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1.
J Fish Dis ; 44(10): 1553-1562, 2021 Oct.
Article in English | MEDLINE | ID: mdl-34160839

ABSTRACT

T-helper cells express CD4 as a co-receptor that binds to major histocompatibility complex class II to synchronize the immune response against upcoming threats via mediating several cytokines. We have previously reported the presence of CD4 homologues in brown trout. The study of cellular immune responses in brown trout is limited by the availability of specific antibodies. We here describe the generation of a polyclonal antibody against CD4-1 that allows for the investigation of CD4+ cells. We used this novel tool to study CD4+ cells in different tissues during viral haemorrhagic septicaemia infection (VHSV) using flow cytometric technique. Flow cytometric analyses revealed an enhanced level of surface CD4-1 expression in the infected group in major lymphoid organs and in the intestine. These results suggest an important role for the T-helper cells within the immune response against viruses, comparable to the immune response in higher vertebrates.


Subject(s)
CD4-Positive T-Lymphocytes/physiology , Fish Diseases/immunology , Hemorrhagic Septicemia, Viral/immunology , Novirhabdovirus/physiology , Trout , Animals , Biomechanical Phenomena , CD4-Positive T-Lymphocytes/virology , Fish Diseases/virology , Hemorrhagic Septicemia, Viral/virology , Kinetics
2.
Hemasphere ; 4(1): e312, 2020 Feb.
Article in English | MEDLINE | ID: mdl-32072137

ABSTRACT

Aberrant activation of key signaling-molecules is a hallmark of acute myeloid leukemia (AML) and may have prognostic and therapeutic implications. AML summarizes several disease entities with a variety of genetic subtypes. A comprehensive model spanning from signal activation patterns in major genetic subtypes of pediatric AML (pedAML) to outcome prediction and pre-clinical response to signaling inhibitors has not yet been provided. We established a high-throughput flow-cytometry based method to assess activation of hallmark phospho-proteins (phospho-flow) in 166 bone-marrow derived pedAML samples under basal and cytokine stimulated conditions. We correlated levels of activated phospho-proteins at diagnosis with relapse incidence in intermediate (IR) and high risk (HR) subtypes. In parallel, we screened a set of signaling inhibitors for their efficacy against primary AML blasts in a flow-cytometry based ex vivo cytotoxicity assay and validated the results in a murine xenograft model. Certain phospho-signal patterns differ between genetic subtypes of pedAML. Some are consistently seen through all AML subtypes such as pSTAT5. In IR/HR subtypes high levels of GM-CSF stimulated pSTAT5 and low levels of unstimulated pJNK correlated with increased relapse risk overall. Combination of GM-CSF/pSTAT5high and basal/pJNKlow separated three risk groups among IR/HR subtypes. Out of 10 tested signaling inhibitors, midostaurin most effectively affected AML blasts and simultaneously blocked phosphorylation of multiple proteins, including STAT5. In a mouse xenograft model of KMT2A-rearranged pedAML, midostaurin significantly prolonged disease latency. Our study demonstrates the applicability of phospho-flow for relapse-risk assessment in pedAML, whereas functional phenotype-driven ex vivo testing of signaling inhibitors may allow individualized therapy.

3.
Blood ; 132(7): 694-706, 2018 08 16.
Article in English | MEDLINE | ID: mdl-29907599

ABSTRACT

Inhibition of Janus-kinase 1/2 (JAK1/2) is a mainstay to treat myeloproliferative neoplasms (MPN). Sporadic observations reported the co-incidence of B-cell non-Hodgkin lymphomas during treatment of MPN with JAK1/2 inhibitors. We assessed 626 patients with MPN, including 69 with myelofibrosis receiving JAK1/2 inhibitors for lymphoma development. B-cell lymphomas evolved in 4 (5.8%) of 69 patients receiving JAK1/2 inhibition compared with 2 (0.36%) of 557 with conventional treatment (16-fold increased risk). A similar 15-fold increase was observed in an independent cohort of 929 patients with MPN. Considering primary myelofibrosis only (N = 216), 3 lymphomas were observed in 31 inhibitor-treated patients (9.7%) vs 1 (0.54%) of 185 control patients. Lymphomas were of aggressive B-cell type, extranodal, or leukemic with high MYC expression in the absence of JAK2 V617F or other MPN-associated mutations. Median time from initiation of inhibitor therapy to lymphoma diagnosis was 25 months. Clonal immunoglobulin gene rearrangements were already detected in the bone marrow during myelofibrosis in 16.3% of patients. Lymphomas occurring during JAK1/2 inhibitor treatment were preceded by a preexisting B-cell clone in all 3 patients tested. Sequencing verified clonal identity in 2 patients. The effects of JAK1/2 inhibition were mirrored in Stat1-/- mice: 16 of 24 mice developed a spontaneous myeloid hyperplasia with the concomitant presence of aberrant B cells. Transplantations of bone marrow from diseased mice unmasked the outgrowth of a malignant B-cell clone evolving into aggressive B-cell leukemia-lymphoma. We conclude that JAK/STAT1 pathway inhibition in myelofibrosis is associated with an elevated frequency of aggressive B-cell lymphomas. Detection of a preexisting B-cell clone may identify individuals at risk.


Subject(s)
Janus Kinase 1/antagonists & inhibitors , Janus Kinase 2/antagonists & inhibitors , Lymphoma, B-Cell/drug therapy , Neoplasm Proteins/antagonists & inhibitors , Primary Myelofibrosis/drug therapy , Protein Kinase Inhibitors/pharmacology , Animals , Cell Line, Tumor , Female , Humans , Janus Kinase 1/genetics , Janus Kinase 1/metabolism , Janus Kinase 2/genetics , Janus Kinase 2/metabolism , Lymphoma, B-Cell/enzymology , Lymphoma, B-Cell/genetics , Lymphoma, B-Cell/pathology , Mice , Mice, Knockout , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Primary Myelofibrosis/enzymology , Primary Myelofibrosis/genetics , Primary Myelofibrosis/pathology , Retrospective Studies
5.
Cancer Cell ; 24(2): 167-81, 2013 Aug 12.
Article in English | MEDLINE | ID: mdl-23948297

ABSTRACT

In contrast to its close homolog CDK4, the cell cycle kinase CDK6 is expressed at high levels in lymphoid malignancies. In a model for p185BCR-ABL+ B-acute lymphoid leukemia, we show that CDK6 is part of a transcription complex that induces the expression of the tumor suppressor p16INK4a and the pro-angiogenic factor VEGF-A. This function is independent of CDK6's kinase activity. High CDK6 expression thus suppresses proliferation by upregulating p16INK4a, providing an internal safeguard. However, in the absence of p16INK4a, CDK6 can exert its full tumor-promoting function by enhancing proliferation and stimulating angiogenesis. The finding that CDK6 connects cell-cycle progression to angiogenesis confirms CDK6's central role in hematopoietic malignancies and could underlie the selection pressure to upregulate CDK6 and silence p16INK4a.


Subject(s)
Cyclin-Dependent Kinase 6/metabolism , Neoplasms/blood supply , Neoplasms/enzymology , Animals , Cell Cycle/physiology , Cyclin-Dependent Kinase 6/genetics , Humans , Leukemia, B-Cell/enzymology , Leukemia, B-Cell/pathology , Lymphoma, B-Cell/enzymology , Lymphoma, B-Cell/pathology , Mice , Mice, Inbred C57BL , Mice, Nude , Mice, Transgenic , Neoplasms/genetics , Neovascularization, Pathologic/enzymology , Neovascularization, Pathologic/pathology
6.
Blood ; 117(12): 3409-20, 2011 Mar 24.
Article in English | MEDLINE | ID: mdl-21220747

ABSTRACT

In BCR-ABL1(+) leukemia, drug resistance is often associated with up-regulation of BCR-ABL1 or multidrug transporters as well as BCR-ABL1 mutations. Here we show that the expression level of the transcription factor STAT5 is another parameter that determines the sensitivity of BCR-ABL1(+) cells against tyrosine kinase inhibitors (TKIs), such as imatinib, nilotinib, or dasatinib. Abelson-transformed cells, expressing high levels of STAT5, were found to be significantly less sensitive to TKI-induced apoptosis in vitro and in vivo but not to other cytotoxic drugs, such as hydroxyurea, interferon-ß, or Aca-dC. The STAT5-mediated protection requires tyrosine phosphorylation of STAT5 independent of JAK2 and transcriptional activity. In support of this concept, under imatinib treatment and with disease progression, STAT5 mRNA and protein levels increased in patients with Ph(+) chronic myeloid leukemia. Based on our data, we propose a model in which disease progression in BCR-ABL1(+) leukemia leads to up-regulated STAT5 expression. This may be in part the result of clonal selection of cells with high STAT5 levels. STAT5 then accounts for the resistance against TKIs, thereby explaining the dose escalation frequently required in patients reaching accelerated phase. It also suggests that STAT5 may serve as an attractive target to overcome imatinib resistance in BCR-ABL1(+) leukemia.


Subject(s)
Drug Resistance, Neoplasm/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Piperazines/therapeutic use , Pyrimidines/therapeutic use , STAT5 Transcription Factor/physiology , Animals , Antineoplastic Agents/therapeutic use , Benzamides , Cells, Cultured , Disease Progression , Gene Expression Regulation, Neoplastic/physiology , Humans , Imatinib Mesylate , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/diagnosis , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, SCID , Mice, Transgenic , Prognosis , STAT5 Transcription Factor/genetics , Treatment Failure , Up-Regulation/genetics , Up-Regulation/physiology , Xenograft Model Antitumor Assays
7.
Blood ; 116(9): 1548-58, 2010 Sep 02.
Article in English | MEDLINE | ID: mdl-20508164

ABSTRACT

Stat5 transcription factors are essential gene regulators promoting proliferation, survival, and differentiation of all hematopoietic cell types. Mutations or fusions of oncogenic tyrosine kinases often result in constitutive Stat5 activation. We have modeled persistent Stat5 activity by using an oncogenic Stat5a variant (cS5). To analyze the hitherto unrecognized role of Stat5 serine phosphorylation in this context, we have generated cS5 constructs with mutated C-terminal serines 725 and 779, either alone or in combination. Genetic complementation assays in primary Stat5(null/null) mast cells and Stat5(DeltaN) T cells demonstrated reconstitution of proliferation with these mutants. Similarly, an in vivo reconstitution experiment of transduced Stat5(null/null) fetal liver cells transplanted into irradiated wild-type recipients revealed that these mutants exhibit biologic activity in lineage differentiation. By contrast, the leukemogenic potential of cS5 in bone marrow transplants decreased dramatically in cS5 single-serine mutants or was completely absent upon loss of both serine phosphorylation sites. Our data suggest that Stat5a serine phosphorylation is a prerequisite for cS5-mediated leukemogenesis. Hence, interference with Stat5a serine phosphorylation might provide a new therapeutic option for leukemia and myeloid dysplasias without affecting major functions of Stat5 in normal hematopoiesis.


Subject(s)
Cell Transformation, Neoplastic , Hematopoiesis/physiology , Leukemia/pathology , STAT5 Transcription Factor/metabolism , Serine/metabolism , Tumor Suppressor Proteins/metabolism , Adult , Aged , Animals , Blotting, Western , Bone Marrow Transplantation , Cell Lineage , Cell Proliferation , Cells, Cultured , Female , Fetus , Flow Cytometry , Humans , Immunoenzyme Techniques , Leukemia/genetics , Leukemia/metabolism , Liver Transplantation , Male , Mast Cells/metabolism , Mice , Mice, Inbred C57BL , Middle Aged , Phosphorylation , Precursor Cells, B-Lymphoid/metabolism , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , STAT5 Transcription Factor/genetics , Serine/genetics , T-Lymphocytes/metabolism , Tumor Suppressor Proteins/genetics
8.
EMBO Mol Med ; 2(3): 98-110, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20201032

ABSTRACT

Tumourigenesis caused by the Bcr/Abl oncoprotein is a multi-step process proceeding from initial to tumour-maintaining events and finally results in a complex tumour-supporting network. A key to successful cancer therapy is the identification of critical functional nodes in an oncogenic network required for disease maintenance. So far, the transcription factors Stat3 and Stat5a/b have been implicated in bcr/abl-induced initial transformation. However, to qualify as a potential drug target, a signalling pathway must be required for the maintenance of the leukaemic state. Data on the roles of Stat3 or Stat5a/b in leukaemia maintenance are elusive. Here, we show that both, Stat3 and Stat5 are necessary for initial transformation. However, Stat5- but not Stat3-deletion induces G(0)/G(1) cell cycle arrest and apoptosis of imatinib-sensitive and imatinib-resistant stable leukaemic cells in vitro. Accordingly, Stat5-abrogation led to effective elimination of myeloid and lymphoid leukaemia maintenance in vivo. Hence, we identified Stat5 as a vulnerable point in the oncogenic network downstream of Bcr/Abl representing a case of non-oncogene addiction (NOA).


Subject(s)
Leukemia/physiopathology , STAT3 Transcription Factor/metabolism , STAT5 Transcription Factor/metabolism , Animals , Apoptosis , Cell Cycle , Gene Deletion , Genes, abl , Mice , Mice, Inbred C57BL , Mice, Knockout , Proto-Oncogene Proteins c-bcr/genetics , STAT3 Transcription Factor/genetics , STAT5 Transcription Factor/genetics
9.
Blood ; 112(12): 4655-64, 2008 Dec 01.
Article in English | MEDLINE | ID: mdl-18684865

ABSTRACT

Specific inhibitors of PI3K isoforms are currently evaluated for their therapeutic potential in leukemia. We found that BCR/ABL(+) human leukemic cells express PI3Kdelta and therefore explored its impact on leukemia development. Using PI3Kdelta-deficient mice, we define a dual role of PI3Kdelta in leukemia. We observed a growth-promoting effect in tumor cells and an essential function in natural killer (NK) cell-mediated tumor surveillance: Abelson-transformed PI3Kdelta-deficient cells induced leukemia in RAG2-deficient mice with an increased latency, indicating that PI3Kdelta accelerated leukemia progression in vivo. However, the absence of PI3Kdelta also affected NK cell-mediated tumor surveillance. PI3Kdelta-deficient NK cells failed to lyse a large variety of target cells because of defective degranulation, as also documented by capacitance recordings. Accordingly, transplanted leukemic cells killed PI3Kdelta-deficient animals more rapidly. As a net effect, no difference in disease latency in vivo was detected if both leukemic cells and NK cells lack PI3Kdelta. Other tumor models confirmed that PI3Kdelta-deficient mice succumbed more rapidly when challenged with T- or B-lymphoid leukemic or B16 melanoma cells. Thus, the action of PI3Kdelta in the NK compartment is as relevant to survival of the mice as the delayed tumor progression. This dual function must be taken into account when using PI3Kdelta inhibitors as antileukemic agents in clinical trials.


Subject(s)
Immunologic Surveillance/genetics , Killer Cells, Natural/immunology , Leukemia/immunology , Phosphatidylinositol 3-Kinases/genetics , Abelson murine leukemia virus/genetics , Animals , Cell Death/genetics , Cell Death/immunology , Cell Line, Transformed , Class I Phosphatidylinositol 3-Kinases , Disease Progression , Gene Expression Regulation, Leukemic , Humans , Jurkat Cells , Killer Cells, Natural/metabolism , Killer Cells, Natural/pathology , Leukemia/genetics , Leukemia/metabolism , Leukemia/mortality , Melanoma, Experimental/genetics , Melanoma, Experimental/immunology , Melanoma, Experimental/mortality , Melanoma, Experimental/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositol 3-Kinases/physiology , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/pharmacology , Tumor Cells, Cultured
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