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The SARS-CoV-2 pandemic has elicited a unique international mobilization of the scientific community to better understand this coronavirus and its associated disease and to develop efficient tools to combat infection. Similar to other coronavirae, SARS-CoV-2 hijacks the host cell complex secretory machinery to produce properly folded viral proteins that will compose the nascent virions; including Spike, Envelope and Membrane proteins, the most exposed membrane viral proteins to the host immune system. Antibody response is part of the anti- viral immune arsenal that infected patients develop to fight viral particles in the body. Herein, we investigate the immunogenic potential of Spike (S), Envelope (E) and Membrane (M) proteins using a human cell-based system to mimic membrane insertion and N-glycosylation. We show that both S and M proteins elicit the production of specific IgG, IgM and IgA in SARS- CoV-2 infected patients. Elevated Ig responses were observed in COVID+ patients with moderate and severe forms of the disease. Finally, when SARS-CoV-2 Spike D614 and G614 variants were compared, reduced Ig binding was observed with the Spike G614 variant. Altogether, this study underlines the needs for including topological features in envelop proteins to better characterize the serological status of COVID+ patients, points towards an unexpected immune response against the M protein and shows that our assay could represent a powerful tool to test humoral responses against actively evolving SARS-CoV-2 variants and vaccine effectiveness. Funding: This work was funded by grants from INSERM, Institut National du Cancer (INCa PLBIO), Fondation pour la Recherche Medicale (FRM, equipe labellisee 2018) to EC and from la Ligue contre le cancer (comite 35, 56 et 85) to TA. Conflict of Interest: EC and LAE are founders of Cell Stress Discoveries Ltd. Ethical Approval: The study was carried out according to the regulation of Rennes Biobank (BRIF number: BB-0033-00056) certified as meeting the requirements of NF S96900 for receipt preparation preservation and provision of biological resources.

Glioblastoma multiforme (GBM) is the most severe primary brain cancer. Despite an aggressive treatment comprising surgical resection and radio/chemotherapy patient’s survival post diagnosis remains short. A limitation for success in finding novel improved therapeutic options for such dismal disease partly lies in the lack of a relevant animal model that accurately recapitulates patient disease and standard of care. In the present study, we have developed a novel immunocompetent GBM model that includes tumor surgery and a radio/chemotherapy regimen resembling the Stupp protocol and we have used this model to test the impact of the pharmacological inhibition of the endoplasmic reticulum (ER) stress sensor IRE1, on treatment efficacy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has elicited a unique mobilization of the scientific community to develop efficient tools to understand and combat the infection. Like other coronavirae, SARS-CoV-2 hijacks host cell secretory machinery to produce viral proteins that compose the nascent virions; including spike (S), envelope (E), and membrane (M) proteins, the most exposed transmembrane proteins to the host immune system. As antibody response is part of the anti-viral immune arsenal, we investigate the immunogenic potential of S, E, and M using a human cell-based system to mimic membrane insertion and N-glycosylation. Both S and M elicit specific Ig production in patients with SARS-CoV-2. Patients with moderate and severe diseases exhibit elevated Ig responses. Finally, reduced Ig binding was observed with spike G614 compared to D614 variant. Altogether, our assay points toward an unexpected immune response against M and represents a powerful tool to test humoral responses against actively evolving SARS-CoV-2 variants and vaccine effectiveness., Graphical abstract, Immunology; Virology

Glioblastoma Multiforme (GBM) is the most severe primary brain tumor and represents more than 15% of all brain tumors. Despite an aggressive treatment comprising surgical resection and radio/chemotherapy, patient’s survival post diagnosis remains short with a median overall survival of 15 months. The lack of efficacy of the current treatments is mostly due to the tumor heterogeneity with different tumor cell types that exhibit various sensitivity to anti-cancer agents and to the diffuse feature of GBM that complicates the efficacy of complete resection. Another limitation for finding new valuable therapy approaches is the lack of relevant animal models that extensively recapitulate the current GBM patients’ standard of care. In the past couple of years, it has been demonstrated that the Unfolded Protein Response (UPR) plays an instrumental role in GBM development. It has been shown that IRE1, the most conserved UPR sensor, signals in tumor cells to reshape the tumor microenvironment to favor tumor growth and most likely to alter the response to treatment. The IRE1/XBP1s signaling axis exhibits pro-oncogenic properties and has a direct impact on patients’ survival. These observations point toward the IRE1/XBP1s axis as a potentially relevant therapeutic target. To further test the potential impact of the pharmacological targeting IRE1/XBP1 signaling, we proposed to use MKC8866, an inhibitors of IRE1, in preclinical models of GBM. As such we developed a novel GBM animal model that recapitulates the different steps engaged in GBM patient clinical handling (including surgical resection, irradiation and chemotherapy), and use this to demonstrate the relevance of IRE1 inhibition in GBM. Considering that IRE1 inhibitors do not cross the blood-brain barrier, we proposed an intraoperative delivery of the drugs though fibrin glue plugs applicated within the resection cavity, and then studied the impact on survival and tumor microenvironment. We showed that local delivery of IRE1 inhibitors combined with radio/chemotherapy had a positive effect on survival and induced tumoral and microenvironment remodeling, pointing at its high therapeutic potential and the need for an early clinical trial.

Background: Mutations in CALR observed in myeloproliferative neoplasms (MPN) were recently shown to be pathogenic via their interaction with MPL and the subsequent activation of the Janus Kinase &ndash, Signal Transducer and Activator of Transcription (JAK-STAT) pathway. However, little is known on the impact of those variant CALR proteins on endoplasmic reticulum (ER) homeostasis. Methods: The impact of the expression of Wild Type (WT) or mutant CALR on ER homeostasis was assessed by quantifying the expression level of Unfolded Protein Response (UPR) target genes, splicing of X-box Binding Protein 1 (XBP1), and the expression level of endogenous lectins. Pharmacological and molecular (siRNA) screens were used to identify mechanisms involved in CALR mutant proteins degradation. Coimmunoprecipitations were performed to define more precisely actors involved in CALR proteins disposal. Results: We showed that the expression of CALR mutants alters neither ER homeostasis nor the sensitivity of hematopoietic cells towards ER stress-induced apoptosis. In contrast, the expression of CALR variants is generally low because of a combination of secretion and protein degradation mechanisms mostly mediated through the ER-Associated Degradation (ERAD)-proteasome pathway. Moreover, we identified a specific ERAD network involved in the degradation of CALR variants. Conclusions: We propose that this ERAD network could be considered as a potential therapeutic target for selectively inhibiting CALR mutant-dependent proliferation associated with MPN, and therefore attenuate the associated pathogenic outcomes.

International audience; Activating transcription factor 6 alpha (referred to as ATF6 hereafter) is an endoplasmic reticulum (ER)-resident glycoprotein and one of the three sensors of the unfolded protein response (UPR). Upon ER stress, ATF6 is exported to the Golgi complex where it is cleaved by the S1P and S2P proteases thus releasing ATF6 cytosolic fragment and leading to the transcription of ATF6 target genes. In this study, we performed a phenotypic small-interfering RNA (siRNA) screening to better characterize the ER mechanisms involved in ATF6 activation upon ER stress. This revealed that silencing of ER-degradation-enhancing alpha-mannosidase-like protein-1 (EDEM1) increased the bioavailability of ER stress-induced ATF6 export to the Golgi complex through the stabilization of the natively unstable ATF6 protein. Moreover, we characterized a somatic variant of EDEM1 (N198I) found in hepatocellular carcinoma that alters ATF6 signaling and might provide a selective advantage to the transforming cells. Hence, our work confirms the natively unstable nature of ATF6 and links this property to potentially associated pro-oncogenic functions.

Purpose: CD90 (Thy-1) is a glycophosphatidylinositol-anchored glycoprotein considered as a surrogate marker for a variety of stem cells, including glioblastoma (GBM) stem cells (GSC). However, the molecular and cellular functions of CD90 remain unclear. Experimental Design: The function of CD90 in GBM was addressed using cellular models from immortalized and primary GBM lines, in vivo orthotopic mouse models, and GBM specimens' transcriptome associated with MRI features from GBM patients. CD90 expression was silenced in U251 and GBM primary cells and complemented in CD90-negative U87 cells. Results: We showed that CD90 is not only expressed on GSCs but also on more differentiated GBM cancer cells. In GBM patients, CD90 expression was associated with an adhesion/migration gene signature and with invasive tumor features. Modulation of CD90 expression in GBM cells dramatically affected their adhesion and migration properties. Moreover, orthotopic xenografts revealed that CD90 expression induced invasive phenotypes in vivo. Indeed, CD90 expression led to enhanced SRC and FAK signaling in our GBM cellular models and GBM patients' specimens. Pharmacologic inhibition of these signaling nodes blunted adhesion and migration in CD90-positive cells. Remarkably, dasatinib blunted CD90-dependent GBM cell invasion in vivo and killed CD90high primary GSC lines. Conclusions: Our data demonstrate that CD90 is an actor of GBM invasiveness through SRC-dependent mechanisms and could be used as a predictive factor for dasatinib response in CD90high GBM patients. Clin Cancer Res; 23(23); 7360–74. ©2017 AACR.