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. 2017 Mar 1;7(3):473-483.
eCollection 2017.

Chk1 inhibition potentiates the therapeutic efficacy of PARP inhibitor BMN673 in gastric cancer

Yuping Yin  1 , Qian Shen  2 , Peng Zhang  3 , Ruikang Tao  4 , Weilong Chang  5 , Ruidong Li  3 , Gengchen Xie  3 , Weizhen Liu  3 , Lihong Zhang  2 , Prabodh Kapoor  6 , Shumei Song  7 , Jaffer Ajani  7 , Gordon B Mills  8 , Jianying Chen  3 , Kaixiong Tao  3 , Guang Peng  9
Affiliations

Chk1 inhibition potentiates the therapeutic efficacy of PARP inhibitor BMN673 in gastric cancer

Yuping Yin et al. Am J Cancer Res. .

Abstract

Globally, gastric cancer is the second leading cause of cancer deaths because of the lack of effective treatments for patients with advanced tumors when curative surgery is not possible. Thus, there is an urgent need to identify molecular targets in gastric cancer that can be used for developing novel therapies and prolonging patient survival. Checkpoint kinase 1 (Chk1) is a crucial regulator of cell cycle transition in DNA damage response (DDR). In our study, we report that Chk1 plays an important role in promoting gastric cancer cell survival and growth, which serves as an effective therapeutic target in gastric cancer. First, Chk1 ablation by small interfering RNA could significantly inhibit cell proliferation and sensitize the effects of ionizing radiation (IR) treatment in both p53 wild type gastric cancer cell line AGS, and p53 mutant cell line MKN1. Secondly, we tested the anticancer effects of Chk1 chemical inhibitor LY2606368, which is a novel Chk1/2 targeted drug undergoing clinical trials in many malignant diseases. We found that LY2606368 can induce DNA damage, and remarkably suppress cancer proliferation and induce apoptosis in AGS and MKN1 cells. Moreover, we identified that LY2606368 can significantly inhibit homologous recombination (HR) mediated DNA repair and thus showed marked synergistic anticancer effect in combination with poly (ADP-ribose) polymerase 1 (PARP1) inhibitor BMN673 in both in vitro studies and in vivo experiments using a gastric cancer PDx model. The synergy between LY2606368 and PARP1 was likely caused by impaired the G2M checkpoint due to LY2606368 treatment, which forced mitotic entry and cell death in the presence of BMN673. In conclusion, we propose that Chk1 is a valued target for gastric cancer treatment, especially Chk1 inhibitor combined with PARP inhibitor may be a more effective therapeutic strategy in gastric cancer.

Keywords: BMN673; Chk1; DNA damage response; LY2606368; gastric cancer.

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Figures

Figure 1
Figure 1
Chk1 ablation can significantly suppress the cell proliferation and sensitize the IR treatment in gastric cancer cells. A, B. Graphical presentation of cell viability at different days examined by MTS assay after Chk1 knockdown. OD values were measured and plotted with respect to time (*P<0.05). C. Graphical presentation of relative (%) cell viability of AGS and MKN1 cells exposed with IR (2 Gy), and with IR (4 Gy) respectively, (*P<0.05). D. Western blot analyses using anti-Chk1 antibodies showing Chk1 siRNA efficiently inhibited the expression of Chk1 in AGS and MKN1 cells. Actin was used as a loading control.
Figure 2
Figure 2
Chk1 inhibitor LY2606368 can induce DNA damage and apoptosis, and can suppress cell proliferation in gastric cancer cells. A. Graphical presentation of % cell viability of AGS and MKN1 cells measured 3 days after treatment with LY2606368. B. Clonogenic assay in AGS and MKN1 cells. Cells were treated with LY2606368 for 3 days. Cell viability in AGS and MKN1 cells were significantly inhibited in a dosage-dependent manner. C. Graphical presentation of relative (%) colony formation of AGS and MKN1 cells in clonogenic assay as described in B after exposure to 25 nM LY2606368 for 24 hours. D. Graphical presentation of apoptosis in AGS and MKN1 cells, measured using Annexin V/PI after exposure to 25 nM LY2606368 for 24 hours. Significant apoptosis was observed in AGS and MKN1 cells staining. E, F. Western blot analyses of AGS and MKN1 cells treated with LY2606368 for 24 hours. Endogenous Chk1, γ-H2AX, cleaved caspase3, and p-Chk1 (Ser345) were detected using their respective antibodies as shown left to each panel.
Figure 3
Figure 3
Chk1 inhibitor LY2606368 can suppress HR repair capacity. DR-GFP cells were used to detect the effect of LY2606368 on HR repair. A. LY2606368 treatment (20 nM) for 24 hours suppresses the HR repair capacity (P<0.05, LY20606368 VS DMSO). Each value was presented with the percentage of the GFP positive cells in pCBASecI transfected cells. The fold change was calculated, and data was showed as Mean ± SD. B. Cell cycle in HR repair assay was analyzed. LY2606368 induces S phase arrest in DR-GFP cells. C. HU was used to synchronize the cell cycle distribution for 16 hours before HR repair analysis, fold change was showed as mean ± SD. D. Cell cycle analysis for HU-synchronized HR repair assays showed there was no significant cell cycle alternation between DMSO control and LY2606368 treated groups after HU synchronization.
Figure 4
Figure 4
LY2606368 can sensitize the anticancer effect of PARP inhibitor BMN673 in gastric cancer cells. A, B. Graphical presentation of relative (%) cell viability of AGS and MKN1 cells after treatment with indicated drugs for 5 days. Cell viability was detected by MTS assays, significant synergistic anticancer effect between LY2606368 and BMN673 was observed in AGS and MKN1 cell, CI value of EC50 is 0.76 in AGS, and 0.41 in MKN1 (CI<1 indicates synergy). C. Clonogenic assay showed that LY2606368 (5 nM) and BMN673 (10 nM) combination inhibits cell survival of AGS and MKN1 cells. D. Graphical presentation of relative (%) colony formation in clonogenic assay as described in C (*P<0.05). E. Graphical presentation of (%) rate of apoptosis in AGS and MKN1 cells after treatment with 5 nM LY2606368, 1 µM BMN673, or both (AGS for 72 hours, MKN1 for 48 h) (*P<0.05). F. Western blot analyses of AGS and MKN1 cells treated with LY2606368, BMN673, or both. Combination of BMN673 and LY2606368 can induce more expression of apoptosis marker cleaved caspase3.
Figure 5
Figure 5
LY2606368 could force mitotic entry of G2M phase cells induced by BMN673 in gastric cancer cells. A, B. Cell cycle analysis of AGS and MKN1 cells after treatment with BMN673 (1 µM), LY2606368 (10 nM), or combination for 24 hours. C, D. Western blot analyses of AGS and MKN1 cells respectively after treatment with BMN673 (1 µM), LY2606368 (10 nM), or combination for 24 hours using antibodies as indicated left to each panel.
Figure 6
Figure 6
LY2606368 and BMN673 combination has synergistic anticancer effect in gastric cancer PDX model. Six weeks old nude mice with subcutaneous implanted gastric cancer PDX tumor were treated with vehicle control, BMN673 (0.33 mg/kg), LY2606368 (2 mg/kg) or combination of these two drugs for 16 days. Statistical significance differences between groups were calculated using unpaired t test. Significant differences were indicated by asterisk (P<0.05) A. Tumor volume of each group was measured in indicated days of treatment. Data were showed as mean ± SD. B. Images of gastric cancer PDX tumor of each group were presented at the same time of study ending. C, D. Tumor volume and tumor weight in each group were measured at the end of the treatment and presented.
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