AZD-7648, a DNA-PK Inhibitor, Induces DNA Damage, Apoptosis, and Cell Cycle Arrest in Chronic and Acute Myeloid Leukemia Cells

Abstract

The non-homologous end joining pathway is vital for repairing DNA double-strand breaks (DSB), with DNA-dependent protein kinase (DNA-PK) playing a critical role. Altered DNA damage response (DDR) in chronic (CML) and acute myeloid leukemia (AML) offers potential therapeutic opportunities. We studied the therapeutic potential of AZD-7648 (DNA-PK inhibitor) in CML and AML cell lines. This study used two CML (K-562 and LAMA-84) and five AML (HEL, HL-60, KG-1, NB-4, and THP-1) cell lines. DDR gene mutations were obtained from the COSMIC database. The copy number and methylation profile were evaluated using MS-MLPA and DDR genes, and telomere length using qPCR. p53 protein expression was assessed using Western Blot, chromosomal damage through cytokinesis-block micronucleus assay, and γH2AX levels and DSB repair kinetics using flow cytometry. Cell density and viability were analyzed using trypan blue assay after treatment with AZD-7648 in concentrations ranging from 10 to 200 µM. Cell death, cell cycle distribution, and cell proliferation rate were assessed using flow cytometry. The cells displayed different DNA baseline damage, DDR gene expressions, mutations, genetic/epigenetic changes, and p53 expression. Only HEL cells displayed inefficient DSB repair. The LAMA-84, HEL, and KG-1 cells were the most sensitive to AZD-7648, whereas HL-60 and K-562 showed a lower effect on density and viability. Besides the reduction in cell proliferation, AZD-7648 induced apoptosis, cell cycle arrest, and DNA damage. In conclusion, these results suggest that AZD-7648 holds promise as a potential therapy for myeloid leukemias, however, with variations in drug sensitivity among tested cell lines, thus supporting further investigation to identify the specific factors influencing sensitivity to this DNA-PK inhibitor.

Keywords: AZD-7648; DNA damage repair; DNA-PK inhibitor; myeloid leukemia; therapeutic target.

Figure 1

Copy number (a) and methylation status (b) of DNA damage repair genes in CML and AML cell lines. MS-MLPA results for seven DNA damage repair genes are presented as a schematic representation of the alterations in copy number and a radar chart showing the methylation status. The deletions (red) were defined as a loss of >0.20, and the amplifications (blue) as a gain of >0.20 in relation to healthy subjects. Samples with a methylation level ≥10% were classified as methylated, and two methylation ranges were determined: levels between 10% and 50% were classified as hemi-methylated, and levels between 51% and 100% were considered methylated.

Figure 2

Expression levels of DDR-related genes in CML and AML cell lines. The expression of CHEK2, PARP1, PRKDC, RAD51, TP53, and XRCC6 was assessed by qPCR. The results are normalized to the HPRT gene and represent the mean ± standard error of the mean (SEM) of 3 independent experiments. Statistical analyses were conducted using one-way ANOVA and Tukey’s multiple comparisons test or Kruskal–Wallis test and Dunn’s multiple comparisons test (TP53). ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 (comparison with HL-60); ** p < 0.01, and *** p < 0.001 (comparison with LAMA-84).

Figure 3

p53 protein expression in CML and AML cell lines. (a) The protein expression was analyzed using Western Blot and the results are presented as the ratio of the fluorescence intensities of p53 versus β-actin and the graphs represent the alteration in relation to the HEL cell line (p53/β-actin equal to 1). Results depict the mean ± SEM of 3 or 2 (NB-4 cells) independent experiments and data were statistically analyzed using the Wilcoxon signed-rank test (comparison with HEL cell line). (b) Representative immunoblot of p53 and β-actin for each cell line.

Figure 4

Double-strand breaks repair kinetics in CML and AML cells following H₂O₂ exposure. The baseline DNA damage levels of the cell lines correspond to the 0 h time point. The results represent the mean ± SEM of γH2AX mean fluorescence intensity (MFI) obtained from 3 independent experiments. Statistical analyses were performed using one-way ANOVA for repeated measures, followed by Tukey’s multiple comparisons test or Friedman test, followed by Dunn’s multiple comparisons test (KG-1, HL-60, and NB-4). * p < 0.05, and ** p < 0.01 (comparison with the 0 h time point); ^# p < 0.05, and ^## p < 0.01 (comparison with the 1 h time point). The “x” represents the absence of significant differences between 0 h and 24 h.

Figure 5

The effect of AZD-7648 (AZD) on cell viability in CML and AML cell lines. (a) Results are expressed as cellular viability, represented as a percentage (%) of viable cells. Results represent the mean ± SEM of 5 independent experiments. Data were statistically analyzed at each time point by comparison of the tested doses with the control using ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test or Kruskal–Wallis test followed by Dunn’s multiple comparisons test. (b) Log dose–response viability curve of AZD-7648 after 24 or 48 (KG-1*) hours of treatment. Results represent the mean ± SEM of 5 independent experiments. ** p < 0.01, and *** p < 0.001 (control vs. 50, 100, 125, 150, and 200 μM); ^$ p < 0.05, and ^$$ p < 0.01 (control vs. 100, 125, 150, and 200 μM); ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 (control vs. 125, 150 and 200 μM); ^¢ p < 0.05, and ^¢¢ p < 0.01 (control vs. 150, and 200 μM); ^££ p < 0.01, and ^£££ p < 0.001 (control vs. 200 μM).

Figure 6

Analysis of cell death induced by AZD-7648 (AZD) in CML and AML cell lines after 24 or 48 (KG-1) hours of treatment. (a) The type of cell death was identified using annexin V/propidium iodide staining and analyzed using flow cytometry. Data are expressed as a percentage (%) of viable, early apoptosis, late apoptosis/necrotic, and necrotic cells and represent mean ± SEM of 5 independent experiments. Statistical analyses were performed by comparison with control, using one-way ANOVA and Dunnett’s multiple comparisons test, Kruskal–Wallis and Dunn’s multiple comparisons test, unpaired t-test, or Mann–Whitney test. (b) Cell morphology was observed by light microscopy (amplification: 1000×), and the most representative image was selected. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 7

The expression levels of cleaved PARP and activated caspase-3 (a) and phosphorylated-H2AX (γH2AX) (b) in LAMA-84, HEL, and KG-1 cell lines after treatment with AZD-7648 (AZD). Results were obtained after 24 or 48 (KG-1) hours of incubation and represent mean ± SEM of 5 independent experiments. Data were statistically analyzed by comparison with control using Kruskal–Wallis followed by Dunn’s multiple comparisons test or one-way ANOVA followed by Dunnett’s multiple comparisons test. ** p < 0.01, and *** p < 0.001.

Figure 8

Effect of AZD-7648 (AZD) on cell proliferation of LAMA-84, HEL, and KG-1 cell lines. Results were obtained after 24 or 48 (KG-1) hours of incubation and represent the percentage (%) of BrdU incorporation. Data are expressed as mean ± SEM of 5 independent experiments and were statistically analyzed by comparison with control, using Kruskal–Wallis followed by Dunn’s multiple comparisons test. * p < 0.05, ** p < 0.01, and *** p < 0.001.