u-0126 and Leukemia--Myeloid--Acute

The transformation of myelodysplastic syndrome (MDS) into acute myeloid leukemia (AML) poses a significant clinical challenge. The trimethylation of H3 on lysine 27 (H3K27me3) methylase and de‑methylase pathway is involved in the regulation of MDS progression. The present study investigated the functional mechanisms of the MEK/ERK and PI3K/AKT pathways in the MDS‑to‑AML transformation. MDS‑AML mouse and SKM‑1 cell models were first established and this was followed by treatment with the MEK/ERK pathway inhibitor, U0126, the PI3K/AKT pathway inhibitor, Ly294002, or their combination. H3K27me3 methylase, enhancer of zeste homolog (EZH)1, EZH2, demethylase Jumonji domain‑containing protein‑3 (JMJD3) and ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX) and H3K27me3 protein levels were determined using western blot analysis. Cell viability, cycle distribution and proliferation were assessed using CCK‑8, flow cytometry, EdU and colony formation assays. The ERK and AKT phosphorylation levels in clinical samples and established models were determined, and SKM‑1 cell behaviors were assessed. The levels of H3K27me3 methylases and de‑methylases and distal‑less homeobox 5 (DLX5) were measured. The results revealed that the ERK and AKT phosphorylation levels were elevated in patients with MDS and MDS‑AML, and in mouse models. Treatment with U0126, a MEK/ERK pathway inhibitor, and Ly294002, a PI3K/AKT pathway inhibitor, effectively suppressed ERK and AKT phosphorylation in mice with MDS‑AML. It was observed that mice with MDS treated with U0126/Ly294002 exhibited reduced transformation to AML, delayed disease transformation and increased survival rates. Treatment of the SKM‑1 cells with U0126/Ly294002 led to a decrease in cell viability and proliferation, and to an increase in cell cycle arrest by suppressing ERK/PI3K phosphorylation. Moreover, treatment with U0126/Ly294002 downregulated EZH2/EZH1 expression, and upregulated JMJD3/UTX expression. The effects of U0126/Ly294002 were nullified when EZH2/EZH1 was overexpressed or when JMJD3/UTX was inhibited in the SKM‑1 cells. Treatment with U0126/Ly294002 also resulted in a decreased H3K27me3 protein level and H3K27me3 level in the DLX5 promoter region, leading to an increased DLX5 expression. Overall, the findings of the present study suggest that U0126/Ly294002 participates in MDS‑AML transformation by modulating the levels of H3K27me3 methylases and de‑methylases, and regulating DLX5 transcripti

Topics: Animals; Histones; Humans; Leukemia, Myeloid, Acute; Mice; Mitogen-Activated Protein Kinase Kinases; Myelodysplastic Syndromes; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction

The drug efflux pump ABCB1 is a key driver of chemoresistance, and high expression predicts treatment failure in acute myeloid leukemia (AML). In this study, we identified and functionally validated the network of enhancers that controls expression of ABCB1. We show that exposure of leukemia cells to daunorubicin activated an integrated stress response-like transcriptional program to induce ABCB1 through remodeling and activation of an ATF4-bound, stress-responsive enhancer. Protracted stress primed enhancers for rapid increases in activity following re-exposure of cells to daunorubicin, providing an epigenetic memory of prior drug treatment. In primary human AML, exposure of fresh blast cells to daunorubicin activated the stress-responsive enhancer and led to dose-dependent induction of ABCB1. Dynamic induction of ABCB1 by diverse stressors, including chemotherapy, facilitated escape of leukemia cells from targeted third-generation ABCB1 inhibition, providing an explanation for the failure of ABCB1 inhibitors in clinical trials. Stress-induced upregulation of ABCB1 was mitigated by combined use of the pharmacologic inhibitors U0126 and ISRIB, which inhibit stress signaling and have potential for use as adjuvants to enhance the activity of ABCB1 inhibitors.

Topics: Acetamides; Activating Transcription Factor 4; Antineoplastic Combined Chemotherapy Protocols; ATP Binding Cassette Transporter, Subfamily B; Butadienes; Cyclohexylamines; Daunorubicin; Drug Resistance, Neoplasm; Enhancer Elements, Genetic; Epigenesis, Genetic; Gene Expression Regulation, Leukemic; Humans; K562 Cells; Leukemia, Myeloid, Acute; Neoplasm Proteins; Nitriles; Up-Regulation

LukS-PV, a component of Panton-Valentine leukocidin, is a pore-forming cytotoxin secreted by Staphylococcus aureus. Here we examined the potential effect of LukS-PV in differentiation of human leukemia cells and the underlying mechanism. We found that LukS-PV could induce differentiation of human acute myeloid leukemia (AML) cells, including AML cell lines and primary AML blasts, as determined by morphological changes, phagocytosis assay and expression of CD14 and CD11b surface antigens. In addition, LukS-PV activated the extracellular signal-regulated kinase (ERK) pathway and significantly upregulated the phosphorylation of c-JUN and c-FOS transcriptional factors in the process of differentiation. Inhibiting ERK pathway activation with U0126 (a MEK1/2 inhibitor) markedly blocked LukS-PV-induced differentiation and decreased the phosphorylation of c-JUN and c-FOS. These findings demonstrate an essential role for the ERK pathway together with c-JUN and c-FOS in the differentiation activity of LukS-PV. Taken together, our data suggest that LukS-PV could be a potential candidate as a differentiation-inducing agent for the therapeutic treatment of AML.

Topics: Bacterial Proteins; Butadienes; CD11b Antigen; Cell Differentiation; Cell Line, Tumor; Humans; Leukemia, Myeloid, Acute; Leukocidins; Lipopolysaccharide Receptors; MAP Kinase Signaling System; Nitriles; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Staphylococcus aureus

Adenosine monophosphate-activated protein kinase (AMPK) is a sensor for cellular energy status. When the cellular energy level is decreased, AMPK is activated and functions to suppress energy-consuming processes, including protein synthesis. Recently, AMPK has received attention as an attractive molecular target for cancer therapy. Several studies have revealed that the activation of AMPK by chemical stimulators, such as metformin, induces apoptosis in a variety of hematologic malignant cells. From another perspective, these results suggest that the function of AMPK is impaired in hematologic tumor cells. However, the precise mechanisms by which this impairment occurs are not well understood. In melanoma cells, oncogenic BRAF constitutively activates the extracellular signal-regulated kinase (ERK) pathway and phosphorylates liver kinase B1, an upstream activator of 5' adenosine monophosphate-activated protein kinase (AMPK), resulting in the inactivation of liver kinase B1 and AMPK. In this study, we analyzed whether ERK is involved in the suppression of AMPK activity using established and primary human leukemia cells. We found an inverse correlation between the intensity of ERK activity and the degree of AMPK activation after stimulation with either glucose deprivation or metformin. We also found that the inhibition of ERK activity by U0126 restored AMPK activation after metformin treatment. Furthermore, a combined treatment with metformin and U0126 enhanced the antileukemic activity of metformin. Importantly, metformin induced ERK activation by suppressing the protein levels of dual specificity phosphatase 6, a negative regulator of ERK. This crosstalk between AMPK and ERK could diminish the antileukemic activity of metformin. Taken together, our present observations suggest a novel therapeutic strategy for improving the efficacy of metformin in treating leukemia.

Topics: Adult; Aged; AMP-Activated Protein Kinase Kinases; AMP-Activated Protein Kinases; Antineoplastic Agents; Apoptosis; Butadienes; Cell Line, Tumor; Drug Interactions; Dual Specificity Phosphatase 6; Enzyme Activation; Feedback, Physiological; Female; Glucose; Humans; Leukemia, Myeloid, Acute; Leukemia, Myelomonocytic, Acute; MAP Kinase Signaling System; Metformin; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Proteins; Nitriles; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; RNA Interference; RNA, Small Interfering; Signal Transduction; Tumor Cells, Cultured

Topics: Butadienes; Extracellular Signal-Regulated MAP Kinases; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Leukemia, Myeloid, Acute; Nitriles; Prenylation; ras Proteins; Simvastatin

Blockade of mitogen-activated protein kinase kinase (MEK1/2), part of the extracellular signal-regulated kinase (ERK) or p44/42 mitogen-activated protein kinase (MAPK) pathway has been shown, in some instances, to cause apoptosis in leukemic blast cells. However, studies are contradictory and have often been based mainly on inhibition of cell growth in a limited number of cell lines. This investigation examined the effect of the potent MEK inhibitor U0126 alone and in combination with Ara-C on apoptosis in acute myeloblastic leukemia (AML) cell lines, patient acute leukemic and nonleukemic samples. Apoptosis was assessed flow cytometrically using Apo2.7 and AnnexinV antibodies which detect apoptosis at the mitochondrial and cell membrane levels, respectively. The proapoptotic effect of the inhibitor varied across the five cell lines tested, from highly significant induction of apoptosis to no apparent response. A possible synergistic effect with the combined use of U0126 and Ara-C was observed in one cell line only. The proapoptotic effect of U0126 in the most sensitive cell line appeared to be related to CD34 positivity. Cells from leukemic patients showed considerable sensitivity in two of four cases with a similar association with CD34 expression being evident. Interestingly, control cells did not show a significant effect when exposed to the inhibitor. These results suggest that U0126 may offer a potential alternative to standard chemotherapy with a particular role in the most primitive types of leukemia, these being often the most resistant to standard chemotherapy.

Topics: Acute Disease; Antineoplastic Agents; Apoptosis; Bone Marrow Cells; Butadienes; Enzyme Inhibitors; Flow Cytometry; Humans; Leukemia; Leukemia, Myeloid, Acute; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Nitriles; Tumor Cells, Cultured

Blockade of mitogen-activated protein kinase kinase (MEK1/2), part of the extracellular signal-regulated kinase (ERK) or p44/42 mitogen-activated protein kinase (MAPK) pathway, has been shown in some instances to cause apoptosis in leukemic blast cells. This investigation examined the effect of the potent MEK/ERK inhibitor U0126 on apoptosis in acute myeloblastic leukemia (AML) cell lines, and acute leukemic and non-leukemic patient samples. The pro-apoptotic effect of the inhibitor varied across the five cell lines tested (KG1a, HEL, TF-1, MO7e, and THP-1) from highly significant induction of apoptosis to no apparent response. The pro-apoptotic effect of U0126 in the most sensitive cell line, KG1a, appeared to be related to its CD34 positivity. Three of five leukemic bone marrow samples showed considerable sensitivity to the inhibitor and a similar association with CD34 expression was evident. Interestingly, control marrow cells from six non-leukemic patients did not show a significant effect when exposed to U0126. These results suggest that this agent may offer a potential alternative to standard chemotherapy with a particular role in the most primitive types of leukemia, these often being the most resistant to standard chemotherapy.

Topics: Butadienes; Cell Line, Tumor; Dimethyl Sulfoxide; Enzyme Inhibitors; Humans; Kinetics; Leukemia, Myeloid, Acute; MAP Kinase Kinase Kinase 1; MAP Kinase Kinase Kinases; Mitogen-Activated Protein Kinases; Nitriles