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J Duyster

Publications and source records attributed to J Duyster.

At least 19 recordsLinked to original sources
PubMed2006

The Bcr-Abl mutations T315I and Y253H do not confer a growth advantage in the absence of imatinib.

Mutations in the Bcr-Abl kinase domain are a frequent cause of imatinib resistance in patients with advanced CML or Ph+ ALL. The impact of these mutations on the overall oncogenic potential of Bcr-Abl and on the clinical course of the disease in the absence of imatinib is presently unclear. In this study, we analyzed the effects of the Bcr-Abl P-loop mutation Y253H and the highly imatinib resistant T315I mutation on kinase activity in vitro and transforming efficiency of Bcr-Abl in vitro and in vivo. Immunoprecipitated Bcr-AblY253H and Bcr-AblT315I proteins displayed similar kinase activities and substrate phosphorylation patterns as Bcr-Abl wildtype. We directly compared the proliferative capacity of mutant to wildtype Bcr-Abl in primary BM cells in vitro and in a murine transplantation model of CML by using a competitive repopulation assay. The results implicate that in the absence of imatinib, there is no growth advantage for cells carrying Bcr-AblT315I or Bcr-AblY253H compared to Bcr-Ablwt, whereas imatinib treatment clearly selects for leukemic cells expressing mutant Bcr-Abl both in vitro and in vivo. Thus, the analysed Bcr-Abl mutants confer imatinib resistance, but do not induce a growth advantage in the absence of imatinib.

Amino Acid Substitution↗
PubMed2005

Phase II study of weekly oxaliplatin plus infusional fluorouracil and folinic acid (FUFOX regimen) as first-line treatment in metastatic gastric cancer.

Oxaliplatin plus fluorouracil/folinic acid (5-FU/FA) every 2 weeks has shown promising activity in advanced gastric cancer. This study assessed the efficacy and safety of weekly oxaliplatin plus 5-FU/FA (FUFOX regimen) in the metastatic setting. Patients with previously untreated metastatic gastric cancer received oxaliplatin (50 mg m(-2)) plus FA (500 mg m(-2), 2-h infusion) followed by 5-FU (2000 mg m(-2), 24-h infusion) given on days 1, 8, 15 and 22 of a 5-week cycle. The primary end point of this multicentre phase II study was the response rate according to RECIST criteria. A total of 48 patients were enrolled. Median age was 62 years and all patients had metastatic disease, with a median number of three involved organs. The most common treatment-related grade 3/4 adverse events were diarrhoea (17%), deep vein thrombosis (15%), neutropenia (8%), nausea (6%), febrile neutropenia (4%), fatigue (4%), anaemia (4%), tumour bleeding (4%), emesis (2%), cardiac ischaemia (2%) and pneumonia (2%). Grade 1/2 sensory neuropathy occurred in 67% of patients but there were no episodes of grade 3 neuropathy. Intent-to-treat analysis showed a response rate of 54% (95% CI, 39-69%), including two complete responses. At a median follow-up of 18.1 months (range 11.2-26.2 months), median survival is 11.4 months (95% CI, 8.0-14.9 months) and the median time to progression is 6.5 months (95% CI, 3.9-9.2 months). The weekly FUFOX regimen is well tolerated and shows notable activity as first-line treatment in metastatic gastric cancer.

Adult↗
PubMed2005

Capecitabine plus docetaxel every 3 weeks in first- and second-line metastatic oesophageal cancer: final results of a phase II trial.

Capecitabine and docetaxel have single-agent activity in upper gastrointestinal tumours, and have together demonstrated preclinical synergy and a survival benefit in breast cancer, and high response rates in first-line metastatic gastric cancer. This trial assessed the efficacy, safety and feasibility of capecitabine in combination with docetaxel in patients with metastatic oesophageal cancer. In all, 24 patients with advanced disease (17 squamous cell carcinoma and seven adenocarcinoma) received oral capecitabine (1000 mg m(-2) twice daily on days 1-14) plus intravenous docetaxel (75 mg m(-2) on day 1) every 3 weeks as first- (n = 16) or second-line (n = 8) therapy. Patients received a median of four cycles of treatment (range, 0-6). The median follow-up is 16.5 months (range, 7.9-21.4 months). Intent-to-treat efficacy analysis showed an overall response rate of 46%. Of the 11 responders (one complete and 10 partial), nine of 16 (56%) received first-line and two of eight (25%) received second-line therapy. The median time to progression was 6.1 months (95% confidence interval (CI), 4.5-7.7 months). The median survival was 15.8 months (95% CI, 7.8-23.9 months). Severe adverse events (grade 3/4) reported were: neutropenia (42%, including febrile neutropenia 8%), hand-foot syndrome (29%), diarrhoea (13%), sensory neuropathy (13%), anaemia (8%) and fatigue (8%). Capecitabine plus docetaxel has a manageable adverse event profile and very promising activity in metastatic oesophageal cancer, at least comparable to other doublet regimens. Therefore, the combination merits further investigation in this setting.

Adenocarcinoma↗
PubMed2005

Compensatory PI3-kinase/Akt/mTor activation regulates imatinib resistance development.

BCR/ABL-kinase mutations frequently mediate clinical resistance to the selective tyrosine kinase inhibitor Imatinib mesylate (IM, Gleevec). However, mechanisms that promote survival of BCR/ABL-positive cells before clinically overt IM resistance occurs have poorly been defined so far. Here, we demonstrate that IM-treatment activated the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTor)-pathway in BCR/ABL-positive LAMA-cells and primary leukemia cells in vitro, as well as in a chronic phase CML patient in vivo. In fact, PI3K/Akt-activation critically mediated survival during the early phase of IM resistance development before manifestation of BCR/ABL-dependent strong IM resistance such as through a kinase mutation. Accordingly, inhibition of IM-induced Akt activation using mTor inhibitors and Akt-specific siRNA effectively antagonized development of incipient IM-resistance in vitro. In contrast, IM-resistant chronic myeloid leukemia (CML) patients with BCR/ABL kinase mutations (n=15), and IM-refractory BCR/ABL-positive acute lymphatic leukemia patients (n=2) displayed inconsistent and kinase mutation-independent autonomous patterns of Akt-pathway activation, and mTor-inhibition overcame IM resistance only if Akt was strongly activated. Together, an IM-induced compensatory Akt/mTor activation may represent a novel mechanism for the persistence of BCR/ABL-positive cells in IM-treated patients. Treatment with mTor inhibitors may thus be particularly effective in IM-sensitive patients, whereas Akt-pathway activation variably contributes to clinically overt IM resistance.

Antineoplastic Agents↗
PubMed2004

[An alternative Abl-kinase inhibitor overcomes imatinib resistance mutations of Bcr-Abl oncogenes].

BACKGROUND AND OBJECTIVE: The tyrosinkinase inhibitor Imatinib is active in Philadelphia-positive (Ph+) leukemia. Mutations within the Bcr-Abl kinase domain represent the major cause for clinical resistance toward imatinib. We aimed to examine, whether the alternative Abl Kinaseinhibitor SKI-DV 2 - 43 may be capable of suppressing the growth of cells expressing mutant forms of Bcr-Abl. METHODS: The proliferation of cells expressing wild-type and mutant forms of Bcr-Abl was measured in the presence of imatinib or the pyrido-pyrimidine SKI-DV 2 - 43. RESULTS: The growth of a cell line expressing wild-type Bcr-Abl was suppressed with higher potency in the presence of SKI-DV 2 - 43 when compared to imatinib. Moreover, SKI-DV 2 - 43 effectively suppressed mutant forms of Bcr-Abl that cause imatinib resistance in patients. CONCLUSION: Therefore, alternative Abl kinase inhibitors might play an important role in the future therapy of Philadelphia-positive leukemias.

Animals↗
PubMed2003

Resistance of Philadelphia-chromosome positive leukemia towards the kinase inhibitor imatinib (STI571, Glivec): a targeted oncoprotein strikes back.

Cancer research within the last decades elucidated signaling pathways and identified genes and proteins that lead or contribute to malignant transformation of a cell. Discovery of the Bcr-Abl oncoprotein as the molecular abnormality causing chronic myeloid leukemia (CML) paved the way for the development of a targeted anticancer therapy. The substantial activity of imatinib mesylate (STI571, Glivec) in CML and Philadelphia (Ph)-chromosome positive acute lymphoblastic leukemia (Ph+ ALL) changed the therapeutic approach to Ph+ leukemia and rang the bell for a new era of anticancer treatment. However, when the phenomenon of relapse occurred despite continued imatinib treatment, we had to learn the lesson that imatinib can select for a resistant disease clone. If such a clone still depends on Bcr-Abl, it either carries a BCR-ABL point mutation that prevents binding of the drug or expresses the fusion protein at high levels. Alternatively, leukemia cells that harbor secondary genetic alterations resulting in Bcr-Abl-independent proliferation are selected for their growth advantage in the presence of imatinib. Point mutations in the BCR-ABL kinase domain prevent binding of imatinib but still allow binding of ATP, thus retaining Bcr-Abl kinase activity. Mutated BCR-ABL is frequently detected in cases of imatinib-resistant Ph+ leukemia and therefore represents the main challenge for the investigation of alternative strategies to either overcome resistance or to prevent the emergence of a resistant leukemic clone.

Antineoplastic Agents↗
PubMed2003

Phosphorylation of tyrosine 393 in the kinase domain of Bcr-Abl influences the sensitivity towards imatinib in vivo.

The Bcr-Abl fusion protein arising through the t(9;22)(q34;q11) reciprocal translocation is the causative agent in chronic myeloid leukemia and a subset of acute lymphocytic leukemia. Imatinib mesylate is a specific inhibitor of the Bcr-Abl kinase and has shown promising results in clinical studies. The structural relation between the Bcr-Abl oncogene and the tyrosine kinase inhibitor imatinib has recently been elucidated by an elegant crystal structure analysis, emphasizing the importance of dephosphorylated tyrosine 393 (Tyr393) in Bcr-Abl for access of the inhibitor to the kinase domain. By mutating this tyrosine to phenylalanine and thereby mimicking a constitutively dephosphorylated state, we now show that Ba/F3 cells transformed by this mutant demonstrate an increased sensitivity towards imatinib in vivo. This effect is not due to an impaired kinase activity of Bcr-Abl Y393F, since a synthetic substrate is phosphorylated with similar kinetics. Treatment of Ba/F3 cells transfected with Bcr-Abl wild type with a phosphatase inhibitor diminished the effect of imatinib, but did not influence the growth of Ba/F3 cells transfected with Bcr-AblY393F. The results support the findings of the crystal structure and indicate that Tyr393 indeed plays a significant role for the sensitivity of Bcr-Abl towards imatinib in vivo. These data implicate the regulation of Tyr393 phosphorylation as a potential mechanism of imatinib resistance.

Antineoplastic Agents↗
PubMed2002

Cell cycle progression of chronic lymphocytic leukemia cells is controlled by cyclin D2, cyclin D3, cyclin-dependent kinase (cdk) 4 and the cdk inhibitor p27.

B-CLL cells are arrested in G0/early G1 phase of the cell cycle and are characterized by a marked hyporesponsiveness towards a variety of polyclonal B cell activators. We have previously demonstrated that costimulation with CpG-ODN and IL-2 can overcome this proliferative defect. Cyclin D3 is the principal D-type cyclin which mediates G1 progression in normal B cells, but in B-CLL cells both cyclin D2 and cyclin D3, were strongly upregulated upon stimulation. Both cyclins were associated with cdk4 but not with cdk6, which is the catalytic partner of D-type cyclins in normal B cells. Moreover, immune complexes consisting of cyclin D2 and cdk4 or cyclin D3 and cdk4 were both functional and phosphorylated the RB protein in vitro. The cell cycle inhibitor p27 plays a pivotal role in cell cycle progression of B lymphocytes and has been shown to be overexpressed in B-CLL cells. P27 was rapidly downregulated in B-CLL cells even when stimulated with a non-CpG-ODN or IL-2 alone, while only moderate regulation could be observed in normal B cells. Taken together, our findings demonstrate that regulation of early cell cycle progression differs between B-CLL cells and normal B cells. These findings do not only contribute to the understanding of B-CLL pathophysiology, but might ultimately lead to the identification of new therapeutic targets.

Apoptosis↗
PubMed2002

Murine stromal cells producing hyper-interleukin-6 and Flt3 ligand support expansion of early human hematopoietic progenitor cells without need of exogenous growth factors.

Expansion of primitive hematopoietic progenitor cells (HPC) is a major challenge in stem cell biology. Stimulation by growth factors (GF) is essential for proliferation of HPC, while the role of stromal cell coculture for maintenance of progenitor/stem cell potential is unclear. We evaluated the potential of a murine stromal cell layer providing hematopoietic GF to support expansion of human CD34(+) cells. Murine MS-5 cells were transfected with the cDNA encoding huFlt3 ligand and the interleukin6/sinterleukin-6R fusion protein hyper-IL-6. Expansion of CFC and week6 CAFC was at least as efficient in transfected clones compared to control cocultures supported with exogenous GF. Cell numbers reached 17.5- to 62.3- (day 14) and 17.4- to 92.4-fold (day 21) of input cells. Expansion of CFU-GM/Mix was 4.0- to 12.8-fold (day 14) and 4.9- to 11.7-fold (day 21). Primitive week6 CAFC were expanded up to 6.5-fold (day 14) and 6.2-fold (day 21) without exogenous GF. When direct contact of HPC and stromal cells was inhibited, a loss of CFC and much more of CAFC potential was observed with unaffected overall cell proliferation. Here, we show the generation of GF producing murine stromal cells which efficiently support early hematopoiesis without exogenous GF. Direct stromal cell-HPC contact is advantageous for maintenance of differentiation potential.

Animals↗
PubMed2002

Effects of imatinib on bone marrow engraftment in syngeneic mice.

Chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemias arise from the genetic reciprocal translocation t(9;22), forming the BCR-ABL fusion gene. These lead to the expression of the constitutively active tyrosine kinase BCR-ABL, which is the causative oncogene for these leukemias. Allogeneic bone marrow transplantation (BMT) or stem cell transplantation (SCT) is currently considered the only curative treatment for chronic myeloid leukemia (CML). Recently, the selective tyrosine kinase inhibitor imatinib mesylate (Glivec, formerly STI-571) has been shown to induce durable hematologic and major cytogenetic responses in a high percentage of patients with chronic phase CML. In patients with advanced disease remissions are transient and most patients relapse despite continued imatinib treatment. Some of these patients go on to receive allogeneic BMT or SCT, during which administration of imatinib is usually discontinued as it is believed to interfere with bone marrow engraftment. In this study, we examined the effect of imatinib on hematopoietic engraftment in a syngeneic mouse model. We found that imatinib has no significant influence on hematopoietic recovery in lethally irradiated mice in vivo. Thus, our results suggest that continued administration of imatinib in the course of BMT or SCT may be a feasible therapeutic regimen.

Animals↗
PubMed2001

Expression of the oncogenic NPM-ALK chimeric protein in human lymphoid T-cells inhibits drug-induced, but not Fas-induced apoptosis.

Anaplastic large cell lymphomas (ALCLs) are frequently associated with the t(2;5)(p23;q35) translocation, leading to the expression of NPM-ALK, a fusion protein linking nucleophosmin and anaplastic lymphoma kinase, a receptor tyrosine kinase. In ALCLs, dimerization of NPM-ALK leads to constitutive autophosphorylation and activation of the kinase, necessary for NPM-ALK oncogenicity. To investigate whether NPM-ALK, like other oncogenic tyrosine kinases, can inhibit drug-induced apoptosis, we permanently transfected NPM-ALK into Jurkat T-cells. As in ALCLs, NPM-ALK was expressed as a constitutively kinase-active 80 kDa protein, and could be detected by immunocytochemistry in nucleoli, nuclei and cytoplasm. Doxorubicin-induced apoptosis (assessed by cell morphology and annexin V-FITC binding) was significantly inhibited in two independent NPM-ALK-expressing clones (5.2+/-1.8 and 7.5+/-0.8% apoptosis), compared to control vector-transduced cells (36+/-6.7%). Similar results were observed with etoposide. In contrast, Fas-induced apoptosis was not inhibited. Cytochrome c release into the cytosol was delayed in doxorubicin-, but not anti-Fas-treated transfectant cells, indicating that apoptosis inhibition occurred upstream of mitochondrial events. Using NPM-ALK mutants, we demonstrated that inhibition of drug-induced apoptosis: (1) requires functional kinase activity, (2) does not involve phospholipase C-gamma, essential for NPM-ALK-mediated mitogenicity and (3) appears to be phosphoinositide 3-kinase independent, despite a strong Akt/PKB activation observed in wild type NPM-ALK-expressing cells. These results suggest that the NPM-ALK antiapoptotic and mitogenic pathways are distinct.

Adenosine Triphosphate↗
PubMed2001

Translocations involving anaplastic lymphoma kinase (ALK).

Anaplastic large-cell lymphoma (ALCL) comprises a group of non-Hodgkin's lymphomas (NHLs) that were first described in 1985 by Stein and co-workers and are characterized by the expression of the CD30/Ki-1 antigen (Stein et al., 1985). Approximately half of these lymphomas are associated with a typical chromosomal translocation, t(2;5)(p23;q35). Much confusion about the exact classification and clinicopathological features of this subgroup of NHL was clarified with the identification of NPM-ALK (nucleophosmin-anaplastic lymphoma kinase) as the oncogene created by the t(2;5) (Morris et al., 1994). With the discovery of NPM-ALK as the specific lymphoma gene mutation, this NHL subtype could be redefined on the molecular level. This achievement was enhanced by the availability of specific antibodies that recognize ALK fusion proteins in paraffin-embedded lymphoma tissues. Several excellent recent reviews have summarized the histopathological and molecular findings of ALCL and their use in the classification of this lymphoma entity (Anagnostopoulos and Stein, 2000; Benharroch et al., 1998; Drexler et al., 2000; Foss et al., 2000; Gogusev and Nezelof, 1998; Kadin and Morris, 1998; Ladanyi, 1997; Morris et al., 2001; Shiota and Mori, 1996; Skinnider et al., 1999; Stein et al., 2000). This review will focus on the molecular function and signal transduction pathways activated by ALK fusion oncogenes, with recent advances and possible clinical implications to be discussed.

Amino Acid Sequence↗
PubMed2001

Adhesion to fibronectin selectively protects Bcr-Abl+ cells from DNA damage-induced apoptosis.

The phenotype of Bcr-Abl-transformed cells is characterized by a growth factor-independent survival and a reduced susceptibility to apoptosis. Furthermore, Bcr-Abl kinase alters adhesion features by phosphorylating cytoskeletal and/or signaling proteins important for integrin function. Integrin-mediated adhesion to extracellular matrix molecules is critical for the regulation of growth and apoptosis. However, effects of integrin signaling on regulation of apoptosis in cells expressing Bcr-Abl are largely unknown. The influence of adhesion on survival and apoptosis in Bcr-Abl+ and Bcr-Abl- BaF3 cells was investigated. p185bcr-abl-transfected BaF3 cells preadhered to immobilized fibronectin had a significant survival advantage and reduced susceptibility to apoptosis following gamma-irradiation when compared with the same cells grown on laminin, on polylysin, or in suspension. Both inhibition of Bcr-Abl kinase by STI571 and inhibition of specific adhesion reversed the fibronectin-mediated antiapoptotic effect in BaF3p185. The DNA damage response of Bcr-Abl- BaF3 cells was not affected by adhesion to fibronectin. In contrast to parental BaF3 cells, BaF3p185 adherent to fibronectin did not release cytochrome c to the cytosol following irradiation. The fibronectin-mediated antiapoptotic mechanism in Bcr-Abl-active cells was not mediated by overexpression of Bcl-XL or Bcl-2 but required an active phosphatidylinositol 3-kinase (PI-3K). Kinase-active Bcr-Abl in combination with fibronectin-induced integrin signaling led to a hyperphosphorylation of AKT. Thus, cooperative activation of PI-3K/AKT by Bcr-Abl and integrins causes synergistic protection of Bcr-Abl+ cells from DNA damage-induced apoptosis.

Animals↗
PubMed2001

Grb4/Nckbeta acts as a nuclear repressor of v-Abl-induced transcription from c-jun/c-fos promoter elements.

Grb4 is an adaptor protein consisting of three src homology (SH) 3 domains and a single SH2 domain. We previously cloned Grb4 as a direct interacting partner of Bcr-Abl and v-Abl via the Grb4 SH2 domain. We now show that overexpression of Grb4 results in significant inhibition of v-Abl-induced transcriptional activation from promitogenic enhancer elements such as activator protein 1 (AP-1) and serum-responsive element (SRE). We demonstrate that the inhibitory activity of Grb4 is independent of the direct interaction of v-Abl and Grb4: a Grb4 mutant that lacks a functional SH2 domain shows an even more pronounced inhibition of AP-1/SRE. Further mutational analysis revealed that the first two SH3 domains primarily mediate the inhibitory function. The inhibitory activity of Grb4 is specific for c-jun/c-fos-regulated promoter elements and is located downstream of MEKK1 and JNK because co-expression of Grb4 resulted in down-regulation of MEKK1-induced AP-1 activity without affecting JNK activity. Thus, the nuclear pool of Grb4 is likely to mediate this inhibition. Indeed, cell fractionation and fluorescence microscopy studies revealed that the stronger inhibitory potential of the Grb4 SH2 mutant occurred in conjunction with increased nuclear localization of this mutant. Our results suggest a novel role for Grb4 in the inhibition of promitogenic enhancer elements such as 12-O-tetradecanoylphorbol-13-acetate-responsive element and SRE.

3T3 Cells↗
PubMed2000

Nucleophosmin-anaplastic lymphoma kinase associated with anaplastic large-cell lymphoma activates the phosphatidylinositol 3-kinase/Akt antiapoptotic signaling pathway.

More than half of anaplastic large-cell lymphomas (ALCLs) have a chromosomal translocation t(2;5) that leads to the expression of a hybrid protein composed of the nucleolar phosphoprotein nucleophosmin (NPM) and the anaplastic lymphoma kinase (ALK) that exhibits an unregulated tyrosine kinase activity. We have previously identified PLC-gamma as a crucial downstream signaling molecule of NPM-ALK that contributes to its mitogenic potential. Here, we show that NPM-ALK recruits the C-terminal SH2 domain of the phosphatidylinositol 3-kinase (PI 3kinase) p85 subunit. PI 3-kinase assays revealed that the kinase is activated by NPM-ALK in vivo, in turn activating PKB/Akt in NPM-ALK-expressing cells. The use of 2 specific PI 3-kinase inhibitors, wortmannin and LY294002, demonstrated the requirement of PI 3-kinase for the growth of NPM-ALK-transformed cell lines, as well as a cell line established from a patient with ALCL. Primary murine bone marrow retrovirally transduced with NPM-ALK showed a transformed phenotype that was reversible on treatment with PI 3-kinase inhibitors. Flow cytometric analysis revealed that wortmannin-treated NPM-ALK-transformed cell lines underwent apoptosis. Furthermore, apoptosis induced by overexpression of the proapoptotic molecule Bad could be partially blocked by the overexpression of NPM-ALK. Thus, NPM-ALK activates the antiapoptotic PI 3-kinase/Akt pathway, which likely contributes to the molecular pathogenesis of ALCL. (Blood. 2000;96:4319-4327)

Amino Acid Substitution↗