Publications by authors named "Allan Sauvat"

38 Publications

High-throughput label-free detection of DNA-to-RNA transcription inhibition using brightfield microscopy and deep neural networks.

Comput Biol Med 2021 Apr 4;133:104371. Epub 2021 Apr 4.

Centre de Recherche des Cordeliers, Equipe labellisée par la Ligue contre le Cancer, Université de Paris, Sorbonne Université, Inserm U1138, Institut Universitaire de France, Paris, France; Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Center, Villejuif, France; Suzhou Institute for Systems Medicine, Chinese Academy of Medical Sciences, Suzhou, China; Po^le de Biologie, Ho^pital Européen Georges Pompidou, AP-HP, Paris, France; Karolinska Institutet, Department of Women's and Children's Health, Karolinska University Hospital, Stockholm, Sweden.

Drug discovery is in constant evolution and major advances have led to the development of in vitro high-throughput technologies, facilitating the rapid assessment of cellular phenotypes. One such phenotype is immunogenic cell death, which occurs partly as a consequence of inhibited RNA synthesis. Automated cell-imaging offers the possibility of combining high-throughput with high-content data acquisition through the simultaneous computation of a multitude of cellular features. Usually, such features are extracted from fluorescence images, hence requiring labeling of the cells using dyes with possible cytotoxic and phototoxic side effects. Recently, deep learning approaches have allowed the analysis of images obtained by brightfield microscopy, a technique that was for long underexploited, with the great advantage of avoiding any major interference with cellular physiology or stimulatory compounds. Here, we describe a label-free image-based high-throughput workflow that accurately detects the inhibition of DNA-to-RNA transcription. This is achieved by combining two successive deep convolutional neural networks, allowing (1) to automatically detect cellular nuclei (thus enabling monitoring of cell death) and (2) to classify the extracted nuclear images in a binary fashion. This analytical pipeline is R-based and can be easily applied to any microscopic platform.
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http://dx.doi.org/10.1016/j.compbiomed.2021.104371DOI Listing
April 2021

Clonogenic Assays to Detect Cell Fate in Mitotic Catastrophe.

Methods Mol Biol 2021 ;2267:227-239

Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université de Paris, Equipe 11 Labellisée par la Ligue Contre le Cancer, Paris, France.

Mitotic catastrophe (MC) is a cell death modality induced by DNA damage that involves the activation of cell cycle checkpoints such as the "DNA structure checkpoint" and "spindle assembly checkpoint" (SAC) leading to aberrant mitosis. Depending on the signal, MC can drive the cell to death or to senescence. The suppression of MC favors aneuploidy. Several cancer therapies, included microtubular poisons and radiations, trigger MC. The clonogenic assay has been used to study the capacity of single cells to proliferate and to generate macroscopic colonies and to evaluate the efficacy of anticancer drugs. Nevertheless, this method cannot analyze MC events. Here, we report an improved technique based on the use of human colon cancer HCT116 stable expressing histone H2B-GFP and DsRed-centrin proteins, allowing to determine the capacity of cells to proliferate, and to determine changes in the nucleus and centrosomes.
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http://dx.doi.org/10.1007/978-1-0716-1217-0_16DOI Listing
January 2021

Metabolomic analyses of COVID-19 patients unravel stage-dependent and prognostic biomarkers.

Cell Death Dis 2021 03 11;12(3):258. Epub 2021 Mar 11.

Département d'Oncologie Médicale, Gustave Roussy Cancer Campus, 94800, Villejuif, France.

The circulating metabolome provides a snapshot of the physiological state of the organism responding to pathogenic challenges. Here we report alterations in the plasma metabolome reflecting the clinical presentation of COVID-19 patients with mild (ambulatory) diseases, moderate disease (radiologically confirmed pneumonitis, hospitalization and oxygen therapy), and critical disease (in intensive care). This analysis revealed major disease- and stage-associated shifts in the metabolome, meaning that at least 77 metabolites including amino acids, lipids, polyamines and sugars, as well as their derivatives, were altered in critical COVID-19 patient's plasma as compared to mild COVID-19 patients. Among a uniformly moderate cohort of patients who received tocilizumab, only 10 metabolites were different among individuals with a favorable evolution as compared to those who required transfer into the intensive care unit. The elevation of one single metabolite, anthranilic acid, had a poor prognostic value, correlating with the maintenance of high interleukin-10 and -18 levels. Given that products of the kynurenine pathway including anthranilic acid have immunosuppressive properties, we speculate on the therapeutic utility to inhibit the rate-limiting enzymes of this pathway including indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase.
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http://dx.doi.org/10.1038/s41419-021-03540-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7948172PMC
March 2021

Oleate-induced aggregation of LC3 at the trans-Golgi network is linked to a protein trafficking blockade.

Cell Death Differ 2020 Dec 17. Epub 2020 Dec 17.

Equipe labellisée par la Ligue contre le cancer, Université de Paris, Sorbonne Université, INSERM UMR1138, Centre de Recherche des Cordeliers, Paris, France.

Oleate, the most abundant endogenous and dietary cis-unsaturated fatty acid, has the atypical property to cause the redistribution of microtubule-associated proteins 1A/1B light chain 3B (referred to as LC3) to the trans-Golgi network (TGN), as shown here. A genome-wide screen identified multiple, mostly Golgi transport-related genes specifically involved in the oleate-induced relocation of LC3 to the Golgi apparatus. Follow-up analyses revealed that oleate also caused the retention of secreted proteins in the TGN, as determined in two assays in which the secretion of proteins was synchronized, (i) an assay involving a thermosensitive vesicular stomatitis virus G (VSVG) protein that is retained in the endoplasmic reticulum (ER) until the temperature is lowered, and (ii) an isothermic assay involving the reversible retention of the protein of interest in the ER lumen and that was used both in vitro and in vivo. A pharmacological screen searching for agents that induce LC3 aggregation at the Golgi apparatus led to the identification of "oleate mimetics" that share the capacity to block conventional protein secretion. In conclusion, oleate represents a class of molecules that act on the Golgi apparatus to cause the recruitment of LC3 and to stall protein secretion.
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http://dx.doi.org/10.1038/s41418-020-00699-3DOI Listing
December 2020

A genome-wide RNA interference screen disentangles the Golgi tropism of LC3.

Autophagy 2021 Mar 23;17(3):820-822. Epub 2020 Dec 23.

Equipe labellisée par la Ligue contre le cancer, Université de Paris, Sorbonne Université, INSERM UMR1138, Centre de Recherche des Cordeliers, Paris, France.

Oleate, the most abundantly occurring -unsaturated fatty acid, has the particularity to induce the accumulation of MAP1LC3B/LC3 (microtubule associated protein 1 light chain 3 beta) at the -Golgi apparatus. A genome-wide RNA interference screen designed to identify the mechanisms of this LC3 redistribution led to the identification of a BECN1-PIK3C3-independent pathway that, however, requires the ATG12-ATG5 and ATG7-dependent conjugation system, and several genes/proteins involved in endoplasmic reticulum (ER)-to-Golgi anterograde protein transport, as well as the unfolded protein response, including the integrated stress response that results in the phosphorylation of EIF2A/eIF2α (eukaryotic translation initiation factor 2A). Functional experiments revealed that oleate blocks conventional protein secretion, stalling the process at the level of the -Golgi network. Oleate-induced blockade of protein secretion occurred even after depletion of ATG5, suggesting that it does not rely on the recruitment of LC3 to the Golgi apparatus (which does require ATG5). Rather, it appears that oleate and other pharmacological inhibitors of protein secretion with a similar mode of action provoke a perturbation of the -Golgi compartment that secondarily results in the local enrichment of LC3.
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http://dx.doi.org/10.1080/15548627.2020.1861836DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8032232PMC
March 2021

Autophagy-mediated metabolic effects of aspirin.

Cell Death Discov 2020 Nov 24;6(1):129. Epub 2020 Nov 24.

Karolinska Institute, Department of Bioscience and Nutrition, Huddinge, Sweden.

Salicylate, the active derivative of aspirin (acetylsalicylate), recapitulates the mode of action of caloric restriction inasmuch as it stimulates autophagy through the inhibition of the acetyltransferase activity of EP300. Here, we directly compared the metabolic effects of aspirin medication with those elicited by 48 h fasting in mice, revealing convergent alterations in the plasma and the heart metabolome. Aspirin caused a transient reduction of general protein acetylation in blood leukocytes, accompanied by the induction of autophagy. However, these effects on global protein acetylation could not be attributed to the mere inhibition of EP300, as determined by epistatic experiments and exploration of the acetyl-proteome from salicylate-treated EP300-deficient cells. Aspirin reduced high-fat diet-induced obesity, diabetes, and hepatosteatosis. These aspirin effects were observed in autophagy-competent mice but not in two different models of genetic (Atg4b or Bcln1) autophagy-deficiency. Aspirin also improved tumor control by immunogenic chemotherapeutics, and this effect was lost in T cell-deficient mice, as well as upon knockdown of an essential autophagy gene (Atg5) in cancer cells. Hence, the health-improving effects of aspirin depend on autophagy.
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http://dx.doi.org/10.1038/s41420-020-00365-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7687910PMC
November 2020

A TLR3 Ligand Reestablishes Chemotherapeutic Responses in the Context of FPR1 Deficiency.

Cancer Discov 2021 Feb 12;11(2):408-423. Epub 2020 Oct 12.

Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université de Paris, Paris, France.

For anthracycline-based chemotherapy to be immunogenic, dying cancer cells must release annexin A1 (ANXA1) that subsequently interacts with the pattern recognition receptor, formyl peptide receptor 1 (FPR1), on the surface of dendritic cells (DC). Approximately 30% of individuals bear loss-of-function alleles of , calling for strategies to ameliorate their anticancer immune response. Here, we show that immunotherapy with a ligand of Toll-like receptor-3, polyinosinic:polycytidylic acid (pIC), restores the deficient response to chemotherapy of tumors lacking ANXA1 developing in immunocompetent mice or those of normal cancers growing in FPR1-deficient mice. This effect was accompanied by improved DC- and T-lymphocyte-mediated anticancer immunity. Of note, carcinogen-induced breast cancers precociously developed in FPR1-deficient mice as compared with wild-type controls. A similar tendency for earlier cancer development was found in patients carrying the loss-of-function allele of . These findings have potential implications for the clinical management of FPR1-deficient patients. SIGNIFICANCE: The loss-of-function variant rs867228 in , harbored by approximately 30% of the world population, is associated with the precocious manifestation of breast, colorectal, esophageal, and head and neck carcinomas. pIC restores deficient chemotherapeutic responses in mice lacking , suggesting a personalized strategy for compensating for the FPR1 defect..
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http://dx.doi.org/10.1158/2159-8290.CD-20-0465DOI Listing
February 2021

An unexpected link between immunogenic cell death and inhibition of gene transcription.

Oncoimmunology 2020 07 8;9(1):1792039. Epub 2020 Jul 8.

Equipe labellisée par la Ligue contre le Cancer, Université de Paris, Sorbonne Université, INSERM UMR1138, Centre de Recherche des Cordeliers, Paris, France.

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http://dx.doi.org/10.1080/2162402X.2020.1792039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7458641PMC
July 2020

On-target versus off-target effects of drugs inhibiting the replication of SARS-CoV-2.

Cell Death Dis 2020 08 19;11(8):656. Epub 2020 Aug 19.

Equipe labellisée par la Ligue contre le cancer, Université de Paris, Sorbonne Université, INSERM UMR1138, Centre de Recherche des Cordeliers, Paris, France.

The current epidemic of coronavirus disease-19 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) calls for the development of inhibitors of viral replication. Here, we performed a bioinformatic analysis of published and purported SARS-CoV-2 antivirals including imatinib mesylate that we found to suppress SARS-CoV-2 replication on Vero E6 cells and that, according to the published literature on other coronaviruses is likely to act on-target, as a tyrosine kinase inhibitor. We identified a cluster of SARS-CoV-2 antivirals with characteristics of lysosomotropic agents, meaning that they are lipophilic weak bases capable of penetrating into cells. These agents include cepharentine, chloroquine, chlorpromazine, clemastine, cloperastine, emetine, hydroxychloroquine, haloperidol, ML240, PB28, ponatinib, siramesine, and zotatifin (eFT226) all of which are likely to inhibit SARS-CoV-2 replication by non-specific (off-target) effects, meaning that they probably do not act on their 'official' pharmacological targets, but rather interfere with viral replication through non-specific effects on acidophilic organelles including autophagosomes, endosomes, and lysosomes. Imatinib mesylate did not fall into this cluster. In conclusion, we propose a tentative classification of SARS-CoV-2 antivirals into specific (on-target) versus non-specific (off-target) agents based on their physicochemical characteristics.
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http://dx.doi.org/10.1038/s41419-020-02842-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7434849PMC
August 2020

Chemical activation of SAT1 corrects diet-induced metabolic syndrome.

Cell Death Differ 2020 Oct 6;27(10):2904-2920. Epub 2020 May 6.

Centre de Recherche des Cordeliers, INSERM U1138, Team "Metabolism, Cancer & Immunity", Sorbonne Université, Université de Paris, Paris, France.

The pharmacological targeting of polyamine metabolism is currently under the spotlight for its potential in the prevention and treatment of several age-associated disorders. Here, we report the finding that triethylenetetramine dihydrochloride (TETA), a copper-chelator agent that can be safely administered to patients for the long-term treatment of Wilson disease, exerts therapeutic benefits in animals challenged with hypercaloric dietary regimens. TETA reduced obesity induced by high-fat diet, excessive sucrose intake, or leptin deficiency, as it reduced glucose intolerance and hepatosteatosis, but induced autophagy. Mechanistically, these effects did not involve the depletion of copper from plasma or internal organs. Rather, the TETA effects relied on the activation of an energy-consuming polyamine catabolism, secondary to the stabilization of spermidine/spermine N-acetyltransferase-1 (SAT1) by TETA, resulting in enhanced enzymatic activity of SAT. All the positive effects of TETA on high-fat diet-induced metabolic syndrome were lost in SAT1-deficient mice. Altogether, these results suggest novel health-promoting effects of TETA that might be taken advantage of for the prevention or treatment of obesity.
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http://dx.doi.org/10.1038/s41418-020-0550-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494776PMC
October 2020

Inhibition of transcription by dactinomycin reveals a new characteristic of immunogenic cell stress.

EMBO Mol Med 2020 05 23;12(5):e11622. Epub 2020 Apr 23.

Equipe labellisée par la Ligue contre le Cancer, Sorbonne Université, INSERM UMR1138, Centre de Recherche des Cordeliers, Université de Paris, Paris, France.

Chemotherapy still constitutes the standard of care for the treatment of most neoplastic diseases. Certain chemotherapeutics from the oncological armamentarium are able to trigger pre-mortem stress signals that lead to immunogenic cell death (ICD), thus inducing an antitumor immune response and mediating long-term tumor growth reduction. Here, we used an established model, built on artificial intelligence to identify, among a library of 50,000 compounds, anticancer agents that, based on their molecular descriptors, were predicted to induce ICD. This algorithm led us to the identification of dactinomycin (DACT, best known as actinomycin D), a highly potent cytotoxicant and ICD inducer that mediates immune-dependent anticancer effects in vivo. Since DACT is commonly used as an inhibitor of DNA to RNA transcription, we investigated whether other experimentally established or algorithm-selected, clinically employed ICD inducers would share this characteristic. As a common leitmotif, a panel of pharmacological ICD stimulators inhibited transcription and secondarily translation. These results establish the inhibition of RNA synthesis as an initial event for ICD induction.
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http://dx.doi.org/10.15252/emmm.201911622DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7207166PMC
May 2020

TiO Nanomaterials Non-Controlled Contamination Could Be Hazardous for Normal Cells Located in the Field of Radiotherapy.

Int J Mol Sci 2020 Jan 31;21(3). Epub 2020 Jan 31.

Commissariat aux Energies Atomique et Alternative, Fundamental Research Division, Jacob Institut, Laboratoire de Cancérologie expérimentale, CEA, F-92265 Fontenay-aux-Roses, France.

Among nanomaterials (NMs), titanium dioxide (TiO) is one of the most manufactured NMs and can be found in many consumers' products such as skin care products, textiles and food (as E171 additive). Moreover, due to its most attractive property, a photoactivation upon non-ionizing UVA radiation, TiO NMs is widely used as a decontaminating agent. Uncontrolled contaminations by TiO NMs during their production (professional exposure) or by using products (consumer exposure) are rather frequent. So far, TiO NMs cytotoxicity is still a matter of controversy depending on biological models, types of TiO NMs, suspension preparation and biological endpoints. TiO NMs photoactivation has been widely described for UV light radiation exposure, it could lead to reactive oxygen species production, known to be both cyto- and genotoxic on human cells. After higher photon energy exposition, such as X-rays used for radiotherapy and for medical imaging, TiO NMs photoactivation still occurs. Importantly, the question of its hazard in the case of body contamination of persons receiving radiotherapy was never addressed, knowing that healthy tissues surrounding the tumor are indeed exposed. The present work focuses on the analysis of human normal bronchiolar cell response after co-exposition TiO NMs (with different coatings) and ionizing radiation. Our results show a clear synergistic effect, in terms of cell viability, cell death and oxidative stress, between TiO NMS and radiation.
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http://dx.doi.org/10.3390/ijms21030940DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7037422PMC
January 2020

Quinacrine-mediated detection of intracellular ATP.

Methods Enzymol 2019 17;629:103-113. Epub 2019 Jun 17.

Faculty of Medicine, University of Paris Sud, Kremlin-Bicêtre, France; Cell Biology and Metabolomics Platforms, Gustave Roussy Cancer Campus, Villejuif, France; Equipe 11 labellisée Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France; Equipe labellisée Ligue Nationale Contre le Cancer, Université Paris Descartes, Université Sorbonne Paris Cité, Université Paris Diderot, Institut National de la Santé et de la Recherche Médicale (INSERM), UMR1138, Centre de Recherche des Cordeliers, Paris, France; Université Paris Descartes, Sorbonne Paris Cité, Paris, France; Université Pierre et Marie Curie, Paris, France. Electronic address:

Several antineoplastic agents are endowed with the ability to induce immunogenic cell death (ICD), a modality of cellular demise that is accompanied by the release of danger associated molecular patterns such as adenosine triphosphate (ATP) into the tumor microenvironment. ATP-mediated ligation of purinergic P2R receptors then facilitates the chemotactic recruitment and activation of innate immune effectors, thus favoring the induction of anticancer immunity. Here, we provide a protocol for the fluorescence microscopy-based quantification of ICD-associated ATP secretion that is amenable to high-throughput screening. As compared to the traditional luciferase-based detection of ATP in cell culture supernatants, the analysis presented here is cost-efficient and can be combined with the parallel assessment of cellular morphology.
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http://dx.doi.org/10.1016/bs.mie.2019.05.050DOI Listing
June 2020

Lurbinectedin synergizes with immune checkpoint blockade to generate anticancer immunity.

Oncoimmunology 2019;8(11):e1656502. Epub 2019 Sep 5.

Metabolomics and Cell Biology Platforms, Gustave Roussy Comprehensive Cancer Institute, Villejuif, France.

Systemic treatment with the active transcription inhibitor lurbinectedin aims at inducing tumor cell death in hyperproliferative neoplasms. Here we show that cell death induced by lurbinectedin reinstates and enhances systemic anticancer immune responses. Lurbinectedin treatment showed traits of immunogenic cell death, including the exposure of calreticulin, the release of ATP, the exodus of high mobility group box 1 (HMGB1) and type 1 interferon responses . Lurbinectedin treated cells induced antitumor immunity when injected into immunocompetent animals and treatment of transplanted fibrosarcomas reduced tumor growth in immunocompetent yet not in immunodeficient hosts. Anticancer effects resulting from lurbinectedin treatment were boosted in combination with PD-1 and CTLA-4 double immune checkpoint blockade (ICB), and lurbinectedin combined with double ICB exhibited strong antineoplastic effects. Cured animals exhibited long term immune memory effects that rendered them resistant to rechallenge with syngeneic tumors underlining the potency of combination therapy with lurbinectedin.
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http://dx.doi.org/10.1080/2162402X.2019.1656502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6791417PMC
September 2019

3,4-Dimethoxychalcone induces autophagy through activation of the transcription factors TFE3 and TFEB.

EMBO Mol Med 2019 11 14;11(11):e10469. Epub 2019 Oct 14.

Gustave Roussy Cancer Campus, Villejuif, France.

Caloric restriction mimetics (CRMs) are natural or synthetic compounds that mimic the health-promoting and longevity-extending effects of caloric restriction. CRMs provoke the deacetylation of cellular proteins coupled to an increase in autophagic flux in the absence of toxicity. Here, we report the identification of a novel candidate CRM, namely 3,4-dimethoxychalcone (3,4-DC), among a library of polyphenols. When added to several different human cell lines, 3,4-DC induced the deacetylation of cytoplasmic proteins and stimulated autophagic flux. At difference with other well-characterized CRMs, 3,4-DC, however, required transcription factor EB (TFEB)- and E3 (TFE3)-dependent gene transcription and mRNA translation to trigger autophagy. 3,4-DC stimulated the translocation of TFEB and TFE3 into nuclei both in vitro and in vivo, in hepatocytes and cardiomyocytes. 3,4-DC induced autophagy in vitro and in mouse organs, mediated autophagy-dependent cardioprotective effects, and improved the efficacy of anticancer chemotherapy in vivo. Altogether, our results suggest that 3,4-DC is a novel CRM with a previously unrecognized mode of action.
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http://dx.doi.org/10.15252/emmm.201910469DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6835206PMC
November 2019

Artificial tethering of LC3 or p62 to organelles is not sufficient to trigger autophagy.

Cell Death Dis 2019 10 10;10(10):771. Epub 2019 Oct 10.

Cell Biology and Metabolomics Platforms, Gustave Roussy Cancer Center, Villejuif, France.

The retention using selective hooks (RUSH) system allows to retain a target protein fused to green fluorescent protein (GFP) and a streptavidin-binding peptide (SBP) due to the interaction with a molar excess of streptavidin molecules ("hooks") targeted to selected subcellular compartments. Supplementation of biotin competitively disrupts the interaction between the SBP moiety and streptavidin, liberating the chimeric target protein from its hooks, while addition of avidin causes the removal of biotin from the system and reestablishes the interaction. Based on this principle, we engineered two chimeric proteins involved in autophagy, namely microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B, best known as LC3) and sequestosome-1 (SQSTM1, best known as p62) to move them as SBP-GFP-LC3 and p62-SBP-GFP at will between the cytosol and two different organelles, the endoplasmic reticulum (ER) and the Golgi apparatus. Although both proteins were functional in thus far that SBP-GFP-LC3 and p62-SBP-GFP could recruit their endogenous binding partners, p62 and LC3, respectively, their enforced relocation to the ER or Golgi failed to induce organelle-specific autophagy. Hence, artificial tethering of LC3 or p62 to the surface of the ER and the Golgi is not sufficient to trigger autophagy.
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http://dx.doi.org/10.1038/s41419-019-2011-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6787181PMC
October 2019

A fluorescent biosensor-based platform for the discovery of immunogenic cancer cell death inducers.

Oncoimmunology 2019;8(8):1606665. Epub 2019 Apr 26.

Metabolomics and Cell Biology Platforms, Gustave Roussy Comprehensive Cancer Institute, Villejuif, France.

Systemic anticancer immunity can be reinstated via the induction of immunogenic cell death (ICD) in malignant cells. Thus, certain classes of cytotoxic compounds, for example, anthracyclines, oxaliplatin and taxanes are endowed with the capacity to act on cancer cells to ignite stress pathways that lead to the surface exposure of calreticulin (CALR) and the cellular release of adenosine triphosphate, annexin A1, high mobility group B1 and type-1 interferons. Altogether, these alterations constitute the hallmarks of ICD. Here we report the design of a discovery pipeline for the identification of novel ICD inducers by means of a phenotypic screening platform. The use of fluorescent biosensors as proxies for the manifestation of ICD hallmarks has enabled the exploration of large collections of chemical compounds by automatized screening routines. Imaging-based assessment and phenotypic selection led to the identification of potential ICD inducers that could be validated further and , confirming that ICD inducers possess the capacity to induce immunological long-term memory and to confer resistance against rechallenge with syngeneic tumors. Machine learning algorithms analyzing the physicochemical properties of ICD inducers can assist in the preselection of compounds with potential ICD-stimulatory properties, further accelerating the screening efforts designed to develop new immunotherapeutic agents.
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http://dx.doi.org/10.1080/2162402X.2019.1606665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6682369PMC
April 2019

Lethal Poisoning of Cancer Cells by Respiratory Chain Inhibition plus Dimethyl α-Ketoglutarate.

Cell Rep 2019 04;27(3):820-834.e9

Centre de Recherche des Cordeliers, INSERM, Sorbonne Université, USPC, Université Paris Descartes, Université Paris Diderot, Equipe 11 labellisée par la Ligue contre le Cancer, 75006 Paris, France; Metabolomics and Cell Biology Platforms, Institut Gustave Roussy, 94805 Villejuif, France. Electronic address:

Inhibition of oxidative phosphorylation (OXPHOS) by 1-cyclopropyl-4-(4-[(5-methyl-3-(3-[4-(trifluoromethoxy)phenyl]-1,2,4-oxadiazol-5-yl)-1H-pyrazol-1-yl)methyl]pyridin-2-yl)piperazine (BAY87-2243, abbreviated as B87), a complex I inhibitor, fails to kill human cancer cells in vitro. Driven by this consideration, we attempted to identify agents that engage in synthetically lethal interactions with B87. Here, we report that dimethyl α-ketoglutarate (DMKG), a cell-permeable precursor of α-ketoglutarate that lacks toxicity on its own, kills cancer cells when combined with B87 or other inhibitors of OXPHOS. DMKG improved the antineoplastic effect of B87, both in vitro and in vivo. This combination caused MDM2-dependent, tumor suppressor protein p53 (TP53)-independent transcriptional reprogramming and alternative exon usage affecting multiple glycolytic enzymes, completely blocking glycolysis. Simultaneous inhibition of OXPHOS and glycolysis provoked a bioenergetic catastrophe culminating in the activation of a cell death program that involved disruption of the mitochondrial network and activation of PARP1, AIFM1, and APEX1. These results unveil a metabolic liability of human cancer cells that may be harnessed for the development of therapeutic regimens.
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http://dx.doi.org/10.1016/j.celrep.2019.03.058DOI Listing
April 2019

Author Correction: Squaramide-based synthetic chloride transporters activate TFEB but block autophagic flux.

Cell Death Dis 2019 Apr 3;10(4):301. Epub 2019 Apr 3.

Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France.

In the version of this article originally submitted, it was stated that the first three authors (Shaoyi_ Than, Yan Wang, Wei Xie) had contributed equally. However, in the published version this information was missing.
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http://dx.doi.org/10.1038/s41419-019-1536-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6447600PMC
April 2019

Squaramide-based synthetic chloride transporters activate TFEB but block autophagic flux.

Cell Death Dis 2019 03 11;10(3):242. Epub 2019 Mar 11.

Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France.

Cystic fibrosis is a disease caused by defective function of a chloride channel coupled to a blockade of autophagic flux. It has been proposed to use synthetic chloride transporters as pharmacological agents to compensate insufficient chloride fluxes. Here, we report that such chloride anionophores block autophagic flux in spite of the fact that they activate the pro-autophagic transcription factor EB (TFEB) coupled to the inhibition of the autophagy-suppressive mTORC1 kinase activity. Two synthetic chloride transporters (SQ1 and SQ2) caused a partially TFEB-dependent relocation of the autophagic marker LC3 to the Golgi apparatus. Inhibition of TFEB activation using a calcium chelator or calcineurin inhibitors reduced the formation of LC3 puncta in cells, yet did not affect the cytotoxic action of SQ1 and SQ2 that could be observed after prolonged incubation. In conclusion, the squaramide-based synthetic chloride transporters studied in this work (which can also dissipate pH gradients) are probably not appropriate for the treatment of cystic fibrosis yet might be used for other indications such as cancer.
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http://dx.doi.org/10.1038/s41419-019-1474-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411943PMC
March 2019

ColocalizR: An open-source application for cell-based high-throughput colocalization analysis.

Comput Biol Med 2019 04 2;107:227-234. Epub 2019 Mar 2.

Université Paris Descartes, Sorbonne Paris Cité, Paris, France; Equipe 11 Labellisée Ligue Nationale Contre le Cancer, Centre de Recherche des Cordeliers, Paris, France; Institut National de la Santé et de la Recherche Médicale, U1138, Paris, France; Université Pierre et Marie Curie, Paris, France; Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France; Faculty of Medicine, University of Paris Sud, Kremlin-Bicêtre, France; Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France; Department of Women's and Children's Health, Karolinska University Hospital, Stockholm, Sweden. Electronic address:

The microscopic assessment of the colocalization of fluorescent signals has been widely used in cell biology. Although imaging techniques have drastically improved over the past decades, the quantification of colocalization by measures such as the Pearson correlation coefficient or Manders overlap coefficient, has not changed. Here, we report the development of an R-based application that allows to (i) automatically segment cells and subcellular compartments, (ii) measure morphology and texture features, and (iii) calculate the degree of colocalization within each cell. Colocalization can thus be studied on a cell-by-cell basis, permitting to perform statistical analyses of cellular populations and subpopulations. ColocalizR has been designed to parallelize tasks, making it applicable to the analysis of large data sets. Its graphical user interface makes it suitable for researchers without specific knowledge in image analysis. Moreover, results can be exported into a wide range of formats rendering post-analysis adaptable to statistical requirements. This application and its source code are freely available at https://github.com/kroemerlab/ColocalizR.
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http://dx.doi.org/10.1016/j.compbiomed.2019.02.024DOI Listing
April 2019

Oncolysis with DTT-205 and DTT-304 generates immunological memory in cured animals.

Cell Death Dis 2018 10 23;9(11):1086. Epub 2018 Oct 23.

Metabolomics and Cell Biology Platforms, Gustave Roussy Comrehensive Cancer Institute, Villejuif, France.

Oncolytic peptides and peptidomimetics are being optimized for the treatment of cancer by selecting agents with high cytotoxic potential to kill a maximum of tumor cells as well as the capacity to trigger anticancer immune responses and hence to achieve long-term effects beyond therapeutic discontinuation. Here, we report on the characterization of two novel oncolytic peptides, DTT-205 and DTT-304 that both selectively enrich in the lysosomal compartment of cancer cells yet differ to some extent in their cytotoxic mode of action. While DTT-304 can trigger the aggregation of RIP3 in ripoptosomes, coupled to the phosphorylation of MLKL by RIP3, DTT-205 fails to activate RIP3. Accordingly, knockout of either RIP3 or MLKL caused partial resistance against cell killing by DTT-304 but not DTT-205. In contrast, both agents shared common features in other aspects of pro-death signaling in the sense that their cytotoxic effects were strongly inhibited by both serum and antioxidants, partially reduced by lysosomal inhibition with bafilomycin A1 or double knockout of Bax and Bak, yet totally refractory to caspase inhibition. Both DTT-304 and DTT-205 caused the exposure of calreticulin at the cell surface, as well as the release of HMGB1 from the cells. Mice bearing established subcutaneous cancers could be cured by local injection of DTT-205 or DTT-304, and this effect depended on T lymphocytes, as it led to the establishment of a long-term memory response against tumor-associated antigens. Thus, mice that had been cured from cancer by the administration of DTT compounds were refractory against rechallenge with the same cancer type several months after the disappearance of the primary lesion. In summary, DTT-205 and DTT-304 both have the capacity to induce immunotherapeutic oncolysis.
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http://dx.doi.org/10.1038/s41419-018-1127-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6199251PMC
October 2018

Recruitment of LC3 to damaged Golgi apparatus.

Cell Death Differ 2019 08 22;26(8):1467-1484. Epub 2018 Oct 22.

Cell Biology and Cancer, Institut Curie, PSL Research University, CNRS UMR144, Paris, France.

LC3 is a protein that can associate with autophagosomes, autolysosomes, and phagosomes. Here, we show that LC3 can also redistribute toward the damaged Golgi apparatus where it clusters with SQSTM1/p62 and lysosomes. This organelle-specific relocation, which did not involve the generation of double-membraned autophagosomes, could be observed after Golgi damage was induced by various strategies, namely (i) laser-induced localized cellular damage, (ii) local expression of peroxidase and exposure to peroxide and diaminobenzidine, (iii) treatment with the Golgi-tropic photosensitizer redaporfin and light, (iv) or exposure to the Golgi-tropic anticancer peptidomimetic LTX-401. Mechanistic exploration led to the conclusion that both reactive oxygen species-dependent and -independent Golgi damage induces a similar phenotype that depended on ATG5 yet did not depend on phosphatidylinositol-3-kinase catalytic subunit type 3 and Beclin-1. Interestingly, knockout of ATG5 sensitized cells to Golgi damage-induced cell death, suggesting that the pathway culminating in the relocation of LC3 to the damaged Golgi may have a cytoprotective function.
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http://dx.doi.org/10.1038/s41418-018-0221-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748261PMC
August 2019

eIF2α phosphorylation: A hallmark of immunogenic cell death.

Oncoimmunology 2018;7(6):e1431089. Epub 2018 Feb 14.

Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus; Villejuif, France.

Immunogenic cell death (ICD) induced by anticancer chemotherapeutics is usually preceded by premortem stress affecting the endoplasmic reticulum (ER). This ER stress does not reflect a canonical unfolded protein response (UPR) but rather manifests solely at the level of the phosphorylation of eIF2α. eIF2α phosphorylation is hence a quintessential hallmark of ICD that can be detected by immunohistochemistry in tumor samples.
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http://dx.doi.org/10.1080/2162402X.2018.1431089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5980344PMC
February 2018

Photodynamic therapy with redaporfin targets the endoplasmic reticulum and Golgi apparatus.

EMBO J 2018 07 28;37(13). Epub 2018 May 28.

Faculty of Medicine, University of Paris Sud, Kremlin-Bicêtre, France

Preclinical evidence depicts the capacity of redaporfin (Redp) to act as potent photosensitizer, causing direct antineoplastic effects as well as indirect immune-dependent destruction of malignant lesions. Here, we investigated the mechanisms through which photodynamic therapy (PDT) with redaporfin kills cancer cells. Subcellular localization and fractionation studies based on the physicochemical properties of redaporfin revealed its selective tropism for the endoplasmic reticulum (ER) and the Golgi apparatus (GA). When activated, redaporfin caused rapid reactive oxygen species-dependent perturbation of ER/GA compartments, coupled to ER stress and an inhibition of the GA-dependent secretory pathway. This led to a general inhibition of protein secretion by PDT-treated cancer cells. The ER/GA play a role upstream of mitochondria in the lethal signaling pathway triggered by redaporfin-based PDT Pharmacological perturbation of GA function or homeostasis reduces mitochondrial permeabilization. In contrast, removal of the pro-apoptotic multidomain proteins BAX and BAK or pretreatment with protease inhibitors reduced cell killing, yet left the GA perturbation unaffected. Altogether, these results point to the capacity of redaporfin to kill tumor cells via destroying ER/GA function.
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http://dx.doi.org/10.15252/embj.201798354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028029PMC
July 2018

Trans-Fats Inhibit Autophagy Induced by Saturated Fatty Acids.

EBioMedicine 2018 Apr 27;30:261-272. Epub 2018 Mar 27.

Université Paris Descartes, Sorbonne Paris Cité, Paris, France; Equipe 11 labellisée Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France; Institut National de la Santé et de la Recherche Médicale, U1138 Paris, France; Université Pierre et Marie Curie, Paris, France; Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France; Faculty of Medicine, University of Paris Sud, Kremlin-Bicêtre, France; Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France; Department of Women's and Children's Health, Karolinska University Hospital, Stockholm, Sweden. Electronic address:

Depending on the length of their carbon backbone and their saturation status, natural fatty acids have rather distinct biological effects. Thus, longevity of model organisms is increased by extra supply of the most abundant natural cis-unsaturated fatty acid, oleic acid, but not by that of the most abundant saturated fatty acid, palmitic acid. Here, we systematically compared the capacity of different saturated, cis-unsaturated and alien (industrial or ruminant) trans-unsaturated fatty acids to provoke cellular stress in vitro, on cultured human cells expressing a battery of distinct biosensors that detect signs of autophagy, Golgi stress and the unfolded protein response. In contrast to cis-unsaturated fatty acids, trans-unsaturated fatty acids failed to stimulate signs of autophagy including the formation of GFP-LC3B-positive puncta, production of phosphatidylinositol-3-phosphate, and activation of the transcription factor TFEB. When combined effects were assessed, several trans-unsaturated fatty acids including elaidic acid (the trans-isomer of oleate), linoelaidic acid, trans-vaccenic acid and palmitelaidic acid, were highly efficient in suppressing autophagy and endoplasmic reticulum stress induced by palmitic, but not by oleic acid. Elaidic acid also inhibited autophagy induction by palmitic acid in vivo, in mouse livers and hearts. We conclude that the well-established, though mechanistically enigmatic toxicity of trans-unsaturated fatty acids may reside in their capacity to abolish cytoprotective stress responses induced by saturated fatty acids.
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http://dx.doi.org/10.1016/j.ebiom.2018.03.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5952403PMC
April 2018

Evaluation of autophagy inducers in epithelial cells carrying the ΔF508 mutation of the cystic fibrosis transmembrane conductance regulator CFTR.

Cell Death Dis 2018 02 7;9(2):191. Epub 2018 Feb 7.

Faculty of Medicine, University of Paris Sud-Saclay, Kremlin-Bicêtre, France.

Cystic Fibrosis (CF) due to the ΔF508 mutation of cystic fibrosis transmembrane conductance regulator (CFTR) can be treated with a combination of cysteamine and Epigallocatechin gallate (EGCG). Since ECGC is not a clinically approved drug, we attempted to identify other compounds that might favourably interact with cysteamine to induce autophagy and thus rescuing the function of ΔF508 CFTR as a chloride channel in the plasma membrane. For this, we screened a compound library composed by chemically diverse autophagy inducers for their ability to enhance autophagic flux in the presence of cysteamine. We identified the antiarrhythmic Ca channel blocker amiodarone, as an FDA-approved drug having the property to cooperate with cysteamine to stimulate autophagy in an additive manner. Amiodarone promoted the re-expression of ΔF508 CFTR protein in the plasma membrane of respiratory epithelial cells. Hence, amiodarone might be yet another compound for the etiological therapy of CF in patients bearing the ΔF508 CFTR mutation.
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http://dx.doi.org/10.1038/s41419-017-0235-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5833759PMC
February 2018

eIF2α phosphorylation is pathognomonic for immunogenic cell death.

Cell Death Differ 2018 08 22;25(8):1375-1393. Epub 2018 Jan 22.

Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France.

The phosphorylation of eIF2α is essential for the endoplasmic reticulum (ER) stress response, the formation of stress granules, as well as macroautophagy. Several successful anticancer chemotherapeutics have the property to induce immunogenic cell death (ICD), thereby causing anticancer immune responses. ICD is accompanied by the translocation of calreticulin (CALR) from the ER lumen to the plasma membrane, which facilitates the transfer of tumor-associated antigens to dendritic cells. Here we systematically investigated the capacity of anticancer chemotherapeutics to induce signs of ER stress. ICD inducers including anthracyclines and agents that provoke tetraploidization were highly efficient in enhancing the phosphorylation of eIF2α, yet failed to stimulate other signs of ER stress including the transcriptional activation of activating transcription factor 4 (ATF4), the alternative splicing of X-box binding protein 1 (XBP1s) mRNA and the proteolytic cleavage of activating transcription factor 6 (ATF6) both in vitro and in cancers established in mice. Systematic analyses of clinically used anticancer chemotherapeutics revealed that only eIF2α phosphorylation, but none of the other signs of ER stress, correlated with CALR exposure. eIF2α phosphorylation induced by mitoxantrone, a prototype ICD-inducing anthracyline, was mediated by eIF2α kinase-3 (EIF2AK3). Machine-learning approaches were used to determine the physicochemical properties of drugs that induce ICD, revealing that the sole ER stress response relevant to the algorithm is eIF2α phosphorylation with its downstream consequences CALR exposure, stress granule formation and autophagy induction. Importantly, this approach could reduce the complexity of compound libraries to identify ICD inducers based on their physicochemical and structural characteristics. In summary, it appears that eIF2α phosphorylation constitutes a pathognomonic characteristic of ICD.
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http://dx.doi.org/10.1038/s41418-017-0044-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6113215PMC
August 2018

The ratio of CD8/FOXP3 T lymphocytes infiltrating breast tissues predicts the relapse of ductal carcinoma .

Oncoimmunology 2016;5(10):e1218106. Epub 2016 Aug 18.

Gustave Roussy Cancer Campus, Villejuif, France; INSERM, U1138, Paris, France; Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Center de Recherche des Cordeliers, Paris, France; Université Paris Descartes, Sorbonne Paris Cité, Paris, France; Université Pierre et Marie Curie, Paris, France; Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France; Karolinska Institute, Department of Women's and Children's Health, Karolinska University Hospital, Stockholm, Sweden.

In a series of 248 tumor samples obtained from image-guided biopsies from patients diagnosed with ductal carcinoma of the breast, we attempted to identify biomarkers that predict microinfiltration at definitive surgery or relapse during follow-up. For this, we used immunohistochemical methods, followed by automated image analyses, to measure the mean diameter of nuclei (which correlates with ploidy), the phosphorylation of eukaryotic initiation factor 2α (eIF2α, which reflects endoplasmic reticulum stress) as well as the density and ratio of CD8 cytotoxic T lymphocytes and FOXP3 regulatory T cells. The median nuclear diameter of malignant cells correlated with eIF2α phosphorylation (in cancerous tissue), which in turn correlated with the density of the CD8 infiltrate and the CD8/FOXP3 ratio (both in cancerous and the adjacent non-cancerous parenchyma). Neither microinfiltration nor lymph node involvement was associated with the probability of relapse. Both correlated positively with the CD8/FOXP3 ratio in the malignant area. In contrast, relapse was associated with a paucity of the CD8 infiltrate as well as an unfavorable CD8/FOXP3 ratio, both in malignant and non-malignant parenchyma. The combined analysis of the CD8/FOXP3 ratio in cancerous and non-cancerous tissues revealed a significant impact of their interaction on the probability of relapse, but not on the presence of microinfiltration or lymph node metastasis. Altogether, these results support the idea of an immunosurveillance system that determines the risk of relapse in ductal carcinoma of the breast.
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http://dx.doi.org/10.1080/2162402X.2016.1218106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5087306PMC
August 2016