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

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.

Noncanonical ß-catenin interactions promote leukemia-initiating activity in early T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a T-cell malignancy characterized by cell subsets and enriched with leukemia-initiating cells (LICs). ß-Catenin modulates LIC activity in T-ALL. However, its role in maintaining established leukemia stem cells remains largely unknown. To identify functionally relevant protein interactions of ß-catenin in T-ALL, we performed coimmunoprecipitation followed by liquid chromatography-mass spectrometry. Here, we report that a noncanonical functional interaction of ß-catenin with the Forkhead box O3 (FOXO3) transcription factor positively regulates LIC-related genes, including the cyclin-dependent kinase 4, which is a crucial modulator of cell cycle and tumor maintenance. We also confirm the relevance of these findings using stably integrated fluorescent reporters of ß-catenin and FOXO3 activity in patient-derived xenografts, which identify minor subpopulations with enriched LIC activity. In addition, gene expression data at the single-cell level of leukemic cells of primary patients at the time of diagnosis and minimal residual disease (MRD) up to 30 days after the standard treatments reveal that the expression of ß-catenin- and FOXO3-dependent genes is present in the CD82+CD117+ cell fraction, which is substantially enriched with LICs in MRD as well as in early T-cell precursor ALL. These findings highlight key functional roles for ß-catenin and FOXO3 and suggest novel therapeutic strategies to eradicate aggressive cell subsets in T-ALL.

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.

TRIM8 interacts with KIF11 and KIFC1 and controls bipolar spindle formation and chromosomal stability.

The faithful inheritance of chromosomes is essential for the propagation of organisms. In eukaryotes, central to this process is the mitotic spindle. Recently, we have identified TRIM8 as a gene aberrantly expressed in gliomas whose expression reduces the clonogenic potential in the patients' glioma cells. TRIM8 encodes an E3 ubiquitin ligase involved in various pathological processes, including hypertrophy, antiviral defense, encephalopathy, and cancer development. To gain insights into the TRIM8 functions, we characterized the TRIM8 interactome in primary mouse embryonic neural stem cells using proteomics. We found that TRIM8 interacts with KIFC1, and KIF11/Eg5, two master regulators of mitotic spindle assembly and cytoskeleton reorganization. By exploring the TRIM8 role in the mitotic spindle machinery, we showed that TRIM8 localizes at the mitotic spindle during mitosis and plays a role in centrosome separation at the beginning of mitosis with a subsequent delay of the mitotic progression and impact on chromosomal stability.

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

Transforming growth factor ß-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.

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

Acute leukemias are aggressive malignancies of developmentally arrested hematopoietic progenitors. We sought here to explore the possibility that changes in hematopoietic stem/progenitor cells during development might alter the biology of leukemias arising from this tissue compartment. Using a mouse model of acute T cell leukemia, we found that leukemias generated from fetal liver (FL) and adult bone marrow (BM) differed dramatically in their leukemia stem cell activity with FL leukemias showing markedly reduced serial transplantability as compared to BM leukemias. We present evidence that this difference is due to NOTCH1-driven autocrine IGF1 signaling, which is active in FL cells but restrained in BM cells by EZH2-dependent H3K27 trimethylation. Further, we confirmed this mechanism is operative in human disease and show that enforced IGF1 signaling effectively limits leukemia stem cell activity. These findings demonstrate that resurrecting dormant fetal programs in adult cells may represent an alternate therapeutic approach in human cancer.

The mechanism of alternative splicing of the X-linked NDUFB11 gene of the respiratory chain complex I, impact of rotenone treatment in neuroblastoma cells.

A study is presented on the regulation of alternative splicing (AS) of the Ndufb11 gene of complex I of the mitochondrial respiratory chain and the impact on this process of rotenone treatment in neuroblastoma cells. In physiological conditions the Ndufb11 gene produces at high level a short transcript isoform encoding for a 153 aa protein. This subunit is essential for the assembly of a functional and stable mammalian complex I. The gene produces also, at low level, a longer transcript isoform encoding for a 163 aa protein whose role is unknown. Evidence is presented here showing that the level of the two isoforms is regulated by three DGGGD ESS elements located in exon 2 which can bind the hnRNPH1 protein. In neuronal cells rotenone treatment affects the Ndufb11 alternative splicing pathway, with the increase of the 163/153 mRNAs ratio. This effect appears to be due to the down-regulation of the hnRNPH1 protein. Since rotenone induces apoptosis in neuronal cells, the post-transcriptional regulation of the Ndufb11 gene can be involved in the programmed cell death process.