ABSTRACT
Precursor-B-cell receptor (pre-BCR) signaling and spleen tyrosine kinase (SYK) recently were introduced as therapeutic targets for patients with B-cell acute lymphoblastic leukemia (B-ALL), but the importance of this pathway in B-ALL subsets and mechanism of downstream signaling have not fully been elucidated. Here, we provide new detailed insight into the mechanism of pre-BCR signaling in B-ALL. We compared the effects of pharmacological and genetic disruption of pre-BCR signaling in vitro and in mouse models for B-ALL, demonstrating exquisite dependency of pre-BCR(+) B-ALL, but not other B-ALL subsets, on this signaling pathway. We demonstrate that SYK, PI3K/AKT, FOXO1 and MYC are important downstream mediators of pre-BCR signaling in B-ALL. Furthermore, we define a characteristic immune phenotype and gene expression signature of pre-BCR(+) ALL to distinguish them from other B-ALL subsets. These data provide comprehensive new insight into pre-BCR signaling in B-ALL and corroborate pre-BCR signaling and SYK as promising new therapeutic targets in pre-BCR(+) B-ALL.
Subject(s)
Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor Cells, B-Lymphoid/chemistry , Receptors, Antigen, B-Cell/metabolism , Signal Transduction , Animals , Cell Line , Forkhead Box Protein O1/metabolism , Heterografts , Humans , Mice , Phosphatidylinositol 3-Kinases/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Proto-Oncogene Proteins c-myc/metabolism , Syk Kinase/metabolismABSTRACT
Syk is a protein tyrosine kinase that couples B-cell receptor (BCR) activation with downstream signaling pathways, affecting cell survival and proliferation. Moreover, Syk is involved in BCR-independent functions, such as B-cell migration and adhesion. In chronic lymphocytic leukemia (CLL), Syk becomes activated by external signals from the tissue microenvironment, and was targeted in a first clinical trial with R788 (fostamatinib), a relatively nonspecific Syk inhibitor. Here, we characterize the activity of two novel, highly selective Syk inhibitors, PRT318 and P505-15, in assays that model CLL interactions with the microenvironment. PRT318 and P505-15 effectively antagonize CLL cell survival after BCR triggering and in nurse-like cell-co-cultures. Moreover, they inhibit BCR-dependent secretion of the chemokines CCL3 and CCL4 by CLL cells, and leukemia cell migration toward the tissue homing chemokines CXCL12, CXCL13, and beneath stromal cells. PRT318 and P505-15 furthermore inhibit Syk and extracellular signal-regulated kinase phosphorylation after BCR triggering. These findings demonstrate that the selective Syk inhibitors PRT318 and P505-15 are highly effective for inhibition of CLL survival and tissue homing circuits, and support the therapeutic development of these agents in patients with CLL, other B-cell malignancies and autoimmune disorders.
Subject(s)
Cell Movement/drug effects , Cyclohexylamines/pharmacology , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrimidines/pharmacology , Apoptosis/drug effects , Blotting, Western , Cell Adhesion/drug effects , Cell Proliferation/drug effects , Chemotaxis , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/enzymology , Phosphorylation/drug effects , Protein-Tyrosine Kinases/metabolism , Stromal Cells/cytology , Stromal Cells/drug effects , Stromal Cells/metabolism , Syk Kinase , Tumor Cells, CulturedABSTRACT
Efalizumab (Raptiva) is a humanized CD11a-specific monoclonal antibody that was recently approved for the treatment of moderate to severe psoriasis. In psoriasis patients, the rate of efalizumab clearance from serum is related to T-cell surface expression of CD11a, suggesting a receptor-mediated clearance model for efalizumab (Bauer et al., 1999). However, limited experimental data are available to explain how the interaction with CD11a results in the systemic clearance of efalizumab. The following studies were designed to test the hypothesis that one mechanism of anti-CD11a antibody clearance is mediated in part by cellular internalization. This was tested in vitro using purified mouse and human T-cells as a model to study the cellular uptake and clearance of anti-CD11a antibodies. Data from these studies suggest that anti-CD11a antibodies are internalized by purified T-cells. Upon internalization, the antibodies appeared to be targeted to lysosomes and were cleared from within the cells in a time-dependent manner. CD11a-mediated internalization and lysosomal targeting of efalizumab may constitute one pathway by which this antibody is cleared in vivo.
Subject(s)
Antibodies, Anti-Idiotypic/metabolism , Antibodies, Monoclonal/metabolism , CD11a Antigen/immunology , Endocytosis/physiology , T-Lymphocytes/metabolism , Animals , Antibodies, Monoclonal, Humanized , Biological Transport , Drug Interactions , Endocytosis/drug effects , Humans , Macrolides/pharmacology , Mice , Subcellular Fractions/metabolism , T-Lymphocytes/drug effectsABSTRACT
Ets factors are members of an ancient multigene family of transcription factors including oncoproteins and possibly tumor suppressors. We previously characterized a novel divergent ets gene, Ehf (ets homologous factor) in mice. Here we report the cDNA sequence, chromosomal location, and tissue/tumor expression patterns of the human EHF gene and the regulatory activity of the EHF protein. EHF maps to 11p12, which is deleted in many prostate, breast, and lung carcinomas and is a hot spot for inherited deletion- or amplification-associated developmental defects. EHF is differentially expressed in normal tissues and carcinomas and between tumor stages and is most highly expressed in the organs known to form carcinomas upon 11p12 deletion. EHF protein represses the ETS-2 induced activity of both stromelysin-1 and collagenase-1 promoters. These data suggest that EHF may contribute to human development and carcinogenesis and is a candidate for the 11p12 tumor suppressor gene.
Subject(s)
Chromosomes, Human, Pair 11 , Gene Expression Regulation, Neoplastic , Neoplasms/metabolism , Transcription Factors/genetics , Amino Acid Sequence , Base Sequence , Chromosome Mapping , DNA, Complementary/analysis , Humans , Matrix Metalloproteinase 1/genetics , Matrix Metalloproteinase 1/metabolism , Matrix Metalloproteinase 3/genetics , Matrix Metalloproteinase 3/metabolism , Molecular Sequence Data , Promoter Regions, Genetic/physiology , Tissue Distribution , Transcription Factors/biosynthesis , Transcription Factors/physiologyABSTRACT
Cocaine and cocaine-associated cues elicit craving in addicts and reinstate cocaine-seeking behavior in rats. Craving and cocaine-seeking behavior may be mediated by withdrawal-induced changes in dopamine (DA) neurotransmission in the amygdala. To examine whether there are concomittant changes in cocaine-seeking behavior and extracellular DA levels during withdrawal, experimental rats were trained to self-administer cocaine (0.75 mg/kg i.v.). After 14 daily 3-hour training sessions, animals underwent either a 1-day, 1-week, or 1-month withdrawal period. Extracellular DA levels were assessed during baseline, extinction, cue reinstatement, and cocaine (15 mg/kg i.p.) reinstatement of cocaine-seeking behavior (i.e., defined as the difference in nonreinforced lever presses on an active minus inactive lever). Cocaine-seeking behavior became more intense during the course of cocaine withdrawal. Additionally, basal and cocaine-induced extracellular DA levels were enhanced after the 1-month withdrawal period. We suggest that the former may reflect a persistent elevation in tonic extracellular DA levels in the amygdala, whereas the latter may reflect a persistent elevation in phasic extracellular DA levels.
