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2.
Blood ; 124(20): 3092-100, 2014 Nov 13.
Article in English | MEDLINE | ID: mdl-25193870

ABSTRACT

JAK3 is a tyrosine kinase that associates with the common γ chain of cytokine receptors and is recurrently mutated in T-cell acute lymphoblastic leukemia (T-ALL). We tested the transforming properties of JAK3 pseudokinase and kinase domain mutants using in vitro and in vivo assays. Most, but not all, JAK3 mutants transformed cytokine-dependent Ba/F3 or MOHITO cell lines to cytokine-independent proliferation. JAK3 pseudokinase mutants were dependent on Jak1 kinase activity for cellular transformation, whereas the JAK3 kinase domain mutant could transform cells in a Jak1 kinase-independent manner. Reconstitution of the IL7 receptor signaling complex in 293T cells showed that JAK3 mutants required receptor binding to mediate downstream STAT5 phosphorylation. Mice transplanted with bone marrow progenitor cells expressing JAK3 mutants developed a long-latency transplantable T-ALL-like disease, characterized by an accumulation of immature CD8(+) T cells. In vivo treatment of leukemic mice with the JAK3 selective inhibitor tofacitinib reduced the white blood cell count and caused leukemic cell apoptosis. Our data show that JAK3 mutations are drivers of T-ALL and require the cytokine receptor complex for transformation. These results warrant further investigation of JAK1/JAK3 inhibitors for the treatment of T-ALL.


Subject(s)
Cell Transformation, Neoplastic/genetics , Disease Models, Animal , Janus Kinase 1/metabolism , Janus Kinase 3/genetics , Leukemia, T-Cell/genetics , Mice , Acute Disease , Animals , Cell Transformation, Neoplastic/drug effects , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Enzyme Activation/drug effects , Janus Kinase 3/antagonists & inhibitors , Janus Kinase 3/metabolism , Leukemia, T-Cell/drug therapy , Leukemia, T-Cell/metabolism , Leukemia, T-Cell/pathology , Male , Mice/genetics , Mice/metabolism , Mice, Inbred BALB C , Mutation , Piperidines/therapeutic use , Pyrimidines/therapeutic use , Pyrroles/therapeutic use , Signal Transduction/drug effects , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , T-Lymphocytes/pathology
3.
Mol Oncol ; 8(3): 728-40, 2014 May.
Article in English | MEDLINE | ID: mdl-24618081

ABSTRACT

Activated forms of the platelet derived growth factor receptor alpha (PDGFRα) have been described in various tumors, including FIP1L1-PDGFRα in patients with myeloproliferative diseases associated with hypereosinophilia and the PDGFRα(D842V) mutant in gastrointestinal stromal tumors and inflammatory fibroid polyps. To gain a better insight into the signal transduction mechanisms of PDGFRα oncogenes, we mutated twelve potentially phosphorylated tyrosine residues of FIP1L1-PDGFRα and identified three mutations that affected cell proliferation. In particular, mutation of tyrosine 720 in FIP1L1-PDGFRα or PDGFRα(D842V) inhibited cell growth and blocked ERK signaling in Ba/F3 cells. This mutation also decreased myeloproliferation in transplanted mice and the proliferation of human CD34(+) hematopoietic progenitors transduced with FIP1L1-PDGFRα. We showed that the non-receptor protein tyrosine phosphatase SHP2 bound directly to tyrosine 720 of FIP1L1-PDGFRα. SHP2 knock-down decreased proliferation of Ba/F3 cells transformed with FIP1L1-PDGFRα and PDGFRα(D842V) and affected ERK signaling, but not STAT5 phosphorylation. Remarkably, SHP2 was not essential for cell proliferation and ERK phosphorylation induced by the wild-type PDGF receptor in response to ligand stimulation, suggesting a shift in the function of SHP2 downstream of oncogenic receptors. In conclusion, our results indicate that SHP2 is required for cell transformation and ERK activation by mutant PDGF receptors.


Subject(s)
Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Receptor, Platelet-Derived Growth Factor alpha/genetics , Receptor, Platelet-Derived Growth Factor alpha/metabolism , mRNA Cleavage and Polyadenylation Factors/genetics , mRNA Cleavage and Polyadenylation Factors/metabolism , Animals , Cell Line , Cell Proliferation , Cells, Cultured , Humans , MAP Kinase Signaling System , Mice , Mutation , Phosphorylation , Protein Binding , STAT5 Transcription Factor/metabolism , Signal Transduction
4.
Haematologica ; 99(1): 85-93, 2014 Jan.
Article in English | MEDLINE | ID: mdl-23872305

ABSTRACT

The NUP214-ABL1 fusion protein is a constitutively active protein tyrosine kinase that is found in 6% of patients with T-cell acute lymphoblastic leukemia and that promotes proliferation and survival of T-lymphoblasts. Although NUP214-ABL1 is sensitive to ABL1 kinase inhibitors, development of resistance to these compounds is a major clinical problem, underlining the need for additional drug targets in the sparsely studied NUP214-ABL1 signaling network. In this work, we identify and validate the SRC family kinase LCK as a protein whose activity is absolutely required for the proliferation and survival of T-cell acute lymphoblastic leukemia cells that depend on NUP214-ABL1 activity. These findings underscore the potential of SRC kinase inhibitors and of the dual ABL1/SRC kinase inhibitors dasatinib and bosutinib for the treatment of NUP214-ABL1-positive T-cell acute lymphoblastic leukemia. In addition, we used mass spectrometry to identify protein interaction partners of NUP214-ABL1. Our results strongly support that the signaling network of NUP214-ABL1 is distinct from that previously reported for BCR-ABL1. Moreover, we found that three NUP214-ABL1-interacting proteins, MAD2L1, NUP155, and SMC4, are strictly required for the proliferation and survival of NUP214-ABL1-positive T-cell acute lymphoblastic leukemia cells. In conclusion, this work identifies LCK, MAD2L1, NUP155 and SMC4 as four new potential drug targets in NUP214-ABL1-positive T-cell acute lymphoblastic leukemia.


Subject(s)
Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/metabolism , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Line, Tumor , Cell Proliferation , Gene Knockdown Techniques , Humans , Phosphorylation , Protein Binding , Protein Interaction Mapping , RNA Interference , src-Family Kinases/antagonists & inhibitors , src-Family Kinases/metabolism
5.
RNA ; 18(6): 1296-306, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22539522

ABSTRACT

RNA interference (RNAi) is one of the processes in the cell that regulates mRNA expression levels. RNAi can be exploited to experimentally knockdown the expression of one or more genes in cell lines or even in cells in vivo and also became an interesting tool to develop new therapeutic approaches. One of the major challenges of using RNAi is selecting effective shRNAs or siRNAs that sufficiently down-regulate the expression of the target gene. Here, we describe a system to select functional shRNAs or siRNAs that makes use of the leukemia cell line Ba/F3 that is dependent on the expression of a mutant form of the PDGFRα kinase for its proliferation and survival. The basis of this system is the generation of an expression construct, where part of the open reading frame of the gene of interest is linked to the mutant PDGFRα. Thus, shRNAs or siRNAs that effectively target the gene of interest also result in a reduction of the expression of the mutant PDGFRα protein, which can be detected by a reduction of the proliferation of the cells. We demonstrate that this validation system can be used for the selection of effective siRNAs as well as shRNAs. Unlike other systems, the system described here is not dependent on obtaining high-transduction efficiencies, and nonspecific effects of the siRNAs or shRNAs can be detected by comparing the effects in the presence or absence of the growth factor interleukin-3.


Subject(s)
RNA, Small Interfering/analysis , Animals , Cell Line, Tumor , Cell Proliferation , Humans , Interleukin-3/metabolism , Mice , Open Reading Frames , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Receptors, Platelet-Derived Growth Factor/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
6.
Cytokine Growth Factor Rev ; 22(5-6): 287-300, 2011.
Article in English | MEDLINE | ID: mdl-22119658

ABSTRACT

Signaling by the many ligands of the TGFß family strongly converges towards only five receptor-activated, intracellular Smad proteins, which fall into two classes i.e. Smad2/3 and Smad1/5/8, respectively. These Smads bind to a surprisingly high number of Smad-interacting proteins (SIPs), many of which are transcription factors (TFs) that co-operate in Smad-controlled target gene transcription in a cell type and context specific manner. A combination of functional analyses in vivo as well as in cell cultures and biochemical studies has revealed the enormous versatility of the Smad proteins. Smads and their SIPs regulate diverse molecular and cellular processes and are also directly relevant to development and disease. In this survey, we selected appropriate examples on the BMP-Smads, with emphasis on Smad1 and Smad5, and on a number of SIPs, i.e. the CPSF subunit Smicl, Ttrap (Tdp2) and Sip1 (Zeb2, Zfhx1b) from our own research carried out in three different vertebrate models.


Subject(s)
Bone Morphogenetic Proteins/metabolism , Smad Proteins/metabolism , Transforming Growth Factor beta/metabolism , Animals , Humans , Signal Transduction
7.
Chem Biol ; 18(3): 277-8, 2011 Mar 25.
Article in English | MEDLINE | ID: mdl-21439469

ABSTRACT

Janus kinases are important signaling proteins implicated in cytokine signaling. In particular, Janus kinase 3 (JAK3) has gained attention as a target for inhibition of the immune system, due to its importance for T and B cell development and function. In this issue however, Haan et al. (2011) show that inhibition of JAK3 activity may not be sufficient for this purpose.

8.
Blood ; 117(15): 4056-64, 2011 Apr 14.
Article in English | MEDLINE | ID: mdl-21325169

ABSTRACT

The genetics of classical Hodgkin lymphoma (cHL) is poorly understood. The finding of a JAK2-involving t(4;9)(q21;p24) in 1 case of cHL prompted us to characterize this translocation on a molecular level and to determine the prevalence of JAK2 rearrangements in cHL. We showed that the t(4;9)(q21;p24) leads to a novel SEC31A-JAK2 fusion. Screening of 131 cHL cases identified 1 additional case with SEC31A-JAK2 and 2 additional cases with rearrangements involving JAK2. We demonstrated that SEC31A-JAK2 is oncogenic in vitro and acts as a constitutively activated tyrosine kinase that is sensitive to JAK inhibitors. In vivo, SEC31A-JAK2 was found to induce a T-lymphoblastic lymphoma or myeloid phenotype in a murine bone marrow transplantation model. Altogether, we identified SEC31A-JAK2 as a chromosomal aberration characteristic for cHL and provide evidence that JAK2 rearrangements occur in a minority of cHL cases. Given the proven oncogenic potential of this novel fusion, our studies provide new insights into the pathogenesis of cHL and indicate that in at least some cases, constitutive activation of the JAK/STAT pathway is caused by JAK2 rearrangements. The finding that SEC31A-JAK2 responds to JAK inhibitors indicates that patients with cHL and JAK2 rearrangements may benefit from targeted therapies.


Subject(s)
Gene Rearrangement/genetics , Hodgkin Disease/genetics , Janus Kinase 2/genetics , Oncogene Proteins, Fusion/genetics , Vesicular Transport Proteins/genetics , Adult , Aged, 80 and over , Animals , Bone Marrow Transplantation , Chromosomes, Human, Pair 4 , Chromosomes, Human, Pair 9 , Disease Models, Animal , Female , Genetic Predisposition to Disease/epidemiology , HEK293 Cells , Hodgkin Disease/epidemiology , Humans , Male , Mice , Middle Aged , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Prevalence , Protein-Tyrosine Kinases/metabolism , Translocation, Genetic , Young Adult
9.
Int J Cardiol ; 131(1): 33-44, 2008 Dec 17.
Article in English | MEDLINE | ID: mdl-18715658

ABSTRACT

With every heartbeat the heart must contract and relax. This seemingly trivial process critically needs tight control of contraction and relaxation phases, and extremely efficient coordination between these two phases to control blood flow and maintain cardiac homeostasis. To achieve this, specialized sensors are required to detect the inherent repeatedly changing environment and needs. One sensor is a stretch-sensor that monitors the filling of the ventricles. Its molecular identity and localization are only partly understood. Here we give a synopsis of the genetic models that leap into our understanding of stretch-sensors. We focus on the widely acknowledged sarcomeric sensor at the Z-disc and the costamere sensor at the sarcolemma. Recently, several novel components of both sensors were discovered. Given that these two sensors seem physically connected, it is likely that these two models are not mutually exclusive and might even communicate. We describe briefly how candidate and known proteins within these sensors receive and transduce mechanical signals in the cardiomyocyte that lead to changes in gene expression underlying homeostasis and its restoration in the heart. Emphasis is placed on the putative link between altered stretch-sensor function and heart failure observed in different genetic mouse models of stretch-sensor components.


Subject(s)
Disease Models, Animal , Heart Failure/genetics , Mechanoreceptors/physiology , Mutation/genetics , Animals , Heart Failure/physiopathology , Humans , Mice
10.
Am J Pathol ; 170(5): 1460-72, 2007 May.
Article in English | MEDLINE | ID: mdl-17456754

ABSTRACT

Smads are intracellular signaling proteins that transduce signals elicited by members of the transforming growth factor (TGF)-beta superfamily. Smad5 and Smad1 are highly homologous, and they mediate primarily bone morphogenetic protein (Bmp) signals. We used the Cre-loxP system and Sm22-Cre and Tie-1-Cre mice to study the function of Smad5 in the developing blood vessel wall. Analysis of embryos demonstrated that deletion of Smad5 in endothelial or smooth muscle cells resulted in a normal organization of embryonic and extra-embryonic vasculature. Angiogenic assays performed in adult mice revealed that mutant mice display a comparable angiogenic and vascular remodeling response to control mice. In Sm22-Cre; Smad5(fl/-) mice, Smad5 is also deleted in cardiomyocytes. Echocardiographic analysis on those 9-month-old female mice demonstrated larger left ventricle internal diameters and decreased fractional shortening compared with control littermates without signs of cardiac hypertrophy. The decreased cardiac contractility was associated with a decreased performance in a treadmill experiment. In isolated cardiomyocytes, fractional shortening was significantly reduced compared with control cells. These data demonstrate that restricted deletion of Smad5 in the blood vessel wall results in viable mice. However, loss of Smad5 in cardiomyocytes leads to a mild heart defect.


Subject(s)
Endothelial Cells/metabolism , Heart/growth & development , Homeostasis/physiology , Myocytes, Smooth Muscle/metabolism , Neovascularization, Physiologic , Smad5 Protein/metabolism , Animals , Animals, Newborn , Blood Vessels/growth & development , Blood Vessels/metabolism , Echocardiography , Embryo, Mammalian , Endothelial Cells/cytology , Female , Gene Expression , Heart/physiology , Immunohistochemistry , Male , Mice , Mice, Knockout , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Myocytes, Smooth Muscle/cytology , Polymerase Chain Reaction , Smad5 Protein/genetics
11.
Neurobiol Dis ; 24(1): 28-40, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16875832

ABSTRACT

The mouse mutant Ozzy, originating from an ENU-mutagenesis programme, displays a head bobbing phenotype. We report here that Ozzy mice show a clear deficit in vestibulo-ocular reflex (VOR). Micro-CT scanning of the inner ears showed narrowing and truncations of at least one of the semicircular canals and loss of the ampullae. Frequency-specific auditory-evoked brainstem response (ABR) tests revealed a slight threshold increase in the middle frequency range compared to wild-type littermates. Linkage analysis localised the gene in a 5.5-cM region on chromosome 2. Subsequently, a 499 T-->A missense mutation was identified in Jag1, leading to a substitution of an evolutionary conserved tryptophane (W167R). Mutations in the human homologue of Jag1 cause Alagille syndrome (AGS), an autosomal dominant disorder associated with liver, heart, eye and skeletal abnormalities, accompanied by a characteristic facies. In human patients, it occasionally affects other organ systems like the kidney or the inner ear. Liver disease is the main diagnostic factor for AGS. Ozzy mice showed significantly less intrahepatic bile ducts than wild-type littermates. Thirty-seven percent of Ozzy mice showed heart defects. No eye or vertebral abnormalities could be detected. In conclusion, Ozzy mice show two of the major and one minor characteristic of AGS.


Subject(s)
Alagille Syndrome/genetics , Alagille Syndrome/physiopathology , Calcium-Binding Proteins/genetics , Intercellular Signaling Peptides and Proteins/genetics , Membrane Proteins/genetics , Mice, Neurologic Mutants/physiology , Vestibule, Labyrinth/physiology , Alagille Syndrome/enzymology , Animals , Bone Diseases/genetics , Chromosome Mapping , Cochlea/pathology , Cochlea/physiology , DNA/genetics , DNA Mutational Analysis , Disease Models, Animal , Evoked Potentials, Auditory, Brain Stem/genetics , Evoked Potentials, Auditory, Brain Stem/physiology , Genetic Linkage , Growth Disorders/genetics , Heart Defects, Congenital/genetics , Jagged-1 Protein , Lectins/metabolism , Liver/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Microscopy, Electron, Scanning , Mutation, Missense/physiology , Psychomotor Performance/physiology , Reflex, Vestibulo-Ocular/genetics , Reflex, Vestibulo-Ocular/physiology , Serrate-Jagged Proteins , Tomography, X-Ray Computed , Vision Disorders/genetics , Visual Perception
12.
Circ Res ; 97(3): 219-26, 2005 Aug 05.
Article in English | MEDLINE | ID: mdl-16037571

ABSTRACT

Endocardial cushions are precursors of mature atrioventricular (AV) valves. Their formation is induced by signaling molecules originating from the AV myocardium, including bone morphogenetic proteins (BMPs). Here, we hypothesized that BMP signaling plays an important role in the AV myocardium during the maturation of AV valves from the cushions. To test our hypothesis, we used a unique Cre/lox system to target the deletion of a floxed Alk3 allele, the type IA receptor for BMPs, to cardiac myocytes of the AV canal (AVC). Lineage analysis indicated that cardiac myocytes of the AVC contributed to the tricuspid mural and posterior leaflets, the mitral septal leaflet, and the atrial border of the annulus fibrosus. When Alk3 was deleted in these cells, defects were seen in the same leaflets, ie, the tricuspid mural leaflet and mitral septal leaflet were longer, the tricuspid posterior leaflet was displaced and adherent to the ventricular wall, and the annulus fibrosus was disrupted resulting in ventricular preexcitation. The defects seen in mice with AVC-targeted deletion of Alk3 provide strong support for a role of Alk3 in human congenital heart diseases, such as Ebstein's anomaly. In conclusion, our mouse model demonstrated critical roles for Alk3 signaling in the AV myocardium during the development of AV valves and the annulus fibrosus.


Subject(s)
Heart Valves/embryology , Heart/embryology , Protein Serine-Threonine Kinases/physiology , Receptors, Growth Factor/physiology , Animals , Bone Morphogenetic Protein Receptors, Type I , DNA-Binding Proteins/genetics , GATA6 Transcription Factor , Heart Valves/abnormalities , Integrases/genetics , Mice , Mice, Transgenic , Myocytes, Cardiac/physiology , Signal Transduction , Transcription Factors/genetics
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