DNA Damage Repair Kinase DNA-Pk and cGAS Synergize To Induce Cancer-Related Inflammation in Glioblastoma

Cytosolic DNA promotes inflammatory responses upon detection by the cyclic GMP-AMP (cGAMP) synthase (cGAS). It has been suggested that cGAS downregulation is an immune escape strategy harnessed by tumor cells. Here, we used glioblastoma cells that show undetectable cGAS levels to address if alternative DNA detection pathways can promote pro-inflammatory signaling. We show that the DNA-PK DNA repair complex (i) drives cGAS-independent IRF3-mediated type I Interferon responses and (ii) that its catalytic activity is required for cGAS-dependent cGAMP production and optimal downstream signaling. We further show that the cooperation between DNA-PK and cGAS favors the expression of chemokines that promote macrophage recruitment in the tumor microenvironment in a glioblastoma model, a process that impairs early tumorigenesis but correlates with poor outcome in glioblastoma patients. Thus, our study supports that cGAS-dependent signaling is acquired during tumorigenesis and that cGAS and DNA-PK activities should be analyzed concertedly to predict the impact of strategies aiming to boost tumor immunogenicity

Élucider la Vulnérabilité des Cellules Souches de Glioblastome aux Dysfonctions Lysosomales

Glioblastoma (GB) is the deadliest and most prevalent primary tumor of the central nervous system (CNS) in adults. Despite invasive treatments of surgical resection followed by radio- and chemotherapy, the median survival of patients hardly reaches 15 months. This aggressiveness is thought to be in part linked to the presence of a subset of cancer stem cells termed glioblastoma stem-like cells (GSCs) within the tumor mass. Involved in the initiation, growth, and recurrence of GB tumors, these cells therefore represent a promising target. In this context, lysosomes are critical for the maintenance of GSCs homeostasis. These organelles, standing at the crossroad between anabolism and catabolism, permit the survival of GSCs in unfavorable conditions. Their destabilization culminates in the specific cell death of GSCs, defining lysosomes as a checkpoint for life-and-death decisions in this cellular context. The MALT1 paracaspase was recently defined as a crucial mediator of lysosomal homeostasis in GSCs. This protease, initially involved in immune responses, restrains the lysosomal compartment, its inhibition resulting in lysosomal-dependent cell death of GSCs through a mechanism involving the RNA binding protein Quaking. However, the events resulting in the lysosomal destabilization and cell death of GSCs remained unclear. In this context, my thesis work allowed the cartography of cellular and organellar events leading to GSC cell death upon MALT1 inhibition and silencing.Le glioblastome (GB) est le cancer du système nerveux central de l’adulte le plus commun et le plus meurtrier. Malgré un traitement invasif de résection chirurgicale suivi de séances de radio- et de chimiothérapies, la survie des patients atteints difficilement les 15 mois. Cette agressivité est considérée comme liée notamment à la présence de cellules souches cancéreuses appelées cellules de type souche de glioblastome, ou GSCs. Ces cellules, impliquées dans l’initiation, la croissance, et la récurrence du GB, sont considérées comme des cibles préférentielles. Les lysosomes jouent un rôle critique dans le maintien de l’homéostasie des GSCs. Ces organelles, agissant à la croisée des mécanismes d’anabolisme et de catabolisme, permettent la survie des GSCs hors de leur niche protectrice. Dans les GSCs, leur déstabilisation culmine en une mort spécifique, définissant ainsi les lysosomes comme un point de contrôle des décisions vie-et- mort dans ce contexte cellulaire. La paracaspase MALT1 a récemment été définie comme un médiateur crucial de l’homéostasie des lysosomes dans les GSCs. Cette protéase, initialement décrite comme impliquée dans les réponses immunitaires, restreint le compartiment lysosomal, son inhibition aboutissant en une mort lysosome-dépendante des GSCs, via un mécanisme impliquant la protéine de liaison à l’ARNm Quaking. Cependant, les événements engendrant la déstabilisation lysosomale ainsi que la mort des GSCs restaient incertains. Ainsi, mon travail de thèse a permis la cartographie des événements au niveau cellulaire et des organelles, participant à la déstabilisation lysosomale suivant le ciblage moléculaire et pharmacologique de la paracaspase MALT1

Élucider la Vulnérabilité des Cellules Souches de Glioblastome aux Dysfonctions Lysosomales

Le glioblastome (GB) est le cancer du système nerveux central de l’adulte le plus commun et le plus meurtrier. Malgré un traitement invasif de résection chirurgicale suivi de séances de radio- et de chimiothérapies, la survie des patients atteints difficilement les 15 mois. Cette agressivité est considérée comme liée notamment à la présence de cellules souches cancéreuses appelées cellules de type souche de glioblastome, ou GSCs. Ces cellules, impliquées dans l’initiation, la croissance, et la récurrence du GB, sont considérées comme des cibles préférentielles. Les lysosomes jouent un rôle critique dans le maintien de l’homéostasie des GSCs. Ces organelles, agissant à la croisée des mécanismes d’anabolisme et de catabolisme, permettent la survie des GSCs hors de leur niche protectrice. Dans les GSCs, leur déstabilisation culmine en une mort spécifique, définissant ainsi les lysosomes comme un point de contrôle des décisions vie-et- mort dans ce contexte cellulaire. La paracaspase MALT1 a récemment été définie comme un médiateur crucial de l’homéostasie des lysosomes dans les GSCs. Cette protéase, initialement décrite comme impliquée dans les réponses immunitaires, restreint le compartiment lysosomal, son inhibition aboutissant en une mort lysosome-dépendante des GSCs, via un mécanisme impliquant la protéine de liaison à l’ARNm Quaking. Cependant, les événements engendrant la déstabilisation lysosomale ainsi que la mort des GSCs restaient incertains. Ainsi, mon travail de thèse a permis la cartographie des événements au niveau cellulaire et des organelles, participant à la déstabilisation lysosomale suivant le ciblage moléculaire et pharmacologique de la paracaspase MALT1.Glioblastoma (GB) is the deadliest and most prevalent primary tumor of the central nervous system (CNS) in adults. Despite invasive treatments of surgical resection followed by radio- and chemotherapy, the median survival of patients hardly reaches 15 months. This aggressiveness is thought to be in part linked to the presence of a subset of cancer stem cells termed glioblastoma stem-like cells (GSCs) within the tumor mass. Involved in the initiation, growth, and recurrence of GB tumors, these cells therefore represent a promising target. In this context, lysosomes are critical for the maintenance of GSCs homeostasis. These organelles, standing at the crossroad between anabolism and catabolism, permit the survival of GSCs in unfavorable conditions. Their destabilization culminates in the specific cell death of GSCs, defining lysosomes as a checkpoint for life-and-death decisions in this cellular context. The MALT1 paracaspase was recently defined as a crucial mediator of lysosomal homeostasis in GSCs. This protease, initially involved in immune responses, restrains the lysosomal compartment, its inhibition resulting in lysosomal-dependent cell death of GSCs through a mechanism involving the RNA binding protein Quaking. However, the events resulting in the lysosomal destabilization and cell death of GSCs remained unclear. In this context, my thesis work allowed the cartography of cellular and organellar events leading to GSC cell death upon MALT1 inhibition and silencing

Élucider la Vulnérabilité des Cellules Souches de Glioblastome aux Dysfonctions Lysosomales

Glioblastoma (GB) is the deadliest and most prevalent primary tumor of the central nervous system (CNS) in adults. Despite invasive treatments of surgical resection followed by radio- and chemotherapy, the median survival of patients hardly reaches 15 months. This aggressiveness is thought to be in part linked to the presence of a subset of cancer stem cells termed glioblastoma stem-like cells (GSCs) within the tumor mass. Involved in the initiation, growth, and recurrence of GB tumors, these cells therefore represent a promising target. In this context, lysosomes are critical for the maintenance of GSCs homeostasis. These organelles, standing at the crossroad between anabolism and catabolism, permit the survival of GSCs in unfavorable conditions. Their destabilization culminates in the specific cell death of GSCs, defining lysosomes as a checkpoint for life-and-death decisions in this cellular context. The MALT1 paracaspase was recently defined as a crucial mediator of lysosomal homeostasis in GSCs. This protease, initially involved in immune responses, restrains the lysosomal compartment, its inhibition resulting in lysosomal-dependent cell death of GSCs through a mechanism involving the RNA binding protein Quaking. However, the events resulting in the lysosomal destabilization and cell death of GSCs remained unclear. In this context, my thesis work allowed the cartography of cellular and organellar events leading to GSC cell death upon MALT1 inhibition and silencing.Le glioblastome (GB) est le cancer du système nerveux central de l’adulte le plus commun et le plus meurtrier. Malgré un traitement invasif de résection chirurgicale suivi de séances de radio- et de chimiothérapies, la survie des patients atteints difficilement les 15 mois. Cette agressivité est considérée comme liée notamment à la présence de cellules souches cancéreuses appelées cellules de type souche de glioblastome, ou GSCs. Ces cellules, impliquées dans l’initiation, la croissance, et la récurrence du GB, sont considérées comme des cibles préférentielles. Les lysosomes jouent un rôle critique dans le maintien de l’homéostasie des GSCs. Ces organelles, agissant à la croisée des mécanismes d’anabolisme et de catabolisme, permettent la survie des GSCs hors de leur niche protectrice. Dans les GSCs, leur déstabilisation culmine en une mort spécifique, définissant ainsi les lysosomes comme un point de contrôle des décisions vie-et- mort dans ce contexte cellulaire. La paracaspase MALT1 a récemment été définie comme un médiateur crucial de l’homéostasie des lysosomes dans les GSCs. Cette protéase, initialement décrite comme impliquée dans les réponses immunitaires, restreint le compartiment lysosomal, son inhibition aboutissant en une mort lysosome-dépendante des GSCs, via un mécanisme impliquant la protéine de liaison à l’ARNm Quaking. Cependant, les événements engendrant la déstabilisation lysosomale ainsi que la mort des GSCs restaient incertains. Ainsi, mon travail de thèse a permis la cartographie des événements au niveau cellulaire et des organelles, participant à la déstabilisation lysosomale suivant le ciblage moléculaire et pharmacologique de la paracaspase MALT1

Protocol for qualitative analysis of lysosome immunoprecipitation from patient-derived glioblastoma stem-like cells

Summary: Lysosomes are critical for the sustenance of glioblastoma stem-like cells (GSCs) properties. We present a protocol to enrich and purify lysosomes from patient-derived GSCs in culture. We describe the steps required to stably express a tagged lysosomal protein in GSCs, mechanically lyse cells, magnetically immunopurify lysosomes, and qualitatively assess these organelles. We then detail the procedure for retrieving intact and purified lysosomes from GSCs. We also specify cell culture conditions, storage procedures, and sample preparation for immunoblotting.For complete details on the use and execution of this protocol, please refer to Maghe et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Lysosomes in glioblastoma: pump up the volume

International audienceLysosomes are acidic, dynamic organelles that supervise catabolism, integrate signaling cascades, and tune cellular trafficking. Moreover, the loss of their integrity may jeopardize cell viability. In cancer cells, lysosomes are qualitatively and quantitatively modified for the tumor's own benefit. For all these reasons, these organelles emerge as appealing intracellular targets to manipulate non-oncogene addiction. This is of particular interest for brain diseases, including neurodegenerative disorders and cancer, in which stem cells are exhausted and transformed, respectively. Recent publications had demonstrated that stem cells displayed disarmed lysosomes in terms of number and functions during aging and oncogenic progression. Likewise, our laboratory identified that the arginine protease MALT1, normally dedicated to the assembly of proper NF-kB activation and processing a number of substrates, arbitrates lysosome biogenesis and mTOR signaling in glioblastoma stem-like cells. Indeed, blocking either the expression or the activity of this enzyme leads to an aberrant increase of lysosomes, alongside of the down-regulation of the mTOR signaling. This surge of lysosomes eradicates glioblastoma stem-like cells. Targeting lysosomes might thus inspire the design of new strategies to face this devastating human cancer. Here, we provide an overview of the functions of the lysosome as well as its role as a cell death initiator, to highlight the potential of lysosomal drugs for glioblastoma therapy

Glioblastome multiforme: Les fleurs du MALT1

International audienceNo abstract availabl

The paracaspase MALT1 controls cholesterol homeostasis in glioblastoma stem-like cells through lysosome proteome shaping

International audienceGlioblastoma stem-like cells (GSCs) compose a tumor-initiating and -propagating population remarkably vulnerable to variation in the stability and integrity of the lysosomal compartment. Previous work has shown that the expression and activity of the paracaspase MALT1 control GSC viability via lysosome abundance. However, the underlying mechanisms remain elusive. By combining RNA sequencing (RNA-seq) with proteome-wide label-free quantification, we now report that MALT1 repression in patient-derived GSCs alters the homeostasis of cholesterol, which accumulates in late endosomes (LEs)-lysosomes. This failure in cholesterol supply culminates in cell death and autophagy defects, which can be partially reverted by providing exogenous membrane-permeable cholesterol to GSCs. From a molecular standpoint, a targeted lysosome proteome analysis unraveled that Niemann-Pick type C (NPC) lysosomal cholesterol transporters are diluted when MALT1 is impaired. Accordingly, we found that NPC1/2 inhibition and silencing partially mirror MALT1 loss-of-function phenotypes. This supports the notion that GSC fitness relies on lysosomal cholesterol homeostasis

Necrosulfonamide causes oxidation of PCM1 and impairs ciliogenesis and autophagy

International audienceCentriolar satellites are high-order assemblies, scaffolded by the protein PCM1, that gravitate as particles around the centrosome and play pivotal roles in fundamental cellular processes notably ciliogenesis and autophagy. Despite stringent control mechanisms involving phosphorylation and ubiquitination, the landscape of post-translational modifications shaping these structures remains elusive. Here, we report that necrosulfonamide (NSA), a small molecule known for binding and inactivating the pivotal effector of cell death by necroptosis MLKL, intersects with centriolar satellites, ciliogenesis, and autophagy independently of MLKL. NSA functions as a potent redox cycler and triggers the oxidation and aggregation of PCM1 alongside select partners, while minimally impacting the overall distribution of centriolar satellites. Additionally, NSA-mediated ROS production disrupts ciliogenesis and leads to the accumulation of autophagy markers, partially alleviated by PCM1 deletion. Together, these results identify PCM1 as a redox sensor protein and provide new insights into the interplay between centriolar satellites and autophagy

The glycoprotein GP130 governs the surface presentation of the G protein–coupled receptor APLNR

International audienceGlioblastoma is one of the most lethal forms of adult cancer, with a median survival of ∼15 mo. Targeting glioblastoma stem-like cells (GSCs) at the origin of tumor formation and relapse may prove beneficial. In situ, GSCs are nested within the vascular bed in tight interaction with brain endothelial cells, which positively control their expansion. Because GSCs are notably addicted to apelin (APLN), sourced from the surrounding endothelial stroma, the APLN/APLNR nexus has emerged as a druggable network. However, how this signaling axis operates in gliomagenesis remains underestimated. Here, we find that the glycoprotein GP130 interacts with APLNR at the plasma membrane of GSCs and arbitrates its availability at the surface via ELMOD1, which may further impact on ARF-mediated endovesicular trafficking. From a functional standpoint, interfering with GP130 thwarts APLNR-mediated self-renewal of GSCs ex vivo. Thus, GP130 emerges as an unexpected cicerone to the G protein–coupled APLN receptor, opening new therapeutic perspectives toward the targeting of cancer stem cells