AZD5438, an inhibitor of Cdk1, 2, and 9, enhances the radiosensitivity of non-small cell lung carcinoma cells

Abstract

Purpose: Radiation therapy (RT) is one of the primary modalities for treatment of non-small cell lung cancer (NSCLC). However, due to the intrinsic radiation resistance of these tumors, many patients experience RT failure, which leads to considerable tumor progression including regional lymph node and distant metastasis. This preclinical study evaluated the efficacy of a new-generation cyclin-dependent kinase (Cdk) inhibitor, AZD5438, as a radiosensitizer in several NSCLC models that are specifically resistant to conventional fractionated RT.

Methods and materials: The combined effect of ionizing radiation and AZD5438, a highly specific inhibitor of Cdk1, 2, and 9, was determined in vitro by surviving fraction, cell cycle distribution, apoptosis, DNA double-strand break (DSB) repair, and homologous recombination (HR) assays in 3 NSCLC cell lines (A549, H1299, and H460). For in vivo studies, human xenograft animal models in athymic nude mice were used.

Results: Treatment of NSCLC cells with AZD5438 significantly augmented cellular radiosensitivity (dose enhancement ratio rangeing from 1.4 to 1.75). The degree of radiosensitization by AZD5438 was greater in radioresistant cell lines (A549 and H1299). Radiosensitivity was enhanced specifically through inhibition of Cdk1, prolonged G(2)-M arrest, inhibition of HR, delayed DNA DSB repair, and increased apoptosis. Combined treatment with AZD5438 and irradiation also enhanced tumor growth delay, with an enhancement factor ranging from 1.2-1.7.

Conclusions: This study supports the evaluation of newer generation Cdk inhibitors, such as AZD5438, as potent radiosensitizers in NSCLC models, especially in tumors that demonstrate variable intrinsic radiation responses.

Fig. 1

Specificity of AZD5438 in human NSCLC cells. (A) Immunoblot analysis of Cdk1, Cdk2, and Cdk9 in NSCLC cells. (B) Clonogenic survival assay for drug sensitivity determination. NSCLC cells were plated with or without AZD5438 (5 nM, 50 nM, 500 nM, 5 µM, and 50 µM). Colonies were counted and percentage of survival was plotted against log [Dose]. (C) AZD5438 prevents Cdk2 activity. H460 cells were treated with nocodazole (50 ng/mL) for 16 h to synchronize cells in M phase. Cells were washed and treated with AZD5438 (435 nM) for 4 h, followed by immunoblot analysis of pRb (Ser₇₈₀). Total Rb protein was used for loading control. (D) AZD5438 does not abrogate Chk1/2 activation. Both A549 (250 nM) and H460 (435 nM) cells were treated with AZD5438 for 24 h and treated with IR (5 Gy) for 30 min. Immunoblot analysis using pChk1(Ser₃₁₇) and pChk2 (T₆₈). Total Chk1 and Chk2 proteins and actin were used for loading control.

Fig. 2

AZD5438 increased the sensitivity of NSCLC cells to IR. (A-C, left panels) Clonogenic survival of NSCLC cells with or without AZD5438. A549 (75 nM), H1299 (50 nM), and H460 (200 nM) cells were treated with AZD5438 for 24 h and treated with IR as indicated. Cells were trypsinized immediately and colony formation was counted. (A-C, right panels) AZD5438 in combination with IR enhanced the tumor growth delay in NSCLC cells: subcutaneous tumors were treated, as indicated, with IR (2 Gy per day, 5 days), AZD5438 (25 mg/kg/day for 5 days), and IR plus AZD5438 (25 mg/kg/day plus 2 Gy per day for 5 days). AZD5438 was administrated po 1 h before IR. Tumor volume (mm³) was measured twice per week and was plotted against days.

Fig. 3

IR sensitization of NSCLC cells transfected with Cdk siRNAs. All 3 NSCLC cells were transfected with either Cdk-specific siRNAs (200 pmol) (A-E) or scrambled siRNAs (200 pmol) using Lipofectamine 2000. After 48 h, cells were trypsinized, counted, and plated for CFA. Remaining cells were lysed and subjected to immunoblot assay for detecting Cdks (insets). Three hours after plating, cells were irradiated, and colonies were counted after 10 days. (A) A549 cells with Cdk1 siRNA; (B) A549 cells with Cdk2 siRNA; (C) A549 cells with Cdk9 siRNA; (D) H460 cells with Cdk1siRNA; (E) H1299 cells with Cdk1siRNA.

Fig. 4

AZD5438 inhibits HR repair. (A) DR-GFP construct schematic for HR repair assay. (B) H1299 cells containing a DR-GFP construct were transfected with phCMV-1-I-SceI (negative control), pCMV3xnls-I-SceI (functional endonuclease), or pGFP (transfection efficiency control), and 75 nM of AZD5438 was added as indicated. GFP signal was assayed at 3 days post-transfection with a FACSCalibur flow cytometer. A total of 50,000 cells were scored per treatment group. (C, left and middle panels) The frequency of recombination events was calculated from the number of GFP-positive cells divided by the number of cells analyzed following correction for transfection efficiency. (Right panel) Immunoblot analysis of Rad51expression in H1299 cells treated with AZD5438 (75 nM) for 72 h, lysed for immunoblotting.

Fig. 5

IR sensitization by AZD5438 was associated with delayed DSB repair in NSCLC cells. (A) Effect of AZD5438 on DNA DSB in 3 NSCLC cell lines. (B) DNA DSB repair kinetics in NSCLC cell lines. Cells were incubated with or without AZD5438 for 24 h (210 nM, 90 nM, and 435 nM for A549, H1299 and H460, respectively), irradiated (2 Gy), immunostained for γH2AX foci, and counted for each time point (average, 50 nuclei). (C) Repair kinetics of NSCLC cells was obtained by plotting the percentage of remaining foci against time; D, AZD5438; R, radiation.

Fig. 6

AZD5438 exhibited G₂-M checkpoint arrest in NSCLC cells. Cells were treated with AZD5438 followed by IR (2 Gy) and collected 8 h post-IR. (A) Cells were stained with PI to detect distribution of cell cycle after the treatments. (B) Quantitation of the perecentage of cells at different phases of the cell cycle in response to drug, IR, and IR plus drug.