pyrimidinones and Leukemia--Myeloid

Interrogation of cellular metabolism with high-throughput screening approaches can unravel contextual biology and identify cancer-specific metabolic vulnerabilities. To systematically study the consequences of distinct metabolic perturbations, we assemble a comprehensive metabolic drug library (CeMM Library of Metabolic Drugs; CLIMET) covering 243 compounds. We, next, characterize it phenotypically in a diverse panel of myeloid leukemia cell lines and primary patient cells. Analysis of the drug response profiles reveals that 77 drugs affect cell viability, with the top effective compounds targeting nucleic acid synthesis, oxidative stress, and the PI3K/mTOR pathway. Clustering of individual drug response profiles stratifies the cell lines into five functional groups, which link to specific molecular and metabolic features. Mechanistic characterization of selective responses to the PI3K inhibitor pictilisib, the fatty acid synthase inhibitor GSK2194069, and the SLC16A1 inhibitor AZD3965, bring forth biomarkers of drug response. Phenotypic screening using CLIMET represents a valuable tool to probe cellular metabolism and identify metabolic dependencies at large.

Topics: Biomarkers, Tumor; Cell Survival; Cluster Analysis; Fatty Acids; Genotype; Humans; Leukemia, Myeloid; Monocarboxylic Acid Transporters; Phenotype; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Pyrimidinones; Pyrrolidines; Signal Transduction; Small Molecule Libraries; Symporters; Systems Analysis; Thiophenes; Triazoles; Tumor Cells, Cultured

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; G1 Phase Cell Cycle Checkpoints; Humans; Leukemia, Myeloid; Macrophages; Male; Mice; Mice, Inbred BALB C; Protein Kinase Inhibitors; Pyrimidinones; RAW 264.7 Cells; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; Xenograft Model Antitumor Assays

Hematologic malignancies arising in the setting of established germ cell tumors have been previously described and have a dismal prognosis. Identification of targetable mutations and pathway dysregulation through massively parallel sequencing and functional assays provides new approaches to disease management.. Herein, we report the case of a 23-year-old male who was diagnosed with a mediastinal germ cell tumor and subsequent acute myeloid leukemia. A shared clonal origin was demonstrated through identification of identical NRAS and TP53 somatic mutations in both malignancies. The patient's leukemia was refractory to standard therapies with short interval relapse. Functional assays demonstrated the patient's blasts to be sensitive to the mitogen-activated protein kinase kinase (MEK) inhibitor trametinib, correlating with the activating NRAS mutation. The patient experienced a sustained partial remission while on trametinib therapy but ultimately suffered relapse of the germ cell tumor. The leukemic clone remained stable and sensitive to trametinib at that time.. This case highlights the potential power of combining genetic sequencing and in vitro functional assays with targeted therapies in the treatment of rare diseases.

Topics: Acute Disease; GTP Phosphohydrolases; High-Throughput Nucleotide Sequencing; Humans; Leukemia, Myeloid; Male; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Mediastinal Neoplasms; Membrane Proteins; Neoplasm Recurrence, Local; Neoplasms, Germ Cell and Embryonal; Point Mutation; Protein Kinase Inhibitors; Pyridones; Pyrimidinones; Treatment Outcome; Tumor Suppressor Protein p53; Young Adult

Modulation of protein kinase C (PKC) activity has been demonstrated to either prevent or enhance drug-induced apoptosis in various tissue types. We tested four novel modulators of PKC activity in comparison to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) for the capability to affect differentiation, cell cycle progression and apoptosis in the human myeloid leukemia cell lines U937 and HL-60. Farnesyl thiotriazole (FTT) and N-(n-heptyl)-5-chloro-1-naphthalenesulfonamide (SC-10) are both direct activators of PKC, whereas 6-(2-(4-[(4-fluorophe-nyl)phenylmethylene]-1-piperidinyl)ethyl)-7-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one (R59022) and [3-[2-[4-(bis-(4-fluorophenyl)methylene]piperidin-1-yl)ethyl]-2,3-dihydro-2-thioxo-4(1H)-quin-azolinone (R59949) are diacyl glycerol kinase inhibitors that activate PKC by enhancing the levels of the endogenous ligand diacyl glycerol. U937 cells displayed a slight reduction in the number of cells in G(2)/M cell cycle phase after exposure to FTT, SC-10, R59022 and R59949, respectively. In contrast, HL-60 cells demonstrated a largely unaltered cell cycle distribution. Whereas TPA treatment resulted in a strong induction of p21(WAF/CIP1), c-Fos and c-Jun levels, neither one of the novel PKC activators altered expression of these proteins. Consequently, we tested the ability of the activators to cause membrane translocation of PKC. While TPA treatment resulted in translocation of the PKC isoforms alpha, delta and epsilon, SC-10 and FTT failed to induce alterations in the PKC content of the membrane and cytosolic fractions, respectively. Expression of the beta(2)-integrin CD11c that is induced during TPA-mediated differentiation remained unaltered after exposure to SC-10 and was partly reduced after treatment with FTT. To further investigate the effect of these activators upon apoptosis in leukemic cells, HL-60 and U937 cells were treated with 1-beta-D-arabinofuranosylcytosine (Ara-C) or etoposide (VP-16). Whereas TPA strongly reduced apoptosis in Ara-C- or VP-16-treated U937 cells, little if any reduction was observed after pretreatment with either FTT, SC-10, R59022 or R59949, respectively, in these cells. In contrast, TPA enhanced apoptosis in Ara-C- or VP-16-treated HL-60 cells. Interestingly, FTT and SC-10 demonstrated a protective effect in Ara-C-treated HL-60 cells. Taken together, these data suggest that the novel PKC activators FTT, SC-10, R59022 and R59949 exhibit modest biological effects upon leukemi

Topics: Antineoplastic Agents; Apoptosis; CD11c Antigen; CD4 Antigens; Cell Cycle; Cell Differentiation; Diacylglycerol Kinase; Enzyme Activators; Enzyme Inhibitors; Farnesol; Flow Cytometry; Genes, fos; Genes, jun; Humans; Immunoblotting; Isoenzymes; Leukemia, Myeloid; Naphthalenes; Oncogene Protein p21(ras); Piperidines; Protein Kinase C; Pyrimidinones; Quinazolines; Quinazolinones; Sulfonamides; Thiazoles; Triazoles; Tumor Cells, Cultured

The photodynamic effects of temoporfin (meso-tetrahydroxyphenylchlorin, mTHPC) and merocyanine 540 (MC540) in murine myeloid leukemia M1 and WEHI 3B (JCS) cells were compared. The mTHPC was found to be more potent and selective. At a lethal dosage of 90% killing (LD90), only 1.3 microM of mTHPC and 4.2 kJ/m2 of light irradiation was required, which was a 20-fold lower drug concentration and 11-fold smaller light dose than that required when using MC540. Meanwhile, three times less, or 15%, of the coincubated erythrocytes were destroyed by mTHPC than by MC540. Confocal micrographs showed that both drugs accumulated diffusely inside the cytoplasm in a very similar fashion, but mTHPC induced a more extensive apoptosis in photosensitized JCS cells. For example, at LD90, mTHPC practically killed all JCS cells via apoptosis and cleaved the DNA to extremely small 150 base-pair fragments. In contrast, among the JCS cells killed by MC540, about 88% died via apoptosis and large DNA fragments were abundant. Relative to MC540, the ability of mTHPC to trigger large-scale and thorough apoptosis in leukemia cells may help explain its potency and selectivity.

Topics: Animals; Apoptosis; Cell Survival; Erythrocytes; Leukemia, Experimental; Leukemia, Myeloid; Light; Mesoporphyrins; Mice; Photochemotherapy; Photosensitizing Agents; Pyrimidinones; Tumor Cells, Cultured

The binding of merocyanine 540 (MC540) to murine myeloid leukemia (M1) cells and normal erythrocytes was measured by fluorescence digital imaging microscopy using an intensified charge-coupled device. It was found that, on average, about three times more MC540 were bound to a unit membrane area of M1 cells than erythrocytes, a result consistent with previous studies. However, it was shown for the first time that MC540 binding varied significantly from one M1 cell to the next, and about 15% of the sensitized M1 cells were as MC540-negative as normal erythrocytes. Using the leukemic inhibitory factor as a differentiation inducer, M1 cells were induced to differentiate into mature macrophage-like cells in vitro. Such treatment lowered the average MC540 binding by about one-third but did not affect the cell-to-cell variation significantly.

Topics: Animals; Erythrocytes; Kinetics; Leukemia, Myeloid; Macrophages, Peritoneal; Mice; Mice, Inbred BALB C; Microscopy, Fluorescence; Phenotype; Photosensitizing Agents; Pyrimidinones; Spectrometry, Fluorescence; Tumor Cells, Cultured

In the presence of albumin Merocyanine 540 (MC540) exhibits a very limited binding to the outer surface of the membrane of normal erythrocytes, whereas pronounced binding is observed to leukemia cells. To find out whether this difference is due to differences in the composition or structural organization of the cell membrane we analyzed effects of a number of covalent and non-covalent perturbations of the red cell membrane on the binding and fluorescence characteristics of membrane-bound MC540. It is shown that exposure of the cells to cationic chlorpromazine, neuraminidase or photodynamic treatment with AlPcS4 as sensitizer caused a limited increase (30-50%) of MC540 binding, together with a red shift of the fluorescence emission maximum and an increase of the relative fluorescence quantum yield of membrane-bound MC540. Other forms of perturbation of the membrane structure, like hyperthermia (48 degrees C) and treatments that produce a decrease of phospholipid asymmetry in addition to accelerated flip-flop, did not result in increased MC540 binding, but did cause a red shift of the fluorescence emission maximum and an increase of the relative fluorescence quantum yield. These changes in fluorescence properties indicate a penetration of the dye into more hydrophobic regions in the membrane. MC540, bound to Brown Norway myelocytic leukemia cells, exhibited a red shift of the fluorescence emission maximum and an increased relative fluorescence quantum yield as compared to MC540 bound to untreated erythrocytes. These changes were of the same order of magnitude as in photodynamically treated red blood cells. Dye binding per surface area, however, was about 3-times higher with these leukemia cells than with photodynamically treated red blood cells. This demonstrates that certain perturbations of the erythrocyte membrane evoked a MC540 binding that became qualitatively comparable to the dye binding to leukemia cells, although dye binding per surface area was still significantly lower.

Topics: Cell Membrane; Chlorpromazine; Cholesterol; Erythrocyte Membrane; Hot Temperature; Humans; Indoles; Leukemia, Myeloid; Light; Lipid Bilayers; Neuraminidase; Organometallic Compounds; Pyrimidinones; Radiation-Sensitizing Agents; Spectrometry, Fluorescence; Tumor Cells, Cultured

The photochemistry of merocyanine 540 (MC 540), a sensitizing dye that binds preferentially to leukemia and electrically excitable cells, has been investigated. MC 540-mediated photooxidation of histidine, arachidonate, and unsaturated phospholipid vesicles was assessed by spin label oximetry and shown to involve type II (singlet oxygen) chemistry. The dye was also shown to be a potent sensitizer of lipid peroxidation in a natural cell membrane, the erythrocyte ghost. Inhibition by azide, stimulation by 2H2O, and identification of the cholesterol product 5 alpha-cholest-6-ene-3 beta,5-diol in this system, all were consistent with singlet oxygen intermediacy. Finally, MC 540 was found to be considerably more phototoxic to K-562 leukemia cells in 2H2O than in H2O. We conclude that singlet oxygen plays a major role in the phototherapeutic effects of this dye.

Topics: Cell Line; Cell Membrane; Dimyristoylphosphatidylcholine; Electron Spin Resonance Spectroscopy; Erythrocyte Membrane; Fluorescent Dyes; Humans; Leukemia, Myeloid; Liposomes; Oxygen; Phosphatidylcholines; Photochemistry; Pyrimidinones; Singlet Oxygen

After fixation in a modified Bouin's solution, the acid dye merocyanine 540 stained granules in granulocytic cells intensely. In immature granulocytes, such as promyelocytes and myelocytes, granules stained pink to violet. In some leukemic myeloblasts, promyelocytes and monocytes, granules also stained deep pink to violet. In more mature granulocytes, such as metamyelocytes, bands, and neutrophils, granules stained bright red to orange. In eosinophils and basophils, granules stained deep red. Granules of the type described were not visualized in normal plasma cells, lymphocytes, monocytes, or megakaryocytes. In normoblasts, cytoplasm stained diffusely red. Cytoplasmic staining in erythroblasts became darker as the cell matured, probably reflecting hemoglobin content. Used as a single agent stain, merocyanine 540 may be useful in distinguishing normal and leukemic granulocytic cells from other types of blood cells.

Topics: Bone Marrow; Bone Marrow Cells; Granulocytes; Humans; Leukemia; Leukemia, Lymphoid; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Pyrimidinones; Reference Values; Staining and Labeling

Merocyanine 540 is a fluorescent dye which stains erythrocytes that have lost their normal membrane phospholipid asymmetry. Because erythrocytes from patients with chronic myelogenous leukemia have been reported to display this abnormal phenotype, peripheral blood erythrocytes from such patients were examined for their ability to stain with the dye. Erythrocytes from all patients with active disease states stained, whereas neither erythrocytes from normal, healthy individuals nor from a patient whose disease symptoms were eliminated by chemotherapy stained. These results suggest that merocyanine 540 may have utility in the clinical evaluation of chronic myelogenous leukemia.

Topics: Adult; Erythrocyte Membrane; Fluorescent Dyes; Humans; Leukemia, Myeloid; Membrane Lipids; Middle Aged; Phospholipids; Pyrimidinones