Abl Protein Tyrosine Kinase Research Articles

Protein tyrosine kinase Abl promotes hepatitis C virus particle assembly via interaction with viral substrate activator NS5A

Previously, we reported that knockdown of Abl protein tyrosine kinase by shRNA or pharmacological inhibition suppresses particle assembly of J6/JFH1 strain–derived hepatitis C virus (HCV) in Huh-7.5 cells. However, the detailed mechanism by which Abl regulates HCV replication remained unclear. In this study, we established Abl-deficient (Abl−) cells through genome editing and compared HCV production between Abl− cells expressing WT or kinase-dead Abl and parental Huh-7.5 cells. Our findings revealed that Abl expression was not required from the stages of virus attachment and entry to viral gene expression; however, the kinase activity of Abl was necessary for the assembly of HCV particles. Reconstitution experiments using human embryonic kidney 293T cells revealed that phosphorylation of Tyr412 in the activation loop of Abl was enhanced by coexpression with the viral nonstructural protein 5A (NS5A) and was abrogated by the substitution of NS5A Tyr330 with Phe (Y330F), suggesting that NS5A functions as a substrate activator of Abl. Abl–NS5A association was also attenuated by the Y330F mutation of NS5A or the kinase-dead Abl, and Abl Tyr412 phosphorylation was not enhanced by NS5A bearing a mutation disabling homodimerization, although the association of Abl with NS5A was still observed. Taken together, these results demonstrate that Abl forms a phosphorylation-dependent complex with dimeric NS5A necessary for viral particle assembly, but that Abl is capable of complex formation with monomeric NS5A regardless of tyrosine phosphorylation. Our findings provide the foundation of a molecular basis for a new hepatitis C treatment strategy using Abl inhibitors.

Galactoxyloglucan-doxorubicin nanoparticles exerts superior cytotoxic effects on cancer cells⿿A mechanistic and in silico approach

Galactoxyloglucan (PST001), isolated from seed kernel of Tamarindus indica is a non-toxic immunostimlatory agent with selective cytotoxicity on cancer cells. Toxicity associated with the chemotherapeutic drug doxorubicin (Dox) is the major barrier in its clinical application. Stable, spherically shaped PST-Dox nanoparticles with an average size of 10nm were prepared via ionic gelation of Dox with PST001 which displayed a pH dependent cumulative Dox release kinetics. PST-Dox nanoparticles demonstrated cancer-specific enhanced cytotoxic effects than PST001 and Dox in cancer cells by enhanced cellular uptake of Dox through the induction of apoptosis, sparing normal cells and RBCs. Elucidation of molecular mechanism by whole genome microarray revealed down-regulation of tyrosine kinase oncogenic pathways as PST-Dox mode of action. An in silico model of PST-Dox was developed and computed the activity against topoisomerase IIÿ, human Abl kinase and protein tyrosine kinases. Computational studies further affirmed the findings of genomic and proteomic investigations with an increased interaction energy between PST-Dox complexes with target system than with Dox and PST001 alone. The important findings and profoundly restrained methodologies highlighted in the current study will accelerate the therapeutic potential of this nanoparticle formulation for substantial clinical studies and testing in several cancers. To conclude, PST-Dox nanoparticles represent a superior drug delivery nanosystem for the effective treatment of cancer even though detailed investigations are warranted.

Abstract B132: Discovery of small molecule allosteric modulators of Abl kinase activity and function

Abstract The Abl protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and forms oncogenic fusion proteins that drive several cancer types. The kinase activity of Abl is repressed by intramolecular interactions involving its regulatory SH3 and SH2 domains. The SH3 domain interacts with a polyproline type II helix formed by the SH2-kinase linker, while the SH2 domain interacts with the back of the C-lobe of the kinase domain. In addition, the myristoylated N-terminal cap region (Ncap) binds a hydrophobic pocket in the C-lobe of the kinase domain, clamping the SH3 and SH2 domains against the back of the kinase domain. Small molecules that allosterically regulate Abl kinase activity through its non-catalytic domains may represent selective probes of Abl function. In this study, we developed a high-throughput screening (HTS) assay to identify chemical modulators of Abl kinase activity that target the regulatory SH3:linker interaction. The fluorescence polarization (FP) assay employs a purified recombinant Abl protein comprised of the regulatory N-cap, SH3 and SH2 domains plus the SH2-kinase linker (Abl N32L protein) and a short fluorescein-labeled peptide probe that selectively binds to the Abl SH3 domain. During assay development, we found that the peptide probe binds to the recombinant Abl N32L protein in vitro, producing a robust FP signal that can be competitively displaced by excess unlabeled peptide. The FP signal is not observed in control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved compounds in the FP assay identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays demonstrated that one of the hits, the antithrombotic drug dipyridamole, enhances Abl kinase activity in vitro to a greater extent than the previously described Abl agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates Abl by disrupting this regulatory interaction. Interestingly, this compound does not significantly affect the kinase activity of Hck, a Src family member that is regulated by similar SH3:linker interactions. Treatment with dipyrimadole in combination with DPH led to strong enhancement of Abl kinase activity in transfected cells. The Abl N32L FP assay has been implemented in a fully automated format and used to screen diversity libraries totaling 60,000 compounds. The Z-factor coefficients from this HTS campaign indicated that the FP assay performed well and several additional scaffolds were identified for future development. These results demonstrate that screening assays that incorporate the non-catalytic domains of Abl can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important kinase system. Citation Format: Prerna Grover, Haibin Shi, Matthew Baumgartner, David Close, Paul A. Johnston, Carlos J. Camacho, Thomas E. Smithgall. Discovery of small molecule allosteric modulators of Abl kinase activity and function. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B132.

Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function.

The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important kinase system.

Src-signaling interference impairs the dissemination of blood-borne tumor cells

Although solid tumors continuously shed cells, only a small fraction of the neoplastic cells that enter the blood stream are capable of establishing metastases. In order to be successful, these cells must attach, extravasate, proliferate and induce angiogenesis. Preclinical studies have shown that small-molecule ATP-competitive Src kinase inhibitors can effectively impair metastasis-associated tumor cell functions in vitro. However, the impact of these agents on the metastatic cascade in vivo is less well understood. In the present studies, we have examined the ability of saracatinib, a dual-specific, orally available inhibitor of Src and Abl protein tyrosine kinases, to interfere with the establishment of lung metastases in mice by tumor cells introduced into the blood stream. The results demonstrate that Src inhibition most effectively interferes with the establishment of secondary tumor deposits when treatments are administered while tumor cells are in the initial phases of dissemination.

Antibody-free lanthanide-based fluorescent probe for determination of protein tyrosine kinase and phosphatase activities

A single-labeled peptide probe for measuring peptide phosphorylation status was developed by using a phosphate sensitive terbium chelate. The activity of Abl protein tyrosine kinase and T-cell protein Tyrosine phosphatase (TC PTP) was monitored in real time. To study the probe design in detail, variable substrate peptide sequences, where the enzyme target site was located from two to five amino acids apart from the nearest tyrosine residue, were synthesized. The maximum change observed in fluorescence intensity after phosphorylation was up to 320%, when the phosphorylated tyrosine was located two amino acids from the lysine coupled to the phosphate sensitive terbium chelate, demonstrating an excellent performance for a homogeneous assay. Also the longer distance of five amino acids between the phosphorylated tyrosine residue and terbium chelate resulted up to 260% change in fluorescence intensity.

Photocleavable Peptide-Conjugated Magnetic Beads for Protein Kinase Assays by MALDI-TOF MS

Peptides were immobilized onto superparamagnetic beads via photocleavable linkers. This enabled simple, rapid, and label-free protein kinase assays via MALDI-TOF MS detection of substrate peptide phosphorylation. Abltide, a model substrate for the Abl protein tyrosine kinase model, was coupled onto amine-terminated beads, incubated with ATP and recombinant c-Abl kinase, and released and further detected to determine phosphorylation. Abltide phosphorylation was found to depend significantly on the length and composition of linkers to the bead surface. Inserting a diblock spacer of poly(glycine) and poly(ethylene glycol) segments markedly enhanced phosphorylation. To validate the assay, the activity of two small-molecule kinase inhibitors, imatinib and dasatinib, which target the oncogenic mutant tyrosine kinase Bcr-Abl to treat chronic myeloid leukemia (CML), was tested. Examining inhibition of the purified c-Abl or Bcr-Abl in K562 CML cell extracts, IC(50) values were determined to be consistent with the literature. This simple, label-free, MALDI-based protein kinase assay can be readily adapted to allow multiplexed assays of multiple peptide substrates and/or analysis of alternative post-translational modifications as a tool for drug discovery and clinical testing.

A conserved salt bridge in the G-loop of multiple protein kinases is essential for catalysis, in vivo Lyn function and prevention of autoimmune disease (84.17)

Abstract The glycine-rich G-loop controls ATP binding and phosphate transfer in protein kinases. Here we show that the functions of Src family and Abl protein tyrosine kinases require an electrostatic interaction between oppositely charged amino acids within their G loops that is conserved in multiple other phylogenetically distinct protein kinases from plants to humans. By limiting G-loop flexibility, it controls ATP binding, catalysis and inhibition by ATP-competitive compounds such as Imatinib. In WeeB mice, mutational disruption of the interaction results in expression of a Lyn protein with reduced catalytic activity, and in perturbed B cell receptor signaling. Like Lyn-/- mice, WeeB mice show profound defects in B cell development and function and succumb to autoimmune glomerulonephritis. This demonstrates the physiological importance of the conserved G-loop salt bridge and at the same time distinguishes the in vivo requirement for the Lyn kinase activity from other potential functions of the protein.

A Conserved Salt Bridge in the G Loop of Multiple Protein Kinases Is Important for Catalysis and for In Vivo Lyn Function

The glycine-rich G loop controls ATP binding and phosphate transfer in protein kinases. Here we show that the functions of Src family and Abl protein tyrosine kinases require an electrostatic interaction between oppositely charged amino acids within their G loops that is conserved in multiple other phylogenetically distinct protein kinases, from plants to humans. By limiting G loop flexibility, it controls ATP binding, catalysis, and inhibition by ATP-competitive compounds such as Imatinib. In WeeB mice, mutational disruption of the interaction results in expression of a Lyn protein with reduced catalytic activity, and in perturbed B cell receptor signaling. Like Lyn(-/-) mice, WeeB mice show profound defects in B cell development and function and succumb to autoimmune glomerulonephritis. This demonstrates the physiological importance of the conserved G loop salt bridge and at the same time distinguishes the in vivo requirement for the Lyn kinase activity from other potential functions of the protein.

Efficacy and safety profile of imatinib mesylate (ST1571) in Japanese patients with advanced gastrointestinal stromal tumors: a phase II study (STI571B1202)

Imatinib mesylate, an inhibitor of KIT, ABL protein, and platelet-derived growth factor receptor alpha (PDGFRalpha) tyrosine kinase, has recently been found to have a dramatic antitumor effect on gastrointestinal stromal tumor (GIST). The aim of this study was to assess the efficacy and safety of imatinib mesylate in Japanese patients with advanced GIST. Patients with measurable lesions were enrolled between April 1, 2002, and September 20, 2002, using a design based on previous phase II studies in the United States and the European Union. The diagnosis of GIST was proven histologically with positive immunostaining for KIT (CD117). Imatinib mesylate was administered at a dose of either 400 mg or 600 mg once a day. Pharmacokinetic parameters and mutation analysis of c-kit were also assessed in a subgroup of patients. A total of 74 patients (28 receiving imatinib mesylate at 400 mg/day; 46 receiving 600 mg/day); median age, 56.0 years, were enrolled. No patient had a complete response, 51 patients (69%) had a partial response, and 19 patients (26%) had stable disease. The median progression-free survival time was 96 weeks. The estimated 3-year overall survival (Kaplan-Meier) rate for all patients was 73.6%. The most frequent adverse effects related to the drug were nausea (78%), diarrhea (70%), dermatitis (62%), facial edema (61%), edema of the lower limbs (58%), vomiting (54%), and eyelid edema (51%). Most of the adverse effects were mild and manageable. Imatinib mesylate is generally safe and has significant activity in the treatment of advanced GIST in Japanese patients.

Long‐term experience with imatinib therapy in chronic phase chronic myelogenous leukemia—Remarkable activity with room for improvement

Long‐term experience with imatinib therapy in chronic phase chronic myelogenous leukemia—Remarkable activity with room for improvement

Structural factors contributing to the Abl/Lyn dual inhibitory activity of 3-substituted benzamide derivatives

To investigate why 3-substituted benzamide derivatives show dual inhibition of Abl and Lyn protein tyrosine kinases, we determined their inhibitory activities against Abl and Lyn, carried out molecular modeling, and conducted a structure–activity relationship study with the aid of a newly determined X-ray structure of the Abl/Lyn dual inhibitor INNO-406 (formerly known as NS-187) bound to human Abl. We found that this series of compounds interacted with both kinases in very similar ways, so that they can inhibit both kinases effectively.

Rb plays a role in survival of Abl-dependent human tumor cells as a downstream effector of Abl tyrosine kinase

The retinoblastoma (Rb) gene product is a tumor suppressor that is mutated or inactivated in many types of human cancers. Although Rb is known to be an upstream negative regulator of Abl protein tyrosine kinase, we propose here that Rb also functions as a downstream effector of Abl that plays a positive role in survival of Abl-dependent human tumor cells, including Bcr/Abl-positive chronic myelogenous leukemia (CML). We show that Rb is constitutively phosphorylated at tyrosine in Abl-dependent tumor cells, and that Abl phosphorylates Rb specifically at Y805 within the C-terminal domain of the molecule. We also show that ectopic expression of Rb induces apoptosis in Abl-dependent tumor cells by inhibiting the Abl tyrosine kinase activity, and that Rb-induced apoptosis is compromised by Abl-catalysed phosphorylation of Rb at Y805. Furthermore, the silencing of endogenous Rb by RNA interference induced apoptosis in Abl-dependent tumor cells. Thus, our findings suggest that Abl-catalysed tyrosine phosphorylation of Rb is necessary for survival of Abl-dependent human tumor cells, and raises the possibility that this phosphorylated Rb can be a molecular target for cancer therapy aimed at inducing apoptosis of Abl-dependent tumor cells, such as Bcr/Abl-positive CML.

The Dual SRC/ABL Kinase Inhibitor BMS-354825 Potently Inhibits the Growth of Myeloid Leukemic Cells Characterized by FLT3-ITD Expression, GM-CSF Dependency, or G-CSF Responsiveness Via an ABL-Independent Mechanism.

BMS-354825 is a dual SRC and ABL inhibitor, which has been shown effective in imatinib-resistant BCR-ABL+ cells, and it is in phase I trials for patients with imatinib-resistant leukemia. We conducted a study to evaluate growth inhibition and inhibition of Src v. Abl protein tyrosine kinases in human myeloid cell lines: MV4-11 expressing an internal tandem duplication of Flt3 (Flt3-ITD), the murine pro-B cell line Ba/F3 that expresses the Flt3-ITD, the GM-CSF dependent Mo7e, and the G-CSF-responsive BaF3-GR (Ba/F3 cells expressing the human G-CSF receptor). We compared BMS-354825 with PP1, a SRC kinase inhibitor with in vitro IC50 at sub-micromolar concentrations. We sought to correlate growth inhibition with SRC or ABL inhibition. In these myeloid cell lines, LYN is the predominant SRC kinase.Methods: Growth inhibition was assessed by Trypan blue exclusion and MTT assay using drug concentrations 0.1 uM – 10 uM. Drugs were added daily to the cell suspension during the 3-day experiment. After a 60 min incubation at concentrations 0.1 nM – 1 uM, SRC or ABL kinase inhibition was analyzed by blotting with a polyclonal phospho-SRC (Tyr416) antibody or a polyclonal phospho-ABL (Tyr245) antibody.Results: In MV4-11 cells BMS-354825 and PP1 caused similar growth inhibition IC50 at 5 uM. By western blotting, inhibition of phospho-Src 416 occurred at 1 nM concentrations of both compounds. Protein expression of ABL was not detected in MV4-11 cells. In Ba/F3-ITD cells, the IC50 for BMS-354825 was 1–10 uM (grown in IL-3) and 0.01 uM (without IL-3). The IC50 for PP1 was 1–10 uM (grown in IL-3) and 0.1 uM (without IL-3). Inhibition of phospho-SRC occurred at 10 nM. In Mo7e cells, grown in the presence of GM-CSF, the IC50 was 5 uM for BMS-354825 v. 10 uM for PP1 by MTT assay. By western blotting, inhibition of phospho-SRC 416 occurred at 1 nM for both BMS-354825 and PP1. To determine specific contribution of LYN to Mo7e growth, we treated Mo7e cells with LYN siRNA. With 70% knock-down of LYN, there was 50% growth inhibition. ABL was present in Mo7e cells, but no phosphoAbl was demonstrated (K562 cells served as positive control). In BaF3-GR cells grown in G-CSF, the IC50 was 5 uM for BMS-354825 vs. 10 uM for PP1 by MTT assay. In western blotting, inhibition of phospho-Src 416 was detected at 10 nM BMS-354825. ABL was present in Ba/F3GR cells, but no phospho-ABL was demonstrated (K562 cells served as positive control).Conclusions: BMS-354825 is more potent than PP1 in causing growth inhibition and SRC kinase inhibition in Mo7e and Ba/F3GR cells that serve as models for acute myeloid leukemia. It is unlikely that ABL is the drug target, because MV4-11 cells do not express ABL and phospho-ABL was not found in Mo7e or Ba/F3 cells. These results suggest that inhibition of SRC tyrosine kinases contributes predominantly to growth inhibition caused by the dual SRC/ABL kinase inhibitor BMS-354825 in myeloid cell lines expressing Flt3-ITD and cytokine-driven proliferation and survival via the IL-3/GM-CSF Receptor or G-CSF Receptor.

A Potent Src/Abl Kinase Inhibitor (Ap23464) Potently Inhibits the Growth of Myeloid Leukemic Cells Characterized by Flt3-Itd Expression, GM-CSF Dependency, or G-CSF Responsiveness Via an Abl-Independent Mechanism.

AP23464 is a potent Src and Abl inhibitor (in vitro IC50 < 1 nM), which has been shown to effectively inhibit growth of imatinib-resistant Bcr-Abl+ cells, and thus, is a promising compound for treating patients with imatinib-resistant leukemia. We conducted a study to evaluate growth inhibition and inhibition of Src versus Abl protein tyrosine kinases in human myeloid cell lines: MV4-11 expressing an internal tandem duplication of Flt3 (Flt3-ITD), the murine pro-B cell line Ba/F3 that expresses the Flt3-ITD, the GM-CSF dependent Mo7e, and the G-CSF-responsive BaF3-GR (Ba/F3 cells expressing the human G-CSF receptor). We compared AP23464 with the PP1, a previously described Src kinase inhibitor (IC50 < 1 uM). We sought to correlate growth inhibition with Src or Abl inhibition.Methods: Growth inhibition was assessed by Trypan blue exclusion and MTT assay using drug concentrations 0.1 uM – 10 uM. Drugs were added daily to the cell suspension during the 3-day experiment. After a 60 min incubation at concentrations 0.1 nM – 1 uM, Src or Abl kinase inhibition was analyzed by blotting with a polyclonal phospho-Src (Tyr416) antibody or a polyclonal phospho-Abl (Tyr245) antibody.Results: In MV4-11 cells AP23464 was more potent than PP1 in causing growth inhibition with IC50 at <1 uM vs 2.5 to 5 uM. By western blotting, inhibition of phospho-Src 416 occurred at the lowest dose of AP23464 and PP1 studied (0.1 nM and 1 nM, respectively). Abl was not detected in MV4-11 cells. In Ba/F3-ITD cells, the IC50 for AP23464 was 1 uM (grown in IL-3) and 0.01–0.1 uM (grown without IL-3). In Ba/F3-ITD cells, IC50 for phosphoSrc was 0.1 uM for both AP23464 and PP1. Abl was present in Ba/F3-ITD cells, but no phospho-Abl was detected. In Mo7e cells grown in the presence of GM-CSF, the IC50 was 1 uM for AP23464 v. 10 uM for PP1. In Mo7e cells treated with Lyn siRNA, there was >50% growth inhibition with 70% knock-down of Lyn. The IC50 for phosphoSrc was 1 nM for both AP23464 and PP1. Abl was present in Mo7e cells, but no phosphoAbl was demonstrated (K562 cells served as positive control). In BaF3-GR cells grown in G-CSF, the IC50 was 1 uM for AP23464 vs. 10 uM for PP1. In western blotting, the IC50 for phospho-Src 416 was detected at 10 nM AP23464. Abl was present in Ba/F3GR cells, but no phosphoAbl was demonstrated (K562 cells served as positive control).Conclusions: AP23464 is more potent than PP1 in causing growth inhibition and Src kinase inhibition in these cell lines that serve as models for acute myeloid leukemia. It is unlikely that Abl is the drug target, because MV4-11 cells do not express detectable Abl, and phospho-Abl was not detected in Mo7e or Ba/F3 cells. These results suggest that inhibition of Src tyrosine kinases contributes predominantly to growth inhibition caused by the Src/Abl kinase inhibitor AP23464 in these cell types.

An improved method for utilization of peptide substrates for antibody characterization and enzymatic assays

Synthetic peptides have become an important tool in antibody production and enzyme characterization. The small size of peptides, however, has hindered their use in assays systems, such as Western blots, and as immunogens. Here, we present a facile method to improve the properties of peptides for multiple applications by ligating the peptides to intein-generated carrier proteins. The stoichiometric ligation of peptide and carrier achieved by intein-mediated protein ligation (IPL) results in the ligation product migrating as a single band on a SDS-PAGE gel. The carrier proteins, HhaI methylase (M.HhaI) and maltose-binding protein (MBP), were ligated to various peptides; the ligated carrier–peptide products gave sharp, reproducible bands when used as positive controls for antibodies raised against the same peptides during Western blot analysis. We further show that ligation of the peptide antigens to a different thioester-tagged carrier protein, paramyosin, produced immunogens for the production of antisera in rabbits or mice. Furthermore, we demonstrate the generation of a substrate for enzymatic assays by ligating a peptide containing the phosphorylation site for Abl protein tyrosine kinase to a carrier protein. This carrier–peptide protein was used as a kinase substrate that could easily be tested for phosphorylation using a phosphotyrosine antibody in Western blot analysis. These techniques do not require sophisticated equipment, reagents, or skills thereby providing a simple method for research and development.

Imatinib mesylate affects the development and function of dendritic cells generated from CD34+ peripheral blood progenitor cells

AbstractImatinib mesylate (STI571) is a competitive Bcr-Abl tyrosine kinase inhibitor and has yielded encouraging results in treatment of chronic myelogenous leukemia (CML) and gastrointestinal stroma tumors (GISTs). Apart from inhibition of the Abl protein tyrosine kinases, it also shows activity against platelet-derived growth factor receptor (PDGF-R), c-Kit, Abl-related gene (ARG), and their fusion proteins while sparing other kinases. In vitro studies have revealed that imatinib mesylate can inhibit growth of cell lines and primitive malignant progenitor cells in CML expressing Bcr-Abl. However, little is known about the effects of imatinib mesylate on nonmalignant hematopoietic cells. In the current study we demonstrate that in vitro exposure of mobilized human CD34+ progenitors to therapeutic concentrations of imatinib mesylate (1-5 μM) inhibits their differentiation into dendritic cells (DCs). DCs obtained after 10 to 16 days of culture in the presence of imatinib mesylate showed concentration-dependent reduced expression levels of CD1a and costimulatory molecules such as CD80 and CD40. Furthermore, exposure to imatinib mesylate inhibited the induction of primary cytotoxic T-lymphocyte (CTL) responses. The inhibitory effects of imatinib mesylate were accompanied by down-regulation of nuclear localized RelB protein. Our results demonstrate that imatinib mesylate can act on normal hematopoietic cells and inhibits the differentiation and function of DCs, which is in part mediated via the nuclear factor κB signal transduction pathway.

Molecular pathogenesis of chronic myeloid leukaemia.

The abnormal haemopoietic precursor cells of chronic myeloid leukaemia (CML) carry the cytogenetic abnormality [t(9;22)(q34;q11)]--a reciprocal translocation that results in the expression of a chimaeric protein derived from the fused BCR and ABL genes. This Bcr-Abl protein tyrosine kinase mediates an array of effects on signal transduction pathways affecting cell survival, proliferation, adhesion and genetic stability. The end-result of these abnormal signalling processes is a bi- or triphasic clinical disease. Initially, CML is characterised by the presence of an excess of myeloid progenitor cells and their mature progeny. This chronic phase of CML is followed, either directly or with an intervening 'accelerated phase', by a stage where primitive blast cells predominate (acute transformation). This review discusses the role of Bcr-Abl-mediated signalling events in cellular transformation, genetic instability and disease progression in CML, and describes current developments in CML treatment using a Bcr-Abl inhibitor.

The effects of STI571 on antigen presentation of dendritic cells generated from patients with chronic myelogenous leukemia.

Chronic myelogenous leukemia is caused by the acquisition of the reciprocal (9;22)(q34;q11) chromosomal translocation in hematopoietic stem cells. The fusion protein showed higher and aberrant tyrosine kinase activity. The inhibition of the tyrosine kinase activity of the protein represents a specific therapeutic strategy for bcr/abl-expressing leukemias. STI571 is a compound of the 2-phenylaminopyrimidine class that selectively inhibits the tyrosine kinase activity of the Abl protein tyrosine kinase. In this study, we evaluated the effects of STI571 on antigen presentation of dendritic cells generated from the patients with CML. The data showed that by the addition of STI571 the dendritic cells derived from CML clone showed an increased expression of CD1a, CD83, CD80 and CD86 by flow cytometry analysis and showed more intense abilities of allogeneic antigen presentation by mixed leukocyte culture, compared with the control cells without STI571. Our results suggested that STI571 not only has a direct cytotoxic effect on bcr-abl gene rearranged cells but also an indirect effect associated with increased anti-leukemic immunological function due to an intensified antigen presentation.

Role of constitutively activated protein tyrosine kinases in malignant myeloproliferative disorders: an update.

Modern molecular technology helped identify more than 10 protein tyrosine kinases related to myeloid malignancies, which allowed the development of small molecule inhibitors targeting deregulated protein tyrosine kinase activity. Protein tyrosine kinase deregulation can occur as a consequence of fusion gene formation because of chromosomal translocations, or as distinct gain-of-function point mutations. Although the tyrosine kinase inhibitor imatinib mesylate (Gleevec) targeting the ABL protein tyrosine kinase has revolutionized current chronic myeloid leukemia therapy, it became rapidly evident that overcoming the multiple cellular resistance mechanisms will be very challenging. To develop efficient therapeutic alternatives, one must understand the complex signal transduction mechanisms involved in transformation by deregulated protein tyrosine kinases. This article reviews the most recently identified molecular mechanisms involved in cell transformation by the BCR/ABL protein tyrosine kinase fusion and presents new members of the increasing family of deregulated protein tyrosine kinases involved in myeloproliferative disorders. In addition, the article discusses new, promising small molecule protein tyrosine kinase inhibitors and the molecular mechanism that may lead to resistance to these drugs. Finally, the article highlights putative alternative strategies that could be used to block signal transduction pathways of deregulated protein tyrosine kinase activity.