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. 2024 Jan 3;137(2):222–231. doi: 10.1097/CM9.0000000000002988

AZD1775 and anti-PD-1 antibody synergistically sensitize hepatoma to radiotherapy

Yichun Yin 1, Jian Wang 2, Junxuan Yi 1, Kaiyue Zhang 1, Zimeng Yin 1, Shunzi Jin 1,3,✉, Baisong Zheng 2,✉
Editor: Yanjie Yin
PMCID: PMC10798739  PMID: 38167245

Abstract

Background:

Radiation (IR)-induced DNA damage triggers cell cycle arrest and has a suppressive effect on the tumor microenvironment (TME). Wee1, a cell cycle regulator, can eliminate G2/M arrest by phosphorylating cyclin-dependent kinase 1 (CDK1). Meanwhile, programed death-1/programed death ligand-1 (PD-1/PDL-1) blockade is closely related to TME. This study aims to investigate the effects and mechanisms of Wee1 inhibitor AZD1775 and anti-PD-1 antibody (anti-PD-1 Ab) on radiosensitization of hepatoma.

Methods:

The anti-tumor activity of AZD1775 and IR was determined by 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) assay on human and mouse hepatoma cells HepG2, Hepa1-6, and H22. The anti-hepatoma mechanism of AZD1775 and IR revealed by flow cytometry and Western blot in vitro. A hepatoma subcutaneous xenograft mice model was constructed on Balb/c mice, which were divided into control group, IR group, AZD1775 group, IR + AZD1775 group, IR + anti-PD-1 Ab group, and the IR + AZD1775 + anti-PD-1 Ab group. Cytotoxic CD8+ T cells in TME were analyzed by flow cytometry.

Results:

Combining IR with AZD1775 synergistically reduced the viability of hepatoma cells in vitro. AZD1775 exhibited antitumor effects by decreasing CDK1 phosphorylation to reverse the IR-induced G2/M arrest and increasing IR-induced DNA damage. AZD1775 treatment also reduced the proportion of PD-1+/CD8+ T cells in the spleen of hepatoma subcutaneous xenograft mice. Further studies revealed that AZD1775 and anti-PD-1 Ab could enhance the radiosensitivity of hepatoma by enhancing the levels of interferon γ (IFNγ)+ or Ki67+ CD8 T cells and decreasing the levels of CD8+ Tregs cells in the tumor and spleen of the hepatoma mice model, indicating that the improvement of TME was manifested by increasing the cytotoxic factor IFNγ expression, enhancing CD8+ T cells proliferation, and weakening CD8+ T cells depletion.

Conclusions:

This work suggests that AZD1775 and anti-PD-1 Ab synergistically sensitize hepatoma to radiotherapy by enhancing IR-induced DNA damage and improving cytotoxic CD8+ T cells in TME.

Keywords: Hepatoma, Radiation, AZD1775, Anti-PD-1 antibody, DNA damage, Tumor microenvironment

Introduction

Hepatocellular carcinoma (hepatoma) is the sixth-most common cancer worldwide, with an extremely poor prognosis.[1] External beam radiotherapy and transarterial radioembolization are commonly used in patients unable to undergo resection or transplantation.[2] However, its effectiveness in larger tumors is limited by the radiosensitivity of normal tissue, including the liver and small bowel.[2,3] A major challenge in the management of patients with hepatoma is that cytotoxic chemotherapy has had disappointing results in clinical trials.[4,5] Novel agents that preferentially sensitize hepatoma vs. normal tissue to the cytotoxic effects of radiotherapy and chemotherapy are greatly needed.

Radiation (IR)-induced DNA damage triggers the DNA damage response (DDR), which has a suppressive effect on the tumor microenvironment (TME). Targeting the response of cancer cells to DNA damage is an attractive strategy for radiosensitization.[6] DNA damage activates cell cycle regulators and arrests the cell cycle in the G1/S and G2/M phases, enabling the action of several intricate mechanisms underlying DNA repair. Additionally, abnormal expression of cell cycle regulators is believed to be a major contributor to hepatocellular carcinogenesis and progression.[7,8] Wee1 is a tyrosine kinase that regulates the G2/M checkpoint by inhibiting cyclin-dependent kinase 1 (CDK1), preventing entry into the mitosis phase in response to DNA damage.[9,10] Inhibition of Wee1 also triggers the phosphorylation of CDK2, which subsequently enhances its activity, leading to abnormal DNA replication and causing double-stranded DNA breaks.[11] High Wee1 expression is reported to be strongly associated with increased cancer metastasis and poor survival in several cancers.[12,13] Moreover, the silencing of Wee1 expression increases cell death, decreases metastasis, and sensitizes cancer cells to radiotherapy and chemotherapy.[14,15] AZD1775 is a potent ATP-competitive inhibitor of Wee1 kinase. Several preclinical studies have demonstrated the synergistic antitumor activity of AZD1775 with chemotherapy in p53-deficient cancer cells.[16,17] Because hepatoma is characterized by a high incidence of P53 mutation and often responds to radiotherapy, Wee1 blockade is expected to be synergistic with radiotherapy for hepatoma. However, few studies have demonstrated such a synergy in hepatoma.

Tumors are strongly affected by TME. TME includes tumor-infiltrating lymphocytes (TILs), inflammatory cells, and immune factors. The presence of activated cytotoxic CD8+ T cells within TME is critical for the eradication of cancer. However, many tumors circumvent cytotoxic CD8+ T cell elimination through mechanisms such as the presentation of molecules that engage immune checkpoints (e.g., programmed death-1 [PD-1]). Therefore, tremendous progress has been made in the development of anti-PD-1-based immunotherapies for cancer in decades. Checkpoint inhibitors such as antibodies against PD-1 have been proven to be partially effective against several malignant tumors, including hepatoma.[18–20] However, there is still no report related to the immunoregulatory effects of IR + AZD1775 + anti-PD-1 antibody (anti-PD-1 Ab) in vivo. Therefore, the development of combination therapies seems necessary because they can provide a direct tumoricidal effect and enhance the antitumor effect of immunotherapy. Therefore, we conducted this study to investigate the potential synergistic antitumor effect by combining radiation with AZD1775 and anti-PD-1 Ab.

Methods

Ethical approval

The study protocol was approved by the Ethics Committee of the Public Health of College, Jilin University (No. 2022-05-30).

Cells, chemicals, and antibodies

Human and mouse hepatoma cells HepG2, Hepa1-6, H22, and human hepatocyte L02 were purchased from ATCC. Cells were cultured in designated mediums supplemented with 10% fetal bovine serum (FBS; Hyclone, GE Healthcare, Chicago, IL, USA), respectively. The cells were incubated in a 95% humidified atmosphere at 37°C in the presence of 5% CO2 to maintain exponential cell growth.

AZD1775 was purchased from Selleck (Shanghai, China), dissolved in dimethyl sulfoxide (DMSO), and added to medium with a final concentration of no more than 0.1% DMSO. Total CDK1, phospho-CDK1 (Tyr15), phospho-H2AX (S139), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Antirabbit and antimouse secondary antibodies were purchased from LI-COR Biosciences (Lincoln, NE, USA).

3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) assay

Human hepatoma cell lines were seeded at a density of 5 × 103 cells per well in 96-well plates and allowed to attach overnight. After treatment under various experimental conditions for 72 h, 50 μL of MTT was added to each well and incubated for 4 h. The supernatant was discarded, and 50 μL of DMSO was added to dissolve the formazan. The optical density at 570 nm was measured.

Animal model and treatments

Female Balb/c mice (6–8 weeks old, body weight: 20 ± 2 g) were purchased from The SPF Biotechnology Co., Ltd. (Beijing, China). All mice were kept in the specific pathogen-free animal laboratory at room temperature (22 ± 1°C), 12/12 h light/night cycle.

Under aseptic conditions, H22 suspension cells were stained with 0.1 mL 0.2% trypan blue solution. The number of viable cells to 5 × 106 cells/mL was adjusted. The axillary skin of the left forelimb of mice was disinfected with conventional alcohol, and then 0.1 mL of diluted tumor cell suspension was injected subcutaneously. The tumor size (a) axis and small (b) axis were measured every day to calculate the tumor volume (V). The formula is: V = ab2/2. When the tumor volume was about 150 mm3, the modeling was considered successful. Mice were randomly allocated into the control group, IR group, AZD1775 group, IR + AZD1775 group, IR + anti-PD-1 Ab group, and the IR + AZD1775 + anti-PD-1 Ab group with at least five mice per group and provided with standard sterile food and sterile water. All tumor-bearing mice were weighed once every day. Mice received 8 Gy radiotherapy once. One hour after IR treatment, mice were treated with AZD1775 (70 mg·kg–1·day–1, once daily) via oral gavage every 2 days. One day after IR treatment, 100 μg of anti-PD-1 Ab (RMP1-14; Bio X Cell, West Lebanon, NH, USA) or immunoglobulin G (IgG) was injected intraperitoneally into mice every 2 days. The mice were sacrificed by cervical dislocation 24 h after administration on the day 10, and the corresponding organs were taken for further use.

Tumor inhibition rate calculation

Tumor inhibition rate = (W1–W2)/W1 × 100%. W1: average tumor weight of the model group (g), W2: average tumor weight of the administration group (g). The general condition of each group of tumor-bearing mice and the growth of the tumor were observed throughout the experiment.

Flow cytometry for IR-induced G2/M phase cell cycle arrest

Cells were seeded into 6-well plates at a density of 200,000 cells per well in 2 mL medium for 16 h. The cells were treated with AZD1775 for 3 h, followed by IR (4 Gy), and then cultured for 24 h. Cells were fixed in 70% ethanol at –20°C and stained with DNA staining solution containing propidium iodide and RNase A overnight. All data were acquired on the LSRII cytometer (BD Bioscience, NJ, USA), and each sample was assessed using a collection of 10,000 events and analyzed using FlowJo software (FlowJo, Ashland, OR, USA).

Flow cytometry for mice spleens and tumor xenografts

After weighing on the 10th day, H22 tumor-bearing mice were killed using the cervical spine method. The tumor tissues and spleens from the mice were removed for flow cytometry detection. Tumor and spleen tissue was harvested into RPMI 1640 medium containing 0.2 mg/mL DNase I and 1 mg/mL collagenase IV and then incubated at 37°C for 30 min. Tissues were dissociated mechanically through a 100-μm cell filter to generate single-cell suspensions, which were then incubated with 3 mL ACK lysis buffer (red blood cell lysis buffer) at 4°C for 30 min. The cell mixture was washed twice with PBS. For cell membrane surface marker analysis, cells were stained with indicated antibodies in 1 × PBS for 30 min at 4°C in the dark. The cells were stained for 30 min with corresponding antibodies for surface antigens at 4°C in PBS. Next, the cells were washed twice with 3 mL 1 × PBS or 1 × permeabilization buffer. Finally, the cells, which were fixed and permeabilized for 30 min in fixation/permeabilization buffer, were stained with intracellular antibodies in 1 × permeabilization buffer in the dark at 4°C for 30 min. All data were acquired on the LSRII cytometer (BD Bioscience, NJ, USA), and each sample was assessed using a collection of 10,000 events and analyzed using FlowJo software.

Western blot

Tumor tissues were harvested and lysed in radio immune precipitation assay lysis buffer (50 mmol/L Tris-HCl [pH 7.5], 150 mmol/L NaCl, and 1% NP-40), and the lysate was cleared by centrifugation at 16,000 × g at 4°C for 5 min. The total cell extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were then transferred onto nitrocellulose membranes. After blocking with 5% nonfat dry milk in Tris-buffered saline with Tween (TBST) for 1 h, the membranes were incubated with the indicated primary antibodies and then with the corresponding alkaline phosphatase (AP)-conjugated secondary antibodies for 1 h. After three washes with TBST, the blots were reacted with nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3′-indolylphosphate (BCIP).

Serum alanine transaminase (ALT) and aspartate transaminase (AST) assessments

Serum ALT (ab285263, Abcam, Shanghai, China) and AST (MBS2019147, MyBioSource, San Diego, CA, USA) were determined using enzyme linked immunosorbent assay (ELISA) kits according to the manufacturer’s protocols.

Statistical analysis

Tests of significance between pairs of data were reported as P-values, derived using the student’s t-test with a two-tailed distribution and calculated at 95% confidence interval. Comparison of multiple sets of data was achieved with analysis of variance (ANOVA) with Tukey’s multiple comparisons. All error bars indicated standard deviation. All analyses were performed using GraphPad Prism v7 (GraphPad Software, San Diego, CA, USA). P <0.05 was considered statistically significant.

Results

Combining IR with AZD1775 synergistically reduces the viability of hepatoma cells

AZD1775, a Wee1 inhibitor, is currently undergoing several clinical trials as a potential drug for cancers such as ovarian cancer and uterine cancer. The reported data indicate that AZD1775 has shown a great response rate with well-tolerant toxicity profiles for many cancers.[21] To determine whether AZD1775 sensitizes hepatoma cell lines to clinical radiotherapy, we first treated three hepatoma cell lines (HepG2, Hepa1-6, and H22) and human hepatocyte L02 with AZD1775 from 25 nmol/L to 800 nmol/L and analyzed the cell viability with an MTT assay to estimate the dose inhibiting proliferation by 50% (half maximal inhibitory concentration [IC50]). As shown in Figure 1A, cell death increased with the AZD1775 dose across the three hepatoma cell lines, and the IC50 for three cells was approximately 120–220 nmol/L. However, the IC50 for L02 was higher than 700 nmol/L, indicating that AZD1775 had a specific killing effect on hepatoma cells. We then treated hepatoma cells using radiation (4 Gy) with or without AZD1775 to verify the synergistic effect of Wee1 inhibition. The results showed the synergistic inhibitory effect of radiation with AZD1775 in the three hepatoma cell lines [Figure 1B].

Figure 1.

Figure 1

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Determination of antitumor effects of AZD1775 alone or in combination with IR. (A) Cell viability was measured by MTT assay in hepatoma cells (HepG2, Hepa1-6, and H22) and human hepatocyte L02 treated with increasing doses of AZD1775. The IC50 values were calculated. (B) Cell viability was measured by MTT assay in hepatoma cells (HepG2, Hepa1-6, and H22) treated with AZD1775 or IR + AZD1775. *P <0.05, †P <0.01. IC50: Half maximal inhibitory concentration; IR: Radiation; MTT: 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide.

AZD1775 reverses the IR-induced G2/M phase cell cycle arrest and enhances IR-induced DNA damage

As we know, Wee1 is a crucial component of the G2/M checkpoint that prevents entry into mitosis in response to cell DNA damage. Thus, we examined whether the radiosensitivity effects of AZD1775 were related to interference from radiation-induced G2/M phase arrest. AZD1755 markedly reversed the accumulation of G2/M phase cells when HepG2, Hepa1-6, and H22 cells were treated with AZD1775 before radiation treatment [Figure 2A]. Wee1 arrested the G2/M checkpoint in response to DNA damage by phosphorylating CDK1. Our findings suggest that phosphorylated CDK1 decreased in both the AZD1775 alone and the IR + AZD1775 groups [Figure 2B]. These results indicate that more cells entered the mitotic phase and did not have sufficient time to repair DNA. We also detected a marker for DNA damage, the phosphorylated expression of H2AX (γH2AX) in three cells. Cells treated with IR + AZD1775 markedly increased the protein levels of γH2AX in the Western blot assay [Figure 2B]. These findings suggest that AZD1775 reverses the radiation-induced G2/M phase cell cycle arrest and enhances radiation-induced DNA damage.

Figure 2.

Figure 2

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AZD1775 reverses the IR-induced G2/M phase arrest and increases DNA damage in vitro. (A) Cell cycle distribution of HepG2, Hepa1-6, and H22 cells after treatments were analyzed by flow cytometry. Bar charts represent the percentage of cells in each phase of the cell cycle. (B) Western blot was used to examine the protein expression of G2/M checkpoint regulators and markers of DNA damage. *P <0.01. CDK1: Cyclin-dependent kinase 1; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; IR: Radiation; γH2AX: Phosphorylated histone H2AX.

AZD1775 increases the sensitivity of hepatoma cells to IR by increasing DNA damage in tumor xenografts

In order to better understand the synergistic sensitization effect, we used Balb/c xenograft mice to verify the efficacy of IR + AZD1775 observed in vitro. Balb/c mice were injected in the hind flank with H22 tumor cells and then treated with 8 Gy IR followed by AZD1775 (70 mg/kg) three times [Figure 3A]. When the defined tumor volume endpoint was reached, all treatment groups showed an obvious tumor growth delay compared with the control group. Importantly, IR + AZD1775 treatment produced the greatest inhibition of tumor growth [Figure 3B]. No significant differences in body weight were observed between the experimental groups [Figure 3C].

Figure 3.

Figure 3

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AZD1775 increases the sensitivity of hepatoma cells to IR by increasing DNA damage in a mice xenograft model. (A) Modeling and treatment flowchart of H22 cells mice xenograft model. (B) Tumor volume was measured during the treatment periods in each group. Representative images of the solid tumors were obtained at the experimental endpoint. (C) The body weights of the mice were monitored during the treatment periods. (D) Western blot was used to examine the protein expression of G2/M checkpoint regulators and markers of DNA damage. *P <0.05. CDK1: Cyclin-dependent kinase 1; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; IR: Radiation; ns: Not significant; γH2AX: Phosphorylated histone H2AX.

Furthermore, in order to determine whether the strengthened antitumor responses observed after combination treatment were mediated by increasing DNA damage, the expression of phosphorylated CDK1 and H2AX was determined by western blotting. As shown in Figure 3D, IR + AZD1775 induced DNA damage (γH2AX) and decreased the levels of the G2/M regulator (p-CDK1) in tumor xenografts. In conclusion, AZD1775 enhances the antitumor efficacy of radiation by disrupting the G2/M cell cycle arrest and increasing DNA damage.

Anti-PD-1 Ab treatment enhances the antitumor efficacy of IR + AZD1775 in vivo

Previous studies have reported that PD-1/programed death ligand-1 (PDL-1) in tumor cells mediates immune tolerance and reduces tumor-infiltrating T-cell killing ability, which may be responsible for resistance to molecular-targeted drugs. To investigate the role of anti-PD-1 immunotherapy in AZD1775-enhancing radiosensitivity of hepatoma, we designed the control, IR + AZD1775, IR + anti-PD-1 Ab, and IR + AZD1775 + anti-PD-1 Ab four groups of Balb/c xenograft mice. Balb/c mice were injected in the hind flank with H22 tumor cells and then treated with 8 Gy IR followed by AZD1775 or anti-PD-1 Ab thrice [Figure 4A]. At the end of the experiment, we found that all treatment groups showed an obvious tumor growth delay compared with the control group [Figure 4B]. Treatment with anti-PD-1 Ab led to marked reductions in xenograft size compared with the IgG antibody-treated mice. Surprisingly, we found that anti-PD-1 Ab therapy in combination with IR + AZD1775 improved antitumor activity in vivo [Figure 4B]. In addition, there was no significant difference in weight between the groups of mice [Figure 4C]. The liver function measurements (AST and ALT) of each group of mice show that our treatment does not cause significant damage to the liver function of the mice [Figure 4D].

Figure 4.

Figure 4

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Anti-PD-1 Ab treatment enhances the antitumor efficacy of IR + AZD1775 in a mice xenograft model. (A) Modeling and treatment flowchart of H22 cells mice xenograft model. (B) Tumor volume was measured during the treatment periods in each group. Representative images of the solid tumors were obtained at the experimental endpoint. (C) The body weights of the mice were monitored during the treatment periods. (D) The liver function indicator (AST and ALT) of each group of mice was measured using ELISA. *P <0.05, †P <0.01. ALT: Alanine transaminase; Anti-PD-1 Ab: Anti-PD-1 antibody; AST: Aspartate transaminase; ELISA: Enzyme linked immunosorbent assay; IR: Radiation; ns: Not significant. PD-1: Programed death-1.

Anti-PD-1 Ab enhances the radiosensitivity of hepatoma cells by improving cytotoxic CD8+ T cells in TME

To explore the mechanisms underlying the enhancement in antitumor ability produced by the combination treatment with anti-PD-1 Ab, AZD1775, and IR, the levels of CD8+ TILs in tumor xenografts from the experiment shown in Figure 4A were measured by flow cytometry. CD8+ TILs mainly function by secreting cytotoxic factors, such as interferon γ (IFNγ). Hence, we investigated the quantities of IFNγ present after treatment. As shown in Figure 5A, tumor-infiltrating IFNγ+/CD8+ T cells significantly increased in the IR + anti-PD-1 Ab and IR + AZD1775 + anti-PD-1 Ab groups, and no obvious changes were observed in the other treatment groups. Similarly, the proportion of Ki67+/CD8+ T cells significantly increased in the IR + anti-PD-1 Ab and IR + AZD1775 + anti-PD-1 Ab groups [Figure 5B], indicating an enhanced proliferative ability of cytotoxic CD8+ T cells. On the contrary, the proportion of CD8+ Tregs in the IR + AZD1775 + anti-PD-1 Ab group decreased the most [Figure 5C]. The decrease in the proportion of CD8+ Tregs indicated a reduction in cytotoxic CD8+ T cell depletion. However, we did not observe the effect of our treatment on PD-1+/CD8+ T cells in tumor xenografts [Figure 5D]. We also observed similar effect of the combination treatment for IFNg+/CD8+ T cells, Ki67+/CD8+ T cells, and CD8+ Tregs in the spleen of mice [Figure 6A–C]. Inconsistent with the results in tumor xenografts, PD-1+/CD8+ T cells of mice spleen in all treatment groups showed an obvious decrease compared with the control group [Figure 6D]. These results suggest that anti-PD-1 Ab enhances the radiosensitivity of hepatoma cells by improving cytotoxic CD8+ T cells in TME.

Figure 5.

Figure 5

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Flow cytometry detection confirms that anti-PD-1 Ab enhances the radiosensitivity of hepatoma cells by improving cytotoxic CD8+ T cells in TME. The levels of IFNγ+/CD8+ T cells (A), Ki67+/CD8+ T cells (B), CD8+ Tregs (C), and PD-1+/CD8+ T cells (D) in xenografts of mice model were determined by flow cytometry. Bar charts represent the percentage of cells. *P <0.05, †P <0.01. Anti-PD-1 Ab: Anti-PD-1 antibody; IFNγ: Interferon γ; IR: Radiation; ns: Not significant; PD-1: Programed death-1; TME: Tumor microenvironment.

Figure 6.

Figure 6

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Flow cytometry detection confirms that anti-PD-1 Ab improves the radiosensitivity of hepatoma cells by regulating cytotoxic CD8+ T cells in the spleen of mice models. The levels of IFNγ+/CD8+ T cells (A), Ki67+/CD8+ T cells (B), CD8+ Tregs (C), and PD-1+/CD8+ T cells (D) in the spleen of mice model were determined by flow cytometry. Bar charts represent the percentage of cells. *P <0.05, †P <0.01. Anti-PD-1 Ab: Anti-PD-1 antibody; IFNγ: Interferon γ; ns: not significant; PD-1: Programed death-1.

Discussion

In this study, we show that the Wee1 inhibitor AZD1775 and anti-PD-1 Ab synergistically radiosensitize human and mouse hepatoma cell lines in vitro and in vivo. Mechanistically, AZD1775 abrogates the G2/M checkpoint and impairs the repair of DNA double-strand breaks. Additionally, we find that the radiosensitizing effect of AZD1775 and anti-PD-1 Ab combination is enhanced by regulating the composition of CD8+ T cells in the tumor immune microenvironment.

Numerous approaches have been tried previously to develop better therapies for hepatoma. Targeting DNA damage repair-related pathways has been a promising therapeutic strategy to sensitize tumor cells to their inherent genomic instability and further improve response rates for the treatment of various types of cancers. Repairing of radiation-induced DDR needs cell cycle checkpoint control and homologous recombination repair. This process has resulted in the development of poly (ADP-ribose) polymerase inhibitors for cancers with breast cancer susceptibility gene 1/2 (BRCA1 or BRCA2) defects and checkpoint kinase 1, Wee1, and ataxia telangiectasia and Rad3-related protein inhibitors that prevent S and G2/M cell cycle arrest after DNA damage.[22] Hence, targeting the response of cancer cells to DNA-damaging agents is an attractive strategy for radiosensitization. Wee1 kinases are critical components in cell cycle arrest post radiotherapy. It has been shown that inhibiting Wee1 results in replication stress, loss of genomic integrity, nucleotide shortage, and subsequent double-strand DNA breaks. Currently, the Wee1 inhibitor, AZD1775, has been combined with other antitumor drugs (cisplatin, gemcitabine, cytarabine, gemcitabine, paclitaxel, temozolomide, 5-fluorouracil, and/or IR) in preclinical and clinical experiments.[17,23,24] Among these combinations, the combination of IR and AZD1775 significantly enhances the efficacy of human breast, prostate, and lung cancer cell killing.[25] Nonetheless, no study has yet been reported to assess its efficacy for treating hepatoma by IR combined with AZD1775. Following this thought, by carefully choosing cell line models that well represent both hepatoma cell lines (HepG2, Hepa1-6, and H22) and human hepatocyte L02, demonstrate that AZD1775 had a specific killing effect on hepatoma cells. AZD1775 increased cell radiosensitivity significantly in the three hepatoma cell lines [Figure 1].

Mechanically, treatment with Wee1 inhibitor allows injured cells to quickly pass through the G2/M repair phase, leading to DNA damage accumulation, which causes mitotic catastrophe and apoptosis.[26,27] In the present study, we demonstrated that AZD1775 potently inhibited the phosphorylation of CDK1 in the absence or presence of radiation [Figure 2B]. Consistent with the role of Wee1 in G2/M checkpoint regulation, AZD1775 prevented IR-induced G2/M phase cell cycle arrest [Figure 2A], which was accompanied by enhanced mitotic catastrophe and γH2AX expression, indicative of enhanced cell death and DNA damage [Figure 2B].

Immune checkpoint blockade (ICB) targeting the programed death (PD) axis is a promising treatment modality that can induce durable tumor control. Nevertheless, the majority of patients with recurrent/metastatic cancer do not respond to PD-1/PDL-1 blockade. A combination of PD-1-axis ICB with cytotoxic treatments designed to enhance the antitumor immunity through the induction of immunogenic cell death may enhance response rates. Here, we explore an alternative combination treatment strategy by addressing a tumor cell-intrinsic mechanism of resistance to IR and cytotoxic T lymphocytes (CTL) killing. We found that the combination of AZD1775 and anti-PD-1 Ab could regulate the tumor immune microenvironment of hepatoma. In addition to its ability to mediate DNA damage-induced cancer cell death, radiotherapy-induced tumor cell DDR activates cytosolic nucleic acid sensor pathways, such as cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING), and propagation of the resulting inflammatory signals remodels the immune contexture of the TME.[28–30] Activation of nucleic acid sensors triggers the production of type I IFNs and inflammatory cytokines, which is frequently referred to as “viral mimicry” with the potential to stimulate antitumor CD8+ T cell responses.[31] Tumor-infiltrating TILs, especially the activated cytotoxic CD8+ T cells within TME, are critical for the eradication of cancer.[32] The confluence of DDR and inflammatory pathway activation has led to numerous recent studies demonstrating that DDR inhibitors can enhance the inflammatory response to radiotherapy. Reports are proving that cancer cells treated with radiation and AZD1775 increase granzyme B-dependent killing of tumor cells by CTLs.[33]

However, many tumors circumvent cytotoxic T-cell elimination through mechanisms such as the reduction of tumor antigen expression, the presentation of molecules that engage immune checkpoints (e.g., PD-1, cytotoxic T lymphocyte-associated protein 4 [CTLA-4]), or the secretion of immunoregulatory molecules.[34–36] Indeed, some of the most impactful cancer treatments are immunotherapies that counteract these immunosuppressive actions, such as CTLA-4 and PD-1/PDL-1 checkpoint inhibitor antibodies, chimeric antigen receptor (CAR)-T-cell therapy, or adoptive transfer of TILs.[37–39] There have been reports proving that the addition of anti-PD-1 Ab to radiotherapy plus Wee1 inhibition significantly increases survival in the mouse oral cancer 1 (MOC1) syngeneic mouse model of head and neck squamous cell carcinoma.[40] In our work, the addition of AZD1775 and anti-PD-1 Ab altered the composition of tumor-infiltrating and mouse spleen cytotoxic CD8+ T cells. Specifically, the proportion of PD-1+/CD8+ T cells in the spleen of tumor-bearing mice in the IR + AZD1775 group was significantly reduced compared to the control group, indicating that AZD1775 could significantly reduce the immunosuppressive effect of hepatoma passing through the PD-1 axis [Figure 6D]. At the same time, the proportion of IFNγ+/CD8+ T cells and Ki67+/CD8+ T cells in the xenograft and the spleen of IR + anti-PD-1 Ab and IR + AZD1775 + anti-PD-1 Ab group mice was relatively higher than in the IR + AZD1775 and control groups. This result proves that blocking the PD-1 axis could enhance the radiosensitivity of hepatoma by increasing the proliferation of CD8+ T cells and IFNγ secreted CD8+ T cells differentiation [Figures 5A,B and 6A,B]. However, AZD1775 treatment could not regulate the proliferation and differentiation of CD8+ T cells compared with the control group, whether in xenografts or spleens (statistical results not shown in Figures 5 and 6). On the contrary, the addition of either AZD1775 or anti-PD-1 Ab alone would induce the decrease of the proportion of CD8+ Tregs cells in the xenograft, thereby reducing the depletion of cytotoxic CD8+ T cells [Figure 5C], indicating that the improvement of cytotoxic CD8+ T cells in TME was more regulated by anti-PD-1 Ab but not by AZD1775. Moreover, AZD1775 could contribute to the radiosensitivity regulated by anti-PD-1 Ab.

In summary, radiotherapy is widely used for many cancer types, PD-1-axis ICB is Food and Drug Administration (FDA)-approved for many cancer types, and AZD1775 is progressing in clinical development. Our data provide important mechanistic insights into how Wee1 kinase inhibition can be rationally combined with other anticancer strategies and provide the preclinical rationale for the combination of these treatments in clinical trials designed to enhance response rates to the PD-axis ICB. The use of three clinically relevant treatments targeting complementary mechanisms for patients with hepatoma is a major strength of our work.

Funding

This work was supported by grants from the Science and Technology Department of Jilin Province (No. YDZJ202201ZYTS590) and the National Natural Science Foundation of China (No. 82173454).

Conflicts of interest

None.

Footnotes

How to cite this article: Yin YC, Wang J, Yi JX, Zhang KY, Yin ZM, Jin SZ, Zheng BS. AZD1775 and anti-PD-1 antibody synergistically sensitize hepatoma to radiotherapy. Chin Med J 2024;137:222–231. doi: 10.1097/CM9.0000000000002988

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