ag-490 and Leukemia--Myelogenous--Chronic--BCR-ABL-Positive

The BCR-ABL tyrosine kinase has been implicated in the dysregulation of oncogenes and tumor suppressor genes involved in chronic myelogenous leukemia (CML). Suppressor of activator protein-1, regulated by interferon (SARI), is a recently identified tumor suppressor gene whose expression has been reported to be suppressed in several malignant neoplasms. However, the expression of SARI in leukemia and the underlying regulatory mechanism remain elusive. In this study, we demonstrated that SARI mRNA expression was low in CML patients. In vitro, BCR-ABL kinase inhibitor imatinib mesylate or siRNA specific to BCR-ABL upregulated SARI mRNA expression in human leukemia cells. In addition, JAK/STAT signaling inhibitor AG490 and RAS/MAPK signaling inhibitor PD98059 upregulated SARI mRNA expression, but PI3K/AKT pathway inhibitor LY294002 had no such effect. Functionally, silencing of SARI in CML-derived cell line K562 partially decreased imatinib mesylate-induced apoptosis. Taken together, these data demonstrate that SARI mRNA expression is suppressed by BCR-ABL through the downstream signaling pathways, suggesting SARI as a potential therapeutic target in CML.

Topics: Adolescent; Adult; Aged; Apoptosis; Basic-Leucine Zipper Transcription Factors; Benzamides; Blotting, Western; Female; Flavonoids; Fusion Proteins, bcr-abl; Gene Expression Regulation, Leukemic; Humans; Imatinib Mesylate; Janus Kinases; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Mitogen-Activated Protein Kinases; Piperazines; Protein Kinase Inhibitors; Pyrimidines; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Transcription, Genetic; Tumor Cells, Cultured; Tumor Suppressor Proteins; Tyrphostins; Young Adult

Chronic myelogenous leukemia (CML) patients treated with imatinib mesylate (IM) become drug resistant by mutations within the kinase domain of Bcr-Abl, and by other changes that cause progression to advanced stage (blast crisis) and increased expression of the Lyn tyrosine kinase, the regulation of which is not understood yet. In Bcr-Abl+ cells inhibition of Jak2, a downstream target of Bcr-Abl, by either Jak2 inhibitors or Jak2-specific short interfering RNA (siRNA) reduced the level of the SET protein, and increased PP2A Ser/Thr phosphatase and Shp1 tyrosine phosphatase activities, which led to decreased levels of activated Lyn. Activation of PP2A combined with Jak2 inhibition enhanced the reduction of activated Lyn kinase compared with Jak2 inhibition alone. In contrast, inhibition of either PP2A or Shp1 combined with Jak2 inhibition interfered with the loss of Lyn kinase activation more so than Jak2 inhibition alone, indicating the involvement of PP2A and Shp1 in the inactivation of the Lyn kinase caused by Jak2 inhibition. Inhibition of Jak2 induced apoptosis and reduced colony formation in IM-sensitive and -resistant Bcr-Abl mutant cell lines. Jak2 inhibition also induced apoptosis in CML cells from blast crisis patients but not in normal hematopoietic cells. These results indicate that Lyn is downstream of Jak2, and Jak2 maintains activated Lyn kinase in CML through the SET-PP2A-Shp1 pathway.

Topics: Animals; Apoptosis; Benzamides; Chromosomal Proteins, Non-Histone; Cyclohexanes; DNA-Binding Proteins; Drug Resistance, Neoplasm; Histone Chaperones; Humans; Imatinib Mesylate; Janus Kinase 2; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Phosphorylation; Piperazines; Protein Phosphatase 2; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Proto-Oncogene Proteins c-akt; Pyrimidines; src-Family Kinases; Transcription Factors; Tyrphostins

The Bcr-Abl tyrosine kinase is the causative factor in most chronic myelogenous leukemia (CML) patients. We have shown that Bcr-Abl is associated with a cluster of signaling proteins, including Janus kinase (Jak) 2, growth factor receptor binding protein 2-associated binder (Gab) 2, Akt, and glycogen synthase kinase (GSK)-3beta. Treatment of CML cell lines and mouse Bcr-Abl+ 32D cells with either Jak2 short interfering RNA or Jak2 kinase inhibitor AG490 inhibited pTyr Gab2 and pSer Akt formation, inhibited the activation of nuclear factor-kappaB, and caused the activation of GSK-3beta, leading to the reduction of c-Myc. Importantly, BaF3 cells expressing T315I and E255K imatinib-resistant mutants of Bcr-Abl underwent apoptosis on exposure to AG490 yet were resistant to imatinib. Similar to wild-type Bcr-Abl+ cells, inhibition of Jak2 by Ag490 treatment resulted in decrease of pSer Akt and c-Myc in imatinib-resistant cells. These results identify Jak2 as a potentially important therapeutic target for CML.

Topics: Animals; Antineoplastic Agents; Apoptosis; Benzamides; Cell Line, Tumor; Enzyme Activation; Enzyme Inhibitors; Fusion Proteins, bcr-abl; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Imatinib Mesylate; Janus Kinase 2; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; NF-kappa B; Phosphorylation; Piperazines; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Pyrimidines; RNA, Small Interfering; Signal Transduction; Transfection; Tyrphostins

We investigated the role of the phosphatidylinositol-3 kinase (PI-3K) pathway in regulating the proliferation of primary chronic myeloid leukaemia (CML) progenitor cells by using imatinib to inhibit the activity of p210(Bcr-Abl). The effect of imatinib on the expression of PI-3K pathway proteins was investigated by kinase assays and Western blotting; PI-3K was inhibited by wortmannin or LY294002, Jak2 by AG490 and farnesylation by FTI II; progenitor cell proliferation (self-renewal) was measured by growing myeloid colonies in vitro, then replating them to observe secondary colony formation. Suppression of p210(Bcr-Abl) with imatinib indirectly suppressed the activity of PI-3K and its downstream targets (Erk, Akt and p70S6 kinase), thereby implicating the PI-3K pathway in p210(Bcr-Abl)-mediated signalling in primary CML progenitor cells. The PI-3K inhibitors, wortmannin and LY294002 reproduced the differential effects of imatinib on normal and CML progenitor cell proliferation in vitro by increasing normal cell (P = 0.001) and reducing CML cell proliferation (P = 0.0003). This differential effect was attributable to dysregulated signalling by granulocyte colony-stimulating factor in CML. The responses of individual patient's cells to wortmannin correlated with their responses to imatinib (P = 0.004) but not their responses to AG490 (Jak2 kinase inhibitor) or FTI II (farnesyltransferase inhibitor). Individual responses to wortmannin also correlated with responses to interferon alpha (IFNalpha) (P = 0.016). Imatinib-resistant K562 cells were sensitive to LY294002. Inhibition of the PI-3K pathway may be common to imatinib and IFNalpha and reflect dysregulated cytokine signalling. As imatinib-resistant cells remained sensitive to wortmannin and LY294002, targeting the PI-3K pathway may provide an alternative therapy for imatinib-resistant patients.

Topics: Alkyl and Aryl Transferases; Androstadienes; Antineoplastic Agents; Benzamides; Blotting, Western; Cell Division; Cells, Cultured; Chromones; Drug Therapy, Combination; Farnesyltranstransferase; Humans; Imatinib Mesylate; Interferon-gamma; Janus Kinase 2; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Piperazines; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Pyrimidines; Signal Transduction; Stem Cells; Tyrphostins; Wortmannin

We have previously shown that the Jak2 tyrosine kinase is activated in Bcr-Abl positive cell lines and blood cells from CML blast crisis patients by tyrosine phosphorylation. We are searching for downstream targets of Jak2 in Bcr-Abl positive cells. It is known that c-Myc expression is required for the oncogenic effects of Bcr-Abl, and that over-expression of c-Myc complements the transformation defect of the Bcr-Abl SH2 deletion mutant. Moreover, the Bcr-Abl SH2 deletion mutant and an Abl C-terminal deletion mutant are deficient in activating c-Myc expression. Since the Jak2 binds to the C-terminal domain of Bcr-Abl and optimal Jak2 activation requires the SH2 domain, we tested whether Jak2 was involved in c-Myc protein induction by Bcr-Abl. We treated the 32Dp210 Bcr-Abl cells with the Jak2 specific tyrosine kinase inhibitor, AG490, and found that this drug, like the Abl tyrosine kinase inhibitor STI-571, inhibited c-Myc protein induction by Bcr-Abl. Treatment of 32Dp210 Bcr-Abl cells with AG490 also inhibited c-MYC RNA expression. It is also known that c-Myc protein is a labile protein that is increased in amounts in response to various growth factors by a mechanism not involving new Myc protein formation. Treatment of 32Dp210 Bcr-Abl cells with both the proteasome inhibitor MG132 and AG490 blocked the reduction of the c-Myc protein observed by AG490 alone. An adaptor protein SH2-Bbeta is involved in the enhancement of the tyrosine kinase activity of Jak2 following ligand/receptor interaction. In this regard we showed that the Jak2/Bcr-Abl complex contains SH2-Bbeta. Expression of the SH2-Bbeta R555E mutant in 32Dp210 Bcr-Abl cells reduced c-Myc expression about 40% compared to a vector control. Interestingly, we found the reduction of the c-Myc protein in several clones of dominant-negative (DN) Jak2 expressing K562 cells correlated very well with the reduction of tumor growth of these cells in nude mice as compared to vector transfected K562 cells. Both STI-571 and AG490 also induced apoptosis in 32Dp210 cells. Of interest, IL-3 containing medium reversed the STI-571 induced apoptosis of 32Dp210 cells but did not reverse the induction of apoptosis by AG490, which strongly supports the specificity of the inhibitory effects of AG490 on the Jak2 tyrosine kinase. In summary, our findings indicate that Jak2 mediates the increase in c-Myc expression that is induced by Bcr-Abl. Our results indicate that activated Jak2 not only mediates an increase of c-MYC

Topics: Adaptor Proteins, Signal Transducing; Carrier Proteins; Cell Line; Fusion Proteins, bcr-abl; Gene Expression Regulation; Genes, myc; Humans; Janus Kinase 2; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leupeptins; Mutagenesis; Protein Structure, Tertiary; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; RNA, Messenger; STAT5 Transcription Factor; Transcription Factors; Tumor Cells, Cultured; Tyrphostins; Xenopus Proteins

Combination of STI571, a tyrosine kinase inhibitor, with other drugs may be beneficial in the treatment of chronic myeloid leukaemia (CML). We measured the effects of STI571, AG490, farnesyltransferase inhibitor (FTI), interferon alpha (IFN-alpha), cytosine arabinoside (Ara-C) and all-trans retinoic acid (ATRA), singly and in combination, on clonogenic leukaemic cell proliferation. STI571, IFN-alpha and ATRA each reduced proliferation by 50-60%; AG490, FTI and Ara-C had less effect. Comparing the observed and expected (i.e. additive) effects of drug combinations showed STI571 + FTI, STI571 + AG490 and IFN-alpha + ATRA were additive; STI571 + IFN-alpha, IFN-alpha + Ara-C and STI571 + AG490 + FTI were less than additive. Thus, STI571 + FTI, STI571 + AG490 and IFN-alpha + ATRA may be better combination therapies for CML than STI571 + IFN-alpha, IFN-alpha + Ara-C or STI571 + AG490 + FTI.

Topics: Alkyl and Aryl Transferases; Antineoplastic Combined Chemotherapy Protocols; Area Under Curve; Benzamides; Cell Division; Cytarabine; Enzyme Inhibitors; Farnesyltranstransferase; Humans; Imatinib Mesylate; Interferon-alpha; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Piperazines; Protein-Tyrosine Kinases; Pyrimidines; Stem Cells; Tretinoin; Tyrphostins

CGP 57148 is a potent inhibitor of the ABL protein tyrosine kinase and a promising new compound for the treatment of a variety of BCR-ABL-positive leukemias. We used this enzyme inhibitor to characterize the biological effects of BCR-ABL in primary cells and two growth factor-dependent BCR-ABL-transfected cell lines. The effect of CGP 57148 on primary cells is dependent on the stage of differentiation. The growth of maturing chronic myeloid leukemia cells is independent of BCR-ABL in the presence of growth factors. However, the proliferation of leukemic immature cobblestone-forming area cells is almost completely blocked after the inhibition of the BCR-ABL kinase. In the BCR-ABL-transfected cell lines, M07/ p210 and Ba/F3/p185, CGP 57148 induces apoptosis by releasing cytochrome c, activating caspase 3, and cleavage of PARP. No alteration of the expression level of the apoptosis regulator BCL-2 was observed. In contrast, BCL-X was down-regulated after exposure to CGP 57148. Inhibitors of signal transduction proteins such as PI-3 kinase, mitogen-activated protein/extracellular signal-regulated kinase kinase, and Janus-activated kinase 2 pathways were not capable of a comparable down-regulation of BCL-X. The Fas/Fas ligand system was not involved either in the induction of apoptosis by CGP 57148. We conclude that the inhibition of the BCR-ABL kinase by CGP 57148 (a) preferentially inhibits the growth of immature leukemic precursor cells, (b) efficiently reverts the antiapoptotic effects of BCR-ABL by down-regulation of BCL-X, and (c) is more effective than the inhibition of the downstream signal transduction pathways of PI-3 kinase, mitogen-activated protein/extracellular signal-regulated kinase kinase, and Janus-activated kinase 2.

Topics: Animals; Apoptosis; bcl-X Protein; Benzamides; Caspase 3; Caspases; Cell Count; Cell Division; Chromones; Cytochrome c Group; Cytosol; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Fas Ligand Protein; fas Receptor; Flavonoids; Fusion Proteins, bcr-abl; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Membrane Glycoproteins; Morpholines; Piperazines; Poly(ADP-ribose) Polymerases; Protein Binding; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-bcl-2; Pyrimidines; Tumor Cells, Cultured; Tyrphostins