phosphothreonine and Leukemia-Lymphoma--Adult-T-Cell

Constitutively activated AKT kinase is a common feature of T-cell acute lymphoblastic leukemia (T-ALL). Here, we report that the novel AKT inhibitor (2S)-1-(1H-indol-3-yl)-3-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxypropan2-amine (A443654) leads to rapid cell death of T-ALL lines and patient samples. Treatment of CEM, Jurkat, and MOLT-4 cells with nanomolar doses of the inhibitor led to AKT phosphorylation accompanied by dephosphorylation and activation of the downstream target, glycogen synthase kinase-3beta. Effects were time- and dose-dependent, resulting in apoptotic cell death. Treatment of Jurkat cells with A443654 resulted in activation of caspase-2, -3, -6, -8, and -9. Apoptotic cell death was mostly dependent on caspase-2 activation, as demonstrated by preincubation with a selective pharmacological inhibitor. It is remarkable that A443654 was highly effective against the drug-resistant cell line CEM-VBL100, which expresses 170-kDa P-glycoprotein. Moreover, A443654 synergized with the DNA-damaging agent etoposide in both drug-sensitive and drug-resistant cell lines when coadministered [combination index (CI) = 0.39] or when pretreated with etoposide followed by A443654 (CI = 0.689). The efficacy of A443654 was confirmed using blasts from six patients with T-ALL, all of whom displayed low levels of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and constitutive phosphorylation of Akt on Ser473. At 1 microM, the inhibitor was able to induce apoptotic cell death of T-ALL blast cells, as indicated by flow cytometric analysis of samples immunostained for active (cleaved) caspase-3. Because activated AKT is seen in a large percentage of patients with T-ALL, A443654, either alone or in combination with existing drugs, may be a useful therapy for primary and drug-resistant T-ALL.

Topics: Apoptosis; Caspases; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Enzyme Activation; Etoposide; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Indazoles; Indoles; Leukemia-Lymphoma, Adult T-Cell; Phosphorylation; Phosphoserine; Phosphothreonine; Proto-Oncogene Proteins c-akt

Macrophage-derived chemokine [CC chemokine ligand 22 (CCL22)] and thymus- and activation-regulated chemokine (CCL17) mediate cellular effects, principally by binding to their receptor CC chemokine receptor 4 (CCR4) and together, constitute a multifunctional chemokine/receptor system with homeostatic and inflammatory roles within the body. This study demonstrates that CCL22 and CCL17 stimulate pertussis toxin-sensitive elevation of intracellular calcium in the CEM leukemic T cell line and human peripheral blood-derived T helper type 2 (Th2) cells. Inhibition of phospholipase C (PLC) resulted in the abrogation of chemokine-mediated calcium mobilization. Chemokine-stimulated calcium responses were also abrogated completely by the inhibition of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor-mediated calcium release. Chemotactic responses of CEM and human Th2 cells to CCL17 and CCL22 were similarly abrogated by inhibition of PLC and inhibition of novel, Ca2+-independent/diacylglycerol-dependent protein kinase C (PKC) isoforms. Inhibition of Ins(1,4,5)P3 receptor-mediated calcium release from intracellular stores had no effect on chemotactic responses to CCR4 ligands. Taken together, this study provides compelling evidence of an important role for PLC and diacylglycerol-dependent effector mechanisms (most likely involving novel PKC isoforms) in CCL17- and CCL22-stimulated, directional cell migration. In this regard, CCL22 stimulates phosphatidylinositol-3 kinase-independent phosphorylation of the novel delta isoform of PKC at threonine 505, situated within its activation loop--an event closely associated with increased catalytic activity.

Topics: Acetophenones; Benzopyrans; Calcium; Calcium Channels; Calcium Signaling; Catalytic Domain; Cell Line, Tumor; Chemokine CCL17; Chemokine CCL22; Chemokines, CC; Chemotaxis; Chromones; Diglycerides; Estrenes; Humans; Indoles; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Leukemia-Lymphoma, Adult T-Cell; Morpholines; Pertussis Toxin; Phosphatidylinositol 3-Kinases; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Phosphothreonine; Protein Processing, Post-Translational; Pyrroles; Pyrrolidinones; Receptors, CCR4; Receptors, Chemokine; Receptors, Cytoplasmic and Nuclear; Recombinant Fusion Proteins; T-Lymphocytes; Th2 Cells