Introduction. Deregulated Notch1 signaling has a fundamental role in the pathogenesis of T cell acute lymphoblastic leukemia (T-ALL), in which the majority of human and murine tumors have acquired acti- vating mutations that lead to aberrant increases in Notch1 signaling. NOTCH1 encodes a transmembrane receptor acting as a ligand-acti- vated transcription factor and playing an important role in cell differ- entiation, proliferation, and apoptosis. Notch1 signaling initiates when the ligand, from either the Jagged or Delta families, binds to the receptor and induces successive proteolytic cleavages, resulting in the release and nuclear translocation of the Notch1 intra-cellular domain (NICD). In the nucleus, NICD assembles a transcriptional complex, leading to de-repression/activation of specific target genes. Besides this “canonical” signaling pathway involving ligand-induced cleavage of Notch for transcriptional regulation, a ligand- or transcription-inde- pendent (non-canonical) function of Notch has also been reported in various systems across species. However, in most cases, the key medi- ators of non-canonical Notch signals are unclear, and the proposed mechanisms appear to vary with context. In T-ALL, Notch non-canon- ical signals and their possible cytoplasmic mediators have been not investigated so far. Methods. To study the non-canonical Notch signal- ing pathway in T-ALL, we used multi-parametric phospho-flow cytometry to simultaneously determine protein expression and pro- tein post-translational modifications (i.e. phosphorylation) at a single cell level in T-ALL cell lines stimulated by the Notch1 ligand Jagged1. Then, protein expression of specific Notch target genes, as readout of Notch1 signaling activation, has been analyzed at slower time points whilst phosphorylation of signaling proteins, which are crucial for T- ALL pathogenesis, has been measured at rapid time points, to identi- fy possible cytoplasmic mediators of the non-canonical Notch1 signaling. Results. We showed that, besides the expression of specific Notch target proteins, Notch1 activation induced the rapid phospho- rylation of cytoplasmic proteins, namely STAT3, Akt, and Rb. Moreover, Notch1 activation evoked heterogeneous signaling profiles across different cell lines, which recapitulate ontogenetic stages in T- ALL. Conclusions. This study showed that “non canonical” cytoplasmic signals are induced in T-ALL in addition to slower transcriptional responses traditionally attributed to Notch activation. In addition, multi-parametric phospho-flow cytometry enables to distinguish Notch signaling network profiles that are associated with the T-ALL ontogeny stages. Identifying cytoplasmic mediators of the Notch non- canonical pathway as biologically relevant signaling hubs may repre- sent new challenges for anti-Notch1 therapy in T-ALL.
NON-CANONICAL NOTCH SIGNALING IN T-ALL: A PHOSPHO-FLOW CYTOMETRY STUDY
CAVALLINI, Chiara;PERBELLINI, Omar;ZORATTI, Elisa;LOVATO, Ornella;PIZZOLO, Giovanni;SCUPOLI, Maria
2014-01-01
Abstract
Introduction. Deregulated Notch1 signaling has a fundamental role in the pathogenesis of T cell acute lymphoblastic leukemia (T-ALL), in which the majority of human and murine tumors have acquired acti- vating mutations that lead to aberrant increases in Notch1 signaling. NOTCH1 encodes a transmembrane receptor acting as a ligand-acti- vated transcription factor and playing an important role in cell differ- entiation, proliferation, and apoptosis. Notch1 signaling initiates when the ligand, from either the Jagged or Delta families, binds to the receptor and induces successive proteolytic cleavages, resulting in the release and nuclear translocation of the Notch1 intra-cellular domain (NICD). In the nucleus, NICD assembles a transcriptional complex, leading to de-repression/activation of specific target genes. Besides this “canonical” signaling pathway involving ligand-induced cleavage of Notch for transcriptional regulation, a ligand- or transcription-inde- pendent (non-canonical) function of Notch has also been reported in various systems across species. However, in most cases, the key medi- ators of non-canonical Notch signals are unclear, and the proposed mechanisms appear to vary with context. In T-ALL, Notch non-canon- ical signals and their possible cytoplasmic mediators have been not investigated so far. Methods. To study the non-canonical Notch signal- ing pathway in T-ALL, we used multi-parametric phospho-flow cytometry to simultaneously determine protein expression and pro- tein post-translational modifications (i.e. phosphorylation) at a single cell level in T-ALL cell lines stimulated by the Notch1 ligand Jagged1. Then, protein expression of specific Notch target genes, as readout of Notch1 signaling activation, has been analyzed at slower time points whilst phosphorylation of signaling proteins, which are crucial for T- ALL pathogenesis, has been measured at rapid time points, to identi- fy possible cytoplasmic mediators of the non-canonical Notch1 signaling. Results. We showed that, besides the expression of specific Notch target proteins, Notch1 activation induced the rapid phospho- rylation of cytoplasmic proteins, namely STAT3, Akt, and Rb. Moreover, Notch1 activation evoked heterogeneous signaling profiles across different cell lines, which recapitulate ontogenetic stages in T- ALL. Conclusions. This study showed that “non canonical” cytoplasmic signals are induced in T-ALL in addition to slower transcriptional responses traditionally attributed to Notch activation. In addition, multi-parametric phospho-flow cytometry enables to distinguish Notch signaling network profiles that are associated with the T-ALL ontogeny stages. Identifying cytoplasmic mediators of the Notch non- canonical pathway as biologically relevant signaling hubs may repre- sent new challenges for anti-Notch1 therapy in T-ALL.
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