A single amino acid exchange inverts susceptibility of related receptor tyrosine kinases for the ATP site inhibitor STI-571

The tyrosine kinase inhibitor STI-571 potently blocks BCR-Abl, platelet-derived growth factor (PDGF) alpha- and beta-receptors, and c-Kit kinase activity. Flt3, a receptor tyrosine kinase closely related to PDGF receptors and c-Kit is, however, not inhibited by STI-571. Sequence alignments of different kinases and indications from the crystal structure of the STI-571 Abl kinase complex revealed amino acid residues that are probably crucial for this activity profile. It was predicted that Flt3 Phe-691 in the beta5 strand may sterically prevent interaction with STI-571. The point mutants Flt3 F691T and PDGFbeta-receptor T681F were constructed, and kinase assays showed that the Flt3 mutant but not the PDGFbeta-receptor mutant is inhibited by STI-571. Docking of STI-571 into computer models of the PDGFbeta-receptor and Flt3 kinase domains and comparison with the crystal structure of the STI-571 Abl kinase complex indicated very similar binding sites among the three nonphosphorylated kinases, suggesting corresponding courses of their Asp-Phe-Gly motifs and activation loops. Accordingly, we observed reduced sensitivity of preactivated compared with nonactivated PDGFR-beta for the inhibition by STI-571. Courses of the activation loop that collide with STI-571 binding explain its inactivity at other kinases as the insulin receptor. The binding site models of PDGFR-beta and Flt3 were applied to predict structural approaches for more selective PDGFbeta-receptor inhibitors.     

Design and synthesis of 3-substituted benzamide derivatives as Bcr-Abl kinase inhibitors

A series of 3-substituted benzamide derivatives structurally related to STI-571 (imatinib mesylate), a Bcr-Abl tyrosine kinase inhibitor used to treat chronic myeloid leukemia (CML), was prepared and evaluated for antiproliferative activity against the Bcr-Abl-positive leukemia cell line K562. About ten 3-halogenated and 3-trifluoromethylated benzamide derivatives were identified as highly potent Bcr-Abl kinase inhibitors. One of these, NS-187 (9b), is a promising new candidate Bcr-Abl inhibitor for the therapy of STI-571-resistant chronic myeloid leukemia.     

9-cis-retinoic acid decreases the level of its cognate receptor, retinoid X receptor, through acceleration of the turnover.

In this study, we investigated the ligand-mediated regulation of retinoid X receptor (RXR) in two human cell lines (HepG2 and JEG-3 cells), which have been reported to express RXRalpha predominantly. Western blot analysis revealed that a treatment with 1 microM 9-cis-retinoic acid (9-cis RA) for 24 h decreased RXRalpha protein level to 72 +/- 9 and 75 +/- 7% in HepG2 and JEG-3 cells, respectively, when the levels in the non-treated cells were expressed as 100%. The decrease was not due to the changes in steady-state level of RXRalpha mRNA or its stability as revealed by Northern blot analysis. However, the 9-cis RA treatment decreased the half-life of RXRalpha protein as determined by pulse-chase analysis. It was thus demonstrated that 9-cis RA downregulates RXRalpha by increasing the turnover of the protein in the two cell lines. The ligand-dependent downregulation of RXRalpha protein may be important for several hormonal signalings, in which the receptors heterodimerize with RXR.     

Structural basis for the autoinhibition and STI-571 inhibition of c-Kit tyrosine kinase

The activity of the c-Kit receptor protein-tyrosine kinase is tightly regulated in normal cells, whereas deregulated c-Kit kinase activity is implicated in the pathogenesis of human cancers. The c-Kit juxtamembrane region is known to have an autoinhibitory function; however the precise mechanism by which c-Kit is maintained in an autoinhibited state is not known. We report the 1.9-A resolution crystal structure of native c-Kit kinase in an autoinhibited conformation and compare it with active c-Kit kinase. Autoinhibited c-Kit is stabilized by the juxtamembrane domain, which inserts into the kinase-active site and disrupts formation of the activated structure. A 1.6-A crystal structure of c-Kit in complex with STI-571 (Imatinib or Gleevec) demonstrates that inhibitor binding disrupts this natural mechanism for maintaining c-Kit in an autoinhibited state. Together, these results provide a structural basis for understanding c-Kit kinase autoinhibition and will facilitate the structure-guided design of specific inhibitors that target the activated and autoinhibited conformations of c-Kit kinase.     

Imatinib mesylate (STI-571) attenuates liver fibrosis development in rats

It is widely recognized that activated hepatic stellate cells (HSC) play a pivotal role in development of liver fibrosis. A platelet-derived growth factor (PDGF) is the most potent mitogen for HSC. The aim of this study was to examine the effect of imatinib mesylate (STI-571, Gleevec), a clinically used PDGF receptor (PDGFR) tyrosine kinase inhibitor, on development of experimental liver fibrosis. The rat model of pig serum-induced hepatic fibrosis was used to assess the effect of daily oral administration of STI-571 on the indexes of fibrosis. STI-571 markedly attenuated development of liver fibrosis and hepatic hydroxyproline and serum fibrosis markers. The number of alpha-smooth muscle actin-positive cells and mRNA expression of alpha2-(I)-procollagen, tissue inhibitor of metalloproteinases-1, and transforming growth factor-beta were also significantly suppressed by STI-571. Our in vitro study showed that STI-571 markedly attenuated PDGF-BB-induced proliferation and migration and alpha-SMA and alpha2-(I)-procollagen mRNA of activated HSC in a dose-dependent manner. STI-571 also significantly attenuated PDGF-BB-induced phosphorylation of PDGFR-beta, MEK1/2, and Akt in activated HSC. Because STI-571 is widely used in clinical practice, it may provide an effective new strategy for antifibrosis therapy.     

Imatinib analogs as potential agents for PET imaging of Bcr-Abl and c-KIT expression at a kinase level

We synthesized two series of imatinib mesylate (STI-571) analogs to develop a Bcr-Abl and c-KIT receptor-specific labeling agent for positron emission tomography (PET) imaging to measure Bcr-Abl and c-KIT expression levels in a mouse model. The methods of molecular modeling, synthesis of STI-571 and its analogs, in vitro kinase assays, and radiolabeling are described. Molecular modeling revealed that these analogs bind the same Bcr-Abl and c-KIT binding sites as those bound by STI-571. The analogs potently inhibit the tyrosine kinase activity of Bcr-Abl and c-KIT, similarly to STI-571. [¹⁸F]-labeled STI-571 was prepared with high specific activity (75 GBq/μmol) by nucleophilic displacement and an average radiochemical yield of 12%. [¹³¹I]-labeled STI-571 was prepared with high purity (>95%) and an average radiochemical yield of 23%. The uptake rates of [¹⁸F]-STI-571 in K562 cells expressing Abl and in U87WT cells overexpressing c-KIT were significantly higher than those in the U87 cell and could be inhibited by STI-71 (confirming the specificity of uptake). PET scans of K562 and U87WT tumor-bearing mice with [¹⁸F]-STI-571 as a contrast agent showed visible tumor uptake and tumor-to-non-target contrast.     

Retinoic acid confers resistance to p53-dependent apoptosis in SH-SY5Y neuroblastoma cells by modulating nuclear import of p53.

Many cell lines derived from neuroblastoma (NB) carry the wild-type p53 gene with a p53-dependent apoptotic pathway that is responsive to DNA damaging agents. A recent study has demonstrated that retinoic acid (RA) pretreatment of NB cells promotes chemoresistance to apoptosis induced by chemotherapeutic agents. We examine here the possible contribution of the p53 pathway to the chemoresistance response associated with the RA treatment in NB cells. Upon treatment with RA (1-10 microM) for 4 days, the human NB cells, SH-SY5Y, developed resistance selectively to p53-dependent apoptotic stimuli including gamma-irradiation, etoposide, and 1-(5-isoquinolinyl sulfonyl)-2-methylpiperazine (H-7). Interestingly, RA affected the ability of H-7 to induce nuclear accumulation of the p53 protein without altering its effect on elevating the steady-state level of p53, suggesting that drug-induced up-regulation and nuclear accumulation of the wild-type p53 protein are separable processes. The modulation of nuclear import of p53 protein by RA may thus represent a potential mechanism by which certain tumor cells with the wild-type p53 gene develop resistance to chemotherapeutic agents.     

ONIOM DFT/PM3 calculation on the interaction between STI-571 and abelson tyrosine kinase

The ONIOM2 (B3LYP/6–31G (d, p): PM3) and B3LYP/6–31G (d, p) methods were applied to investigate the interaction between STI-571 and abelson tyrosine kinase binding site. The complex of N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)- phenyl]-benzamide (part of STI-571) and related 16 amino acid residues were found at B3LYP/6–31G (d, p) level to have hydrogen bonds and π....π stacking interaction, their binding energy via HAF optimization was −20.4 kcal mol⁻¹. The results derived from this study agreed well with the reported observation. Figure Optimized structure of STI-571 and Thr315 in abelson tyrosine kinase based on ONIOM2 method     

A myristoyl/phosphotyrosine switch regulates c-Abl

The c-Abl tyrosine kinase is inhibited by mechanisms that are poorly understood. Disruption of these mechanisms in the Bcr-Abl oncoprotein leads to several forms of human leukemia. We found that like Src kinases, c-Abl 1b is activated by phosphotyrosine ligands. Ligand-activated c-Abl is particularly sensitive to the anti-cancer drug STI-571/Gleevec/imatinib (STI-571). The SH2 domain-phosphorylated tail interaction in Src kinases is functionally replaced in c-Abl by an intramolecular engagement of the N-terminal myristoyl modification with the kinase domain. Functional studies coupled with structural analysis define a myristoyl/phosphotyrosine switch in c-Abl that regulates docking and accessibility of the SH2 domain. This mechanism offers an explanation for the observed cellular activation of c-Abl by tyrosine-phosphorylated proteins, the intracellular mobility of c-Abl, and it provides new insights into the mechanism of action of STI-571.     

Disabling poxvirus pathogenesis by inhibition of Abl-family tyrosine kinases

The Poxviridae family members vaccinia and variola virus enter mammalian cells, replicate outside the nucleus and produce virions that travel to the cell surface along microtubules, fuse with the plasma membrane and egress from infected cells toward apposing cells on actin-filled membranous protrusions. We show that cell-associated enveloped virions (CEV) use Abl- and Src-family tyrosine kinases for actin motility, and that these kinases act in a redundant fashion, perhaps permitting motility in a greater range of cell types. Additionally, release of CEV from the cell requires Abl- but not Src-family tyrosine kinases, and is blocked by STI-571 (Gleevec), an Abl-family kinase inhibitor used to treat chronic myelogenous leukemia in humans. Finally, we show that STI-571 reduces viral dissemination by five orders of magnitude and promotes survival in infected mice, suggesting possible use for this drug in treating smallpox or complications associated with vaccination. This therapeutic approach may prove generally efficacious in treating microbial infections that rely on host tyrosine kinases, and, because the drug targets host but not viral molecules, this strategy is much less likely to engender resistance compared to conventional antimicrobial therapies.     

Tyrosine kinases. New target of anticancer therapy

Recently, clinical studies of new drugs development to target specific forms of cancer were reported. Herceptin, a monoclonal antibody against the Her2/neu receptor tyrosine kinase, prolonged the survival of women with Her2/neu positive metastatic breast cancer. STI571, a small molecule inhibitor of the BCR/ABL, c-Kit and platelet derived growth factor receptor tyrosine kinase, produced pronounced clinical responses in patients with BCR/ABL positive chronic myeloid leukemia and c-Kit positive gastrointestial stromal tumors. In order to consider the use of the inhibitor of tyrosine kinases activity as anticancer drug, their mechanisms of the oncogenic activation and their impact on tumor transformation should be studied. The treatment with tyrosine kinase inhibitors such as STI571 or herceptin was a spectacular clinical success which stimulated research on the structure and function of both kinases and their inhibitors.     

The alpha subunit of the granulocyte-macrophage colony-stimulating factor receptor interacts with c-Kit and inhibits c-Kit signaling

The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates hematopoiesis and the function of mature host defense cells through the GM-CSF receptor (GMR), which is composed of alpha (alphaGMR) and beta (betaGMR) subunits. Stem cell factor is another important hematopoietic cytokine that signals through c-Kit, a receptor tyrosine kinase, and regulates hematopoietic stem cell maintenance and erythroid development. Like other cytokine receptors, GMR and c-Kit are generally deemed as independent adaptor molecules capable of transducing cytokine-specific signals. We found that the alphaGMR directly interacts with c-Kit and that the interaction is mediated by the cytoplasmic domains. Furthermore, alphaGMR inhibited c-Kit auto-phosphorylation induced by the ligand stem cell factor. Consistent with the inhibitory effect, the expression of alphaGMR was suppressed in cells whose viability was dependent on c-Kit signaling. In contrast, the alternatively spliced alpha2 isoform of the alphaGMR could not inhibit c-Kit signaling, providing a rationale for the existence of the alpha2 isoform. Our results suggest that in addition to having the commonly appreciated roles in cytokine signal transduction, the receptors alphaGMR and c-Kit could interact to coordinate their signal initiation.     

3-Deazauridine triggers dose-dependent apoptosis in myeloid leukemia cells and enhances retinoic acid-induced granulocytic differentiation of HL-60 cells

Therapeutic nucleoside analogue 3-deazauridine (DU) exerts cytotoxic activity against cancer cells by disruption of DNA synthesis resulting in cell death. The present study evaluates whether DU alone at doses 2.5-15 microM or in combination with all trans retinoic acid (RA) or dibutyryl cAMP (dbcAMP) is effective against myelogenous leukemia. The data of this study indicate that DU induces dose-dependent cell death by apoptosis in myeloid leukemia cell lines HL-60, NB4, HEL and K562 as demonstrated by cell staining or flow cytometry and agarose gel electrophoresis. 24h-treatment with DU produced dose-dependent HL-60 cell growth inhibition and dose-independent S phase arrest that was not reversed upon removal of higher doses of DU (10-15 microM). Exposition to nontoxic dose of DU (2.5 microM) for 24h followed by RA or dbcAMP and 96 h-cotreatment with DU significantly enhanced RA- but not dbcAMP-mediated granulocytic differentiation. Cell maturation was paralleled with an increase in the proportion of cells in G1 or G2+M phase. We conclude that, depending on the dose or the sequence of administration with RA, an inhibitor of DNA replication, DU triggers a process of either differentiation or apoptosis in myeloid leukemia cells.     

Roles of stem cell factor/c-Kit and effects of Glivec/STI571 in human uveal melanoma cell tumorigenesis

The B-Raf(V599E)-mediated constitutive activation of ERK1/2 is involved in establishing the transformed phenotype of some uveal melanoma cells (Calipel, A., Lefevre, G., Pouponnot, C., Mouriaux, F., Eychene, A., and Mascarelli, F. (2003) J. Biol. Chem. 278, 42409-42418). We have shown that stem cell factor (SCF) is involved in the proliferation of normal uveal melanocytes and that c-Kit is expressed in 75% of primary uveal melanomas. This suggests that the acquisition of autonomous growth during melanoma progression may involve the SCF/c-Kit axis. We used six human uveal melanoma tumor-derived cell lines and normal uveal melanocytes to characterize the SCF/c-Kit system and to assess its specific role in transformation. We investigated the possible roles of activating mutations in c-KIT, the overexpression of this gene, and ligand-dependent c-Kit overactivation in uveal melanoma cell tumorigenesis. Four cell lines (92.1, SP6.5, Mel270, and TP31) expressed both SCF and c-Kit, and none harbored the c-KIT mutations in exons 9, 11, 13, and 17 that have been shown to induce SCF-independent c-Kit activation. Melanoma cell proliferation was strongly inhibited by small interfering RNA-mediated depletion of c-Kit in these cells, despite the presence of (V599E)B-Raf in SP6.5 and TP31 cells. We characterized the signaling pathways involved in SCF/c-Kit-mediated cell growth and survival in normal and tumoral melanocytes and found that constitutive ERK1/2 activation played a key role in both the SCF/c-Kit autocrine loop and the gain of function of (V599E)B-Raf for melanoma cell proliferation and transformation. We also provide the first evidence that Glivec/STI571, a c-Kit tyrosine kinase inhibitor, could be used to treat uveal melanomas.     

Rational therapeutic intervention in cancer: kinases as drug targets.

Landmark clinical studies of new drugs developed to target specific forms of cancer were reported in 2001. Herceptin, a monoclonal antibody against the Her2/neu receptor tyrosine kinase, prolonged the survival of women with Her-2/neu positive metastatic breast cancer, when combined with chemotherapy. STI-571, a small molecule inhibitor of the Bcr-Abl, c-kit and platelet derived growth factor receptor tyrosine kinases, produced dramatic clinical responses in patients with Bcr-Abl positive chronic myeloid leukemia and c-kit positive gastrointestinal stromal tumors. These examples have galvanized the cancer research community to extend kinase-inhibitor therapy to other cancers.     

STI-571: an anticancer protein-tyrosine kinase inhibitor

STI-571 (imatinib, Gleevec, Glivec, CGP 57148) is an inhibitor of the Abl group of protein-tyrosine kinases. One of these enzymes, the Bcr-Abl oncoprotein, results from the fusion of the BCR and ABL genes that result from the reciprocal chromosomal translocation that forms the Philadelphia chromosome. The Philadelphia chromosome occurs in 95% of people with chronic myeloid leukemia. ABL is the cellular homologue of the oncogene found in murine Abelson leukemia virus, and BCR refers to breakpoint cluster region. The Bcr-Abl oncoprotein exhibits elevated protein-tyrosine kinase activity, which is strongly implicated in the mechanism of development of chronic myeloid leukemia. STI-571 is effective in the treatment of the stable phase of chronic myeloid leukemia. The c-Abl protein kinase domain exists in an active and inactive conformation. STI-571 binds only to the inactive state of the enzyme as shown by X-ray crystallography. The drug binds to a portion of the ATP-binding site and extends from there into adjacent hydrophobic regions. STI-571 is a competitive inhibitor of Abl kinase with respect to ATP. Resistance to STI-571 is often the result of mutations in residues of the Bcr-Abl kinase that ordinarily bind to the drug. Inhibition of target protein kinases represents an emerging therapeutic strategy for the treatment of cancer.     

Inhibition of Axl improves the targeted therapy against ALK-mutated neuroblastoma

Neuroblastoma (NB) patients harboring mutated ALK can be expected to potentially benefit from targeted therapy based on ALK tyrosine kinase inhibitor (TKI), such as crizotinib and ceritinib. However, the effect of the treatment varies with different individuals, although with the same genic changes. Axl receptor tyrosine kinase is expressed in a variety of human cancers, but little data are reported in NB, particularly in which carrying mutated ALK. In this study, we focus on the roles of Axl in ALK-mutated NB for investigating rational therapeutic strategy. We found that Axl is expressed in ALK-positive NB tissues and cell lines, and could be effectively activated by its ligand GAS6. Ligand-dependent Axl activation obviously rescued crizotinib-mediated suppression of cell proliferation in ALK-mutated NB cells. Genetic inhibition of Axl with specific small interfering RNA markedly increased the sensitivity of cells to ALK-TKIs. Furthermore, a small-molecule inhibitor of Axl significantly enhanced ALK-targeted therapy, as an increased frequency of apoptosis was observed in NB cells co-expressing ALK and Axl. Taken together, our results demonstrated that activation of Axl could lead to insensitivity to ALK inhibitors, and dual inhibition of ALK and Axl might be a potential therapeutic strategy against ALK-mutated NB.     

Arsenic trioxide inhibits neuroblastoma growth in vivo and promotes apoptotic cell death in vitro

Recent clinical studies have shown that inorganic arsenic trioxide (As(2)O(3)) at low concentrations induces complete remission with minimal toxicity in patients with refractory acute promyelocytic leukemia (APL). Preclinical studies suggest that As(2)O(3) induces apoptosis and possibly differentiation in APL cells. Like APL cells, neuroblastoma (NB) cells are thought to be arrested at an early stage of differentiation, and cells of highly malignant tumors fail to undergo spontaneous maturation. Both APL and NB cells can respond with differentiation to retinoic acid (RA) treatment in vitro and probably also in vivo. For that reason we investigated the effect of As(2)O(3) alone and in combination with RA on NB cell lines. In vitro, the number of viable NB cells was reduced at As(2)O(3) concentrations around 1 microM after 72 h exposure. The IC50 in six different cell lines treated for 3 days was in the 1.5 to 5 microM concentration interval, the most sensitive being SK-N-BE(2) cells derived from a chemotherapy resistant tumor. The combined treatment with RA (1 and 3 microM) showed no consistent additional effect with regard to induced cell death. The effect of As(2)O(3) on NB cell number involved As(2)O(3)-induced apoptotic pathways (decreased expression of Bcl-2 and stimulation of caspase-3 activity) with no clear evidence of induced differentiation. The in vivo effect of As(2)O(3) on NB growth was also investigated in nude mice bearing tumors of xenografted NB cells. Although tumor growth was reduced by As(2)O(3) treatment, complete remission was not achieved at the concentrations tested. We suggest that As(2)O(3), in combination with existing treatment modalities, might be a treatment approach for high risk NB patients.