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.

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.

Background: Cancer cells alter their metabolism to sustain high levels of growth and proliferation. One of the most common alterations is the metabolic shift of cancer cells from oxidative phosphorylation (OXPHOS) to glycolysis, regardless of oxygen presence (aerobic glycolysis), resulting in lower ATP production. This alteration is associated with up‐regulation of glucose transporters (GLUTs) and glycolytic enzymes, such as hexokinases, and enhanced lactate fermentation. This shift is mainly regulated by HIF‐1, p53 and MYC. Hence, metabolism constitute a new target in acute myeloid leukemia (AML), where mutations in metabolism [isocitrate dehydrogenase 1/2 (IDH1/2)] were already describe. AML is a heterogeneous hematologic neoplasia characterized by myeloid differentiation arrest and uncontrolled proliferation.Aims: This study aimed to metabolically characterize AML cell lines and assess the potential of glycolysis and OXPHOS metabolism as a therapeutic target in AML using in vitro models.Methods: Six AML cell lines of different subtypes and with different genetic backgrounds were used (HEL, HL‐60, K‐562, KG‐1, NB‐4 and THP‐1). The presence of IDH1/2 exon 4 mutations was assessed using Sanger sequencing. Intracellular peroxides, superoxide anion and mitochondrial membrane potential levels were measured by fluorimetry using DCFH₂‐DA, DHE and JC‐1 probes, respectively. Cells were incubated in presence or absence of 2‐deoxy‐D‐glucose (2‐DG; glycolysis inhibitor) or oligomycin complex (OXPHOS inhibitor). Metabolic activity was assessed by resazurin assay and cell death by flow cytometry (FC) using Annexin V and 7‐AAD double staining. Cell cycle was also assessed by FC, using propidium iodide/RNase. Glucose intake was measured by ¹⁸F‐FDG uptake and expression levels of 8 SLC2A, 2 hexokinases and HIF‐1α were assessed using RT‐PCR.Results: NB‐4 cell line has the highest ¹⁸F‐FDG uptake, whereas HL‐60 and THP‐1 cells showed the lowest. HEL, K‐562 and KG‐1 had similar ¹⁸F‐FDG uptake. THP‐1 cells expressed the 8 tested SLC2A genes. NB‐4 cell line also had the highest peroxide and superoxide anion levels. K‐562 and KG‐1 cells had similar ROS levels and IDH1 genotype (single nucleotide variant rs11554137 in heterozygosity). Glycolysis inhibition reduced metabolic activity in a dose‐ and cell line dependent manner and HEL and KG‐1 were the most sensitive cell lines to 2‐DG. OXPHOS inhibition led to a decrease in metabolic activity in a cell line‐dependent manner. K‐562 cells were the most sensitive to this inhibition and THP‐1 cells were the most resistant to both compounds (2‐DG and oligomycim). These inhibitors induce cell death by apoptosis and cell cycle arrest in G₀/G₁ phase. 2‐DG induced a more evident cytotoxic effect on K‐562 and NB‐4 and a cytostatic effect on KG‐1 and THP‐1 cells. Conversely, oligomycin had a cytotoxic effect on K‐562 and KG‐1 cells and a cytostatic effect on NB‐4, THP‐1 and K 562. Moreover, 2‐DG decreased ¹⁸F‐FDG uptake, mainly in KG‐1 and NB‐4 cell lines, inducing up‐regulation of SLC2A11 and down‐regulation of HK1 gene expression. Lastly, oligomycin increased ¹⁸F‐FDG uptake, particularly in K‐562 cells, with up‐regulation of SLC2A1 and HK2 gene expression.Summary/Conclusion: AML cell lines have different metabolic preferences, with HEL, KG‐1 and NB‐4 cells appearing to be more glycolytic, whereas K‐562 cells more dependent on OXPHOS. Contrary, THP‐1 cells seems to be OXPHOS independent. These cell lines seem to reprogram their metabolism in order to overcome OXPHOS inhibition, suggesting that glycolysis could be a better therapeutic target in AML.