Depression in Patients with Mastocytosis: Prevalence, Features and Effects of Masitinib Therapy

Depression in patients with mastocytosis is often reported but its prevalence and characteristics are not precisely described. In addition, the impact of therapies targeting mast cells proliferation, differentiation and degranulation on psychic symptoms of depression have never been investigated. Our objective was to determine the prevalence and to describe features of depression in a large cohort of mastocytosis patients (n = 288) and to investigate the therapeutic impact of the protein kinase inhibitor masitinib in depression symptoms. The description of depression was based on the analysis of a database with Hamilton scores using Principal Component Analysis (PCA). Efficacy of masitinib therapy was evaluated using non parametric Wilcoxon test for paired data within a three months period (n = 35). Our results show that patients with indolent mastocytosis present an elevated prevalence of depression (64%). Depression was moderate in 56% but severe in 8% of cases. Core symptoms (such as psychic anxiety, depressed mood, work and interests) characterized depression in mastocytosis patients. Masitinib therapy was associated with significant improvement (67% of the cases) of overall depression, with 75% of recovery cases. Global Quality of Life slightly improved after masitinib therapy and did not predicted depression improvement. In conclusion, depression is very frequent in mastocytosis patients and masitinib therapy is associated with the reduction its psychic experiences. We conclude that depression in mastocytosis may originate from processes related to mast cells activation. Masitinib could therefore be a useful treatment for mastocytosis patients with depression and anxiety symptoms

Molecular mechanisms involved in glioblastoma stem cell neurovascular plasticity

Les Glioblastomes (GBM, grade IV selon l’OMS) sont les tumeurs cérébrales primaires les plus agressives et sont caractérisées par une néovascularisation importante associée à l’hypoxie et à la nécrose. L’origine cellulaire des GBM est controversée, mais des sous-populations de cellules multipotentes ont été identifiées au sein des tumeurs, et seraient responsables de la radio/chimiorésistance des GBM. Ces cellules souches de glioblastome (GSC) contrôlent activement la vascularisation tumorale par leur interaction étroite avec les cellules vasculaires composant les niches tumorales. La voie Notch est une signalisation canonique essentielle au développement et à l’homéostasie du système nerveux central et son réseau vasculaire associé. Dans le contexte des GBM, cette cascade serait nécessaire à la gliomagénèse, par le maintien du réservoir de GSC au sein de la niche périvasculaire. Cependant, le mode d’action moléculaire de Notch dans les GBM reste encore à démontrer, du fait de résultats divergents observés dans plusieurs études. Dans la première partie de mon travail de thèse, j’ai contribué à l’exploration de la signalisation Notch1 dans des cultures de GSC établies et caractérisées au sein du laboratoire. Le niveau basal d’activation de Notch1 étant faible dans nos GSC, l’approche a été d’activer constitutivement cet axe par transduction lentivirale. Suite à cette activation forcée, les GSC subissent un changement phénotypique majeur et se différencient en cellules périvasculaires ou cellules « pericyte-like ». Cette transition neurovasculaire des GSC promeut la vascularisation active des tumeurs par la normalisation du réseau vasculaire in vivo. Par la suite, j’ai posé la question des mécanismes moléculaires en aval de Notch1 ; par l’étude des facteurs de transcription TAL1 et SLUG, deux candidats potentiels au contrôle de cette plasticité neurovasculaire. Dans ce but, j’ai examiné leur contribution au phénotype vasculaire des GSC dans un modèle in vitro de la niche périvasculaire ; et in vivo par l’analyse d’échantillons humains de GBM. Enfin, j’ai également observé que l’activation de Notch1 module l’activité de la machinerie du protéasome, ce qui pourrait contribuer activement à la transition moléculaire observée dans les GSC. Ces travaux mettent en avant la plasticité phénotypique des GSC: une meilleure compréhension de ces processus pourrait mener à la conception de thérapies ciblant efficacement les GSC et leur vascularisation associée.Glioblastomas (GBM, WHO grade IV) are highly aggressive brain tumors in which extensive vascularization is associated with hypoxia and necrosis. GBM cell of origin is controversial; however multipotent stem-like subpopulations have been identified within tumors, and could account for GBM radio/chemoresistance. These glioblastoma stem-like cells (GSC) actively promote tumoral vascularization processes by closely interacting with vascular cells composing tumoral niches. The Notch cascade is a canonical signaling pathway required during developmental stages and adult homeostasis of the central nervous system and the associated vascular network. In the context of GBM, this molecular axis could induce gliomagenesis by promoting GSC maintenance in the perivascular niche. However, Notch-induced molecular mechanisms controlling GBM progression still remain elusive, due to divergent results observed in numerous reports. During the first part of my thesis work, I contributed to the assessment of Notch1 functions in GSC cultures isolated and characterized in our lab. Given a low Notch1 basal activation status in our GSCs, our approach was to constitutively activate this axis via lentiviral transduction. Following this forced activation, GSCs undergo drastic phenotypic changes and differenciate into perivascular-like or “pericyte-like” cells. This neurovascular transition of GSCs induces active tumoral vascularization by promoting normalization of the vascular network in vivo. Consequently, I questioned the molecular mechanisms downstream of Notch1 by focusing on TAL1 and SLUG transcription factors, two potential candidates controlling this neurovascular plasticity. For this purpose, I examined their contribution to the GSC vascular-like phenotype in an in vitro model of the perivascular niche; and in vivo by analyzing human GBM samples. Finally, I also observed that Notch1 activation modulates the activity of the proteasomal machinery, which could actively contribute to the molecular transition occurring in GSCs. This work highlights GSC phenotypic plasticity: a better understanding of these processes could lead to the design of therapies efficiently targeting GSCs and their associated vasculature

Mécanismes moléculaires impliqués dans la plasticité neurovasculaire des cellules souches de glioblastome

Glioblastomas (GBM, WHO grade IV) are highly aggressive brain tumors in which extensive vascularization is associated with hypoxia and necrosis. GBM cell of origin is controversial; however multipotent stem-like subpopulations have been identified within tumors, and could account for GBM radio/chemoresistance. These glioblastoma stem-like cells (GSC) actively promote tumoral vascularization processes by closely interacting with vascular cells composing tumoral niches. The Notch cascade is a canonical signaling pathway required during developmental stages and adult homeostasis of the central nervous system and the associated vascular network. In the context of GBM, this molecular axis could induce gliomagenesis by promoting GSC maintenance in the perivascular niche. However, Notch-induced molecular mechanisms controlling GBM progression still remain elusive, due to divergent results observed in numerous reports. During the first part of my thesis work, I contributed to the assessment of Notch1 functions in GSC cultures isolated and characterized in our lab. Given a low Notch1 basal activation status in our GSCs, our approach was to constitutively activate this axis via lentiviral transduction. Following this forced activation, GSCs undergo drastic phenotypic changes and differenciate into perivascular-like or “pericyte-like” cells. This neurovascular transition of GSCs induces active tumoral vascularization by promoting normalization of the vascular network in vivo. Consequently, I questioned the molecular mechanisms downstream of Notch1 by focusing on TAL1 and SLUG transcription factors, two potential candidates controlling this neurovascular plasticity. For this purpose, I examined their contribution to the GSC vascular-like phenotype in an in vitro model of the perivascular niche; and in vivo by analyzing human GBM samples. Finally, I also observed that Notch1 activation modulates the activity of the proteasomal machinery, which could actively contribute to the molecular transition occurring in GSCs. This work highlights GSC phenotypic plasticity: a better understanding of these processes could lead to the design of therapies efficiently targeting GSCs and their associated vasculature.Les Glioblastomes (GBM, grade IV selon l’OMS) sont les tumeurs cérébrales primaires les plus agressives et sont caractérisées par une néovascularisation importante associée à l’hypoxie et à la nécrose. L’origine cellulaire des GBM est controversée, mais des sous-populations de cellules multipotentes ont été identifiées au sein des tumeurs, et seraient responsables de la radio/chimiorésistance des GBM. Ces cellules souches de glioblastome (GSC) contrôlent activement la vascularisation tumorale par leur interaction étroite avec les cellules vasculaires composant les niches tumorales. La voie Notch est une signalisation canonique essentielle au développement et à l’homéostasie du système nerveux central et son réseau vasculaire associé. Dans le contexte des GBM, cette cascade serait nécessaire à la gliomagénèse, par le maintien du réservoir de GSC au sein de la niche périvasculaire. Cependant, le mode d’action moléculaire de Notch dans les GBM reste encore à démontrer, du fait de résultats divergents observés dans plusieurs études. Dans la première partie de mon travail de thèse, j’ai contribué à l’exploration de la signalisation Notch1 dans des cultures de GSC établies et caractérisées au sein du laboratoire. Le niveau basal d’activation de Notch1 étant faible dans nos GSC, l’approche a été d’activer constitutivement cet axe par transduction lentivirale. Suite à cette activation forcée, les GSC subissent un changement phénotypique majeur et se différencient en cellules périvasculaires ou cellules « pericyte-like ». Cette transition neurovasculaire des GSC promeut la vascularisation active des tumeurs par la normalisation du réseau vasculaire in vivo. Par la suite, j’ai posé la question des mécanismes moléculaires en aval de Notch1 ; par l’étude des facteurs de transcription TAL1 et SLUG, deux candidats potentiels au contrôle de cette plasticité neurovasculaire. Dans ce but, j’ai examiné leur contribution au phénotype vasculaire des GSC dans un modèle in vitro de la niche périvasculaire ; et in vivo par l’analyse d’échantillons humains de GBM. Enfin, j’ai également observé que l’activation de Notch1 module l’activité de la machinerie du protéasome, ce qui pourrait contribuer activement à la transition moléculaire observée dans les GSC. Ces travaux mettent en avant la plasticité phénotypique des GSC: une meilleure compréhension de ces processus pourrait mener à la conception de thérapies ciblant efficacement les GSC et leur vascularisation associée

Vascular Transdifferentiation in the CNS: A Focus on Neural and Glioblastoma Stem-Like Cells

International audienceGlioblastomas are devastating and extensively vascularized brain tumors from which glioblastoma stem-like cells (GSCs) have been isolated by many groups. These cells have a high tumorigenic potential and the capacity to generate heterogeneous phenotypes. There is growing evidence to support the possibility that these cells are derived from the accumulation of mutations in adult neural stem cells (NSCs) as well as in oligodendrocyte progenitors. It was recently reported that GSCs could transdifferentiate into endothelial-like and pericyte-like cells both in vitro and in vivo, notably under the influence of Notch and TGFβ signaling pathways. Vascular cells derived from GBM cells were also observed directly in patient samples. These results could lead to new directions for designing original therapeutic approaches against GBM neovascularization but this specific reprogramming requires further molecular investigations. Transdifferentiation of nontumoral neural stem cells into vascular cells has also been described and conversely vascular cells may generate neural stem cells. In this review, we present and discuss these recent data. As some of them appear controversial, further validation will be needed using new technical approaches such as high throughput profiling and functional analyses to avoid experimental pitfalls and misinterpretations

SLUG and Truncated TAL1 Reduce Glioblastoma Stem Cell Growth Downstream of Notch1 and Define Distinct Vascular Subpopulations in Glioblastoma Multiforme

International audienceGlioblastomas (GBM) are high-grade brain tumors, containing cells with distinct phenotypes and tumorigenic potentials, notably aggressive and treatment-resistant multipotent glioblastoma stem cells (GSC). The molecular mechanisms controlling GSC plasticity and growth have only partly been elucidated. Contact with endothelial cells and the Notch1 pathway control GSC proliferation and fate. We used three GSC cultures and glioma resections to examine the expression, regulation, and role of two transcription factors, SLUG (SNAI2) and TAL1 (SCL), involved in epithelial to mesenchymal transition (EMT), hematopoiesis, vascular identity, and treatment resistance in various cancers. In vitro, SLUG and a truncated isoform of TAL1 (TAL1-PP22) were strongly upregulated upon Notch1 activation in GSC, together with LMO2, a known cofactor of TAL1, which formed a complex with truncated TAL1. SLUG was also upregulated by TGF-β1 treatment and by co-culture with endothelial cells. In patient samples, the full-length isoform TAL1-PP42 was expressed in all glioma grades. In contrast, SLUG and truncated TAL1 were preferentially overexpressed in GBMs. SLUG and TAL1 are expressed in the tumor microenvironment by perivascular and endothelial cells, respectively, and to a minor extent, by a fraction of epidermal growth factor receptor (EGFR) -amplified GBM cells. Mechanistically, both SLUG and truncated TAL1 reduced GSC growth after their respective overexpression. Collectively, this study provides new evidence for the role of SLUG and TAL1 in regulating GSC plasticity and growth

Asymmetric Distribution of GFAP in Glioma Multipotent Cells

International audienceAsymmetric division (AD) is a fundamental mechanism whereby unequal inheritance of various cellular compounds during mitosis generates unequal fate in the two daughter cells. Unequal repartitions of transcription factors, receptors as well as mRNA have been abundantly described in AD. In contrast, the involvement of intermediate filaments in this process is still largely unknown. AD occurs in stem cells during development but was also recently observed in cancer stem cells. Here, we demonstrate the asymmetric distribution of the main astrocytic intermediate filament, namely the glial fibrillary acid protein (GFAP), in mitotic glioma multipotent cells isolated from glioblastoma (GBM), the most frequent type of brain tumor. Unequal mitotic repartition of GFAP was also observed in mice non-tumoral neural stem cells indicating that this process occurs across species and is not restricted to cancerous cells. Immunofluorescence and videomicroscopy were used to capture these rare and transient events. Considering the role of intermediate filaments in cytoplasm organization and cell signaling, we propose that asymmetric distribution of GFAP could possibly participate in the regulation of normal and cancerous neural stem cell fate

Notch1 Stimulation Induces a Vascularization Switch With Pericyte-Like Cell Differentiation of Glioblastoma Stem Cells

International audienceGlioblastoma multiforms (GBMs) are highly vascularized brain tumors containing a subpopulation of multipotent cancer stem cells. These cells closely interact with endothelial cells in neurovascular niches. In this study, we have uncovered a close link between the Notch1 pathway and the tumoral vascularization process of GBM stem cells. We observed that although the Notch1 receptor was activated, the typical target proteins (HES5, HEY1, and HEY2) were not or barely expressed in two explored GBM stem cell cultures. Notch1 signaling activation by expression of the intracellular form (NICD) in these cells was found to reduce their growth rate and migration, which was accompanied by the sharp reduction in neural stem cell transcription factor expression (ASCL1, OLIG2, and SOX2), while HEY1/2, KLF9, and SNAI2 transcription factors were upregulated. Expression of OLIG2 and growth were restored after termination of Notch1 stimulation. Remarkably, NICD expression induced the expression of pericyte cell markers (NG2, PDGFRβ, and α-smooth muscle actin [αSMA]) in GBM stem cells. This was paralleled with the induction of several angiogenesis-related factors most notably cytokines (heparin binding epidermal growth factor [HB-EGF], IL8, and PLGF), matrix metalloproteinases (MMP9), and adhesion proteins (vascular cell adhesion molecule 1 [VCAM1], intercellular adhesion molecule 1 [ICAM1], and integrin alpha 9 [ITGA9]). In xenotransplantation experiments, contrasting with the infiltrative and poorly vascularized tumors obtained with control GBM stem cells, Notch1 stimulation resulted in poorly disseminating but highly vascularized grafts containing large vessels with lumen. Notch1-stimulated GBM cells expressed pericyte cell markers and closely associated with endothelial cells. These results reveal an important role for the Notch1 pathway in regulating GBM stem cell plasticity and angiogenic properties

Mapping the N-Terminal Hexokinase-I Binding Site onto Voltage-Dependent Anion Channel-1 To Block Peripheral Nerve Demyelination

International audienceThe voltage-dependent anion channel (VDAC), the most abundant protein on the outer mitochondrial membrane, is implicated in ATP, ion and metabolite exchange with cell compartments. In particular, the VDAC participates in cytoplasmic and mitochondrial Ca2+ homeostasis. Notably, the Ca2+ efflux out of Schwann cell mitochondria is involved in peripheral nerve demyelination that underlies most peripheral neuropathies. Hexokinase (HK) isoforms I and II, the main ligands of the VDAC, possess a hydrophobic N-terminal structured in α-helix (NHKI) that is necessary for the binding to the VDAC. To gain further insight into the molecular basis of HK binding to the VDAC, we developed and optimized peptides based on the NHKI sequence. These modifications lead to an increase of the peptide hydrophobicity and helical content that enhanced their ability to prevent peripheral nerve demyelination. Our results provide new insights into the molecular basis of VDAC/HK interaction that could lead to the development of therapeutic compounds for demyelinating peripheral neuropathies

Transformation Foci in IDH1-mutated Gliomas Show STAT3 Phosphorylation and Downregulate the Metabolic Enzyme ETNPPL, a Negative Regulator of Glioma Growth

International audienceIDH1-mutated gliomas are slow-growing brain tumours which progress into high-grade gliomas. The early molecular events causing this progression are ill-defined. Previous studies revealed that 20% of these tumours already have transformation foci. These foci offer opportunities to better understand malignant progression. We used immunohistochemistry and high throughput RNA profiling to characterize foci cells. These have higher pSTAT3 staining revealing activation of JAK/STAT signaling. They downregulate RNAs involved in Wnt signaling (DAAM2, SFRP2), EGFR signaling (MLC1), cytoskeleton and cell-cell communication (EZR, GJA1). In addition, foci cells show reduced levels of RNA coding for Ethanolamine-Phosphate Phospho-Lyase (ETNPPL/AGXT2L1), a lipid metabolism enzyme. ETNPPL is involved in the catabolism of phosphoethanolamine implicated in membrane synthesis. We detected ETNPPL protein in glioma cells as well as in astrocytes in the human brain. Its nuclear localization suggests additional roles for this enzyme. ETNPPL expression is inversely correlated to glioma grade and we found no ETNPPL protein in glioblastomas. Overexpression of ETNPPL reduces the growth of glioma stem cells indicating that this enzyme opposes gliomagenesis. Collectively, these results suggest that a combined alteration in membrane lipid metabolism and STAT3 pathway promotes IDH1-mutated glioma malignant progression

A novel 3D nanofibre scaffold conserves the plasticity of glioblastoma stem cell invasion by regulating galectin-3 and integrin-β1 expression

International audienceGlioblastoma Multiforme (GBM) invasiveness renders complete surgical resection impossible and highly invasive Glioblastoma Initiating Cells (GICs) are responsible for tumour recurrence. Their dissemination occurs along pre-existing fibrillary brain structures comprising the aligned myelinated fibres of the corpus callosum (CC) and the laminin (LN)-rich basal lamina of blood vessels. The extracellular matrix (ECM) of these environments regulates GIC migration, but the underlying mechanisms remain largely unknown. In order to recapitulate the composition and the topographic properties of the cerebral ECM in the migration of GICs, we have set up a new aligned polyacrylonitrile (PAN)-derived nanofiber (NF) scaffold. This system is suitable for drug screening as well as discrimination of the migration potential of different glioblastoma stem cells. Functionalisation with LN increases the spatial anisotropy of migration and modulates its mode from collective to single cell migration. Mechanistically, equally similar to what has been observed for mesenchyma I migration of GBM in vivo, is the upregulation of galectin-3 and integrin-beta 1 in Gli4 cells migrating on our NF scaffold. Downregulation of Calpain-2 in GICs migrating in vivo along the CC and in vitro on LN-coated NF underlines a difference in the turnover of focal adhesion (FA) molecules between single-cell and collective types of migration