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Imatinib (STI571 or CGP57148B) is an innovative treatment for tumours with a constitutively activated form of c-ABL, c-KIT, or PDGFR. Such tumours include Philadelphia-chromosome-positive (Ph-positive) leukaemias, gastrointestinal stromal tumours, and PDGFR-positive leukaemias. Diseases such as primary hypereosinophilia and dermatofibrosarcoma protuberans also seem to respond to imatinib. Clinical trials assessing the therapeutic effects of imatinib have shown that the drug is highly effective with few associated side-effects, achieving durable cytogenetic responses in many patients with chronic-phase BCR-ABL-positive leukaemias. However, the emergence of resistance, particularly in patients with acute leukaemias, has prompted intense research, and many are concerned about the future prospects for imatinib. The resistance has been found in patients with acute-phase disease, but may also occur in patients with chronic-phase disease. Two cellular mechanisms for resistance to imatinib have been identified: amplification of BCR-ABL gene and mutations in the catalytic domain of the protein. In addition, suboptimum inhibition of BCR-ABL in vivo could contribute to the selection of resistant cells. We have summarised all currently available data on resistance to imatinib, both published and unpublished, including the mechanisms of resistance identified so far, and their clinical relevance to the different forms of Ph-positive leukaemias is discussed. Furthermore, we discuss strategies to overcome or prevent the development of resistance. [ABSTRACT FROM AUTHOR]

Background: Resistance to imatinib is an important clinical issue in the treatment of Philadelphia chromosome-positive leukemias which is being tackled by the development of new, more potent drugs, such as the dual Src/Abl tyrosine kinase inhibitors dasatinib and bosutinib and the imatinib analog nilotinib. In the current study we describe the design, synthesis and biological properties of an imatinib analog with a chlorine-substituted benzamide, namely compound 584 (cmp-584)., Design and Methods: To increase the potency, we rationally designed cmp-584, a compound with enhanced shape complementarity with the kinase domain of Abl. cmp-584 was synthesized and characterized in vitro against a panel of 67 serine/threonine and tyrosine kinases using radioactive and enzyme-linked immunosorbent kinase assays. We studied inhibitory cellular activity using Bcr/Abl-positive human cell lines, murine transfectants in proliferation experiments, and a murine xenotrans-planted model. Kinase assays on isolated Bcr/Abl protein were also performed. Finally, we used a wash-out approach on whole cells to study the binding kinetics of the inhibitor., Results: cmp-584 showed potent anti-Abl activity both on recombinant protein (IC(50): 8 nM) and in cell-based assays (IC(50): 0.1-10 nM). The drug maintained inhibitory activity against platelet-derived growth factor receptors and c-KIT and was also active against Lyn (IC(50): 301 nM). No other kinase of the panel was inhibited at nanomolar doses. cmp-584 was 20- to 300-fold more active than imatinib in cells. This superior activity was evident in intact cells, in which full-length Bcr-Abl is present. In vivo experiments confirmed the activity of cmp-584. Wash-out experiments showed that short exposure to the drug impaired cell proliferation and Bcr-Abl phosphorylation for a substantially longer period of time than imatinib., Conclusions: The present results suggest a slower off-rate (dissociation rate) of cmp-584 compared to imatinib as an explanation for the increased cellular activity of the former.

NPM/ALK is an oncogenic fusion protein expressed in approximately 50% of anaplastic large cell lymphoma cases. It derives from the t(2;5)(p23;q35) chromosomal translocation that fuses the catalytic domain of the tyrosine kinase, anaplastic lymphoma kinase (ALK), with the dimerization domain of the ubiquitously expressed nucleophosmin (NPM) protein. Dimerization of the ALK kinase domain leads to its autophosphorylation and constitutive activation. Activated NPM/ALK stimulates downstream survival and proliferation signaling pathways leading to malignant transformation. Herein, we investigated the molecular mechanisms of autoactivation of the catalytic domain of ALK. Because kinases are typically regulated by autophosphorylation of their activation loops, we systematically mutated (Tyr --> Phe) three potential autophosphorylation sites contained in the "YXXXYY" motif of the ALK activation loop, and determined the effect of these mutations on the catalytic activity and biological function of NPM/ALK. We observed that mutation of both the second and third tyrosine residues (YFF mutant) did not affect the kinase activity or transforming ability of NPM/ALK. In contrast, mutation of the first and second (FFY), first and third (FYF), or all three (FFF) tyrosine residues impaired both kinase activity and transforming ability of NPM/ALK. Furthermore, a DFF mutant, in which the aspartic residue introduces a negative charge similar to a phosphorylated tyrosine, possessed catalytic activity similar to the YFF mutant. Together, our findings indicate that phosphorylation of the first tyrosine of the YXXXYY motif is necessary for the autoactivation of the ALK kinase domain and the transforming activity of NPM/ALK.

A new mechanistic principle for reporting the phosphorylation of tyrosine is described, which should prove applicable to even the most fastidious of protein tyrosine kinases, as demonstrated by the acquisition of a fluorescent sensor for the extraordinarily demanding anaplastic lymphoma kinase.

Deregulated activation of protein tyrosine kinases (PTKs) is a frequent event underlying malignant transformation in many types of cancer. The formation of oncogenic fusion tyrosine kinases (FTKs) resulting from genomic rearrangements, represents a common mechanism by which kinases escape the strict controls that normally regulate their expression and activation. FTKs are typically composed of an N-terminal dimerisation domain, provided by the fusion partner protein, fused to the kinase domain of receptor or non-receptor tyrosine kinases (non-RTKs). Since FTKs do not contain extracellular domains, they share many characteristics with non-RTKs in terms of their properties and approaches for therapeutic targeting. FTKs are cytoplasmic or sometimes nuclear proteins, depending on the normal distribution of their fusion partner. FTKs no longer respond to ligand and are instead constitutively activated by dimerisation induced by the fusion partner. Unlike RTKs, FTKs cannot be targeted by therapeutic antibodies, instead they require agents that can cross the cell membrane as with non-RTKs. Here we review the PTKs known to be expressed as FTKs in cancer and the strategies for molecularly targeting these FTKs in anti-cancer therapy.

The oncogenic fusion tyrosine kinase nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) induces cellular transformation in anaplastic large-cell lymphomas (ALCLs) carrying the t(2;5) chromosomal translocation. Protein-protein interactions involving NPM/ALK are important for the activation of downstream signaling pathways. This study was aimed at identifying novel NPM/ALK-binding proteins that might contribute to its oncogenic transformation. Using a proteomic approach, several RNA/DNA-binding proteins were found to coimmunoprecipitate with NPM/ALK, including the multifunctional polypyrimidine tract binding proteinassociated splicing factor (PSF). The interaction between NPM/ALK and PSF was dependent on an active ALK kinase domain and PSF was found to be tyrosine-phosphorylated in NPM/ALK-expressing cell lines and in primary ALK(+) ALCL samples. Furthermore, PSF was shown to be a direct substrate of purified ALK kinase domain in vitro, and PSF Tyr293 was identified as the site of phosphorylation. Y293F PSF was not phosphorylated by NPM/ALK and was not delocalized in NPM/ALK(+) cells. The expression of ALK fusion proteins induced delocalization of PSF from the nucleus to the cytoplasm and forced overexpression of PSF-inhibited proliferation and induced apoptosis in cells expressing NPM/ALK. PSF phosphorylation also increased its binding to RNA and decreased the PSF-mediated suppression of GAGE6 expression. These results identify PSF as a novel NPM/ALK-binding protein and substrate, and suggest that PSF function may be perturbed in NPM/ALK-transformed cells.

Anaplastic lymphoma kinase (ALK) is a valid target for anticancer therapy; however, potent ALK inhibitors suitable for clinical use are lacking. Because the majority of described kinase inhibitors bind in the ATP pocket of the kinase domain, we have characterized this pocket in ALK using site-directed mutagenesis, inhibition studies, and molecular modeling. Mutation of the gatekeeper residue, a key structural determinant influencing inhibitor binding, rendered the fusion protein, NPM/ALK, sensitive to inhibition by SKI-606 in the nanomolar range, while PD173955 inhibited the NPM/ALK mutant at micromolar concentrations. In contrast, both wild type and mutant NPM/ALK were insensitive to imatinib. Computer modeling indicated that docking solutions obtained with a homology model representing the intermediate conformation of the ALK kinase domain reflected closely experimental data. The good agreement between experimental and virtual results indicate that the ALK molecular models described here are useful tools for the rational design of ALK selective inhibitors. In addition, 4-phenylamino-quinoline compounds may have potential as templates for ALK inhibitors.

The anaplastic lymphoma kinase (ALK), whose constitutively active fusion proteins are responsible for 5-10% of non-Hodgkin's lymphomas, shares with the other members of the insulin receptor kinase (IRK) subfamily an activation loop (A-loop) with the triple tyrosine motif Y-x-x-x-Y-Y. However, the amino acid sequence of the ALK A-loop differs significantly from the sequences of both the IRK A-loop and the consensus A-loop for this kinase subfamily. A major difference is the presence of a unique "RAS" triplet between the first and second tyrosines of the ALK A-loop, which in IRK is replaced by "ETD". Here we show that a peptide reproducing the A-loop of ALK is readily phosphorylated by ALK, while a homologous IRK A-loop peptide is not unless its "ETD" triplet is substituted by "RAS". Phosphorylation occurs almost exclusively at the first tyrosine of the Y-x-x-x-Y-Y motif, as judged by Edman analysis of the phosphoradiolabeled product. Consequently, a peptide in which the first tyrosine had been replaced by phenylalanine (FYY) was almost unaffected by ALK. In contrast, a peptide in which the second and third tyrosines had been replaced by phenylalanine (YFF) was phosphorylated more rapidly than the parent peptide (YYY). A number of substitutions in the YFF peptide outlined the importance of Ile and Arg at positions n - 1 and n + 6 in addition to the central triplet, to ensure efficient phosphorylation by ALK. Such a peculiar substrate specificity allows the specific monitoring of ALK activity in crude extracts of NPM-ALK positive cells, using the YFF peptide, which is only marginally phosphorylated by a number of other tyrosine kinases.

A crucial issue in the development of molecularly-targeted anticancer therapies is the identification of appropriate molecules whose targeting would result in tumour regression with a minimal level of systemic toxicity. Anaplastic lymphoma kinase (ALK) is a transmembrane receptor tyrosine kinase, normally expressed at low levels in the nervous system. As a consequence of chromosomal translocations involving the alk gene (2p23), ALK is also aberrantly expressed and constitutively activated in approximately 60% of CD30+ anaplastic large cell lymphomas (ALCLs). Due to the selective overexpression of ALK in tumour cells, its direct involvement in the process of malignant transformation and its frequent expression in ALCL patients, the authors recognise ALK as a suitable candidate for the development of molecularly targeted strategies for the therapeutic treatment of ALK-positive lymphomas. Strategies targeting ALK directly or indirectly via the inhibition of the protein networks responsible for ALK oncogenic signalling are discussed.

Deregulated apoptosis is a common finding in tumorigenesis. The oncogenic tyrosine kinase nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) delivers a strong survival signal in anaplastic large cell lymphomas (ALCLs). Although NPM/ALK activates multiple antiapoptotic pathways, the biologic relevance and therapeutic potential of more downstream apoptotic effectors are mostly unknown. In this report, the NPM/ALK-mediated induction of Bcl-XL (but not of Bcl-2) was identified in human ALCL-derived cells. NPM/ALK kinase activity was required to promote Bcl-XL expression and its protective effect on mitochondrial homeostasis. Down-regulation of Bcl-XL significantly reduced the antiapoptotic potential of NPM/ALK in both transformed murine Ba/F3 pro-B cells and human ALCL-derived KARPAS-299 cells. To elucidate the role of Bcl-XL in vivo, Ba/F3-NPM/ALK+ cells expressing a doxycycline (Dox)-inducible Bcl-XL antisense transgene (pTet-ON) were injected into nude mice. Doxycycline administration prevented a fatal systemic disease in 15 of 15 intravenously injected mice and the appearance of subcutaneous tumor xenografts in 9 of 12 mice; in vivo down-regulation of Bcl-XL was also documented. Our results show a pivotal role for Bcl-XL in ALK-mediated oncogenicity; a single protein placed downstream of a known oncogene can be crucial for the survival of neoplastic cells both in vitro and in vivo. Bcl-XL deserves further investigation as a possible therapeutic target in ALK+ ALCLs.

Background and Objectives: Chronic myelomonocytic leukemia (CMML) is a myelodysplastic syndrome that has been associated with the expression of platelet-derived growth factor b receptor (PDGFRbeta) fusion proteins, namely TEL/PDGFRbeta. These fusion proteins possess a constitutive PDGFRbeta tyrosine kinase activity, leading to aberrant PDGFRbeta signaling and cellular transformation. The expression of PDGFRbeta fusions in CMML could have therapeutic relevance, as PDGFRb is inhibited by the selective tyrosine kinase inhibitor, imatinib. Here, we investigated the possibility of employing imatinib to treat CMML., Design and Methods: We assessed the effect of imatinib on TEL/PDGFRbeta transformed cells in terms of proliferation, by trypan blue exclusion and 3H-thymidine uptake, and TEL/PDGFRbeta autophosphorylation by anti-phosphotyrosine immunoblotting. TEL/PDGFRbeta expression in mononuclear cells from the peripheral blood of 27 clinically diagnosed CMML patients was determined by reverse transcriptase-polymerase chain reaction., Results: Imatinib potently inhibited the proliferation of TEL/PDGFRbeta transformed cells (IC50=7.5 nM), and TEL/PDGFRbeta kinase activity. However, TEL/PDGFRbeta expression was detected in only 1 of 27 CMML patients (4%, confidence intervals: 0-13%). Additionally, another PDGFRbeta fusion protein, Hip1/PDGFRbeta, had a similarly low incidence in the same samples: 1 of 25 (4%, confidence intervals: 0-14%)., Interpretation and Conclusions: Although imatinib represents an attractive therapeutic agent for neoplasias associated with abnormal PDGFRbeta signaling, the low frequency of the TEL/PDGFRbeta and Hip1/PDGFRbeta fusion proteins in CMML suggests that its application to this disease maybe limited. Detection of PDGFRbeta fusion genes in individual patients is necessary in order to employ this drug rationally in CMML.

Kinetic analysis of dexamethasone-induced apoptosis in the human lymphoblastoid cell line CCRF CEM C7A has revealed a point when cells, morphologically indistinguishable from untreated cells, have irreversibly engaged a program leading to death, measured by a loss of clonogenicity. Since all cells that fail to clone eventually died through apoptosis, measurements of clonogenicity in this system provide an accurate measure of commitment to apoptotic death. Inhibition of caspases by peptide inhibitors blocked proteolysis of endogenous substrates and reduced nuclear condensation yet did not alter either dexamethasone-induced changes in clonogenicity or mitochondrial membrane potential. In contrast to the results with caspase inhibitors, expression of BCL-2 in CCRF CEM C7A cells proved sufficient to block all changes associated with apoptosis, including loss of both clonogenicity and changes in mitochondrial membrane potential. These results demonstrate that commitment to cell death can precede the key biochemical or morphological features of apoptosis by several hours and indicate that separate regulators govern cellular commitment to clonogenic death and the subsequent execution phase characterised as apoptosis.

Signals transduced through CD40 rescue cells of the Ramos-Burkitt lymphoma (Ramos-BL) B cell line from surface immunoglobulin M (sIgM)-triggered growth arrest and apoptosis. This study investigates whether protein tyrosine kinase (PTK) activity and tyrosine phosphorylation on p95(vav) and on the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3 kinase) play a role in the regulation of Ramos-BL B cell survival. The PTK inhibitor herbimycin A (HA) triggers significant growth arrest prior to apoptosis from the G1-phase of the cell cycle, indicating that tyrosine phosphorylation of key proteins is critical for Ramos-BL cell cycle progression and survival. Indeed, signals transduced through CD40 fail to rescue Ramos-BL B cells from HA-triggered growth arrest and apoptosis. Since Vav and PI3 kinase are intimately involved in the regulation of cellular growth, their tyrosine phosphorylation status was determined in unstimulated and anti-IgM- and anti-CD40-treated Ramos-BL B cells: Vav and p85 are devoid of tyrosine-phosphorylated epitopes in control cells whereas p85, but not Vav, is significantly phosphorylated following ligation of sIgM and anti-CD40 triggers tyrosine phosphorylation on both proteins. Thus, tyrosine-phosphorylated Vav may be a critical effector of CD40-mediated survival. As tyrosine-phosphorylated PI3 kinase is common to both sIgM-triggered death and CD40-triggered survival pathways, its lipid kinase activity was correlated with tyrosine phosphorylation on p85: Ramos-BL B cells exhibit high basal levels of PI3 kinase activity, determined by immunoprecipitation with anti-p85 and 32P incorporation into phosphatidylinositol, which is not significantly affected by stimulation with anti-IgM but which is elevated by 36 +/- 2.9% following ligation of CD40. Thus, tyrosine phosphorylation on p85 correlates with the CD40-triggered increase in PI3 kinase activity but not with basal levels nor with sIgM-triggered levels of enzymatic activity: these data suggest the presence of different PI3 kinase isoforms or the existence of multiple regulatory pathways for the same PI3 kinase isotype in Ramos-BL B cells.