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

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.

Antitumor Effect of Brusatol in Acute Lymphoblastic Leukemia Models Is Triggered by Reactive Oxygen Species Accumulation.

Acute lymphoblastic leukemia (ALL) is one of the most common hematological malignancies at pediatric ages and is characterized by different chromosomal rearrangements and genetic abnormalities involved in the differentiation and proliferation of lymphoid precursor cells. Brusatol is a quassinoid plant extract extensively studied due to its antineoplastic effect through global protein synthesis and nuclear factor erythroid 2-related factor-2 (NRF2) signaling inhibition. NRF2 is the main regulator of cellular antioxidant response and reactive oxygen species (ROS), which plays an important role in oxidative stress regulation. This study aimed to evaluate the effect of brusatol in in vitro models of ALL. KOPN-8 (B-ALL), CEM (T-ALL), and MOLT-4 (T-ALL) cell lines were incubated with increasing concentrations of brusatol, and the metabolic activity was evaluated using the resazurin assay. Flow cytometry was used to evaluate cell death, cell cycle, mitochondrial membrane potential (Δψmit), and to measure ROS and reduced glutathione (GSH) levels. Our results show that brusatol promoted a decrease in metabolic activity in ALL cell lines in a time-, dose-, and cell-line-dependent manner. Brusatol induced a cytostatic effect by cell cycle arrest in G0/G1 in all cell lines; however, cell death mediated by apoptosis was only observed in T-ALL cells. Brusatol leads to an oxidative stress imbalance by the increase in ROS levels, namely, superoxide anion. Redox imbalance and cellular apoptosis induced by brusatol are highly modulated by mitochondria disruption as a decrease in mitochondrial membrane potential is detected. These data suggest that brusatol might represent a new therapeutic approach for acute lymphoblastic leukemia, particularly for ALL T-cell lineage.

Zinc Prevents DNA Damage in Normal Cells but Shows Genotoxic and Cytotoxic Effects in Acute Myeloid Leukemia Cells.

Genomic instability is prevented by the DNA damage response (DDR). Micronutrients, like zinc (Zn), are cofactors of DDR proteins, and micronutrient deficiencies have been related to increased cancer risk. Acute myeloid leukemia (AML) patients commonly present Zn deficiency. Moreover, reports point to DDR defects in AML. We studied the effects of Zn in DDR modulation in AML. Cell lines of AML (HEL) and normal human lymphocytes (IMC) were cultured in standard culture, Zn depletion, and supplementation (40 µM ZnSO4) conditions and exposed to hydrogen peroxide (H2O2) or ultraviolet (UV) radiation. Chromosomal damage, cell death, and nuclear division indexes (NDI) were assessed through cytokinesis-block micronucleus assay. The phosphorylated histone H2AX (yH2AX) expression was monitored at 0 h, 1 h, and 24 h after exposure. Expression of DDR genes was evaluated by quantitative real time polymerase chain reaction (qPCR). Zn supplementation increased the genotoxicity of H2O2 and UV radiation in AML cells, induced cytotoxic and antiproliferative effects, and led to persistent yH2AX activation. In contrast, in normal lymphocytes, supplementation decreased damage rates, while Zn depletion favored damage accumulation and impaired repair kinetics. Gene expression was not affected by Zn depletion or supplementation. Zn presented a dual role in the modulation of genome damage, preventing damage accumulation in normal cells and increasing genotoxicity and cytotoxicity in AML cells.

Acute myeloid leukemia sensitivity to metabolic inhibitors: glycolysis showed to be a better therapeutic target.

Cancer cells alter their metabolism by switching from glycolysis to oxidative phosphorylation (OXPHOS), regardless of oxygen availability. Metabolism may be a molecular target in acute myeloid leukemia (AML), where mutations in metabolic genes have been described. This study evaluated glycolysis and OXPHOS as therapeutic targets. The sensitivity to 2-deoxy-D-glucose (2-DG; glycolysis inhibitor) and oligomycin (OXPHOS inhibitor) was tested in six AML cell lines (HEL, HL-60, K-562, KG-1, NB-4, THP-1). These cells were characterized for IDH1/2 exon 4 mutations, reactive oxygen species, and mitochondrial membrane potential. Metabolic activity was assessed by resazurin assay, whereas cell death and cell cycle were assessed by flow cytometry. Glucose uptake and metabolism-related gene expression were analyzed by 18F-FDG and RT-PCR/qPCR, respectively. No IDH1/2 exon 4 mutations were detected. HEL cells had the highest 18F-FDG uptake and peroxides/superoxide anion levels, whereas THP-1 showed the lowest. 2-DG reduced metabolic activity in all cell lines with HEL, KG-1, and NB-4 being the most sensitive cells. Oligomycin decreased metabolic activity in a cell line-dependent manner, the THP-1 resistant and HL-60 being the most sensitive. Both inhibitors induced apoptosis and cell cycle arrest in a cell line- and compound-dependent manner. 2-DG decreased 18F-FDG uptake in HEL, HL-60, KG-1, and NB-4, while oligomycin increased the uptake in K-562. Metabolism gene expression had different responses to treatments. In conclusion, HEL and KG-1 show to be more glycolytic, whereas HL-60 was more OXPHOS dependent. Results suggest that AML cells reprogram their metabolism to overcome OXPHOS inhibition suggesting that glycolysis may be a better therapeutic target.