Activation of FLT3-Associated Signaling Pathways in Quizartinib-Resistant Macrophage-Like Cells of Acute Myeloid Leukemia

Although environmental factors such as cytokines and stroma are recognized to influence the growth and chemosensitivity of acute myeloid leukemia (AML) cells, little is known about the effect of physical forces such as pressure. The pressure in the marrow of AML patients can be 10–20 fold higher compared to patients with solid tumors or non-malignant conditions,1 possibly due to increased cellularity and fibrosis. Therefore, we sought to establish whether the pressure in the marrow is another environmental factor that can influence chemosensitivity. Using AML cell lines and primary patient samples, we show that high pressure promotes a transition to a gel-like plasma membrane that reduces the intracellular accumulation of daunorubicin in AML cells. Therefore, biomechanical stimulus such as pressure is another environmental factor that can influence chemosensitivity. We designed and manufactured pressure chambers to deliver different levels of physiological pressure to AML cells while maintaining continuous gas exchange (Online Supplementary Figure S1). Two AML cell lines, HL60 and TEX, were cultured at pressures up to 310 mmHg above atmospheric pressure (atm). A pressure of 310 mmHg above atm corresponds to less than 0.5% of the maximum pressure acting on the head of the femur of an upright 160 lb person, and therefore, would be physiologically relevant as erosion of the trabeculae from leukemic infiltration increases sensitivity to external forces.1,2 We found that the growth and viability of these cell lines did not change at high pressure (Figure 1A). Next, we explored the impact of increased pressure on the sensitivity of these cells to three anthracyclines used in the treatment of AML: daunorubicin, idarubicin, and mitoxantrone.3,4 HL60 and TEX cells grown at increased static pressure were more resistant to daunorubicin, (Figure 1B), but not idarubicin (Figure 1C) or mitoxantrone (Figure 1D and Online Supplementary Figure S2). Furthermore, the effects of increased pressure on chemosensitivity were reversible. Figure 1. Acute myeloid leukemia (AML) cells at increased pressure display chemoresistance to danorubicin. (A). Cell viability measured by trypan blue exclusion assay of HL60 and TEX leukemia cells cultured over 4 days at 37°C at atmospheric pressure (atm) ... We also tested the impact of increased static pressure on samples from 5 patients with acute leukemia (4=AML, 1=T-ALL) that were sensitive to daunorubicin (patients’ characteristics are shown in Online Supplementary Table S1). Similar to the cell lines, increased pressure did not alter the growth and viability of the primary cells, but 3 of 5 samples (3 of 4 AML samples) had reduced sensitivity to daunorubicin at high pressure (Figure 1E and Online Supplementary Figure S3). To investigate the biological effects of increased pressure that may influence chemosensitivity, we measured the intracellular accumulation of daunorubicin. Compared to cells at atm, cells cultured at increased pressure accumulated less daunorubicin (Figure 2A and Online Supplementary Figure S4). In contrast, we observed no difference in the accumulation of [3H] mitoxantrone in TEX cells treated at increased pressure (Figure 2B). Figure 2. AML cells have reduced daunorubicin accumulation at increased pressure. (A). HL60 and TEX cells were cultured at 37°C at atm and 310 mmHg above atm for 3 days and then treated with 80, 160 and 320 nM of [3H] daunorubicin for 3 hours at the same ... No prior studies have investigated the impact of static pressure on plasma membrane dynamics in cancer cells. However, studies on red blood cells,5,6 bacterial membranes7 and synthetic lipid vesicles8,9 demonstrate that increased pressure alters membrane dynamics and influences drug and particle uptake. In these systems, the acyl chains of the phospholipids in the membrane straighten under increased pressure causing the membrane to become thicker and transition from a fluid liquid-crystalline state to a more solid gel-like state. The thicker gel-like membrane can reduce the permeability of the membrane to small molecules and can also impair the activity of membrane-associated drug uptake channels. Notably, these changes in membrane dynamics are rapidly reversible upon returning to normal pressure. To determine if plasma membrane dynamics are altered in AML cells at increased pressure, HL60, TEX, and primary patient AML cells were cultured at high pressure and stained with the lipophilic probe, laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) (see Online Supplementary Appendix for description of method). The fluorescence spectrum of laurdan is sensitive to the physical state of membrane phospholipids, and has been used to evaluate plasma membrane dynamics in red blood cells exposed to increased pressure6 and in K562 leukemia cells exposed to increased temperatures.10 Using two- photon microscopy at 37°C, atm, and within 10 min of removing from the pressure chamber, we calculated the generalized polarization (GP) factor of laurdan and observed an increase in the gel-like state of the membrane in both HL60 and TEX cells cultured at high pressure (Figure 2C). Moreover, primary AML cells from patients 1, 2, and 3 that demonstrated a change in chemosensitivity to daunorubicin at higher pressure also showed an increase in the gel-like state in their membrane at increased pressure. In contrast, primary cells from patients 4 (T-ALL) and 5 (AML) that did not display a change in chemosensitivity at increased pressure also showed no change in membrane dynamics at increased pressure (Figure 2D and Online Supplementary Figure S5). Thus, in a subset of AML cells, increased pressure promotes a transition to a gel-like membrane that can reduce the intracellular accumulation of select anthracyclines. Cholesterol is an important regulator of plasma membrane structure and consistency, and the amount of cholesterol in the membrane can vary over 10-fold between cells. Prior studies have evaluated the impact of cholesterol content on membrane dynamics after exposure to biomechanical forces. For example, addition of cholesterol to endothelial cells decreased membrane fluidity and dampened the impact of sheer stress on the plasma membrane.11 Similarly, using synthetic lipid vesicles, as the amount of cholesterol in the vesicle increased, the membrane became more gel-like and little further change was observed upon exposure to increased pressure.12 Conversely, membranes with the least amount of cholesterol are the most liquid and show the greatest change in their membrane dynamics upon exposure to increased pressure. However, it is unknown whether cells alter their membrane composition in response to changes in biomechanical pressure. It is worthy of note that the tested anthracyclines vary in their hydrophobicity. Of the three agents, daunorubicin has the highest logP (logarithm of partition coefficient) value of 1.83 indicating that it is the most hydrophobic, while idarubicin and mitoxantrone have lower logP values of 0.2 and -3.1, respectively. Changes in membrane dynamics would, therefore, preferentially affect the more hydrophobic daunorubicin compared to the other agents. Our study is not without limitations. First, membrane phase transitions are generally rapidly reversible and the imaging was done at atm. Although we imaged the cells within 10 min of removal from the pressure chamber, some of the changes in membrane dynamics may have reversed before imaging could be completed. Second, the increased pressure could have decreased daunorubicin solubility and thus resulted in decreased sensitivity to the drug, although this is very unlikely as we did not detect precipitation of daunorubicin. In summary, our study shows that increased static pressure renders AML cells resistant to daunorubicin by reducing intracellular drug levels, but does not affect sensitivity to idarubicin and mitoxantrone. Thus, these results highlight biomechanical stimuli such as pressure as another environmental factor that can influence chemosensitivity. Moreover, it suggests that some AML patients might benefit from chemotherapeutic regimens that contain anthracyclines such as idarubicin or mitoxantrone rather than daunorubicin.

ObjectiveThe aim of this study was to investigate the mechanism by which quercetin (Que) affects apoptosis and autophagy in pediatric acute myeloid leukaemia (AML) cells by inhibiting the activation of the PI3K/AKT signaling pathway through the regulation of the miR-224-3p/PTEN axis.MethodsBlood samples were collected from AML children and healthy volunteers. miR-224-3p and PTEN expression levels were measured. AML cells were pre-treated with Que. MiR-224-3p and PTEN expression levels in AML cells were altered via plasmid transfection. After intervention, PI3K/AKT phosphorylation, AML cell proliferation and apoptosis, concentrations of interleukin-1 β (IL-1β) and tumor necrosis factor-α (TNF-α) in AML cell culture supernatant, apoptosis-related genes Bax and Bcl-2, and autophagy markers LC3-I and LC3-II were tested. The targeting relationship between miR-224-3p and PTEN was identified.ResultsMiR-224-3p expression was elevated in AML children, while PTEN was decreased. Que was available to accelerate AML cell apoptosis and restrain its autophagy. Que inhibited miR-224-3p expression and promoted PTEN expression. Upregulating miR-224-3p or downregulating PTEN weakened the effect of Que on AML cell apoptosis and autophagy. MiR-224-3p negatively modulated PTEN expression. Up-regulation of PTEN reversed the effects of up-regulation of miR-224-3p on apoptosis and autophagy in AML cells. In addition, Que inhibited PI3K/AKT signaling pathway activation, while up-regulation of miR-224-3p or down-regulation of PTEN could attenuate the inhibitory effect of Que on PI3K/AKT signaling pathway. Moreover, up-regulation of PTEN reversed the effect of up-regulation of miR-224-3p on the PI3K/AKT signaling pathway.ConclusionQue affects apoptosis and autophagy in pediatric AML cells by inhibiting PI3K/AKT signaling pathway activation through regulation of miR-224-3p/PTEN axis.

Targeting mTORC1/2 by a mTOR Kinase Inhibitor (PP242) induces Apoptosis In AML Cells Under Conditions Mimicking Bone Marrow Microenvironment

Cyclopamine Induced Apoptosis in Primary CD34+ Acute Leukemic Cells.

BackgroundMicroRNA (miR)-containing exosomes released by acute myeloid leukemia (AML) cells can be delivered into hematopoietic progenitor cells to suppress normal hematopoiesis. Herein, our study was performed to evaluate the effect of exosomal miR-4532 secreted by AML cells on hematopoiesis of hematopoietic stem cells.MethodsFirstly, differentially expressed miRs related to AML were identified using microarray analysis. Subsequently, AML cell lines were collected, and CD34+ HSCs were isolated from healthy pregnant women. Then, miR-4532 expression was measured in AML cells and AML cell-derived exosomes and CD34+ HSCs, together with evaluation of the targeting relationship between miR-4532 and LDOC1. Then, AML cells were treated with miR-4532 inhibitor, and exosomes were separated from AML cells and co-cultured with CD34+ HSCs. Gain- and loss-function approaches were employed in CD34+ HSCs. Colony-forming units (CFU) and expression of dickkopf-1 (DKK1), a hematopoietic inhibiting factor associated with pathogenesis of AML, were determined in CD34+ HSCs, as well as the extents of JAK2 and STAT3 phosphorylation and LDOC1 expression.ResultsmiR-4532 was found to be upregulated in AML cells and AML cell-derived exosomes, while being downregulated in CD34+ HSCs. In addition, exosomes released by AML cells targeted CD34+ HSCs to decrease the expression of CFU and increase the expression of DKK1. miR-4532 was delivered into CD34+ HSCs to target LDOC1 via AML cell-released exosomes. AML cell-derived exosomes containing miR-4532 inhibitor increased CFU but reduced DKK1 in CD34+ HSCs. Inhibition of miR-4532 or JAK2, or ectopic expression of LDOC1 upregulated CFU and downregulated DKK1 expression as well as the extents of JAK2 and STAT3 phosphorylation in CD34+ HSCs.ConclusionIn conclusion, AML cell-derived exosomes carrying miR-4532 repress normal HSC hematopoiesis via activation of the LDOC1-dependent STAT3 signaling pathway.

miR-183-5p Inhibits Occurrence and Progression of Acute Myeloid Leukemia via Targeting Erbin.

MLN0128, a Second-Generation mTOR Kinase Inhibitor, Disrupts Survival Signaling and Triggers Apoptosis in AML

Pharmacologic inhibition or molecular down-regulation of topoisomerase 2β (TOP2β) potentiates all trans retinoic acid (ATRA)-induced differentiation and apoptosis in acute myeloid leukemia (AML) cells. Since ATRA-induced myeloid differentiation involves activation of the MAPK/ERK signaling pathway and aberrant activation of MAPK/ERK is frequently observed in AML we tested the role of this pathway in modulating the cellular effects of ATRA when TOP2β is inhibited with the bisdioxopiperazines, ICRF 187 (dexrazoxane, Zinecard™) or ICRF193. Treatment of HL-60 AML (M2) cells with ATRA increased the percentage of differentiated cells, as assessed by the nitroblue tetrazolium reduction assay, and enhanced levels of phosphorylated ERK. Paradoxically, while combination treatment with ATRA/ICRF187 enhanced differentiation (P<0.05), ERK phosphorylation was attenuated compared to treatment with ATRA alone. Moreover, pretreatment with the MEK inhibitor PD98059, which completely blocks ATRA-induced differentiation and ERK phosphorylation, only partially blocked (46% reduction) differentiation induced by ATRA/ICRF187 (P<0.01) and had no effect on ATRA/ICRF187-induced apoptosis. In agreement with this observation, combination treatment with ATRA/ICRF187 induced apoptosis (P<0.05) in KG-1 (M1) AML cells and primary AML cells from patients, that do not phosphorylate ERK or differentiate in response to ATRA treatment. In summary, our data suggest that inhibition of TOP2β sensitizes AML cells to ATRA induced differentiation and apoptosis primarily via an ERK-independent mechanism. Further, combination treatment ATRA and ICRF187 may be clinically relevant to overcome ATRA resistance in AML patients with a deregulated MAPK/ERK signaling pathway. Citation Format: Eric J. Norris, Amy Dorszynski, Aaron Lucander, Darla Destephanis, Ram Ganapathi, Mahrukh Ganapathi. Inhibition of topoisomerase 2β sensitizes acute myeloid leukemia cells to ATRA induced apoptosis independent of the MAPK/ERK pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1186.

Atiprimod blocks phosphorylation of JAK-STAT and inhibits proliferation of acute myeloid leukemia (AML) cells

Inhibition of the Mitochondrial Protein Import Machinery Is Selectively Cytotoxic to Acute Myeloid Leukemia (AML) Cells and Stem Cells

Acute Myeloid Leukemia Cells Rely on the Glycolytic Enzyme PGK1 to Support Energy Production and Amino Acid Metabolism

Hypoxia-Induced Chemoresistance to Doxorubicin in Acute Myeloid Leukemia (AML) Cells Occurs Via HIF-1α Independent Mechanism.

Up-Regulation of Immune Tolerance Genes in Leukemic Mesenchymal Stromal Cells Is Induced By Acute Myeloid Leukemia Cells through an IFN-Gamma-Dependent Inflammatory Signaling

Bone Marrow Stromal Cell Mediated Drug Resistance in AML Cells: Role of Extracellular Vesicles and AKT Signaling

Inhibition of GSK-3 Signalling Enhances Sensitivity of Non-Promyelocitic Acute Myeloid Leukemia (AML) Cell to Chemotherapy