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Biomedical subjects

J D Griffin

Publications and source records attributed to J D Griffin.

At least 19 recordsLinked to original sources
PubMed2006

AMN107 (nilotinib): a novel and selective inhibitor of BCR-ABL.

Chronic myelogenous leukaemia (CML) and Philadelphia chromosome positive (Ph+) acute lymphoblastic leukaemia (ALL) are caused by the BCR-ABL oncogene. Imatinib inhibits the tyrosine kinase activity of the BCR-ABL protein and is an effective, frontline therapy for chronic-phase CML. However, accelerated or blast-crisis phase CML patients and Ph+ ALL patients often relapse due to drug resistance resulting from the emergence of imatinib-resistant point mutations within the BCR-ABL tyrosine kinase domain. This has stimulated the development of new kinase inhibitors that are able to over-ride resistance to imatinib. The novel, selective BCR-ABL inhibitor, AMN107, was designed to fit into the ATP-binding site of the BCR-ABL protein with higher affinity than imatinib. In addition to being more potent than imatinib (IC50< 30 nM) against wild-type BCR-ABL, AMN107 is also significantly active against 32/33 imatinib-resistant BCR-ABL mutants. In preclinical studies, AMN107 demonstrated activity in vitro and in vivo against wild-type and imatinib-resistant BCR-ABL-expressing cells. In phase I/II clinical trials, AMN107 has produced haematological and cytogenetic responses in CML patients, who either did not initially respond to imatinib or developed imatinib resistance. Dasatinib (BMS-354825), which inhibits Abl and Src family kinases, is another promising new clinical candidate for CML that has shown good efficacy in CML patients. In this review, the early characterisation and development of AMN107 is discussed, as is the current status of AMN107 in clinical trials for imatinib-resistant CML and Ph+ ALL. Future trends investigating prediction of mechanisms of resistance to AMN107, and how and where AMN107 is expected to fit into the overall picture for treatment of early-phase CML and imatinib-refractory and late-stage disease are discussed.

Animals↗
PubMed2005

Signal transduction and cellular functions of the TEL/ARG oncoprotein.

The TEL/ARG oncogene is formed by t(1;12)(q25;p13) reciprocal translocation and is associated with human leukemia. We have previously demonstrated that the expression of TEL/ARG in Ba/F3 cells results in prolonged viability and hyper-responsiveness to IL-3. To determine the molecular mechanisms, a series of mutants of TEL/ARG were generated, and each cDNA was expressed in Ba/F3 or CHO cells. The PNT domain in TEL and K317 in ARG were essential for both signaling and biological effects. The SH3 domain in ARG was required for hyper-responsiveness to IL-3, but not for prolonged viability. The opposite was true for the SH2 domain in ARG. Mutation of Y314 in TEL, a putative GRB2-binding site, led to reduced viability, and loss of hyper-responsiveness to IL-3. All biological functions were profoundly impaired with deletion of the C-terminus in ARG, despite maintaining high levels of its kinase activity. When expressed in CHO cells, wild-type TEL/ARG induced the formation of fillopodia, in a fashion dependent on the C-terminal portion and intact kinase activity. Thus, these results suggest several critical domains within TEL/ARG necessary for function, and indicate that the signaling pathways necessary for viability, growth factor hyper-responsiveness and cytoskeletal reorganization are likely to be separate.

Actin Cytoskeleton↗
PubMed2004

Modern diagnostics in chronic myeloproliferative diseases (CMPDs).

According to the new WHO classification a group of chronic myeloproliferative diseases (CMPDs) were defined: chronic myeloid leukemia (CML), chronic neutrophilic leukemia (CNL), chronic eosinophilic leukemia and hypereosinophilic syndrome (CEL/HES), polycythemia vera (PV), chronic idiopathic myelofibrosis (with extramedullary hematopoiesis, CIMF), essential thrombocythemia (ET), and so called CMPD/unclassifiable. As clinical features and laboratory findings differ widely between these diseases several diagnostic approaches are mandatory at diagnosis for classification and are needed also for follow up studies, especially for the measurement of minimal residual disease (MRD). We here outline the laboratory set up at diagnosis and during follow up in CMPDs with specific focus on the respective therapeutical consequences. Only by using a comprehensive diagnostic panel including cytomorphology, cytogenetics, and molecular genetic methods establishing the correct diagnosis, optimizing treatment as well as evaluating treatment response is possible in CMPDs today.

Blood Cell Count↗
PubMed2004

Histone deacetylase inhibitor NVP-LAQ824 has significant activity against myeloid leukemia cells in vitro and in vivo.

NVP-LAQ824 is a novel potent hydroxamic acid-derived histone deacetylase inhibitor that induces apoptosis in nanomolar concentrations in myeloid leukemia cell lines and patient samples. Here we show the activity of NVP-LAQ824 in acute myeloid leukemia cells and BCR/ABL-expressing cells of mouse and human origin, both sensitive and resistant to imatinib mesylate (Gleevec, STI-571). Whereas imatinib inhibited overall cellular tyrosine phosphorylation in Ba/F3.p210 cells, NVP-LAQ824 did not inhibit tyrosine phosphorylation, and did not affect BCR/ABL or ABL protein expression. Neither compound was able to inhibit cellular tyrosine phosphorylation in the imatinib-resistant Ba/F3.p210-T315I cell line. These data taken together suggest that BCR/ABL kinase activity is not a direct target of NVP-LAQ824. Synergy between NVP-LAQ824 and imatinib was demonstrated against BCR/ABL-expressing K562 myeloid leukemia cell lines. In addition, we show that NVP-LAQ824 was well tolerated in vivo in a pre-clinical murine leukemia model, with antileukemia activity resulting in significant prolongation of the survival of mice when treated with NVP-LAQ824 compared to control mice. Taken together, these findings provide the framework for NVP-LAQ824 as a novel therapeutic in myeloid malignancies.

Acetylation↗
PubMed2002

Brain armadillo protein delta-catenin interacts with Abl tyrosine kinase and modulates cellular morphogenesis in response to growth factors.

delta-Catenin associates with adhesive junctions and facilitates cellular morphogenesis (Lu et al., 1999). Here we show that delta-catenin colocalizes with actin filaments and Abl tyrosine kinase in the growth cones of cultured hippocampal neurons. PC12 cells induced to express delta-catenin show accelerated neurite extension upon nerve growth factor (NGF) stimulation. STI571, an Abl family kinase inhibitor, further accentuates these stimulatory effects. delta-Catenin is a potent substrate for Abl in vitro using an immunocomplex assay and most of the Abl-induced tyrosine phosphorylation within cells is present in the N-terminus of delta-catenin. When delta-catenin-expressing epithelial cells are induced to scatter in response to hepatocyte growth factor (HGF), STI571 leads to the rapid redistribution of delta-catenin and changes in cellular morphology. We suggest that delta-catenin is a possible Abl substrate and acts downstream of Abl to orchestrate actin-based cellular morphogenesis.

Actin Cytoskeleton↗
PubMed2001

Synaptic and morphological characteristics of temperature-sensitive and -insensitive rat hypothalamic neurones.

1. Intracellular recordings were made from neurones in rat hypothalamic tissue slices, primarily in the preoptic area and anterior hypothalamus, a thermoregulatory region that integrates central and peripheral thermal information. The present study compared morphologies and local synaptic inputs of warm-sensitive and temperature-insensitive neurones. 2. Warm-sensitive neurones oriented their dendrites perpendicular to the third ventricle, with medial dendrites directed toward the periventricular region and lateral dendrites directed toward the medial forebrain bundle. In contrast, temperature-insensitive neurones generally oriented their dendrites parallel to the third ventricle. 3. Both warm-sensitive and temperature-insensitive neurones displayed excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs). In most cases, EPSP and IPSP frequencies were not affected by temperature changes, suggesting that temperature-insensitive neurones are responsible for most local synapses within this hypothalamic network. 4. Two additional neuronal groups were identified: silent neurones having no spontaneous firing rates and EPSP-driven neurones having action potentials that are primarily dependent on excitatory synaptic input from nearby neurones. Silent neurones had the most extensive dendritic trees, and these branched in all directions. In contrast, EPSP-driven neurones had the fewest dendrites, and usually the dendrites were oriented in only one direction (either medially or laterally), suggesting that these neurones receive more selective synaptic input.

Animals↗
PubMed2001

The role of protein composition in specifying nuclear inclusion formation in polyglutamine disease.

Intracellular inclusions are a unifying feature of polyglutamine (polyQ) neurodegenerative diseases, yet each polyQ disease displays a unique pattern of neuronal degeneration. This implies that the protein context of expanded polyQ plays an important role in establishing selective neurotoxicity. Here, in studies of the spinocerebellar ataxia type 3 disease protein ataxin-3, we demonstrate that the protein sequence surrounding polyQ specifies the constituents of nuclear inclusions (NI) formed by the disease protein. The nuclear proteins cAMP response element-binding protein-binding protein (CBP) and Mastermind-like-1 strongly colocalize only to NI formed by full-length ataxin-3, whereas the splicing factor SC35 colocalizes only to NI formed by a polyQ-containing, carboxyl-terminal fragment of ataxin-3. These differences in NI formation reflect specific protein interactions normally undertaken by ataxin-3, as both normal and mutant full-length ataxin-3 co-immunoprecipitate with CBP and sediment on density gradients as macromolecular complexes. Moreover, normal ataxin-3 represses cAMP response element-binding protein-mediated transcription, indicating a functional consequence of ataxin-3 interactions with CBP. Finally, we show that mutant ataxin-3 forms insoluble intranuclear complexes, or microaggregates, before NI can be detected, implying a precursor-product relationship. These results suggest that protein context-dependent recruitment of nuclear proteins to intranuclear microaggregates, and subsequently to NI, may contribute to selective neurotoxicity in polyQ diseases.

Ataxin-3↗
PubMed2001

STI571 inactivation of the gastrointestinal stromal tumor c-KIT oncoprotein: biological and clinical implications.

Mutations in the c-KIT receptor occur somatically in many sporadic Gastrointestinal Stromal Tumors (GIST), and similar mutations have been identified at the germline level in kindreds with multiple GISTs. These mutations activate the tyrosine kinase activity of c-KIT and induce constitutive signaling. To investigate the function of activated c-KIT in GIST, we established a human GIST cell line, GIST882, which expresses an activating KIT mutation (K642E) in the first part of the cytoplasmic split tyrosine kinase domain. Notably, the K642E substitution is encoded by a homozygous exon 13 missense mutation, and, therefore, GIST882 cells do not express native KIT. GIST882 c-KIT protein is constitutively tyrosine phosphorylated, but tyrosine phosphorylation was rapidly and completely abolished after incubating the cells with the selective tyrosine kinase inhibitor STI571. Furthermore, GIST882 cells evidenced decreased proliferation and the onset of apoptotic cell death after prolonged incubation with STI571. Similar results were obtained after administering STI571 to a primary GIST cell culture that expressed a c-KIT exon 11 juxtamembrane mutation (K558NP). These cell-culture-based studies support an important role for c-KIT signaling in GIST and suggest therapeutic potential for STI571 in patients afflicted by this chemoresistant tumor.

Apoptosis↗
PubMed2001

ARG tyrosine kinase activity is inhibited by STI571.

The tyrosine kinase inhibitor STI571 inhibits BCR/ABL and induces hematologic remission in most patients with chronic myeloid leukemia. In addition to BCR/ABL, STI571 also inhibits v-Abl, TEL/ABL, the native platelet-derived growth factor (PDGF)beta receptor, and c-KIT, but it does not inhibit SRC family kinases, c-FMS, FLT3, the epidermal growth factor receptor, or multiple other tyrosine kinases. ARG is a widely expressed tyrosine kinase that shares substantial sequence identity with c-ABL in the kinase domain and cooperates with ABL to regulate neurulation in the developing mouse embryo. As described here, ARG has recently been implicated in the pathogenesis of leukemia as a fusion partner of TEL. A TEL/ARG fusion was constructed to determine whether ARG can be inhibited by STI571. When expressed in the factor-dependent murine hematopoietic cell line Ba/F3, the TEL/ARG protein was heavily phosphorylated on tyrosine, increased tyrosine phosphorylation of multiple cellular proteins, and induced factor-independent proliferation. The effects of STI571 on Ba/F3 cells transformed with BCR/ABL, TEL/ABL, TEL/PDGFbetaR, or TEL/ARG were then compared. STI571 inhibited tyrosine phosphorylation and cell growth of Ba/F3 cells expressing BCR/ABL, TEL/ABL, TEL/PDGFbetaR, and TEL/ARG with an IC(50) of approximately 0.5 microM in each case, but it had no effect on untransformed Ba/F3 cells growing in IL-3 or on Ba/F3 cells transformed by TEL/JAK2. Culture of TEL/ARG-transfected Ba/F3 cells with IL-3 completely prevented STI571-induced apoptosis in these cells, similar to what has been observed with BCR/ABL- or TEL/ABL-transformed cells. These results indicate that ARG is a target of the small molecule, tyrosine kinase inhibitor STI571.

Adenosine Triphosphate↗
PubMed2001

Mechanisms of transformation by the BCR/ABL oncogene.

The Philadelphia chromosome generates a chimeric oncogene in which the BCR and c-ABL genes are fused. The product of this oncogene, BCR/ABL, has elevated ABL tyrosine kinase activity, relocates to the cytoskeleton, and phosphorylates multiple cellular substrates. BCR/ABL transforms hematopoietic cells and exerts a wide variety of biological effects, including reduction in growth factor dependence, enhanced viability, and altered adhesion of chronic myelocytic leukemia (CML) cells. Elevated tyrosine kinase activity of BCR/ABL is critical for activating downstream signal transduction and for all aspects of transformation. This review will describe mechanisms of transformation by the BCR/ABL oncogene and opportunities for clinical intervention with specific signal transduction inhibitors such as STI-571 in CML.

Benzamides↗
PubMed2001

Phosphatidyl inositol signaling by BCR/ABL: opportunities for drug development.

The t(9;22) translocation associated with chronic myelogenous leukemia (CML) fuses the c-ABL gene on chromosome 9 with the BCR gene on chromosome 22, resulting in the production of one or more of a family of chimeric oncoproteins, p190, p210, or p230 BCR/ABL. These proteins have activated ABL kinase activity and are located in the cytoplasm of CML cells, predominantly in the cytoskeleton. Recent studies have led to the identification of numerous potential substrates for BCR/ABL, including many proteins that normally function in signal transduction pathways downstream from hematopoietic growth factor receptors. BCR/ABL is autophosphorylated on tyrosine residues and attracts a variety of adapter proteins and other signaling proteins, setting up large signaling complexes that ultimately result in growth. viability, and adhesion signals. Using new in vitro and animal model systems, it is now becoming possible to link specific signaling pathways to biological abnormalities in CML cells. Furthermore, the relative importance of some BCR/ABL-activated pathways is becoming clear. In vivo studies in certain lines of transgenic mice suggest that the antiapoptotic effect of Bcr/Abl is more important than previously thought. Our current studies indicate important roles for phosphoinositide 3-kinase/Akt and for STAT molecules. As a result of these more detailed biochemical analyses of BCR/ABL function, new targets for future drug development have been identified.

Animals↗
PubMed2001

Notch receptors and hematopoiesis.

Notch receptors are involved in a variety of cell-fate decisions that affect the development and function of many organs, including hematopoiesis and the immune system. There are four mammalian Notch receptors that have only partially overlapping functions despite sharing similar structures and ligands. The ligands for Notch are transmembrane proteins expressed on adjacent cells, including Jagged and Delta, and it is quite possible that signaling is bidirectional. A large Notch precursor protein is proteolytically cleaved to form the mature cell-surface receptor. Ligand binding induces additional proteolytic events followed by translocation of the intracellular domain to the nucleus. There, Notch interacts with transcription factors such as RBPJ kappa, activating transcription of basic helix-loop-helix genes such as HES1. These in turn regulate expression of tissue-specific transcription factors that influence lineage commitment and other events. In this review, the details of Notch signaling will be discussed, with a focus on what is known about the role of Notch in hematopoiesis.

Animals↗
PubMed2001

Role of the tyrosine phosphatase SHP-1 in K562 cell differentiation.

The erythro-megakaryoblastic leukemia cell line K562 undergoes erythroid or myeloid differentiation in response to treatment with various inducing agents. We observed that expression of the SH2-containing protein tyrosine phosphatase SHP-1 was induced upon exposure of K562 cells to differentiating agents. Under the same conditions, expression of SHP-2, a close relative of SHP-1, and the more distantly related PTP-1 B remained unchanged. Induction of SHP-1 expression correlates with dephosphorylation of a specific and limited set of tyrosyl phosphoproteins, suggesting that dephosphorylation of these proteins may be important for the differentiation process. Importantly, expression of exogenous SHP-1 inhibits K562 proliferation and alters the adhesion properties of these cells, indicating a more differentiated phenotype. Moreover, SHP-1 is found in a complex with both p210 Bcr-Abl and p190 Bcr-Abl, suggesting that it may regulate Bcr-Abl or Bcr-Abl-associated phosphotyrosyl proteins. Our results indicate that induction of SHP-1 expression is important for K562 differentiation in response to various inducers and raise the possibility that functional inactivation of SHP-1 may play a role in progression to blast crisis in chronic myelogenous leukemia.

Cell Adhesion↗
PubMed2001

Mechanisms of resistance imatinib (STI571) in preclinical models and in leukemia patients.

The tyrosine kinase inhibitor imatinib (STI571, Glivec) blocks the activity of the BCR/ABL oncogene and induces hematologic remissions in the majority of patients with chronic myeloid leukemia (CML). Glivec is an aminopyrimidine derivative that interacts with the ATP-binding site within the kinase domain of ABL and several other tyrosine kinases, including c-KIT, PDGF beta receptor, and ARG. The compound is currently in phase III clinical trials. Although patients with chronic phase CML have been found to develop drug resistance only rarely so far, patients in more advanced phases of the leukemia develop resistance frequently. The available information on Glivec resistance will be reviewed.

Animals↗
PubMed2000

BCR/ABL regulates expression of the cyclin-dependent kinase inhibitor p27Kip1 through the phosphatidylinositol 3-Kinase/AKT pathway.

Deregulation of cell cycle checkpoints is an almost universal abnormality in human cancers and is most often due to loss-of-function mutations of tumor suppressor genes such as Rb, p53, or p16(INK4a). In this study, we demonstrate that BCR/ABL inhibits the expression of a key cell cycle inhibitor, p27(Kip1), by signaling through a pathway involving phosphatidylinositol 3-kinase (PI3K). p27(Kip1) is a widely expressed inhibitor of cdk2, an essential cell cycle kinase regulating entry into S phase. We demonstrate that the decrease of p27(Kip1) is directly due to BCR/ABL in hematopoietic cells by two different approaches. First, induction of BCR/ABL by a tetracycline-regulated promoter is associated with a reversible down-regulation of p27(Kip1). Second, inhibition of BCR/ABL kinase activity with the Abl tyrosine kinase inhibitor STI571 rapidly increases p27(Kip1) levels. The PI3K inhibitor LY-294002 blocks the ability of BCR/ABL to induce p27(Kip1) down-regulation and inhibits BCR/ABL-induced entry into S phase. The serine/threonine kinase AKT/protein kinase B is a known downstream target of PI3K. Transient expression of an activated mutant of AKT was found to decrease expression of p27(Kip1), even when PI3K was inhibited by LY-294002. The mechanism of p27(Kip1) regulation is primarily related to protein stability, since inhibition of proteasome activity increased p27(Kip1) levels in BCR/ABL-transformed cells, whereas very little change in p27 transcription was found. Overall, these data are consistent with a model in which BCR/ABL suppresses p27(Kip1) protein levels through PI3K/AKT, leading to accelerated entry into S phase. This activity is likely to explain in part previous studies showing that activation of PI3K was required for optimum transformation of hematopoietic cells by BCR/ABL in vitro and in vivo.

Animals↗