Olaparib (cat. no. HY-10162), veliparib (cat. no. HY-10129), talazoparib (cat. no. HY-16106) and AZD6738 (cat. no. HY-19323) were purchased from MedChemExpress. Olaparib and veliparib were dissolved in 100% dimethyl sulfoxide (DMSO; cat. no. A3672,0250; PanReac AppliChem; ITW Reagents Division) to create a 100 mM stock solution, while talazoparib and AZD6738 were dissolved in DMSO to a concentration of 50 mM. The drug stocks were kept at −80°C until used.

The MMNK-1 (cat. no. JCRB1554; immortalized human cholangiocyte) (29) and HuH-28 (cat. no. JCRB0426; cholangiocarcinoma) (30) cell lines were obtained from the Japanese Collection of Research Bioresources Cell Bank (JCRB), while the TFK-1 (cell no. RCB2537; cholangiocarcinoma) cell line (31) was received from the RIKEN BioResource Center. SiSP-K01 and SiSP-K05 primary cell lines were a gift from Professor Seiji Okada of Kumamoto University (Kumamoto, Japan) (32). SiSP-K01 was derived from a 64-year-old female with intrahepatic, moderately differentiated CCA and was used at passage 51. SiSP-K05 was derived from another female patient, age 53 years, with intrahepatic, moderately differentiated CCA and was also used at passage 51. The origin of each cell line is summarized in Table SI. The experimental protocol was approved by the Human Research Ethics Committee of the Faculty of Medicine, Ramathibodi Hospital, Mahidol University (Bangkok, Thailand; approval no. MURA2023/155).

The MMNK-1 and TFK-1 cell lines were maintained in DMEM/F12 (cat. no. 12400024; Gibco; Thermo Fisher Scientific, Inc.) and RPMI-1640 medium (cat. no. 11875093; Gibco; Thermo Fisher Scientific, Inc.), respectively. The HuH-28, SiSP-K01 and SiSP-K05 cell lines were cultured in DMEM (cat. no. 12800017; Gibco; Thermo Fisher Scientific, Inc.). All cell lines were supplemented with 1% penicillin/streptomycin (cat. no. 15140122; Invitrogen; Thermo Fisher Scientific, Inc.) and 10% fetal bovine serum (FBS; cat. no. ES-009-B; Merck KGaA), with the exception of MMNK-1, which was supplemented with 15% FBS. The cells were incubated at 37°C with 5% CO₂.

The genetic profiles of the TFK-1 and HuH-28 cell lines were obtained by next-generation sequencing, as described in Jamnongsong et al (33). The genetic profiles of SiSP-K01 (https://www.ncbi.nlm.nih.gov/sra/SRR31111387), and SiSP-K05 (https://www.ncbi.nlm.nih.gov/sra/SRR31111386) were also obtained by next-generation sequencing and are available under BioProject ID PRJNA1176211 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1176211/). The set of 27 DDR genes was based on those in the study by Bezrookove et al (26); AT-rich interaction domain 1A/B (ARID1A and ARID1B), ataxia telangiectasia mutated (ATM), ATR, ATRX chromatin remodeler (ATRX), BRCA1 associated deubiquitinase 1 (BAP1), BRCA1 associated RING domain 1 (BARD1), BLM RecQ like helicase (BLM), BRCA1, BRCA2, BRCA1 interacting DNA helicase 1 (BRIP1), checkpoint kinase 2 (CHEK2), FA complementation group A/C/D2/E/F/G/L (FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG and FANCL), MRE11 homolog, double strand break repair nuclease (MRE11), nibrin (NBN), partner and localizer of BRCA2 (PALB2), RAD50 double strand break repair (RAD50), RAD51 recombinase (RAD51), RAD51 paralog B/C (RAD51B and RAD51C) and WRN RecQ like helicase (WRN). To assess the pathogenicity of the identified exonic mutations that led to changes in amino acids, the Franklin tool by Genoox (http://franklin.genoox.com; accessed on September 26, 2023) was employed. This analysis was conducted in accordance with the Association for Molecular Pathology (AMP) classification guidelines. These guidelines stratify variants into four tiers based on their clinical relevance to bile duct cancer, as discussed by Li et al (34). Tier 1 includes variants with strong clinical significance, tier 2 comprises variants with potential significance, tier 3 encompasses variants of uncertain significance, and tier 4 contains variants that are benign or likely to be benign.

Cells were seeded into 96-well plates at a final concentration of 3,000 cells/well and incubated for 24 h before the medium was replaced with that containing the drugs of interest. Treatments were with either a PARP inhibitor (olaparib, veliparib, and talazoparib) or the ATR inhibitor AZD6738 alone, or a combination of PARP inhibitor and AZD6738, for a duration of 120 h at 37°C. A mock treatment was also conducted, in which the concentration of DMSO was >0.016%. Following treatment, cell viability was assessed using the CellTiter-Glo^® Luminescent Cell Viability Assay according to the manufacturer's protocol (cat. no. G7572; Promega Corporation). Survival percentages were determined by normalizing the luminescent signal to that of untreated cells. These percentages were then plotted and half maximal inhibitory concentration (IC₅₀) values were calculated using GraphPad Prism software, version 9.5.1 (Dotmatics). The CI values were subsequently calculated according to the Chou-Talalay method (35) as shown below: CI=(IC50c)1(IC50a)1+(IC50c)2(IC50a)2

In this formula, (IC_50c)₁ is the IC₅₀ of AZD6738 used in combination; (IC_50a)₁ is the IC₅₀ of AZD6738 used alone; (IC_50c)₂ is the IC₅₀ of the PARP inhibitor used in combination and (IC_50a)₂ is the IC₅₀ of the PARP inhibitor used alone.

The CI values were categorized as follows (36): 0.1–0.3, strong synergism; 0.3–0.7, synergism; 0.7–0.85, moderate synergism; 0.85–0.9, slight synergism; 0.9–1.1, nearly additive; 1.1–1.2, slight antagonism; 1.2–1.45, moderate antagonism. The experiments were conducted in three biological replicates.

Cells were seeded in 6-well plates at a density of 600 cells/well for MMNK-1 or 1,000 cells/well for SiSP-K01 and SiSP-K05 and incubated at 37°C with 5% CO₂ for 24 h. They were then exposed to AZD6738, various concentrations of PARP inhibitors, or a combination of both, for 120 h at 37°C. The media was subsequently replaced, and the cells were incubated for 7–10 days until colonies formed. For visualization, a 0.5% w/v solution of crystal violet (cat. no. C077; Sigma-Aldrich; Merck KGaA) in 40% v/v methanol in water was added and incubated for 10 min at room temperature. The plates were then washed and air-dried, and images were acquired using a ChemiDoc^™ MP Imaging System (Bio-Rad Laboratories, Inc.). All the images were exported as tif files, and the intensity of each well was measured using ImageJ 1.53n software (37) as previously described (38). The experiments were performed on three biological replicates.

Cells were seeded in a slide chamber (Lab-Tek^™, cat. no. 154526; Thermo Fisher Scientific, Inc.) and allowed to grow until they reached 80% confluence. Subsequently, the medium was replaced with fresh media containing AZD6738, PARP inhibitor or a combination of AZD6738 and PARP inhibitor. The cells were then incubated for an additional 24 h at 37°C in a 5% CO₂ atmosphere. Fluorescence staining was performed using a method modified from that in previous studies (39,40). Briefly, cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. After another wash with PBS, cells were permeabilized with 0.5% Triton X-100 in PBS for 15 min. Non-specific binding was blocked using Intercept^® (PBS) blocking buffer (cat. no. 927-70001; LI-COR Biosciences) for 1 h at room temperature. For the detection of γ-H2AX foci, cells were incubated with a mouse monoclonal antibody against γ-H2AX (Ser139; 1:1,000; cat. no. 80312; Cell Signaling Technology, Inc.) at 4°C overnight. Alexa Fluor^® 488 donkey anti-mouse IgG (1:500; cat. no. A21202; Thermo Fisher Scientific, Inc.) was used as the secondary antibody and was incubated with the cells for 1 h at room temperature. Micronuclei and nuclei were visualized with Hoechst 33342 solution (1:2,000; cat. no. H3570; Thermo Fisher Scientific, Inc.) for 5 min at room temperature. Images were acquired with a fluorescence microscope (ECLIPSE Ci; Nikon Corporation). All samples were visualized using the same intensity and exposure time, and images were analyzed using ImageJ 1.53n software (37). At least 225 nuclei were analyzed for both micronuclei and γ-H2AX foci formation. The experiments were performed with at least two biological replicates.

The IC₅₀ and CI results were reported as the mean ± standard deviation. The IC₅₀ values and relative intensities of colonies across each CCA cell line were statistically compared with those of the MMNK-1 cholangiocyte cell line, utilizing multiple t-tests with the Holm-Sidak method in GraphPad Prism software, version 9.5.1 (Dotmatics). P<0.05 was considered to indicate a statistically significant difference. The same statistical analysis was applied to assess significant differences in the average number of micronuclei per cell between treatments with AZD6738 or PARP inhibitors alone or in combination. The results of the γ-H2AX foci formation assay are presented as medians and were analyzed using the Mann-Whitney test.

As shown in Table I, the numbers of mutated genes differed among the CCA cell lines. Among the four CCA cell lines, TFK-1 had the fewest DDR mutated genes (n=9), whereas HuH-28 had the same number of DDR mutated genes as SiSP-K01 (n=11). SiSP-K05 contained the highest number of mutated genes (n=15). Fig. 1 demonstrates the distribution of DDR mutated genes in each CCA cell line. In all CCA cell lines analyzed, mutations in ATR, BRCA2 and WRN were detected. In addition, a subset of three CCA cell lines exhibited mutations in eight different genes: ARID1A, ARID1B, ATM, ATRX, BARD1, BRIP1, FANCA and PALB2. Three other mutated genes, BLM, BRCA1 and RAD51B, were found in two CCA cell lines; HuH-28 and SiSP-K05 for BLM and BRCA1, and RAD51B for SiSP-K01 and SiSP-K05. There were four mutated genes, namely BAP1, CHEK2, FANCF and RAD50, that were only observed in a single CCA cell line. However, there were nine genes associated with DDR, specifically FANCC, FANCD2, FANCE, FANCG, FANCL, MRE11, NBN, RAD51 and RAD51C, that were not detected in any of the CCA cell lines. Details of the genetic variants in DDR genes that were identified are presented in Table SII. Next, the classification of genetic variants in DDR genes was determined according to the guidelines of the AMP, as indicated in Table II. SiSP-K01 exhibited a range of variants spanning the clinically relevant tiers 2–4, whereas SiSP-K05 had a range of variants spanning tiers 2 and 3. Notably, the TFK-1 and HuH-28 cell lines only demonstrated variants exclusively from tiers 3 and 4. Additionally, it was observed that variants in BRCA1 and PALB2 genes were confined to tiers 2 and 3, whereas those in the BRCA2 and BRIP1 genes spanned tiers 2–4. Variants in other genes fell into tier 3 and/or tier 4. Collectively, these findings suggest that the CCA cell lines are promising candidates for further testing with PARP inhibitors and AZD6738.

To assess cell viability in the four CCA cell lines with distinct DDR mutated backgrounds, TFK-1, HuH-28, SiSP-K01 and SiSP-K05 cells were treated with AZD6738, PARP inhibitors alone, or their combinations, and their responses to treatment were compared with those of the immortalized cholangiocyte cell line MMNK-1. Fig. 2 and Table III display the dose-response curves from the sensitivity assay and the IC_50a profiles of all the cell lines in response to AZD6738 and the various PARP inhibitors, respectively.

Comparison of AZD6738 and the PARP inhibitors indicated that AZD6738 was the most toxic to the cell lines. This is evidenced by AZD6738 having the lowest IC_50a values, both minimal and maximal, in comparison with the PARP inhibitors. The IC_50a values for AZD6738 ranged from 0.554±0.020 µM (in SiSP-K05) to 15.633±5.324 µM (in SiSP-K01). By contrast, the ranges of the IC_50a values for the PARP inhibitors were as follows: Olaparib, from 19.740±16.283 µM (in HuH-28) to 121.067±5.140 µM (in SiSP-K01); veliparib, from 67.607±3.466 µM (in SiSP-K05) to 256.800±14.127 µM (in MMNK-1); and talazoparib, from 1.095±0.920 µM (in HuH-28) to 127.767±39.302 µM (in MMNK-1).

Next, the IC_50a profiles of CCA cell lines when treated with AZD6738 or various PARP inhibitors alone were compared with those of MMNK-1 cholangiocytes (Table III). For AZD6738, only SiSP-K05 (IC_50a, 0.554±0.020 µM) was significantly more sensitive (P=0.017) than the MMNK-1 cholangiocytes (IC_50a, 0.997±0.141 µM). By contrast, SiSP-K01 cells (IC_50a, 15.633±5.324 µM; P=0.018) exhibited significantly lower sensitivity than MMNK-1 cholangiocytes to AZD6738, while TFK-1 and HuH-28 cells both displayed sensitivity levels similar to those of the cholangiocytes. For the PARP inhibitors, three CCA cell lines, namely TFK-1 (IC_50a, 25.667±3.661 µM; P<0.001), HuH-28 (IC_50a, 19.740±16.283 µM; P=0.010) and SiSP-K05 (IC_50a, 46.790±16.939 µM; P=0.021), demonstrated significantly greater sensitivity than MMNK-1 cells (IC_50a, 85.033±5.664 µM) to olaparib, while HuH-28 (IC_50a, 145.950±10.112 µM; P=0.010), SiSP-K01 (IC_50a, 143.833±20.857 µM; P=0.005) and SiSP-K05 cells (IC_50a, 67.607±3.466 µM; P<0.001) were significantly more sensitive than MMNK-1 cells (IC_50a, 256.800±14.127 µM) to veliparib. Additionally, the sensitivity of HuH-28 and SiSP-K05 cells to talazoparib was heightened compared with that of MMNK-1 cells. SiSP-K01 cells (IC_50a, 121.067±5.140 µM; P=0.005) were significantly less sensitive than MMNK-1 cells to olaparib, whereas TFK-1 cells displayed veliparib sensitivity comparable to that of MMNK-1 cells. The sensitivity to talazoparib of TFK-1 and SiSP-K01 cells was similar to that of MNNK-1 cells. Among the cell lines with significantly greater sensitivity than MNNK-1 cells to PARP inhibitors, talazoparib was the most potent, as evidenced by its low IC_50a. The IC_50a values for the PARP inhibitors were as follows: Olaparib, MMNK-1 (85.033±5.664 µM) compared with TFK-1 (25.667±3.661 µM), HuH-28 (19.740±16.283 µM) and SiSP-K05 (46.790±16.939 µM); veliparib, MMNK-1 (256.800±14.127 µM) compared with HuH-28 (145.950±10.112 µM), SiSP-K01 (143.833±20.857 µM) and SiSP-K05 (67.607±3.466 µM); and talazoparib, MMNK-1 (127.767±39.302 µM) compared with HuH-28 (1.095±0.920 µM) and SiSP-K05 (4.378±1.977 µM).

The clonogenicity of the two primary CCA cell lines and MMNK-1 cholangiocytes when treated individually with AZD6738 and of PARP inhibitors at the respective IC_50a was evaluated (Fig. S1). The results demonstrate that all inhibitors inhibited clonogenicity. The inhibitory effect on clonogenic survival was >50% when compared with the clonogenicity of the untreated group. These results confirm that at the IC_50a, these drugs are capable of inhibiting the clonogenicity of primary cell lines.

Collectively, these observations suggest that, compared with AZD6738, PARP inhibitors exhibit a broader range of effectiveness in CCA cell lines with diverse genetic backgrounds and less toxicity. Among the tested PARP inhibitors, talazoparib is the most potent.

To determine whether combining AZD6738 with PARP inhibitors increases DNA damage compared with the individual effect of each drug at its IC_50a, micronuclei formation was evaluated in the CCA cell lines subjected to these treatments. The results showed that the drug combinations induced more DNA damage than each drug did on its own, particularly when cells were treated with the olaparib (Fig. 3A) and talazoparib (Fig. 3C) combinations. Specifically, in the case of olaparib (Fig. 3A), there were significant differences in the extent of damage when MMNK-1, TFK-1, SiSP-K01 and SiSP-K05 cell lines were treated with the combination of olaparib and AZD6738 compared with olaparib alone. For the TFK-1 cell line, a significant difference was also observed when the effect of combination treatment was compared with that of AZD6738 alone. With talazoparib (Fig. 3C), the SiSP-K01 cell line exhibited a significantly higher average number of micronuclei per cell when treated with the combination than with individual AZD6738 or talazoparib treatments. By contrast, SiSP-K05 presented a significant increase only when the combination was compared with talazoparib alone; no such increase was observed when compared with AZD6738 alone. However, in the context of veliparib (Fig. 3B), combining AZD6738 with veliparib did not result in a significant increase in micronuclei across all cell lines tested, with the exception of the SiSP-K01 cell line, in which a significant difference was found between the combination treatment and veliparib alone. Representative images of micronuclei for each condition are presented in Fig. S2, Fig. S3, Fig. S4.

The investigation was expanded to determine if the increased DNA damage seen following treatment with drug combinations could be attributed to a rise in DNA double-strand breaks, as compared with the individual effects of the drugs at their IC_50a (Fig. 4). The number of cell lines with significant increases in γ-H2AX foci, a marker of DNA double-strand breaks, following combination treatments when compared with AZD6738 alone was greater than that when compared with PARP inhibitor alone. Specifically, with olaparib and talazoparib (Fig. 4A and C), a significant increase in γ-H2AX foci was observed in all CCA cell lines and in normal cholangiocytes when these were compared with the effects of AZD6738 alone. However, with veliparib, the significant increase was noted only in CCA cell lines, not in cholangiocytes. Furthermore, the CCA cell lines HuH-28, SiSP-K01 and SiSP-K05 exhibited a significant increase in γ-H2AX when treated with combinations of olaparib or talazoparib, compared with these PARP inhibitors alone. With regard to veliparib, two CCA cell lines, HuH-28 and SiSP-K05, demonstrated a significant increase in γ-H2AX for the combination compared with veliparib alone. Representative images of γ-H2AX foci formation, indicative of DNA damage, are presented in Fig. S5, Fig. S6, Fig. S7.

These observations highlight the enhanced efficacy of combination treatments in inducing DNA damage, as compared with the effects of individual drug treatments. The pronounced increase in DNA double-strand breaks observed across various cell lines suggests the potential for synergistic interactions between AZD6738 and the PARP inhibitors.

The effects of combinations of AZD6738 and different PARP inhibitors on cell viability were investigated, to determine which combinations yielded synergistic effects and dose reductions. The CI, a quantitative tool, was used to assess whether the drug interactions were synergistic, antagonistic or additive (36). First, the IC_50c profile of AZD6738 when combined with the PARP inhibitors olaparib, veliparib and talazoparib was determined as shown in Table IV, and the dose-response curves are presented in Fig. S8. Following this, the CIs were calculated, which are presented in Table V and visualized in Fig. 5.

The data reveal that all drug combinations were most effective against the SiSP-K05 cell line, with the IC_50c for each drug combination being lower for SiSP-K05 cells than for MMNK1 cells, as detailed in Table IV. Specifically, the combinations of AZD6738 with olaparib, veliparib and talazoparib consistently showed lower IC_50c values in SiSP-K05 cells compared with MMNK-1 cells. AZD6738 and olaparib had the following IC_50c values: MMNK-1, 1.238±0.083 and 1.655±0.161 µM; SiSP-K05, 0.641±0.116 µM (P=0.003) and 0.538±0.106 µM (P=0.002), respectively. For AZD6738 and veliparib, the IC_50c values were: MMNK-1, 1.307±0.118 and 1.438±0.179 µM; SiSP-K05, 0.612±0.103 µM (P=0.003) and 0.495±0.132 µM (P=0.002), respectively. AZD6738 and talazoparib had the following IC_50c values; MMNK-1, 1.178±0.084 and 0.633±0.090 µM; SiSP-K05, 0.319±0.007 µM (P<0.001) and 0.158±0.009 µM (P=0.002), respectively. By contrast, the SiSP-K01 cell line exhibited lower sensitivity than MMNK-1 cells to all these drug combinations, with IC_50c values as follows: AZD6738 and olaparib, 5.804±1.415 µM (P=0.005) and 33.953±17.481 µM (P=0.033); AZD6738 and veliparib; 6.802±0.172 µM (P<0.001) and 46.230±2.546 µM (P<0.001); AZD6738 and talazoparib; 2.703±0.373 µM (P=0.005) and 9.195±1.692 µM (P=0.002), respectively. Furthermore, HuH-28 cells were less sensitive than MMNK-1 cells to the combination of AZD6738 and veliparib, but both cell lines displayed a similar response to the combination of AZD6738 and olaparib. For the AZD6738 and talazoparib combination, the IC_50c of AZD6738 for both TFK-1 and HuH-28 cells differed significantly from that for MMNK-1 cells. However, no such difference was observed for talazoparib between these two cell lines. Notably, the IC_50c values for all PARP inhibitors were considerably lower when used in combination than when administered alone.

The CI analysis (Table V), revealed that synergistic effects were more prevalent in CCA cell lines when AZD6738 was combined with either olaparib or talazoparib, in comparison to its combination with veliparib. These findings are visually represented in Fig. 5. Specifically, the combination of AZD6738 and olaparib displayed synergistic effects in TFK-1, HuH-28 and SiSP-K01 cell lines. These effects varied, with values ranging from 0.623±0.097 in HuH-28 cells to 0.887±0.047 in TFK-1 cells. The combination of AZD6738 and talazoparib produced even stronger synergistic effects, with values spanning from 0.283±0.079 in SiSP-K01 cells to 0.619±0.051 in SiSP-K05 cells. Notably, the combinations comprising AZD6738 with either olaparib or talazoparib both showed synergistic effects in TFK-1 and SiSP-K01 cells. However, synergism in the SiSP-K05 cell line was only observed for the AZD6738 and talazoparib combination. Notably, all tested combinations exhibited synergistic effects in the SiSP-K01 cell line, with particularly strong synergism observed for the AZD6738 and talazoparib combination.

Overall, while synergistic effects were observed in SiSP-K01 cells for all drug combinations, the IC_50c profile indicates potential toxicity from these combinations. By contrast, the combination of AZD6738 and talazoparib appears to offer better efficacy for SiSP-K05 cells. This is evidenced by the IC_50c values of AZD6738 and talazoparib in SiSP-K05 cells being lower than those in MMNK-1 cells, and the observed synergistic effect of this combination.