ag-490 and Leukemia--Myeloid

Several cytokines and growth factors that stimulate the proliferation of acute myelogenous leukemia (AML) cells transduce their signals by activating the transcription factor Janus-activated kinase 2 (JAK2). Accordingly, the inhibition of JAK2 or of its downstream signaling pathways suppresses the proliferation of AML cells. Because (E)-3(6-bromopyridin-2-yl)-2-cyano-N-((S0-1-phenylethyl)acrylamide) (WP1066) is a novel analogue of the JAK2 inhibitor AG490, we tested its activity in AML cells and investigated its mechanism of action. Using clonogenic assays, we found that although WP1066 had a marginal effect on normal marrow progenitors, it inhibited the proliferation of AML colony-forming cells obtained from patients with newly diagnosed AML and that of the AML cell lines OCIM2 and K562. WP1066 inhibited OCIM2 cell multiplication by inducing accumulation of cells at the G(0)-G(1) phase of the cell cycle. Similar to its parent compound AG490, WP1066 inhibited the phosphorylation of JAK2, but unlike AG490, WP1066 also degraded JAK2 protein, thereby blocking its downstream signal transducer and activator of transcription (STAT) and phosphoinositide-3-kinase pathways. These effects resulted in the activation of the caspase pathway. Incubation of both OCIM2 and K562 cells with WP1066 activated caspase-3, induced cleavage of poly(ADP-ribose) polymerase, and caused caspase-dependent apoptotic cell death. Thus, WP1066 is a potent JAK2 inhibitor whose effects in AML and other hematologic malignancies merit further investigation.

Topics: Acute Disease; Adult; Aged; Apoptosis; Caspases; Cell Cycle; Cell Proliferation; Colony-Forming Units Assay; Enzyme Inhibitors; Female; Humans; Immunoblotting; Janus Kinase 2; Leukemia, Myeloid; Male; Middle Aged; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein-Tyrosine Kinases; Pyridines; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; STAT3 Transcription Factor; Trans-Activators; Tumor Cells, Cultured; Tyrphostins

High levels of cytokines are associated with a poor prognosis in acute myeloid leukemia (AML). However, cytokines may induce, on one hand, survival factor expression and cell proliferation and, on the other hand, expression of inhibitory signals such as up-regulation of suppressors of cytokine signaling (SOCS) and induce apoptotic cell death. Because blasts from patients with AML express high procaspase protein levels, we asked whether granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances procaspase protein production in AML cells. In the GM-CSF-responsive OCIM2 AML cell line, GM-CSF induced signal transducer and activator of transcription 5 (Stat 5) phosphorylation, up-regulated cyclin D2, and stimulated cell cycle progression. Concurrently, GM-CSF stimulated expression of SOCS-2 and -3 and of procaspases 2 and 3 and induced caspase 3 activation, poly(ADP[adenosine 5'-diphosphate]-ribose) polymerase (PARP) cleavage, and apoptotic cell death. The Janus kinase (Jak)-Stat inhibitor AG490 abrogated GM-CSF-induced expression of procaspase 3 and activation of caspase 3. Under the same conditions GM-CSF up-regulated production of BAX as well as Bcl-2, Bcl-XL, survivin, and XIAP. GM-CSF also increased procaspase 3 protein levels in OCI/AML3 and Mo7e cells, suggesting that this phenomenon is not restricted to a single leukemia cell line. Our data suggest that GM-CSF exerts a dual effect: it stimulates cell division but contemporaneously up-regulates Jak-Stat-dependent proapoptotic proteins. Up-regulation of procaspase levels in AML is thus a beacon for an ongoing growth-stimulatory signal.

Topics: Acute Disease; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Caspases; Cell Division; DNA-Binding Proteins; Enzyme Induction; Enzyme Inhibitors; Enzyme Precursors; Gene Expression Regulation, Neoplastic; Genes, bcl-2; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Inhibitor of Apoptosis Proteins; Leukemia, Erythroblastic, Acute; Leukemia, Myeloid; Microtubule-Associated Proteins; Neoplasm Proteins; Poly(ADP-ribose) Polymerases; Protein Biosynthesis; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Recombinant Proteins; Repressor Proteins; Signal Transduction; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins; Survivin; Trans-Activators; Transcription Factors; Tumor Cells, Cultured; Tumor Stem Cell Assay; Tyrphostins; X-Linked Inhibitor of Apoptosis Protein

Cells from patients with MDS-derived AML display heterogeneous proliferative responses to transforming growth factor beta (TGF beta). We analyzed growth inhibition and SMAD2 phosphorylation by TGF beta in CD34+ cells from nine patients, as compared to normal controls. While TGF beta consistently inhibited thymidine incorporation of normal cells (41% of control, P < 0.05), cells from patients with AML were growth-inhibited in only four of seven cases (40%), whereas TGF beta stimulated thymidine incorporation in the three other samples (166%). Remarkably, TPO reverted the stimulatory effect of TGF beta to profound growth inhibition. Upon exposure to TGF beta, SMAD2 protein was phosphorylated in normal CD34+ cells (n = 3), CD34+ leukemic blasts from all examined patients with AML (n = 4), and in the myeloid leukemic cell lines M-07e and HEL. TGF beta inhibited TPO-mediated thymidine incorporation, cell proliferation and survival in all samples analyzed. In M-07e cells and CD34+ cells from healthy donors, this inhibition was enhanced by an antagonist of JAK2 (AG490), but not a MEK-1 antagonist (PD098059). Conversely, in CD34+ cells from a patient with AML, both AG490 and PD098059 significantly enhanced TGF beta-mediated suppression of TPO-induced thymidine incorporation. Thus, in MDS-derived AML, altered responses to TGF beta may be due to defects downstream of SMAD2 and may involve MAPK activation.

Topics: Acute Disease; Adult; Antigens, CD34; Cell Division; Disease Progression; DNA Replication; DNA-Binding Proteins; DNA, Neoplasm; Enzyme Inhibitors; Flavonoids; Hematopoietic Stem Cells; Humans; Interleukin-3; Janus Kinase 2; Leukemia, Myeloid; MAP Kinase Kinase 1; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Myelodysplastic Syndromes; Neoplasm Proteins; Neoplastic Stem Cells; Phosphorylation; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Reverse Transcriptase Polymerase Chain Reaction; Smad2 Protein; Thrombopoietin; Thymidine; Trans-Activators; Transforming Growth Factor beta; Tyrphostins

Hematopoietic growth factors (HGF) are essential for proliferation and differentiation of hematopoietic precursors and activate a distinct set of JAK-STAT (Janus kinases-signal transducers and activators of transcription) proteins. Previous results from our group have shown a strong expression of JAK-STAT proteins in primary acute myelogenous leukemia (AML) blasts and AML cell lines. Here, we asked whether a constitutive activation of the JAK-STAT pathway might be involved in the pathogenesis of AML. We could demonstrate a constitutive activation of STAT1, 3 and 5 by immunoprecipitation of the tyrosine phosphorylated proteins in different human AML cell lines. Three patterns of STAT activation were found: (I) activation of only STAT1, (II) activation of STAT1 in combination with STAT3, and (III) activation of STAT1, 3 and 5. Furthermore, STAT1 and 3 formed stable heterodimers only in cell lines with constitutive STAT3 activation. In all cell lines analyzed, tyrosine phosphorylation of the four known Janus kinases could not be detected, although JAK1 was stably associated with STAT3. To further analyze whether a constitutive activation of tyrosine kinases might contribute to the autonomous growth of AML blasts, inhibitor studies were performed. The tyrphostin AG490, an inhibitor of the JAK-STAT pathway, but not A1, an inactive tyrphostin induced a time- and dose-dependent growth arrest without overt morphological signs of differentiation in AML cell lines. Our results show that STAT transcription factors are constitutively activated in human AML cell lines and might contribute to the autonomous proliferation of AML blasts. Inhibition of this pathway might be of interest for the establishment of more specific antileukemic strategies.

Topics: Acute Disease; Alternative Splicing; Cell Division; Dimerization; DNA-Binding Proteins; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation, Leukemic; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; HL-60 Cells; Humans; Interleukin-3; Interleukin-6; Janus Kinase 1; K562 Cells; Leukemia, Myeloid; Milk Proteins; Neoplasm Proteins; Phosphorylation; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Proteins; Signal Transduction; STAT1 Transcription Factor; STAT3 Transcription Factor; STAT5 Transcription Factor; Trans-Activators; Transcription, Genetic; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; TYK2 Kinase; Tyrphostins; U937 Cells