The Notch1/c-Myc Pathway in T Cell Leukemia

The transmembrane receptor Notch1 is a member of the evolutionarily conserved family of developmental regulators originally identified in Drosophila melanogaster. Notch signaling plays essential roles in regulating cell fate in thymic, intestinal, vascular and neuronal development (1-5). Recent studies detect mutations in the Notch1 receptor in roughly half of patients with T cell acute lymphoblastic leukemia (T-ALL) (6). Although expression of an activated Notch1 allele has been shown to cause leukemia in mice, the molecular mechanisms whereby Notch1 mediates cellular transformation are unknown (7). To understand how Notch1 contributes to T cell leukemogenesis, we generated mouse leukemic cell lines where the expression of activated Notch1 was doxycycline-regulated. This cell line was used for gene expression profiling to specifically identify Notch1-regulated genes in leukemia. These studies revealed that Notch1 directly induces the expression of c-myc and that inhibition of Notch1 results in cell cycle arrest and apoptosis and decreased c-myc levels (8). These studies and those performed by Aster, Pear and colleagues in human T-ALL cell lines demonstrate that the direct Notch1-mediated activation of c-myc is required to maintain leukemic growth (8-10). Interestingly, the Notch1/c-Myc oncogenic pathway does not appear limited to T-ALL, as studies by the Efstratiadis group show that expression of intracellular Notch1 leads to mammary tumorigenesis and importantly, transformation appears at least partially c-myc dependent (11). Collectively, these studies begin to delineate how Notch1 mediates cellular transformation and raises the possibility that the Notch1/c-Myc pathway may contribute to human breast cancer and potentially other solid tumors.     

KSHV Manipulates Notch Signaling by DLL4 and JAG1 to Alter Cell Cycle Genes in Lymphatic Endothelia

Increased expression of Notch signaling pathway components is observed in Kaposi sarcoma (KS) but the mechanism underlying the manipulation of the canonical Notch pathway by the causative agent of KS, Kaposi sarcoma herpesvirus (KSHV), has not been fully elucidated. Here, we describe the mechanism through which KSHV directly modulates the expression of the Notch ligands JAG1 and DLL4 in lymphatic endothelial cells. Expression of KSHV-encoded vFLIP induces JAG1 through an NFκB-dependent mechanism, while vGPCR upregulates DLL4 through a mechanism dependent on ERK. Both vFLIP and vGPCR instigate functional Notch signalling through NOTCH4. Gene expression profiling showed that JAG1- or DLL4-stimulated signaling results in the suppression of genes associated with the cell cycle in adjacent lymphatic endothelial cells, indicating a role for Notch signaling in inducing cellular quiescence in these cells. Upregulation of JAG1 and DLL4 by KSHV could therefore alter the expression of cell cycle components in neighbouring uninfected cells during latent and lytic phases of viral infection, influencing cellular quiescence and plasticity. In addition, differences in signaling potency between these ligands suggest a possible complementary role for JAG1 and DLL4 in the context of KS.     

Hes1 expression and CYLD repression are essential events downstream of Notch1 in T-cell leukemia

Notch activation is a current event in T Acute Lymphoblastic Leukemia (T-ALL) but the downstream elements that are able to support Notch-dependent leukemias are not well characterized. We have recently shown that the Notch-Hes1-CYLD-NFκB axis is crucial in the maintenance of T-ALL, but detailed evaluation of the contribution of each one of these elements is still missing. Here we use a Notch1-induced leukemia in vivo model to study the effect of silencing the Notch-target gene, Hes1 or overexpressing the Hes1-target, CYLD. We here show that both strategies completely abolish the ability of constitutive active Notch1 to generate T-ALL.     

Hes1 expression and CYLD repression are essential events downstream of Notch1 in T-cell leukemia

Notch activation is a current event in T Acute Lymphoblastic Leukemia (T-ALL) but the downstream elements that are able to support Notch-dependent leukemias are not well characterized. We have recently shown that the Notch-Hes1-CYLD-NFκB axis is crucial in the maintenance of T-ALL, but detailed evaluation of the contribution of each one of these elements is still missing. Here we use a Notch1-induced leukemia in vivo model to study the effect of silencing the Notch-target gene, Hes1 or overexpressing the Hes1-target, CYLD. We here show that both strategies completely abolish the ability of constitutive active Notch1 to generate T-ALL.     

Notch signaling via Hes1 transcription factor maintains survival of melanoblasts and melanocyte stem cells

Melanoblasts (Mbs) are thought to be strictly regulated by cell-cell interactions with epidermal keratinocytes, although the precise molecular mechanism of the regulation has been elusive. Notch signaling, whose activation is mediated by cell-cell interactions, is implicated in a broad range of developmental processes. We demonstrate the vital role of Notch signaling in the maintenance of Mbs, as well as melanocyte stem cells (MSCs). Conditional ablation of Notch signaling in the melanocyte lineage leads to a severe defect in hair pigmentation, followed by intensive hair graying. The defect is caused by a dramatic elimination of Mbs and MSCs. Furthermore, targeted overexpression of Hes1 is sufficient to protect Mbs from the elimination by apoptosis. Thus, these data provide evidence that Notch signaling, acting through Hes1, plays a crucial role in the survival of immature Mbs by preventing initiation of apoptosis.     

PIM Kinases as Potential Therapeutic Targets in a Subset of Peripheral T Cell Lymphoma Cases

Currently, there is no efficient therapy for patients with peripheral T cell lymphoma (PTCL). The Proviral Integration site of Moloney murine leukemia virus (PIM) kinases are important mediators of cell survival. We aimed to determine the therapeutic value of PIM kinases because they are overexpressed in PTCL patients, T cell lines and primary tumoral T cells. PIM kinases were inhibited genetically (using small interfering and short hairpin RNAs) and pharmacologically (mainly with the pan-PIM inhibitor (PIMi) ETP-39010) in a panel of 8 PTCL cell lines. Effects on cell viability, apoptosis, cell cycle, key proteins and gene expression were evaluated. Individual inhibition of each of the PIM genes did not affect PTCL cell survival, partially because of a compensatory mechanism among the three PIM genes. In contrast, pharmacological inhibition of all PIM kinases strongly induced apoptosis in all PTCL cell lines, without cell cycle arrest, in part through the induction of DNA damage. Therefore, pan-PIMi synergized with Cisplatin. Importantly, pharmacological inhibition of PIM reduced primary tumoral T cell viability without affecting normal T cells ex vivo. Since anaplastic large cell lymphoma (ALK+ ALCL) cell lines were the most sensitive to the pan-PIMi, we tested the simultaneous inhibition of ALK and PIM kinases and found a strong synergistic effect in ALK+ ALCL cell lines. Our findings suggest that PIM kinase inhibition could be of therapeutic value in a subset of PTCL, especially when combined with ALK inhibitors, and might be clinically beneficial in ALK+ ALCL.     

Adropin preserves the blood‐brain barrier through a Notch1/Hes1 pathway after intracerebral hemorrhage in mice

Adropin is expressed in the CNS and plays a crucial role in the development of stroke. However, little is currently known about the effects of adropin on the blood‐brain barrier (BBB) function after intracerebral hemorrhage (ICH). In this study, the role of adropin in collagenase‐induced ICH was investigated in mice. At 1‐h post‐ICH, mice were administered with recombinant human adropin by intranasal. Brain water +content, BBB permeability, and neurological function were measured at different time intervals. Proteins were quantified using western blot analysis, and the localizations of adropin and Notch1 were visualized via immunofluorescence staining. It is shown that adropin reduced brain water content and improved neurological functions. Adropin preserved the functionality of BBB by increasing N‐cadherin expression and reducing extravasation of albumin. Moreover, in vivo knockdown of Notch1 and Hes1 both abolished the protective effects of adropin. Taken together, our data demonstrate that adropin constitutes a potential treatment value for ICH by preserving BBB and improving functional outcomes through the Notch1 signaling pathway.

     

R-Loops Enhance Polycomb Repression at a Subset of Developmental Regulator Genes

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Caveolin-1 promote astroglial differentiation of neural stem/progenitor cells through modulating Notch1/NICD and Hes1 expressions

In the present study, we aim to elucidate the role of caveolin-1 in modulating astroglial differentiation of neural progenitor cells (NPCs) and the potential mechanisms involved. We first investigated astroglial differentiation and Notch signaling by detecting the expressions of S100β, GFAP, NICD and hairy enhancer of split 1 (Hes1) in the brains of wild-type and caveolin-1 knockout mice. Caveolin-1 knockout mice revealed remarkably less astroglial differentiation and lower levels of NICD and Hes1 expressions than wild type mice. We then studied the potential roles of caveolin-1 in modulating NICD and Hes1 expressions and astroglial differentiation in isolated cultured NPCs by using caveolin-1 peptide and caveolin-1 RNA silencing. In the differentiating NPCs, caveolin-1 peptide markedly promoted astroglial formation and up-regulated the expressions of NICD and Hes1. In contrast, the knockdown of caveolin-1 inhibited astroglial differentiation of NPCs and the expressions of NICD and Hes1. Taken together, these results provide strong evidence that caveolin-1 can promote astroglial differentiation of NPCs through modulating Notch1/NICD and Hes1 expressions.