR/Cas9 plasmid transfection. A length of 402 bp DNA sequence covering the targeted locus was amplified and subjected to T7E1 assay, which could digest the mismatched heteroduplex formed by the hybridization of the wild-type and mutant HPRT gene and generate two smaller fragments (approximate 130 bp and 270 bp). As shown in Fig. 1B, the mutation frequency reached peak levels at 24 hours posttransfection and decreased thereafter, which might due to the proliferation of the cells harboring a wild-type HPRT. Herein, the genomic DNA was collected 24 hours after transfection in the following assay.

Although T7E1 assay could estimate the targeted mutation efficiency (24), analysis of a large amount of samples by PCR, enzymatic digestion, and electrophoresis is time-consuming. HRM analysis is an efficient technology for large-scale sequence variant screening by measuring the changes of the Tm of the real-time PCR-amplified fragments. We obtained HRM data from the 157-bp amplicon containing the sgHPRT-targeted site, and the melting curves were converted into a subtractive difference plot to show the fluorescence difference to the references (control plasmid–transfected cells). As shown in Fig. 1C, HRM analysis clearly revealed the disparity of the melting curve between sgHPRT-treated cells and the control group. Parallel experiments, targeting HBEGF locus, were also performed, and the results of HRM analysis showed the same pattern as that of sgHPRT (Supplementary Fig. S1). In addition, the flexibility and validity of this mutation exploring approach were confirmed by using T98G and HeLa cells (Supplementary Fig. S2).

To further examine the robustness of HRM analysis as a quantitative approach, a range of sgHPRT plasmid (from 0 to 8 μg) were mixed with control plasmid to provide a total of 8 μg per aliquot and transfected into HEK293T cells (70% cell confluent in a 60-mm diameter culture dish). It was assumed that transfection of more sgHPRT plasmid could create more DSBs, thus lead to higher mutation efficiency at the targeted site via NHEJ repair. Figure 1D showed the gradual shift of the subtractive fluorescence difference (ΔF) corresponding to the mutation efficiencies induced by different proportions of CRISPR/Cas9 plasmids. Importantly, the logarithm of ΔFmax and the mutation efficiency displayed a strong positive linear correlation (Fig. 1E). These results indicate the usefulness of HRM analysis as a quantitative means to determine the CRISPR/Cas9 plasmid–induced mutation.

Establishment of a screening for NHEJ inhibitors

When different cell lines were transfected with different CRISPR/Cas9 plasmids, different values of ΔFmax were obtained (Fig. 1C; Supplementary Figs. S1B and S2A–S2C). This phenomenon appeared to be related to the variation of the transfection efficiency and chromatin accessibility of sgRNA and Cas9 nuclease at the targeted site, as well as the intrinsic capacity of HRM analysis to resolve the mutation frequency of a given PCR amplicon. An elevated ΔFmax value might improve the sensitivity and resolution of the assay. Therefore, the screening was performed using the sgHPRT plasmid and HEK293T cells, by which the highest ΔFmax value could be achieved (Fig. 1C; Supplementary Fig. S2C).

The experimental procedures of screening for NHEJ inhibitors were optimized and depicted in Fig. 2A. Five hours after the transfection of sgHPRT plasmid, HEK293T cells were subcultured into 96-well plates at a density of 2 × 104 cells per well. Simultaneously, pharmaceutical compounds were added into the individual wells of the 96-well plates, and incubated for 24 hours. Then genomic DNA was extracted by a rapid Chelex 100-based method, and targeted locus was amplified and subjected to HRM analysis.

Figure 2.

Design and validation of the screening for NHEJ inhibitors. A, Schematic depicting the screening procedures. HEK293T cells were transfected with sgHPRT plasmid for 5 hours, and subcultured into 96-well plates in the presence of the screening compounds. Twenty-four hours later, genomic DNA was extracted and subjected to HRM analysis. Two DNA-PKcs inhibitors, NU7441 and KU-0060648, were incubated with cells in 96-well plates. Three-dimensional plots show the HRM data of different concentrations of NU7441 (B) and KU-0060648 (D). Linear correlation between the concentrations and ΔFmax for NU7441 (C) and KU-0060648 (E).

Figure 2.

Design and validation of the screening for NHEJ inhibitors. A, Schematic depicting the screening procedures. HEK293T cells were transfected with sgHPRT plasmid for 5 hours, and subcultured into 96-well plates in the presence of the screening compounds. Twenty-four hours later, genomic DNA was extracted and subjected to HRM analysis. Two DNA-PKcs inhibitors, NU7441 and KU-0060648, were incubated with cells in 96-well plates. Three-dimensional plots show the HRM data of different concentrations of NU7441 (B) and KU-0060648 (D). Linear correlation between the concentrations and ΔFmax for NU7441 (C) and KU-0060648 (E).

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To confirm the feasibility of the screening workflow, we executed the experimental protocols using two DNA-PKcs inhibitors, NU7441 and KU-0060648, which have suppressive effects on NHEJ repair (35, 36). As shown in Fig. 2B and D, both of the two DNA-PKcs inhibitors declined the ΔFmax value in a concentration-dependent manner, implying the reduction of NHEJ-mediated mutations. A significant correlation between the concentrations and the logarithm of ΔFmax values were observed (Fig. 2C and E). The coefficients of determination R2 were 0.987 and 0.961 for NU7441 and KU-0060648, respectively. Taken together, these results suggest our screening approach is capable to identify NHEJ inhibitors.

Screening of an approved drug library for NHEJ inhibitors

We performed a screening of an approved drug library, including 1,540 compounds for NHEJ inhibitors. According to the pharmacologic actions, the compounds in the library can be divided into 10 different categories. Multiple colors and numbers (Category no. 1 to 10) were assigned to different categories and the composition of the drug library was demonstrated in a circle chart (Fig. 3A). To monitor the screening quality, each screening plate contained control plasmid–transfected cells as references, while sgHPRT plasmid-transfected cells treated with 1% DMSO or 2 μmol/L NU7441 were regarded as negative and positive controls, respectively. The final concentration of the screening compounds is 5 μmol/L. The range of Z' factor value was 0.498−0.860 (mean value, 0.645; 95% confidence interval, 0.592–0.699), suggesting the excellent screening quality. A full list of the screening results of ΔFmax is shown in Supplementary Table S1, and the sorted data are graphically plotted in Fig. 3B.

Figure 3.

Results of the screening of an approved drug library. A, The drugs were classified according to their pharmacologic action. The composition of the 10 categories was shown in the circle chart. B, Scatter plots of the screening results of 1,540 compounds. Tables of top hits of Category no. 7 (cardiovascular system; C) and Category no. 8 (nervous system; D).

Figure 3.

Results of the screening of an approved drug library. A, The drugs were classified according to their pharmacologic action. The composition of the 10 categories was shown in the circle chart. B, Scatter plots of the screening results of 1,540 compounds. Tables of top hits of Category no. 7 (cardiovascular system; C) and Category no. 8 (nervous system; D).

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Intriguingly, compounds from different categories showed dissimilar distribution of ΔFmax values, implying certain pharmacologic mechanisms probably underlie the NHEJ inhibitory capacity. Category no. 5 (antineoplastic agents) had the most NHEJ-inhibiting drugs (Supplementary Fig. S3B), because most of them were known as DNA-damaging chemicals that could inhibit DNA biosynthesis, or prevent the DNA double helix from being resealed, and thereby impede the NHEJ repair of the HPRT locus (37, 38). Remarkably, four cardiac glycosides from Category no. 7 (cardiovascular system) greatly reduced the ΔFmax values, that were digtoxin, digoxin, lanatoside C, and ouabain (Fig. 3C). In addition, ΔFmax values were decreased by a considerable number of compounds from Category no. 8 (nervous system), most of which were neuroleptics and antidepressants (Fig. 3D). Some potential NHEJ inhibitors from other categories were also listed in Supplementary Fig. S3A, S3C–S3E. Concerning the potency of the NHEJ-inhibiting effects, we next focused on cardiac glycosides and compounds from Category no. 8 in the following validating investigation.

Screening hits validation

To affirm the screening results, sgHPRT plasmid–transfected HEK293T cells were incubated with different concentration of the screening hits and HRM analysis were repeated, meanwhile cytotoxic effects of these compounds were evaluated by CCK-8 cell viability assay. We identified ouabain and penfluridol as effective NHEJ inhibitors, which could significantly decrease ΔFmax values under the non-cytotoxic concentrations (Fig. 4A and B; and J. Du, unpublished data), which were 0.1 μmol/L for ouabain and 5 μmol/L for penfluridol.

Figure 4.

Dose–response analysis of the screening hits. The chemical structures, and the cytotoxic and NHEJ-inhibiting effects of ouabain (A) and penfluridol (B) were shown in a dose-dependent manner. HEK293T cells were treated with different concentration of ouabain or penfluridol for 24 hours. C, Measurement of NHEJ and HR activities by using the NHEJ and HR reporter plasmids. HEK293T cells were transfected with I-SceI–linearized plasmids and incubated with ouabain or penfluridol for 24 hours. A DsRed-expressing plasmid was cotransfected as transfection control. Recircularized plasmids by NHEJ or HR have the intact GFP gene that can be assayed by flow cytometry. *, P < 0.05; **, P < 0.01 versus DMSO-treated group, n = 4.

Figure 4.

Dose–response analysis of the screening hits. The chemical structures, and the cytotoxic and NHEJ-inhibiting effects of ouabain (A) and penfluridol (B) were shown in a dose-dependent manner. HEK293T cells were treated with different concentration of ouabain or penfluridol for 24 hours. C, Measurement of NHEJ and HR activities by using the NHEJ and HR reporter plasmids. HEK293T cells were transfected with I-SceI–linearized plasmids and incubated with ouabain or penfluridol for 24 hours. A DsRed-expressing plasmid was cotransfected as transfection control. Recircularized plasmids by NHEJ or HR have the intact GFP gene that can be assayed by flow cytometry. *, P < 0.05; **, P < 0.01 versus DMSO-treated group, n = 4.

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Next, the effects of ouabain and penfluridol on NHEJ or HR activities were further verified by using engineered GFP gene–based plasmids (28, 29). The GFP gene in the NHEJ or HR reporter plasmid is inactivated by an additional exon or by mutations. And recognition sites for a rare-cutting I-SceI endonuclease are inserted into the GFP gene for the induction of DSBs. After digested with I-SceI, the linearized plasmids were transfected into HEK293T cells together with a DsRed plasmid for normalizing transfection efficiency. Successful repair of the I-SceI–induced DSBs by NHEJ or HR could recover the functional GFP gene. As shown in Fig. 4C, NU7441 inhibited NHEJ repair and shifted the balance of DSB repair toward HR. Both NHEJ and HR activities were suppressed by ouabain treatment, and only NHEJ seemed to be affected by penfluridol treatment.

Evaluation of ouabain and penfluridol as radiosensitizers

The suppressive effects on NHEJ repair of ouabain and penfluridol were merely verified at the CRISPR/Cas9 plasmid target site and the extrachromosomal I-SceI recognition sites. Next, we sought to determine whether these compounds could block the NHEJ repair of the IR-induced DSBs. HeLa cells were pretreated with 0.1 μmol/L ouabain or 5 μmol/L penfluridol for 2 hours, then cells were exposed to 8 Gy of X-ray, and the compounds were further incubated with the cells for 3 hours. Neutral comet assay was performed 12 hours postirradiation to measure the unrepaired DNA fragments. As shown in Fig. 5A, the nuclei of control cells displayed a spherical morphology, indicating the IR-induced DSBs were restored successfully. On the other hand, in the presence of ouabain or penfluridol, the amount of broken DNA was significantly increased, evidenced by the elevated DNA content in the comet tails (Fig. 5A and B). Consistently, colony formation assay distinctly showed the radiosensitizing effects of ouabain and penfluridol (Fig. 5C–F). Collectively, the NHEJ inhibitors revealed by our screening method could impair IR-induced DNA damage repair, hence aggravating the cytotoxicity of IR.

Figure 5.

Radiosensitizing effects of ouabain and penfluridol. A, HeLa cells were incubated with 0.1 μmol/L ouabain or 5 μmol/L penfluridol for 2 hours, then exposed to 8 Gy of X-ray, and further incubated with these drugs for 3 hours. Neutral comet assay was performed 12 hours postirradiation. B, The percentage of DNA content in the tails were analyzed; *, P < 0.05 versus DMSO-treated group, n = 3. Representative images of the colony formation assay of ouabain (C) and penfluridol (E) were shown. Treatment of ouabain (D) and penfluridol (F) increased the radiosensitivity of HeLa cells. *, P < 0.05 versus DMSO-treated group, n = 3.

Figure 5.

Radiosensitizing effects of ouabain and penfluridol. A, HeLa cells were incubated with 0.1 μmol/L ouabain or 5 μmol/L penfluridol for 2 hours, then exposed to 8 Gy of X-ray, and further incubated with these drugs for 3 hours. Neutral comet assay was performed 12 hours postirradiation. B, The percentage of DNA content in the tails were analyzed; *, P < 0.05 versus DMSO-treated group, n = 3. Representative images of the colony formation assay of ouabain (C) and penfluridol (E) were shown. Treatment of ouabain (D) and penfluridol (F) increased the radiosensitivity of HeLa cells. *, P < 0.05 versus DMSO-treated group, n = 3.

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Inhibition of DSB repair by ouabain and penfluridol

Finally, we assessed the nuclear γH2AX and 53BP1 foci formations, both of which were the markers of DSBs. HeLa cells were treated with X-ray and NHEJ inhibitors as that of the neutral comet assay. As shown in Fig. 6A, the γH2AX and 53BP1 foci of ouabain or penfluridol-treated cells failed to resolve after 12 hours postirradiation compared with the control cells. Western blotting analysis further revealed that ouabain or penfluridol treatment significantly restrained the expression of phosphorylated DNA-PKcs induced by IR (Fig. 6B). However, these NHEJ inhibitors did not seem to interact directly with DNA-PKcs, because they failed to show any inhibitory effects on DNA-PK kinase activity in vitro (Supplementary Fig. S4). Moreover, they might not suppress the activation of ATM in response to IR, as well as its downstream target, Chk2. These data suggest that ouabain and penfluridol might interrupt DSB repair by preventing DNA-PKcs from being activated.

Figure 6.

Impairment of DSB repair by ouabain and penfluridol. A, HeLa cells were incubated with 0.1 μmol/L ouabain or 5 μmol/L penfluridol for 2 hours, then exposed to 8 Gy of X-ray, and further incubated with these drugs for 3 hours. Twelve hours postirradiation, γH2AX and 53BP1 foci formations were visualized by immunofluorescent staining. B, Four hours postirradiation, the expressions of phosphorylated DNA-PKcs, ATM, and Chk2 were analyzed by Western blotting. *, P < 0.05 versus DMSO-treated group, n = 3.

Figure 6.

Impairment of DSB repair by ouabain and penfluridol. A, HeLa cells were incubated with 0.1 μmol/L ouabain or 5 μmol/L penfluridol for 2 hours, then exposed to 8 Gy of X-ray, and further incubated with these drugs for 3 hours. Twelve hours postirradiation, γH2AX and 53BP1 foci formations were visualized by immunofluorescent staining. B, Four hours postirradiation, the expressions of phosphorylated DNA-PKcs, ATM, and Chk2 were analyzed by Western blotting. *, P < 0.05 versus DMSO-treated group, n = 3.

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Highlighting the essential role of the NHEJ pathway to resolve highly cytotoxic DSBs, identification of novel NHEJ inhibitors holds great promise for augmenting cancer therapeutic efficiency of genotoxic treatment (2, 3). Here, we designed and carried out an NHEJ inhibitor screening, which mainly involved the following two steps: first create a genomic specific DSB by CRISPR/Cas9 system, second evaluate the mutation frequencies of the targeted site by HRM analysis. Our results affirmed a positive correlation between NHEJ activities and mutation frequencies of the targeted site. By means of this approach, several hits were emerged as novel NHEJ inhibitors. The most noteworthy result was the inhibition of NHEJ by cardiac glycosides and a number of compounds affecting neurotransmitters. Further examination revealed ouabain and penfluridol had the suppressive effects on IR-induced DSB repair, thus increased the radiosensitivity of HeLa cells. Preliminary evidences indicated the inhibitory effects of these compounds on the DNA-PKcs phosphorylation induced by IR exposure.

Owing to the complexity of the HR and NHEJ pathways, several cell-based systems have been developed to screening the novel DSB repair inhibitors. In these approaches, DSBs were mainly introduced by IR or rare cutting meganuclease I-SceI (39–42), and the consequences of DSB repair were deduced by the results of neutral single-cell electrophoresis (39), colony formation assay (40), expressions of DSB repair-dependent fluorescent protein (41) or fluorescent staining of HR- and NHEJ-specific foci (42). Because NHEJ is the predominant DSB repair pathway, there is rationale in monitoring its activity to facilitate the screening of radiosensitizers. Theoretically, higher mutation frequencies at the DSB sites correlate the higher levels of NHEJ activities. But it is unachievable to locate the NHEJ-mediated mutational sites in the genome because of the stochastic DSBs induced by IR.

By virtue of the exquisite specificity of Cas9-induced DSBs, the repair consequences of the targeted loci can be analyzed directly and quantitatively. For example, a sequence trace decomposition strategy has been developed for the identification of short sequence deletions and insertions (43), and it has been reported that a digital PCR-based assay enabled the simultaneous detection of HR and NHEJ of the nuclease-induced DSBs (44). It is not surprising that both of these approaches generally relied on PCR amplification of the targeted sequences. We also used a PCR-based method termed HRM analysis, which involves only about 30 minutes of automatic fluorescence acquisition after the PCR reaction in the presence of a suitable saturating dye (21, 22). And mutation frequencies assessed by subtractive transformation of the melting fluorescent data could distinguish the altered levels of NHEJ activities. Treatment of NU7441 and KU-0060648, known to block NHEJ repair via DNA-PKcs inhibition, exhibited a dose-dependent reduction of the mutation frequencies, reflecting by the ΔFmax values. This suggests that NHEJ inhibitory capacity could be deciphered by ΔFmax assessment. This rapid and closed-tube technique enabled the screening of a large number of samples for NHEJ inhibitors. Although we used only HEK293T cells for the screening study, this approach can be applied to other cell lines, such as the tumor cells with a given genetic background, therefore, the cell type–specific NHEJ inhibitors or novel NHEJ mechanisms might be disclosed.

A limitation of our screening assay is that the status of HR repair remains elusive. Because HR repair preserves the genomic integrity, HRM analysis is unable to detect the unaltered sequence information. Indeed, Cas9-induced DSBs have an eventual tendency toward NHEJ repair, because if a given DSB is repaired by HR, the locus will be continuously recut by Cas9 nuclease until the protospacer adjacent motif or sgRNA-binding sequences are disrupted through NHEJ repair (45). HR repair directed by a third donor template may prevent the cycles of cleavage and ligation; however, the donor template is extrachromosomal and dominant in quantity, whereas the endogenous template for HR repair is only on the equivalent position of the sister chromatid during the S and G2 phases, hence the HR activity is difficult to estimate in the normal context of chromatin. By using a GFP-based plasmid religation assay, it was showed that ouabain could inhibit HR activities, suggesting that the NHEJ inhibitors revealed by our screening assay might also affect HR repair through frustrating the upstream signaling events responsible for both NHEJ and HR.

To better understand the NHEJ-inhibitory capacity of the screening drugs, the data were generally classified by their pharmacologic activities. Consistent with a previous screening study (42), our results suggest that cardiac glycosides might serve as promising DSB repair inhibitors. Cardiac glycosides are well-established Na+/K+-ATPase inhibitors and have been widely used in the treatment of congestive heart failure and atrial arrhythmia for a long time (46). Most notably, numerous reports have suggested that cancer cells are susceptible to cardiac glycosides and several derivatives have entered clinical trials either as monotherapy or combined with conventional regimens (47–50). It has been also uncovered that cardiac glycosides impaired DNA damage repair and diminish radioresistance of cancer cells (51–54). Given the relative low cytotoxicity, ouabain might be therapeutically favorable radiosensitizer, and the effects of ouabain should be further investigated in xenograft models. Although the detailed molecular mechanisms of cardiac glycoside–induced radiosensitization have not been fully understood, our results suggest that ouabain could prevent DNA-PKcs activation, thus inhibit NHEJ activities.

Another kind of NHEJ inhibitors revealed by the screening were the drugs for the treatment of psychiatric disorders. Among them, penfluridol appeared to be a potential candidate for radiosensitization. Interestingly, besides the NHEJ-inhibitory effects displayed by our results, multiple mechanisms of antitumor function of penfluridol have been reported, including production of reactive oxygen species, suppression of metastasis, and induction of autophagy-mediated apoptosis (55–57). In addition, trifluoperazine, a member of the phenothiazines sharing the inhibitory activity against dopamine D2 receptor with penfluridol, has NHEJ-suppressive capacity via DNA-PK inhibition (58, 59). This strongly suggests that antipsychotics could be readily repurposed as radiosensitizers.

In conclusion, CRISPR/Cas9 system, the highly efficient and versatile tool for genome editing, functioned as a pair of precise scissors in the screening assay. Then, the DNA breakage was sewn together by the error-prone NHEJ. Finally, HRM analysis, the quality inspector, rigidly discriminated the patchy DNA. Through screening an approved drug library, several drugs were identified as potential NHEJ inhibitors, and the radiosensitizing effects of ouabain and penfluridol were clearly demonstrated in vitro. Despite the fact that the molecular mechanisms exquisitely governing NHEJ repair have not been completely elucidated, the robust and flexible CRISPR/Cas9–based NHEJ inhibitor screening strategy might pave the way for exploring novel radiosensitizers. Mechanistic investigation of the new NHEJ inhibitors will provide new insights into how cells orchestrate the DNA damage response and allow the identification of vulnerable targets for cancer therapy. Importantly, it is necessary to put more efforts to optimize the inhibitory specificity against NHEJ and minimize the undesirable pharmacologic actions. Detailed exploration of the activities of the NHEJ inhibitor analogues and derivatives might represent a feasible solution for successful translation of these inhibitors into clinical applications.

No potential conflicts of interest were disclosed.

Conception and design: J. Yu, F. Liu

Development of methodology: J. Du, H. Zhang

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J. Du, J. Shang, F. Chen

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J. Du, Y. Zhang, N. Yin, H. Zhang, J. Yu

Writing, review, and/or revision of the manuscript: J. Yu, F. Liu

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): T. Xie

Study supervision: F. Liu

This work was supported by Natural Science Foundation of China (grant numbers 31270897 and 81271682, to F. Liu; and 81202149, to J. Yu).

We would like to thank Dr. Vera Gorbunova (University of Rochester) and Dr. Mao Zhiyong (Tongji University) for kindly providing the NHEJ and HR reporter plasmids. We acknowledge the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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