scholarly journals Development of Notch-dependent T-cell leukemia by deregulated Rap1 signaling

Blood ◽  
2008 ◽  
Vol 111 (5) ◽  
pp. 2878-2886 ◽  
Author(s):  
Shu-Fang Wang ◽  
Misayo Aoki ◽  
Yasuhiro Nakashima ◽  
Yoriko Shinozuka ◽  
Hiroki Tanaka ◽  
...  
Keyword(s):  
T Cell ◽  
Target Genes ◽  
Lymphoblastic Leukemia ◽  
Cell Receptor ◽  
Nucleotide Exchange ◽  
Double Positive ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Enhanced Expression ◽  
Rap1 Activation

SPA-1 (signal-induced proliferation associated gene-1) functions as a suppressor of myeloid leukemia by negatively regulating Rap1 signaling in hematopoietic progenitor cells (HPCs). Herein, we showed that transplantation of HPCs expressing farnesylated C3G (C3G-F), a Rap1 guanine nucleotide exchange factor, resulted in a marked expansion of thymocytes bearing unique phenotypes (CD4/CD8 double positive [DP] CD3− TCRβ−) in irradiated recipients. SPA-1−/− HPCs expressing C3G-F caused a more extensive expansion of DP thymocytes, resulting in lethal T-cell acute lymphoblastic leukemia (T-ALL) with massive invasion of clonal T-cell blasts into vital organs. The C3G-F+ blastic thymocytes exhibited constitutive Rap1 activation and markedly enhanced expression of Notch1, 3 as well as the target genes, Hes1, pTα, and c-Myc. All the T-ALL cell lines from C3G-F+ SPA-1−/− HPC recipients expressed high levels of Notch1 with characteristic mutations resulting in the C-terminal truncation. This proliferation was inhibited completely in the presence of a γ-secretase inhibitor. Transplantation of Rag2−/− SPA-1−/− HPCs expressing C3G-F also resulted in a marked expansion and transformation of DP thymocytes. The results suggested that deregulated constitutive Rap1 activation caused abnormal expansion of DP thymocytes, bypassing the pre-T-cell receptor and eventually leading to Notch1 mutations and Notch-dependent T-ALL.

NOTCH1 pathway activation is an early hallmark of SCL T leukemogenesis

Blood ◽  
2007 ◽  
Vol 110 (10) ◽  
pp. 3753-3762 ◽  
Author(s):  
Joachim R. Göthert ◽  
Rachael L. Brake ◽  
Monique Smeets ◽  
Ulrich Dührsen ◽  
C. Glenn Begley ◽  
...  
Keyword(s):  
T Cell ◽  
Target Genes ◽  
Lymphoblastic Leukemia ◽  
Cell Receptor ◽  
Surface Expression ◽  
Normal Surface ◽  
Double Positive ◽  
Functional Link ◽  
Pathway Activation ◽  
Cell Acute Lymphoblastic Leukemia

Abstract The acquired activation of stem cell leukemia (SCL) during T lymphopoiesis is a common event in T-cell acute lymphoblastic leukemia (T-ALL). Here, we generated tamoxifen (TAM)–inducible transgenic mice (lck-ERT2-SCL) to study the consequences of acquired SCL activation during T-cell development. Aberrant activation of SCL in thymocytes resulted in the accumulation of immature CD4+CD8+ (double-positive, DP) cells by preventing normal surface expression of the T-cell receptor αβ (TCRαβ) complex. SCL-induced immature DP cells were further characterized by up-regulated NOTCH1 and generated noncycling polyclonal CD8+TCRβlow cells. The prevalence of these cells was SCL dependent because TAM withdrawal resulted in their disappearance. Furthermore, we observed that SCL activation led to a dramatic up-regulation of NOTCH1 target genes (Hes-1, Deltex1, and CD25) in thymocytes. Strikingly, NOTCH1 target gene up-regulation was already observed after short-term SCL induction, implying that enhanced NOTCH signaling is mediated by SCL and is not dependent on secondary genetic events. These data represent the basis for a novel pathway of SCL-induced leukemogenesis and provide a functional link between SCL and NOTCH1 during this process.

A Novel Cellular Pathway of SCL T-Leukemogenesis Revealed by a Conditional Transgenic Mouse Model.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1835-1835
Author(s):  
Joachim R. Gothert ◽  
Rachael Brake ◽  
C. Glenn Begley ◽  
David J. Izon
Keyword(s):  
T Cell ◽  
Mouse Model ◽  
Transgenic Mouse ◽  
Trichostatin A ◽  
Lymphoblastic Leukemia ◽  
Cell Receptor ◽  
Surface Expression ◽  
Transgenic Mouse Model ◽  
Tamoxifen Treatment ◽  
Double Positive

Abstract The acquired activation of stem cell leukemia (SCL) during T-lymphopoiesis is a common event in T-cell acute lymphoblastic leukemia. Here, we generated a novel tamoxifen-inducible transgenic mouse model (lck-ERT2-SCL) to study the cellular targets of acquired SCL activation during T-cell development. Upon tamoxifen treatment we observed the thymic emergence of abnormal, non-cycling CD8+TCRβlow and immature CD4+CD8+ (double-positive, DP) cells displaying increased viability. Unexpectedly, fetal thymic organ culture analysis of lck-ERT2-SCL thymi revealed the development of DP cells before the emergence of CD8+TCRβlow cells, which implied the derivation of CD8+TCRβlow cells from DPs rather than immature CD8 single-positive (SP) thymocytes. Interestingly, histone deacetylase (HDAC) inhibition with trichostatin A (TSA) had a divergent effect on SCL perturbed thymopoiesis: TSA increased T-cell receptor surface expression within DP and CD8 SP cells however did not alter the CD8 shifted CD4/CD8-ratio. Furthermore, we studied the expression of NOTCH1 in SCL induced TCRβlow thymocytes. Strikingly, we found that SCL induced NOTCH1-upregulation in DP TCRβlow cells. We therefore conclude that SCL promotes the emergence of abnormal CD8+TCRβlow cells by an only partially HDAC dependent mechanism from DP TCRβlow cells. Moreover, SCL induced DP TCRβlow cells are characterized by upregulated NOTCH1, which in turn might promote the effect of acquired NOTCH1 mutations during T-leukemogenesis.

scholarly journals Disregulated expression of the transcription factor ThPOK during T-cell development leads to high incidence of T-cell lymphomas

2015 ◽  
Vol 112 (25) ◽  
pp. 7773-7778 ◽  
Author(s):  
Hyung-Ok Lee ◽  
Xiao He ◽  
Jayati Mookerjee-Basu ◽  
Dai Zhongping ◽  
Xiang Hua ◽  
...  
Keyword(s):  
Transcription Factor ◽  
T Cell ◽  
Lymphoblastic Leukemia ◽  
T Cell Development ◽  
Cell Receptor ◽  
Cell Development ◽  
Thymocyte Development ◽  
Tcr Signaling ◽  
Double Positive ◽  
Human T Cell

The transcription factor T-helper-inducing POZ/Krueppel-like factor (ThPOK, encoded by the Zbtb7b gene) plays widespread and critical roles in T-cell development, particularly as the master regulator of CD4 commitment. Here we show that mice expressing a constitutive T-cell–specific ThPOK transgene (ThPOKconst mice) develop thymic lymphomas. These tumors resemble human T-cell acute lymphoblastic leukemia (T-ALL), in that they predominantly exhibit activating Notch1 mutations. Lymphomagenesis is prevented if thymocyte development is arrested at the DN3 stage by recombination-activating gene (RAG) deficiency, but restored by introduction of a T-cell receptor (TCR) transgene or by a single injection of anti-αβTCR antibody into ThPOKconst RAG-deficient mice, which promotes development to the CD4+8+ (DP) stage. Hence, TCR signals and/or traversal of the DN (double negative) > DP (double positive) checkpoint are required for ThPOK-mediated lymphomagenesis. These results demonstrate a novel link between ThPOK, TCR signaling, and lymphomagenesis. Finally, we present evidence that ectopic ThPOK expression gives rise to a preleukemic and self-perpetuating DN4 lymphoma precursor population. Our results collectively define a novel role for ThPOK as an oncogene and precisely map the stage in thymopoiesis susceptible to ThPOK-dependent tumor initiation.

scholarly journals Wnt5a enhances proliferation of chronic lymphocytic leukemia and ERK1/2 phosphorylation via a ROR1/DOCK2-dependent mechanism

Leukemia ◽  
2020 ◽  
Author(s):  
Md Kamrul Hasan ◽  
Emanuela M. Ghia ◽  
Laura Z. Rassenti ◽  
George F. Widhopf ◽  
Thomas J. Kipps
Keyword(s):  
Cell Proliferation ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Receptor ◽  
High Plasma ◽  
Lymphocytic Leukemia ◽  
Nucleotide Exchange ◽  
Rich Domain ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Receptor Ligation

Abstract Patients with chronic lymphocytic leukemia (CLL) have high plasma-levels of Wnt5a, which can induce phosphorylation of ERK1/2 and enhance CLL-cell proliferation. Such effects could be inhibited by treatment with an ERK1/2 inhibitor, ERK1/2-specific siRNA, or cirmtuzumab, an anti-ROR1 mAb. The CLL-derived line, MEC1, expresses Wnt5a, but not ROR1. MEC1 cells transfected to express ROR1 (MEC1-ROR1) had higher levels of phosphorylated ERK1/2 than parental MEC1, or MEC1 transfected with ROR1ΔPRD, a truncated ROR1 lacking the cytoplasmic proline-rich domain (PRD), or ROR1P808A a mutant ROR1 with a P→A substitution at 808, which is required for complexing with the Rac-specific-guanine-nucleotide-exchange factor DOCK2 upon stimulation with Wnt5a. We silenced DOCK2 with siRNA and found this repressed the capacity of Wnt5a to induce ERK1/2 phosphorylation in MEC1-ROR1 or CLL cells. CLL cells that expressed ROR1 had higher levels of phosphorylated ERK1/2 or DOCK2 than CLL cells lacking ROR1. Although we found ibrutinib could inhibit the phosphorylation of ERK1/2 and DOCK2 induced by B-cell-receptor ligation, we found that this drug was unable to inhibit Wnt5a-induced, ROR1-dependent phosphorylation of ERK1/2 or DOCK2. This study demonstrates that Wnt5a can induce activation of ERK1/2 and enhance CLL-cell proliferation via a ROR1/DOCK2-dependent pathway independent of BTK.

scholarly journals p95vav associates with tyrosine-phosphorylated SLP-76 in antigen-stimulated T cells.

1996 ◽  
Vol 184 (3) ◽  
pp. 1161-1166 ◽  
Author(s):  
L Tuosto ◽  
F Michel ◽  
O Acuto
Keyword(s):  
T Cell ◽  
Gene Activation ◽  
Cell Receptor ◽  
Pleckstrin Homology Domain ◽  
Binding Motif ◽  
Nucleotide Exchange ◽  
Tcr Signaling ◽  
Nucleotide Exchange Factor ◽  
Independent Observations

p95vav, the product of the vav protooncogene, has been implicated in the T cell receptor (TCR)-mediated signaling cascade p95vav is phosphorylated on tyrosine residues after TCR stimulation by anti-TCR/CD3 antibodies and possesses a number of landmark features of signaling molecules such as a putative guanine nucleotide exchange factor domain, a pleckstrin homology domain, and an Sre homology (SH) 2 and two SH3 domains, which provide the capacity to form multimeric signaling complexes. However, the precise role of p95vav in TCR signaling remains unclear. In this work we show that physiological stimulation of T cell hybridomas with antigen presented by major histocompatibility complex class II molecules leads to a strong tyrosine phosphorylation of p95vav and its association with tyrosine-phosphorylated SLP-76. SLP-76 is a newly described SH2-containing protein that has been previously found to bind to the adapter molecule Grb2. Moreover, we provide evidence that p95vav-SI P-76 association is SH2-mediated by demonstrating that this interaction can be inhibited by a phosphopeptide containing a putative p95vav-SH2-binding motif (pYESP) present in SLP-76. Furthermore, in vitro experiments show that after antigen stimulation, phosphorylated p95vav-SLP-76 can bind to Grb2 in a complex that contains pp36/38 and pp116 proteins. Our data provide a clue to explain recent independent observations that overexpression of p95vav or SLP-76 enhances TCR-mediated gene activation.

scholarly journals Early Thymocyte Development Is Regulated by Modulation of E2a Protein Activity

2001 ◽  
Vol 194 (6) ◽  
pp. 733-746 ◽  
Author(s):  
Isaac Engel ◽  
Carol Johns ◽  
Gretchen Bain ◽  
Richard R. Rivera ◽  
Cornelis Murre
Keyword(s):  
T Cell ◽  
Target Genes ◽  
Cell Lineage ◽  
Cell Receptor ◽  
Binding Activity ◽  
Mitogen Activated Protein Kinase ◽  
Thymocyte Development ◽  
Protein Activity ◽  
Double Positive ◽  
Dna Binding Activity

The E2A gene encodes the E47 and E12 basic helix-loop-helix (bHLH) transcription factors. T cell development in E2A-deficient mice is partially arrested before lineage commitment. Here we demonstrate that E47 expression becomes uniformly high at the point at which thymocytes begin to commit towards the T cell lineage. E47 protein levels remain high until the double positive developmental stage, at which point they drop to relatively moderate levels, and are further downregulated upon transition to the single positive stage. However, stimuli that mimic pre-T cell receptor (TCR) signaling in committed T cell precursors inhibit E47 DNA-binding activity and induce the bHLH inhibitor Id3 through a mitogen-activated protein kinase kinase–dependent pathway. Consistent with these observations, a deficiency in E2A proteins completely abrogates the developmental block observed in mice with defects in TCR rearrangement. Thus E2A proteins are necessary for both initiating T cell differentiation and inhibiting development in the absence of pre-TCR expression. Mechanistically, these data link pre-TCR mediated signaling and E2A downstream target genes into a common pathway.

Identification of CalDAG-GEFI as an intracellular target for the vicinal dithiol binding agent phenylarsine oxide in human platelets

2014 ◽  
Vol 111 (05) ◽  
pp. 892-901 ◽  
Author(s):  
Chih-Yun Kuo ◽  
Hui-Chun Wang ◽  
Po-Hsiung Kung ◽  
Chi-Yu Lu ◽  
Chieh-Yu Liao ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Human Platelets ◽  
Nucleotide Exchange ◽  
Phenylarsine Oxide ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Intracellular Target ◽  
Potential Strategy ◽  
Rap1 Activation

SummaryCalDAG-GEFI, a guanine nucleotide exchange factor activating Rap1, is known to play a key role in Ca2+-dependent glycoprotein (GP)IIb/IIIa activation and platelet aggregation. Although inhibition of CalDAG-GEFI could be a potential strategy for antiplatelet therapy, no inhibitor of this protein has been identified. In the present study, phenylarsine oxide (PAO), a vicinal dithiol blocker, potently prevented Rap1 activation in thrombin-stimulated human platelets without significantly inhibiting intracellular Ca2+ mobilisation and protein kinase C activation. PAO also prevented the Ca2+ ionophore-induced Rap1 activation and platelet aggregation, which are dependent on CalDAG-GEFI. In the biotin-streptavidin pull-down assay, CalDAG-GEFI was efficiently pull-downed by streptavidin beads from the lysates of biotin-conjugated PAO-treated platelets, suggesting that PAO binds to intracellular CalDAG-GEFI with high affinity. The above effects of PAO were reversed by a vicinal dithiol compound 2,3-dimercaptopropanol. In addition, CalDAG-GEFI formed disulfide-linked oligomers in platelets treated with the thiol-oxidant diamide, indicating that CalDAG-GEFI contains redox-sensitive thiols. In a purified recombinant protein system, PAO directly inhibited CalDAG-GEFI-stimulated GTP binding to Rap1. Using CalDAG-GEFI and Rap1-overexpressed human embryonic kidney 293T cells, we further confirmed that PAO abolished Ca2+-mediated Rap1 activation. Taken together, these results have demonstrated that CalDAG-GEFI is one of the targets of action of PAO, and propose an important role of vicinal cysteines for the functions of CalDAG-GEFI.

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