Effects of Combined Therapeutic Targeting of AXL and ATR on Pleural Mesothelioma Cells

Abstract

Few treatment options exist for pleural mesothelioma (PM), which is a progressive malignant tumor. However, the efficacy of molecular-targeted monotherapy is limited, and further therapeutic strategies are warranted to treat PM. Recently, the cancer cell-cycle checkpoint inhibitors have attracted attention because they disrupt cell-cycle regulation. Here, we aimed to establish a novel combinational therapeutic strategy to inhibit the cell-cycle checkpoint kinase, ATR in PM cells. The siRNA screening assay showed that anexelekto (AXL) knockdown enhanced cell growth inhibition when exposed to ATR inhibitors, demonstrating the synergistic effects of the ATR and AXL combination in some PM cells. The AXL and ATR inhibitor combination increased cell apoptosis via the Bim protein and suppressed cell migration when compared with each monotherapy. The combined therapeutic targeting of AXL and ATR significantly delayed regrowth compared with monotherapy. Thus, optimal AXL and ATR inhibition may potentially improve the PM outcome.

Introduction

Malignant mesothelioma (MM) is a progressive malignant tumor derived from the serous mesothelial cells lining the body cavity, mainly arising from the pleura, peritoneum, and pericardium. In particular, pleural mesothelioma (PM) accounts for 80% to 85% of all MMs, followed by peritoneal and other MMs (1). Long-term exposure to asbestos is thought to be involved in PM development, which is known to occur 25 to 50 years after exposure. The three major pathologic PM types are epithelial (60%), sarcomatoid (10%), and biphasic (30%) PMs. A combination of the cytotoxic anticancer drugs cisplatin and pemetrexed has been used for systemic treatment of inoperable PM (2). Several clinical trials, such as the CONFIRM and MERIT studies have demonstrated the efficacy of nivolumab (the immune checkpoint inhibitor) monotherapy as the second-line treatment for relapsed PM (3, 4). Recently, the combination of nivolumab and ipilimumab was approved by the FDA as the first-line regimen of patients with advanced-stage PM based on the results of the phase III trial CheckMate 743 (5). Thus, the treatment options for advanced PM have gradually increased in recent years but are limited and unsatisfactory compared with those for advanced lung cancer. Concerning molecularly targeted agents, the multi-kinase inhibitor nintedanib has been reported to reduce the risk of PM progression when used in combination with conventional chemotherapy (6). In contrast, the results of the P3 trial on the aforementioned nintedanib in combination with cytotoxic anticancer drugs reported no significant difference in PFS between the nintedanib and placebo groups (7). None of the molecular-targeted agents has been approved for PM; therefore, developing novel treatments is warranted to improve clinical prognosis. Generally, the malignant transformation of PM mainly occurs through multistage carcinogenesis, and few reports on the involvement of driver oncogenes exist, suggesting that regulating tumor progression by molecular-targeted monotherapy might be challenging (8).

Recently, cell-cycle checkpoint inhibitors in cancer have attracted attention because they cause inhibition of tumor growth by disrupting cell-cycle regulation. Of these, ATR protein kinases play an important role in regulating the cell cycle in DNA replication, DNA repair, and replication stress, making ATR inhibition a promising strategy for cancer therapy (9). The effects of ATR inhibitors or their downstream Chk1 inhibitors in combination with other drugs are the focus of study in several cancer fields, such as ovarian and lung cancers (10–13).

Anexelekto (AXL) is a tyrosine kinase receptor that belongs to the TAM protein family and is usually expressed in several types of malignancies, including PM (14–20). AXL plays a pivotal role in proliferation, migration, survival, and drug resistance in malignant tumors; its overexpression is consequently correlated with poor prognosis in several cancers (21–25). Therefore, regulating the AXL pathway is a promising therapeutic strategy against the progression of several cancers. This study aimed to examine a novel combinational therapeutic strategy by inhibiting ATR and AXL and to reveal the underlying mechanisms that describe the efficacy of the combination on PM cells.

Materials and Methods

Cell lines and reagents

Fourteen PM cell lines were used in this study, of which MSTO-211H (211H, RRID: CVCL_1430), NCI-H28 (H28, RRID: CVCL_1555), NCI-H226 (H226, RRID: CVCL_1544), NCI-H2052 (H2052, RRID: CVCL_1518), NCI-H2373 (H2373, RRID: CVCL_A533), and NCI-H2452 (H2452, RRID: CVCL_1553) were purchased from the ATCC. ACC-MESO-1 (ACC1, RRID: CVCL_5113), ACC-MESO-4 (ACC4, RRID: CVCL_5114), Y-MESO-8A (Y8A, RRID: CVCL_5188), Y-MESO-9 (Y9, RRID: CVCL_5190), Y-MESO-12 (Y12, RRID: CVCL_5178), Y-MESO-14 (Y14, RRID: CVCL_5179), Y-MESO-25 (Y25, RRID: CVCL_5182), and Y-MESO-30 (Y30, RRID: CVCL_5187) were established in the laboratory of Dr. Yoshitaka Sekido and have been deposited in RIKEN BioResource Research Center (26). Met-5A (RRID: CVCL_3749), an epithelial cell line from the mesothelium, was purchased from ATCC. All cell lines were maintained in a humidified incubator at 37°C with 5% CO₂ and were cultured in RPMI1640 medium with 10% FBS and 1% penicillin–streptomycin. Cell lines were authenticated using DNA fingerprinting and the cell lines were routinely checked for mycoplasma infection using the MycoAlertTM Mycoplasma detection Kit. AZD6738, an ATR inhibitor (27), ONO7475, a specific AXL and MER tyrosine kinase inhibitor (28, 29), and NPS1034, a dual inhibitor of AXL and MET (30), were purchased from SelleckChem and ChemieTek.

Cell viability assay

The MTT dye reduction method was used for testing cell viability. Tumor cells (2–4 × 10³ cells/100 μL/well) in RPMI1640 medium supplemented with 10% FBS were plated in 96-well plates and cultured with the indicated compound for 72 hours. Then, 50 μL of MTT solution (2 mg/mL, Sigma) were added to each well. The plates were incubated for 2 hours, the medium was removed, and the dark blue crystals in each well were dissolved in 100 mL of DMSO. Absorbance was measured using a microplate reader at a test wavelength of 550 nm and a reference wavelength of 630 nm. The percentage of growth was determined relative to the untreated controls. Experiments were repeated at least thrice with triplicate samples. For the long-term cell culture assay, cells were treated with ONO7475, AZD6738, or their combination for 9 days, where the drugs were replenished every 72 hours. The plates were stained with crystal violet and visually examined.

Antibodies and Western blotting

Protein aliquots (25 μg each) were resolved by SDS-PAGE (Bio-Rad). The electrophoresed protein samples were transferred onto polyvinylidene difluoride membranes (Bio-Rad). After washing thrice, the membranes were incubated with blotting-grade blocker (Bio-Rad) for 1 hour at 37°C and then overnight at 4°C with primary antibodies against p-AXL (Tyr702) (D12B2), AXL (C44G1), p-Chk1 (Ser345), Chk1 (2G1D5), ATR (E1S3S), p-RPA32 (Ser4 and Ser8), p-histone H2A.X (Ser139), Bim (C34C5), Bak (D2D3), Bax (D2E11), Bcl-xL (54H6), Bcl-2 (124), Mcl-1 (D35A5), cleaved-PARP (Asp214) (D64E10), β-actin (13E5) (1:1,000 dilution; Cell Signaling Technology). After washing thrice, the membranes were incubated for 1 hour at 37°C with HRP-conjugated species-specific secondary antibodies. Immunoreactive bands were visualized using the SuperSignal West Dura Extended Duration Substrate Enhanced Chemiluminescent Substrate (Pierce Biotechnology). Each experiment was independently performed at least thrice.

Transfection of siRNAs

Duplexed Silencer Select siRNAs against AXL [s1845(#1) and s1846(#2); Invitrogen, ATR (s536(#1) and s57272(#2); Invitrogen], and Bim (s195011; Invitrogen) were transfected into cells using Lipofectamine RNAi-MAX (Invitrogen) according to the manufacturer's instructions. The Silencer Select Negative Control No. 1 siRNA (siSCR, Invitrogen) was used as a scrambled control in all the experiments. AXL and ATR knockdowns were confirmed using Western blotting. Each sample was tested in triplicates in three independent assays. To perform a comprehensive siRNA screening analysis, the Silencer Select human kinase siRNA library V4 (Ambion, 4397918) was used according to the manufacturer's instructions.

Migration assay

For transwell migration assays, approximately 5 × 10⁴ cells in 100 μL of serum-free RPMI1640 containing 0.1% BSA were seeded onto filter inserts in a 24-well plate, which was placed in RPMI1640 with 10% FBS on the bottom side of the membrane. Cells were incubated at 37°C for 8 to 48 hours, depending on the cell line under study (8, 24, and 48 for Y30, H2373, and H226 cells, respectively). The membrane was then ethanol-fixed and treated with the Giemsa stain. The upper side of the membrane was removed and the cells remaining on the lower side of the membrane were observed under a microscope. Cells were counted in one field of view at 400× magnification at four different locations, and the average value was calculated. The membranes were photographed under a microscope at 100× magnification.

Cell-cycle analysis and apoptosis analysis

For cell-cycle analysis, cells were harvested 48 hours after treatment with 1 μmol/L of AZD6738 and/or 1 μmol/L of ONO7475, washed twice with ice-cold PBS, collected by centrifugation, and resuspended at 1 × 10⁵ cells/mL in propidium iodide (PI) staining buffer (0.1% Triton X-100 and 50 μg/mL PI in PBS). For analyzing apoptosis, cells were harvested 48 hours after drug treatment, washed twice with ice-cold PBS, and incubated with annexin V-fluorescein isothiocyanate and PI for 15 minutes at 37°C. The cells were analyzed using a BD Accuri C6 Plus Flow Cytometer and FlowJo software in both analyses. At least 1 × 10⁴ events were recorded for each culture. Cellular apoptosis was analyzed using Caspase-Glo 3/7 Assay Kits (Promega) that measure caspase-3/7 activity (31).

Cell line-derived xenograft models

Suspensions of 5 × 10⁶ Y30 cells in 100 μL of PBS were injected subcutaneously into the flanks of 6-week-old male mice with severe combined immunodeficiency (SCID; Clea). Once the mean tumor volume reached approximately 100 to 200 mm³, five or six mice were randomized into four groups and treated with vehicle, 25 mg/kg AZD6738, 10 mg/kg ONO7475, or both drugs (29, 32–34). Drugs were administered daily by oral gavage, and the general condition of the mice and the body weight were monitored daily and twice a week, respectively. Tumors were measured twice weekly using calipers and their volumes were calculated as width² × length/2.

After 38 days of AZD6738 and/or ONO7475 treatment, the mice were euthanized. Tumors were collected and their weights were measured. This study, which included animal experiments, was approved by the Institutional Review Board of the University Hospital, Kyoto Prefectural University of Medicine (approval no. M2021–104). The mice were housed in an animal facility free from specific pathogens. According to the institutional guidelines, animal surgery was performed after the animals were anesthetized with sodium pentobarbital, and efforts were made to minimize animal suffering. The mice were sacrificed by cutting the subclavian artery, and the subcutaneous tumor was removed with a scalpel and a pair of scissors.

Tumor histologic analyses

As reported previously (29), formalin-fixed, paraffin-embedded tissue sections (4 μmol/L thick) were deparaffinized and subjected to antigen retrieval by microwaving the tissue sections in 10 mmol/L citrate buffer (pH 6.0). Proliferating cells were detected by incubation at 37°C in 5% CO₂ with Ki-67 antibodies (Clone MIB-1; GA62661–2; Agilent). Apoptosis was quantitated using the terminal deoxynucleotidyl TUNEL method, according to the manufacturer's instructions.

Statistical analysis

Data from the MTT assays were expressed as the mean ± SD. The differences were statistically analyzed by ANOVA using Prism 9.0 (GraphPad). Statistical significance was set at P < 0.05. The combination index (CI) was evaluated using Compusyn (35). CI < 0.8, 0.8 ≤ CI ≤ 1.2, and CI > 1.2 were considered as having synergistic, additive, and antagonistic effects, respectively. All CI plot analyses for each cell line were calculated using Compusyn with cell viability as the effect when exposed to the drug at 72 hours.

Data availability

The data generated in this study are available within the article and its supplementary data files.

Results

AXL inhibition enhanced the efficacy of ATR inhibitor on cell viability of PM cells

To evaluate the impact of ATR inhibition in PM cells, we examined the efficacy of the ATR inhibitor AZD6738 on cell growth inhibition in 14 PM cell lines, including the epithelial ACC1, ACC4, H226, Y9, Y12, Y25, and Y30 cells; the biphasic 211H, H2452, Y8A, and Y14 cells; and the sarcomatoid H28, H2052, and H2373 cells (36–39). AZD6738 monotherapy showed varying effects on the viabilities of the 14 PM cell lines. However, most PM cells did not achieve the IC₅₀s when 1 μmol/L of AZD6738 was used, except for the 211H cells (Fig. 1A). The efficacy of AZD6738 was not associated with the PM cell phenotype. To determine the mechanism by which these cells were less effective against AZD6738, we further investigated the effect of knockdown of specific genes using an siRNA Screening Library with 1 μmol/L AZD6738. Among the 56 receptor-type tyrosine kinase siRNAs, we focused on the knockdown of AXL due to its promising effect in reducing the viability of H2373 and Y30 cells (Fig. 1B; Supplementary Table S1). We confirmed that AXL knockdown enhanced the inhibitory effect of the ATR inhibitor AZD6738 on the viabilities of several PM cell lines (Fig. 1C; Supplementary Fig. S1A). Western blotting showed that an increase in phosphorylation and the total number of AXL proteins is promoted by treating 0.1 μmol/L of AZD6738 for 24 hours in H2373, Y30, and H226 cell lines (Fig. 1D; Supplementary Fig. S2).

Figure 1.

AXL inhibition enhanced the efficacy of ATR inhibitor on cell viability of pleural mesothelioma cells. A, The pleural mesothelioma 14 cell lines were treated with 1 μmol/L AZD6738 for 72 hours, and the cell viability was assessed using MTT assays (n = 5, mean ± SD). B, H2373 and Y30 cell lines treated with nonspecific control siRNA or 56 types of RTK-specific siRNAs, and incubated with 1 μmol/L AZD6738 for 72 hours. Rate of the cell viability of each RTK-specific siRNA against nonspecific control siRNA was assessed using MTT assays. (n = 5, mean). C, H226, H2373, and Y30 cells treated with nonspecific control siRNA or AXL-specific siRNA and incubated with or without 1 μmol/L AZD6738 for 72 hours, and the cell viability was assessed using MTT assays. (n = 5, mean ± SD; *, P < 0.05). D, The indicated cells were incubated with 0.1 μmol/L AZD6738 for 4 or 24 hours. The cells were lysed, and the indicated proteins were detected using Western blotting.

These results showed that AXL inhibition plays a pivotal role in enhancing the sensitivity of ATR inhibitors as a feedback loop in the three PM cell lines.

Synergistic effect between AXL and ATR inhibitors on cell viability of PM cells

To evaluate the impact of AXL inhibition on PM cells, we examined the efficacy of ONO7475 on cell growth inhibition in the 14 PM cell lines. Various efficacies of ONO7475 monotherapy existed on PM cell viability. However, most PM cell lines did not achieve IC₅₀s when 1 μmol/L of ONO7475 was used, except for the Y8A and Y14 cell lines. The efficacy of ONO7475 was not related to the phenotypes of the PM cell lines (Fig. 2A). We confirmed that ATR knockdown using specific siRNA enhanced the cytotoxicity of ONO7475 on several PM cell lines, such as H2373, Y30, and H226 (Fig. 2B; Supplementary Fig. S1B).

Figure 2.

Synergistic effect between AXL and ATR inhibitors on cell viability of pleural mesothelioma cells. A, The 14 pleural mesothelioma cell lines were treated with 1 μmol/L ONO7475 for 72 hours, and the cell viability was assessed using MTT assays (n = 5, mean ± SD). B, H226, H2373, and Y30 cells treated with nonspecific control siRNA or ATR-specific siRNA and incubated with or without 1 μmol/L ONO7475 for 72 hours, and the cell viability was assessed using MTT assays (n = 5, mean ± SD). C, Three cell lines were treated with 1 μmol/L ONO7475 or 1 μmol/L AZD6738, or a combination of these agents for 72 hours, and the cell viability was assessed using MTT assays (n = 5, mean ± SD). D, The analysis was performed with ONO7475 and AZD6738 at three concentrations of 0.3, 1, and 3 μmol/L and their combination treatment for 72 hours, respectively. CI values in the combination were calculated with CompuSyn software at various Fa points. E, The indicated cells were incubated with 1 μmol/L ONO7475 or/and 1 μmol/L AZD6738 for 24 hours. The cells were lysed, and the indicated proteins were detected by Western blotting. F, Cells were treated with medium, 1 μmol/L ONO7475 or 1 μmol/L AZD6738, or a combination for 9 days where the drugs were replenished every 72 hours. The plates were stained with crystal violet and visually examined. A plate representative of three independent experiments is shown. Data are represented as *, P < 0.05.

To identify a potential novel treatment for patients with PM, we evaluated the effect of AZD6738 in combination with ONO7475 on the viability of the 14 PM cell lines. AZD6738 in combination with ONO7475 showed a significant difference in the viability of 9 PM cell lines, including H226, H2373, Y30, Y9, ACC1, 211H, H28, Y8A, and H2452 when compared with using AZD6738 or ONO7475 alone (Fig. 2C; Supplementary Fig. S3B). In addition, the H2373, Y30, and H226 cell lines showed significant cell growth inhibition with NPS1034, another AXL inhibitor, and AZD6738 (Supplementary Fig. S3A). Contrastingly, the effects of the combinational therapy on five PM cell lines were not significantly different compared with those of each monotherapy (Supplementary Fig. S3B). To investigate the optimal concentration for the combination, we also tested a low-dose combination of AZD6738 and ONO7475. Although there was a significant difference in the effect of the combination at a low dose, the effect was strongest when using 1 μmol/L of AZD6738 and ONO7475 (Supplementary Table S2). To evaluate the synergistic effect, we tested cell viability with the combination and determined the CI using the method followed by Chou and Talalay (40). The analysis was performed with ONO7475 and AZD6738 at three concentrations of 0.3, 1, and 3 μmol/L and their combination (Supplementary Table S2). Our data showed that ONO7475 and AZD6738 treatment resulted in reduced cell viability with CI values <0.8 indicating synergy for 6 of the 14 PM cell lines that showed significant differences in the combined effect (Fig. 2D; Supplementary Fig. S3B). In addition, the CI value for immortalized normal mesothelial cells, Met-5A, was more than 1.2, indicating antagonism (Fig. 2D; Supplementary Table S2). Western blots of the three cell lines exposed to ONO7475, AZD6738, and the combination for 24 hours are shown in Fig. 2E. Monotherapy with AZD6738 increased AXL phosphorylation and total AXL, whereas ONO7475 alone and in combination with AZD6738 effectively suppressed AXL phosphorylation. AZD6738 alone and in combination with ONO7475 suppressed the Chk1 phosphorylation, a downstream molecule of ATR. PM cell lines treated with the combination showed the highest p-RPA32 (S4/S8) and γH2Ax protein levels, which is indicative of cellular stress. The continuous ONO7475 and AZD6738 co-treatment of three PM cell lines for 9 days visually reduced cell viability compared with ONO7475 or AZD6738 monotherapy (Fig. 2F).

Collectively, cell line-based analysis revealed the synergistic effects of AXL and ATR inhibitors on the viability of some PM cell lines.

Combination therapy of AXL and ATR inhibitors reduced cell motility and promoted cell apoptosis via Bim in PM cell lines

We examined the effects of AXL and/or ATR inhibitors on the motility of PM cell lines using a cell migration assay. Microscopic observation showed that AZD6738 and ONO7475 combination remarkably decreased the number of migrating PM cells compared with each monotherapy and control (Fig. 3A). All three cell lines showed a significant decrease in migrating cells in combination therapy compared with the AXL inhibitor monotherapy (Fig. 3B; Supplementary Fig. S4). These observations indicated that the combination therapy could contribute to the antitumor effects in PM cells, via cell motility. We investigated the impact of AXL and/or ATR inhibitors on the cell cycle of PM cells using FACS analysis. ATR is activated in response to DNA damage and replication stress, and its activation was reported to repair DNA damage in the G₁ phase (41). The cell numbers in G₁ phase in H226, H2373, and Y30 were significantly increased when exposed to AZD6738 monotherapy and in combination with ONO7475 for 48 hours compared with the control (Fig. 4A; Supplementary Fig. S5A). To assess the apoptosis of PM cell lines, we further performed an apoptosis assay using annexin V. ONO7475 in combination with AZD6738 showed a significant increase in the apoptosis of H226, H2373, and Y30 cell lines when compared with either ONO7475 or AZD6738 alone for 48 hours (Fig. 4B; Supplementary Fig. S5B). To further investigate the detailed mechanisms of apoptosis, apoptosis-related protein expression was examined. Of these, Bim and cleaved-PARP were elevated in all three PM cells in the combination therapy compared with each monotherapy (Fig. 4C). Knockdown of Bim by siRNAs attenuated caspase-3/7 activity when treated with the combination compared with the control in H226, H2373, and Y30 cell lines (Fig. 4D; Supplementary Fig. S1C). These results suggest that combined therapy with ONO7475 and AZD6738 promoted cell apoptosis through Bim.

Figure 3.

Combination therapy of AXL and ATR inhibitors reduced cell motility in pleural mesothelioma cells. A, H226, H2373, and Y30 cell lines were treated with 1 μmol/L of ONO7475, 1 μmol/L of AZD6738, or a combination of these agents for 48, 24, or 8 hours. Representative images of HE of the membrane. Scale bar, 200 μmol/L. B, The cell migration was assessed using migration assays. The membranes were photographed under a microscope at 400× mean of four evaluated areas (mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 4.

Effect of combination of ONO7475 and AZD6738 on cell cycle and apoptosis in pleural mesothelioma cells. A, H226, H2373, and Y30 cell lines were treated with 1 μmol/L of ONO7475 or 1 μmol/L of AZD6738, or a combination of these agents for 48 hours, then harvested and analyzed by flow cytometry (n = 3; *, P < 0.01; **, P < 0.001). B, Induction of apoptosis in H226, H2373, and Y30 cell lines by 48 hours treatment with 1 μmol/L of ONO7475, 1 μmol/L of AZD6738, or a combination, assessed by flow cytometric analysis after Annexin-V FITC staining (n = 3; *, P < 0.05). C, The indicated cells were incubated with 1 μmol/L of ONO7475 and/or 1 μmol/L of AZD6738 for 48 hours. The cells were lysed, and the indicated proteins were detected by Western blotting (D) H226, H2373, and Y30 cells treated with nonspecific control siRNA or Bim-specific siRNA and incubated with or without 1 μmol/L of ONO7475 and 1 μmol/L AZD6738 for 48 hours, and apoptosis was analyzed by measurement of caspase-3/7 activity (n = 3; *, P < 0.001). Data are represented as mean ± SD.

Combination of AZD6738 and ONO7475 decreased tumor growth in a xenograft model of PM better than either drug alone

Next, we examined the antitumor potential of AZD6738 in combination with ONO7475 using a cell line-derived xenograft (CDX) model. The Y30 cell line was implanted into the flanks of SCID mice. When tumors reached approximately 200 mm³, the mice were treated daily with vehicle, AZD6738, ONO7475, or their combination. Treatment with any of the single agents slightly delayed Y30 tumor growth. Notably, AZD6738 and ONO7475 combination significantly inhibited Y30 tumor growth after day 31 compared with either monotherapy (Fig. 5A). In addition, the tumor weight on day 38 posttumor removal in the combination group was significantly reduced compared with either ONO7475 or AZD6738 monotherapy (Fig. 5B). The number of Ki-67-positive proliferating tumor cells was significantly lower in the combination-treated tumors than in those treated with AZD6738 or ONO7475 alone (Fig. 5C and D). TUNEL-positive apoptotic tumor cells did not differ significantly among the treatment with AZD6738 or ONO7475 alone or with the combination in tumors derived from Y30 cells. We also examined the protein levels of γH2Ax in the tumors using Western blot analysis. The expression of cleaved-PARP and γH2Ax proteins was upregulated by the combination therapy, consistent with our in vitro results (Fig. 5E). During treatment with AZD6738 or ONO7475, either alone or in combination, no evidence of severe loss in body weight was detected, indicating that the combination was well-tolerated (Supplementary Fig. S6). These results of CDX models suggest that the AZD6738 and ONO7475 combination may be a potential therapeutic strategy against PM.

Figure 5.

Initial combination therapy of ONO7475 and AZD6738 delayed regrowth of cell line–derived xenograft (CDX) tumors. A, Y30 CDX tumors were treated with vehicle (control), ONO7475 10 mg/kg, AZD6738 25 mg/kg, or AZD6738 25 mg/kg plus ONO7475 10 mg/kg (combination; control n = 4, ONO7475 n = 4, AZD6738 n = 4, and combination n = 5 per group). Tumor volumes were measured over time from the start of treatment; results are shown (mean ± SD; *, P < 0.05). B, Y30 CDX tumors were treated with vehicle (control), ONO7475 10 mg/kg, AZD6738 25 mg/kg, or AZD6738 25 mg/kg plus ONO7475 10 mg/kg (combination) for 38 days and weight of resection tumor were measured (control n = 4, ONO7475 n = 4, AZD6738 n = 4, and combination n = 5 per group, mean ± SD; *, P < 0.05). C, Representative images of Y30 xenografts IHC stained with antibodies specific for human Ki-67 and TUNEL. Bar, 50 μmol/L. D, Quantification of proliferating cells, as determined by their Ki-67–positive proliferation index (percentage of Ki-67–positive cells) and TUNEL assays as described in the Materials and Methods. Columns (n = 5, mean ± SD; *, P < 0.001). E, Y30 CDX tumors were treated with vehicle (control), ONO7475 at 10 mg/kg, AZD6738 at 25 mg/kg, or AZD6738 at 25 mg/kg plus ONO7475 at 10 mg/kg (combination) for 4 days were lysed, and indicated proteins were detected by Western blotting.

Discussion

Chemotherapy for advanced PM has been the only cytotoxic anticancer drug for a long time. Recently, a combination of the first-line immune checkpoint inhibitors nivolumab and ipilimumab has been approved in several countries based on the results of the CheckMate 743 clinical trial (5), and the number of treatment options is currently increasing. However, therapeutic options remain limited, and various studies are underway to improve the clinical outcomes of patients with advanced PM.

DNA damage is one of the crucial factors in cancer development and progression. Cytotoxic chemotherapy and radiotherapy contribute to the induction of cell death through DNA damage. However, tumor cells promote DNA repair pathways to resist these antitumor drugs. Therefore, inhibiting molecular targets involved in DNA damage repair, such as PARP and ATR, plays an important role in inducing cell apoptosis, and their combination is expected to have promising therapeutic effects for refractory malignant tumors. ATR is a serine–threonine kinase that activates cell-cycle checkpoints in response to DNA damage and acts as a tumor suppressor gene. ATR inhibitors suppress ovarian and lung tumors in combination with radiotherapy and cytotoxic anticancer agents, such as topotecan and gemcitabine (10–13). In this study, we first focused on the effect of ATR inhibition on the viability of PM cell lines; however, the effect of ATR inhibitor monotherapy is limited. Therefore, the purpose of this study was to find RTK molecules that potentiate the effects of ATR inhibitors and to determine their combined effects with these inhibitors in vitro and in vivo. Contrastingly, the siRNA screening assay showed the impact of AXL knockdown in combination with an ATR inhibitor on the growth inhibition of PM cell lines. Among them, we selected three PM cells lines in which the AXL feedback mechanism was strongly induced by ATR inhibition and proceeded with subsequent research. Previous reports showed that combination therapy with ATR and AXL inhibitors is a promising therapeutic option for lung cancer, which acts by increasing RPA32 hyperphosphorylation, inducing DNA double-strand breaks, and initiating a mitotic catastrophe (42). The efficacy of molecular targeted therapy of tumors with driver oncogenes is limited due to the reactivation of specific signaling pathways via multiple feedback mechanisms. Our preclinical study reported that the EGFR tyrosine kinase inhibitor (EGFR-TKI), osimertinib, adversely activated AXL by shutting off the negative feedback loop to SPRY4, which suppressed AXL phosphorylation (43). Previous reports have shown that aberrant GAS6-AXL activation is related to the progression of MM cells (44). Nevertheless, the fundamental mechanisms of AXL activation in PM cells remain unclear. We found that AXL expression is upregulated by exposure to ATR inhibitors. AXL upregulation is caused by a feedback loop of ATR inhibition, suggesting that this might be one of the evasion mechanisms of PM cells to escape cell death, consistent with EGFR-mutated lung cancer treated with EGFR-TKIs. In this study, some PM cell lines showed synergistic effects of dual inhibition of ATR and AXL on cell proliferation, migration, and apoptosis. Identifying responders of PM patients to treatment with a combination of ATR and AXL inhibitors may be worthwhile. However, promising biomarkers for responders to molecular-targeted therapy in PM cells have not been identified. A previous report showed that high AXL protein levels were associated with resistance to ATR inhibition in lung cancer (42). No apparent correlation between the expression of AXL and ATR-related proteins and the efficacy of the combination therapy was detected, although PM cells with a high level of AXL tended to be effective for this combination therapy and we consider this as a limitation of our preclinical study on PM cells. Further large-cohort investigations are warranted to examine predictive biomarkers for detecting responders in combination therapy with ATR and AXL. γH2Ax has attracted attention as a biomarker for DNA damage response (DDR) inhibitors. Various predictive biomarkers for the therapeutic efficacy of ATR inhibitors, such as ATM, TP53, and ARID1A, are currently being investigated. More recently, SLFEN11 has been reported as a potential biomarker for monotherapy with ATR inhibitors and combination therapy with other drugs in lung cancer (42, 45–47). However, we could not find any factors that could be used as predictive biomarkers, which is one of the limitations of our study (Supplementary Fig. S7). Moreover, the results of analysis using the CDX tumor model substantiated the activation of apoptosis through increased levels of cleaved-PARP proteins in response to the combination therapy. However, although there was a tendency of increased apoptosis under the combination therapy, as determined using the TUNEL assay, a significant difference was observed. These observations imply that the in vivo tumor microenvironment and drug delivery might have influenced the outcomes. In this study, the CDX tumor model was evaluated using only one PM cell line. Our study has limitations in terms of obtaining a comprehensive understanding of this disparity in outcomes. In addition, it is unclear whether the drug concentrations of each medication are suitable for their concurrent use in vivo, which is another limitation of this study. However, when the optimal in vivo dose was determined on the basis of previous reports (29, 32–34), fatal side effects were not observed, suggesting that the dose is well tolerated. We successfully demonstrated the significant apoptotic effect of the combined treatment of AZD6738 and ONO7475 through Bim-mediated mechanisms in PM cells. A previous study indicated that inhibiting Bcl-XL and MCL-1 in combination suppressed the proliferation of PM cells (48). However, our analysis revealed no significant trends in the expression of these proteins. These findings suggest that distinct types of PM cells may exhibit varying dependencies on apoptosis-inducing signals.

In this study, the combined effect of the AXL and ATR inhibitors enhanced the antitumor effects in in vivo experiments, compared with that in in vitro experiments. AXL in malignant tumors plays a pivotal role in the proliferation, migration, survival, and epithelial-to-mesenchymal transition (EMT; ref. 29). In addition to the impact on cell viability and cell apoptosis, the migration assay showed that the combination of AXL and ATR inhibitors significantly suppressed the invasion of PM cells. PM tumors are characterized by aggressive local invasion and progression by dissemination into the intrathoracic viscera rather than by distant metastasis (49). Therefore, the AXL inhibitor combination, which has a high inhibitory effect on invasion, is expected to be a promising therapeutic strategy for suppressing tumor invasion in PM. Conversely, increased toxicity is a concern in the clinical application of combination therapies, although this combination was well tolerated in the CDX model. The safety of combination therapy needs to be evaluated in further clinical investigations.

Here, a novel combination therapy targeting AXL and ATR had synergistic effects on cell growth, apoptosis, and migration in PM cell lines. CDX models showed that this novel combination significantly attenuated tumor growth compared with each monotherapy. Our observations showed that optimal AXL and ATR inhibition may potentially improve the outcome of patients with PM.

Supplementary Material

Supplementary Table1

Supplementary Table1

Supplementary Table2

Supplementary Table2

Figure S1

Figure S1

Figure S2

Figure S2

Figure S3

Figure S3

Figure S4

Figure S4

Figure S5

Figure S5

Figure S6

Figure S6

Figure S7

Figure S7

Authors' Disclosures

T. Yamada reports grants from Pfizer, Ono Pharmaceutical, Janssen Pharmaceutical K.K., AstraZeneca, Takeda Pharmaceutical Company Limited, and personal fees from Eli Lilly outside the submitted work. K. Takayama reports personal fees from AstraZeneca, Chugai-Roche, Boehringer-Ingelheim, MSD-Merck, and Daiichi-Sankyo; grants and personal fees from Ono-Pharmaceutical and Taiho Pharmaceutical outside the submitted work. No disclosures were reported by the other authors.

Authors' Contributions

S. Hirai: Resources, data curation, formal analysis, investigation, methodology, writing–original draft, project administration. T. Yamada: Conceptualization, formal analysis, supervision, funding acquisition, validation, investigation, methodology, writing–original draft, project administration, writing–review and editing. Y. Katayama: Resources, investigation, methodology. M. Ishida: Resources, investigation. H. Kawachi: Resources, investigation. Y. Matsui: Resources, investigation. R. Nakamura: Resources, investigation. K. Morimoto: Resources, investigation. M. Horinaka: Resources, investigation. T. Sakai: Resources, investigation. Y. Sekido: Resources, investigation. S. Tokuda: Resources, investigation. K. Takayama: Supervision, investigation.