Extracellular vesicle microRNAs contribute to Notch signaling pathway in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive T-cell malignancy characterized by genotypically-defined and phenotypically divergent cell populations, governed by adaptive landscapes. Clonal expansions are associated to genetic and epigenetic events, and modulation of external stimuli that affect the hierarchical structure of subclones and support the dynamics of leukemic subsets. Recently, small extracellular vesicles (sEV) such as exosomes were also shown to play a role in leukemia. Here, by coupling miRNome, bulk and single cell transcriptome profiling, we found that T-ALL-secreted sEV contain NOTCH1-dependent microRNAs (EV-miRs), which control oncogenic pathways acting as autocrine stimuli and ultimately promoting the expansion/survival of highly proliferative cell subsets of human T-cell leukemias. Of interest, we found that NOTCH1-dependent EV-miRs mostly comprised members of miR-17-92a cluster and paralogues, which rescued in vitro the proliferation of T-ALL cells blocked by γ-secretase inhibitors (GSI) an regulated a network of genes characterizing patients with relapsed/refractory early T-cell progenitor (ETP) ALLs. All these findings suggest that NOTCH1 dependent EV-miRs may sustain the growth/survival of immunophenotypically defined cell populations, altering the cell heterogeneity and the dynamics of T-cell leukemias in response to conventional therapies

Epigenetic Restoration of Fetal-like IGF1 Signaling Inhibits Leukemia Stem Cell Activity

n/

Characterization of the potential role of myosin IIa in parkin translocation during mitopahgy

Mitochondria are important organelles of eukaryotic cells that provide energy through cellular respiration. Cells have evolved several quality control mechanisms to preserve functional mitochondria and avoid cell damage. Damaged mitochondria are recognized and removed by mitophagy to avoid the production of reactive oxygen species. A major signal for the recognition of damaged mitochondria is the electrical membrane potential depolarization, which leads to the recruitment of PTEN-induced putative kinase 1 (PINK1), followed by the recruitment of the E3 ubiquitin ligase parkin at the outer mitochondrial membrane. Parkin ubiquitinates numerous mitochondrial outer membrane proteins and initiates mitophagy. Mutations in the gene encoding parkin are frequently found in familiar forms of Parkinson’s disease. Although several factors involved in parkin mitochondrial recruitment have been characterized, additional proteins may be involved. The aim of this work was to determine whether other factors may be involved in colocalizing parkin to damaged mitochondria. Following up a SILAC-immunoprecipitation experiment, we hypothesized that an unconventional myosin (myosin IIa) may be involved in the recruitment of parkin to the mitochondria. Myosins are a large family of actin-based cytoskeletal motors that use energy derived from ATP hydrolysis to generate movement. Non-conventional myosins are well studied for their contribution to synaptic function in neuronal cells. MYH9 was identified as potential interactor of parkin by mass spectrometry. This interaction was validated through co-immunoprecipitation and immunofluorescence experiments. In addition, myosin alteration with small chemicals that depolymerize actin filament or inhibit myosin activity, impaired parkin localization to mitochondria upon stress. This study potentially implicates myosin IIa as a modulator of parkin recruitment to the mitochondria, and may thus open the door to new therapeutic strategies for Parkinson’s disease.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat

The Notch1 signaling pathway directly modulates the human RANKL-induced osteoclastogenesis

Abstract Notch signaling is an evolutionary conserved pathway with a key role in tissue homeostasis, differentiation and proliferation. It was reported that Notch1 receptor negatively regulates mouse osteoclast development and formation by inhibiting the expression of macrophage colony-stimulating factor in mesenchymal cells. Nonetheless, the involvement of Notch1 pathway in the generation of human osteoclasts is still controversial. Here, we report that the constitutive activation of Notch1 signaling induced a differentiation block in human mononuclear CD14+ cells directly isolated from peripheral blood mononuclear cells (PBMCs) upon in vitro stimulation to osteoclasts. Additionally, using a combined approach of single-cell RNA sequencing (scRNA-Seq) simultaneously with a panel of 31 oligo-conjugated antibodies against cell surface markers (AbSeq assay) as well as unsupervised learning methods, we detected four different cell stages of human RANKL-induced osteoclastogenesis after 5 days in which Notch1 signaling enforces the cell expansion of specific subsets. These cell populations were characterized by distinct gene expression and immunophenotypic profiles and active Notch1, JAK/STAT and WNT signaling pathways. Furthermore, cell–cell communication analyses revealed extrinsic modulators of osteoclast progenitors including the IL7/IL7R and WNT5a/RYK axes. Interestingly, we also report that Interleukin-7 receptor (IL7R) was a downstream effector of Notch1 pathway and that Notch1 and IL7R interplay promoted cell expansion of human RANKL-induced osteoclast progenitors. Taken together, these findings underline a novel cell pattern of human osteoclastogenesis, outlining the key role of Notch1 and IL-7R signaling pathways

TAB2 c.1398dup variant leads to haploinsufficiency and impairs extracellular matrix homeostasis

Transforming growth factor β-activated kinase 1 (TAK1) mediates multiple biological processes through the nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and the mitogen-activated protein kinase (MAPK) signaling pathways. TAK1 activation is tightly regulated by its binding partners (TABs). In particular, binding with TAB2 is crucial for cardiovascular development and extracellular matrix (ECM) homeostasis. In our previous work, we reported a novel multisystem disorder associated with the heterozygous TAB2 c.1398dup variant. Here, we dissect the functional effects of this variant in order to understand its molecular pathogenesis. We demonstrate that TAB2 c.1398dup considerably undergoes to nonsense-mediated messenger RNA decay and encodes a truncated protein that loses its ability to bind TAK1. We also show an alteration of the TAK1 autophosphorylation status and of selected downstream signaling pathways in patients' fibroblasts. Immunofluorescence analyses and ECM-related polymerase chain reaction-array panels highlight that patient fibroblasts display ECM disorganization and altered expression of selected ECM components and collagen-related pathways. In conclusion, we deeply dissect the molecular pathogenesis of the TAB2 c.1398dup variant and show that the resulting phenotype is well explained by TAB2 loss-of-function. Our data also offer initial insights on the ECM homeostasis impairment as a molecular mechanism probably underlying a multisystem disorder linked to TAB2