Publications by authors named "Marion Leduc"

25 Publications

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Cancer cell-autonomous overactivation of PARP1 compromises immunosurveillance in non-small cell lung cancer.

J Immunother Cancer 2022 06;10(6)

Equipe 11 labellisée par la Ligue contre le Cancer, Université de Paris Cité, Sorbonne Université, Centre de Recherche des Cordeliers, INSERM UMR1138, Paris, France

Background: High activity of poly(ADP-ribose) polymerase-1 (PARP1) in non-small cell lung cancer (NSCLC) cells leads to an increase in immunohistochemically detectable PAR, correlating with poor prognosis in patients with NSCLC, as well as reduced tumor infiltration by cytotoxic T lymphocytes (CTLs). Intrigued by this observation, we decided to determine whether PARP1 activity in NSCLC cells may cause an alteration of anticancer immunosurveillance.

Methods: Continuous culture of mouse NSCLC cells in the presence of cisplatin led to the generation of cisplatin-resistant PAR clones. As compared with their parental controls, such PAR cells formed tumors that were less infiltrated by CTLs when they were injected into immunocompetent mice, suggesting a causative link between high PARP1 activity and compromised immunosurveillance. To confirm this cause-and-effect relationship, we used CRISPR/Cas9 technology to knock out PARP1 in two PAR NSCLC mouse cell lines (Lewis lung cancer [LLC] and tissue culture number one [TC1]), showing that the removal of PARP1 indeed restored cisplatin-induced cell death responses.

Results: PARP1 knockout (PARP1) cells became largely resistant to the PARP inhibitor niraparib, meaning that they exhibited less cell death induction, reduced DNA damage response, attenuated metabolic shifts and no induction of PD-L1 and MHC class-I molecules that may affect their immunogenicity. PAR tumors implanted in mice responded to niraparib irrespective of the presence or absence of T lymphocytes, suggesting that cancer cell-autonomous effects of niraparib dominate over its possible immunomodulatory action. While PAR NSCLC mouse cell lines proliferated similarly in immunocompetent and T cell-deficient mice, PARP1 cells were strongly affected by the presence of T cells. PARP1 LLC tumors grew more quickly in immunodeficient than in immunocompetent mice, and PARP1 TC1 cells could only form tumors in T cell-deficient mice, not in immunocompetent controls. Importantly, as compared with PAR controls, the PARP1 LLC tumors exhibited signs of T cell activation in the immune infiltrate such as higher inducible costimulator (ICOS) expression and lower PD-1 expression on CTLs.

Conclusions: These results prove at the genetic level that PARP1 activity within malignant cells modulates the tumor microenvironment.
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http://dx.doi.org/10.1136/jitc-2021-004280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9247697PMC
June 2022

BCG therapy downregulates HLA-I on malignant cells to subvert antitumor immune responses in bladder cancer.

J Clin Invest 2022 06;132(12)

INSERM U1015, Gustave Roussy, Université Paris-Saclay, Villejuif, France.

Patients with high-risk, nonmuscle-invasive bladder cancer (NMIBC) frequently relapse after standard intravesical bacillus Calmette-Guérin (BCG) therapy and may have a dismal outcome. The mechanisms of resistance to such immunotherapy remain poorly understood. Here, using cancer cell lines, freshly resected human bladder tumors, and samples from cohorts of patients with bladder cancer before and after BCG therapy, we demonstrate 2 distinct patterns of immune subversion upon BCG relapse. In the first pattern, intracellular BCG infection of cancer cells induced a posttranscriptional downregulation of HLA-I membrane expression via inhibition of autophagy flux. Patients with HLA-I-deficient cancer cells following BCG therapy had a myeloid immunosuppressive tumor microenvironment (TME) with epithelial-mesenchymal transition (EMT) characteristics and dismal outcomes. Conversely, patients with HLA-I-proficient cancer cells after BCG therapy presented with CD8+ T cell tumor infiltrates, upregulation of inflammatory cytokines, and immune checkpoint-inhibitory molecules. The latter patients had a very favorable outcome. We surmise that HLA-I expression in bladder cancers at relapse following BCG does not result from immunoediting but rather from an immune subversion process directly induced by BCG on cancer cells, which predicts a dismal prognosis. HLA-I scoring of cancer cells by IHC staining can be easily implemented by pathologists in routine practice to stratify future treatment strategies for patients with urothelial cancer.
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http://dx.doi.org/10.1172/JCI145666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9197524PMC
June 2022

Everolimus and plicamycin specifically target chemoresistant colorectal cancer cells of the CMS4 subtype.

Cell Death Dis 2021 10 21;12(11):978. Epub 2021 Oct 21.

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

Colorectal cancers (CRC) can be classified into four consensus molecular subtypes (CMS), among which CMS1 has the best prognosis, contrasting with CMS4 that has the worst outcome. CMS4 CRC is notoriously resistant against therapeutic interventions, as demonstrated by preclinical studies and retrospective clinical observations. Here, we report the finding that two clinically employed agents, everolimus (EVE) and plicamycin (PLI), efficiently target the prototypic CMS4 cell line MDST8. As compared to the prototypic CMS1 cell line LoVo, MDST8 cells treated with EVE or PLI demonstrated stronger cytostatic and cytotoxic effects, increased signs of apoptosis and autophagy, as well as a more pronounced inhibition of DNA-to-RNA transcription and RNA-to-protein translation. Moreover, nontoxic doses of EVE and PLI induced the shrinkage of MDST8 tumors in mice, yet had only minor tumor growth-reducing effects on LoVo tumors. Altogether, these results suggest that EVE and PLI should be evaluated for their clinical activity against CMS4 CRC.
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http://dx.doi.org/10.1038/s41419-021-04270-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8531384PMC
October 2021

High throughput screening for autophagy.

Methods Cell Biol 2021 23;165:89-101. Epub 2021 Jan 23.

Centre de Recherche des Cordeliers, Équipe 11 Labellisée par la Ligue Contre le Cancer, Université de Paris, Sorbonne Université, Inserm U1138, Institut Universitaire de France, Paris, France; Université Paris Sud, Paris Saclay, Faculty of Medicine, Kremlin-Bicêtre, France. Electronic address:

Robotized high throughput screening allows for the assessment of autophagy in a large number of samples. Here, we describe a drug discovery platform for the phenotypic identification of novel autophagy inducers by means of automated cell biology workflows employing robotized cell culture, sample preparation and data acquisition. In this setting, fluorescent biosensor cells that express microtubule-associated proteins 1A/1B light chain 3B (best known as LC3) conjugated to green fluorescent protein (GFP), are utilized together with automated high content microscopy for the image-based assessment of autophagy. In sum, we detail a drug discovery screening workflow from high throughput sample preparation and processing to data acquisition and analysis.
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http://dx.doi.org/10.1016/bs.mcb.2020.12.011DOI Listing
November 2021

IGF1 receptor inhibition amplifies the effects of cancer drugs by autophagy and immune-dependent mechanisms.

J Immunother Cancer 2021 06;9(6)

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

Background: Pharmacological autophagy enhancement constitutes a preclinically validated strategy for preventing or treating most major age-associated diseases. Driven by this consideration, we performed a high-content/high-throughput screen on 65 000 distinct compounds on a robotized fluorescence microscopy platform to identify novel autophagy inducers.

Results: Here, we report the discovery of picropodophyllin (PPP) as a potent inducer of autophagic flux that acts on-target, as an inhibitor of the tyrosine kinase activity of the insulin-like growth factor-1 receptor (IGF1R). Thus, PPP lost its autophagy-stimulatory activity in cells engineered to lack IGF1R or to express a constitutively active AKT serine/threonine kinase 1 (AKT1) mutant. When administered to cancer-bearing mice, PPP improved the therapeutic efficacy of chemoimmunotherapy with a combination of immunogenic cytotoxicants and programmed cell death 1 (PDCD1, better known as PD-1) blockade. These PPP effects were lost when tumors were rendered PPP-insensitive or autophagy-incompetent. In combination with chemotherapy, PPP enhanced the infiltration of tumors by cytotoxic T lymphocytes, while reducing regulatory T cells. In human triple-negative breast cancer patients, the activating phosphorylation of IGF1R correlated with inhibited autophagy, an unfavorable local immune profile, and poor prognosis.

Conclusion: Altogether, these results suggest that IGF1R may constitute a novel and druggable therapeutic target for the treatment of cancer in conjunction with chemoimmunotherapies.
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http://dx.doi.org/10.1136/jitc-2021-002722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8204183PMC
June 2021

Multifaceted modes of action of the anticancer probiotic Enterococcus hirae.

Cell Death Differ 2021 07 11;28(7):2276-2295. Epub 2021 May 11.

Gustave Roussy Cancer Campus (GRCC), Villejuif, France.

A deviated repertoire of the gut microbiome predicts resistance to cancer immunotherapy. Enterococcus hirae compensated cancer-associated dysbiosis in various tumor models. However, the mechanisms by which E. hirae restored the efficacy of cyclophosphamide administered with concomitant antibiotics remain ill defined. Here, we analyzed the multifaceted modes of action of this anticancer probiotic. Firstly, E. hirae elicited emigration of thymocytes and triggered systemic and intratumoral IFNγ-producing and CD137-expressing effector memory T cell responses. Secondly, E. hirae activated the autophagy machinery in enterocytes and mediated ATG4B-dependent anticancer effects, likely as a consequence of its ability to increase local delivery of polyamines. Thirdly, E. hirae shifted the host microbiome toward a Bifidobacteria-enriched ecosystem. In contrast to the live bacterium, its pasteurized cells or membrane vesicles were devoid of anticancer properties. These pleiotropic functions allow the design of optimal immunotherapies combining E. hirae with CD137 agonistic antibodies, spermidine, or Bifidobacterium animalis. We surmise that immunological, metabolic, epithelial, and microbial modes of action of the live E. hirae cooperate to circumvent primary resistance to therapy.
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http://dx.doi.org/10.1038/s41418-021-00753-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8257780PMC
July 2021

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

Comput Biol Med 2021 06 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
June 2021

Lysosomotropic agents including azithromycin, chloroquine and hydroxychloroquine activate the integrated stress response.

Cell Death Dis 2021 01 6;12(1). Epub 2021 Jan 6.

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.

The integrated stress response manifests with the phosphorylation of eukaryotic initiation factor 2α (eIF2α) on serine residue 51 and plays a major role in the adaptation of cells to endoplasmic reticulum stress in the initiation of autophagy and in the ignition of immune responses. Here, we report that lysosomotropic agents, including azithromycin, chloroquine, and hydroxychloroquine, can trigger eIF2α phosphorylation in vitro (in cultured human cells) and, as validated for hydroxychloroquine, in vivo (in mice). Cells bearing a non-phosphorylatable eIF2α mutant (S51A) failed to accumulate autophagic puncta in response to azithromycin, chloroquine, and hydroxychloroquine. Conversely, two inhibitors of eIF2α dephosphorylation, nelfinavir and salubrinal, enhanced the induction of such autophagic puncta. Altogether, these results point to the unexpected capacity of azithromycin, chloroquine, and hydroxychloroquine to elicit the integrated stress response.
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http://dx.doi.org/10.1038/s41419-020-03324-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790317PMC
January 2021

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

Cell Death Differ 2021 05 17;28(5):1733-1752. 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
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8167183PMC
May 2021

Phosphorylation of eukaryotic initiation factor-2α (eIF2α) in autophagy.

Cell Death Dis 2020 06 8;11(6):433. Epub 2020 Jun 8.

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

The integrated stress response is characterized by the phosphorylation of eukaryotic initiation factor-2α (eIF2α) on serine 51 by one out of four specific kinases (EIF2AK1 to 4). Here we provide three series of evidence suggesting that macroautophagy (to which we refer to as autophagy) induced by a variety of distinct pharmacological agents generally requires this phosphorylation event. First, the induction of autophagic puncta by various distinct compounds was accompanied by eIF2α phosphorylation on serine 51. Second, the modulation of autophagy by >30 chemically unrelated agents was partially inhibited in cells expressing a non-phosphorylable (S51A) mutant of eIF2α or lacking all four eIF2α kinases, although distinct kinases were involved in the response to different autophagy inducers. Third, inhibition of eIF2α phosphatases was sufficient to stimulate autophagy. In synthesis, it appears that eIF2α phosphorylation is a central event for the stimulation of autophagy.
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http://dx.doi.org/10.1038/s41419-020-2642-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7280501PMC
June 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

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

AIF-regulated oxidative phosphorylation supports lung cancer development.

Cell Res 2019 07 27;29(7):579-591. Epub 2019 May 27.

Department of Thoracic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China.

Cancer is a major and still increasing cause of death in humans. Most cancer cells have a fundamentally different metabolic profile from that of normal tissue. This shift away from mitochondrial ATP synthesis via oxidative phosphorylation towards a high rate of glycolysis, termed Warburg effect, has long been recognized as a paradigmatic hallmark of cancer, supporting the increased biosynthetic demands of tumor cells. Here we show that deletion of apoptosis-inducing factor (AIF) in a Kras-driven mouse lung cancer model resulted in a marked survival advantage, with delayed tumor onset and decreased malignant progression. Mechanistically, Aif deletion leads to oxidative phosphorylation (OXPHOS) deficiency and a switch in cellular metabolism towards glycolysis in non-transformed pneumocytes and at early stages of tumor development. Paradoxically, although Aif-deficient cells exhibited a metabolic Warburg profile, this bioenergetic change resulted in a growth disadvantage of Kras-driven as well as Kras wild-type lung cancer cells. Cell-autonomous re-expression of both wild-type and mutant AIF (displaying an intact mitochondrial, but abrogated apoptotic function) in Aif-knockout Kras mice restored OXPHOS and reduced animal survival to the same level as AIF wild-type mice. In patients with non-small cell lung cancer, high AIF expression was associated with poor prognosis. These data show that AIF-regulated mitochondrial respiration and OXPHOS drive the progression of lung cancer.
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http://dx.doi.org/10.1038/s41422-019-0181-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6796841PMC
July 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

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

The oncolytic compound LTX-401 targets the Golgi apparatus.

Cell Death Differ 2016 12 2;23(12):2031-2041. Epub 2016 Sep 2.

Metabolomics and Cell Biology Platforms, Gustave Roussy Comprehensive Cancer Institute, Villejuif 94805, France.

LTX-401 is an oncolytic amino acid derivative with potential immunogenic properties. Here, we demonstrate that LTX-401 selectively destroys the structure of the Golgi apparatus, as determined by means of ultrastructural analyses and fluorescence microscopic observation of cells expressing Golgi-targeted GFP reporters. Subcellular fractionation followed by mass spectrometric detection revealed that LTX-401 selectively enriched in the Golgi rather than in mitochondria or in the cytosol. The Golgi-dissociating agent Brefeldin A (BFA) reduced cell killing by LTX-401 as it partially inhibited LTX-401-induced mitochondrial release of cytochrome c and the activation of BAX. The cytotoxic effect of LTX-401 was attenuated by the double knockout of BAX and BAK, as well as the mitophagy-enforced depletion of mitochondria, yet was refractory to caspase inhibition. LTX-401 induced all major hallmarks of immunogenic cell death detectable with biosensor cell lines including calreticulin exposure, ATP release, HMGB1 exodus and a type-1 interferon response. Moreover, LTX-401-treated tumors manifested a strong lymphoid infiltration. Altogether these results support the contention that LTX-401 can stimulate immunogenic cell death through a pathway in which Golgi-localized LTX-401 operates upstream of mitochondrial membrane permeabilization.
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http://dx.doi.org/10.1038/cdd.2016.86DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5136493PMC
December 2016

Overexpression of smad7 blocks primary tumor growth and lung metastasis development in osteosarcoma.

Clin Cancer Res 2014 Oct 8;20(19):5097-112. Epub 2014 Aug 8.

INSERM, UMR 957, Equipe labellisée Ligue contre le Cancer 2012, Nantes, France. Université de Nantes, Laboratoire de Physiopathologie de la Résorption Osseuse et Thérapie des Tumeurs Osseuses Primitives, Nantes, France. CHU Hôtel Dieu, Nantes, France.

Purpose: Osteosarcoma is the main malignant primary bone tumor in children and adolescents for whom the prognosis remains poor, especially when metastasis is present at diagnosis. Because transforming growth factor-β (TGFβ) has been shown to promote metastasis in many solid tumors, we investigated the effect of the natural TGFβ/Smad signaling inhibitor Smad7 and the TβRI inhibitor SD-208 on osteosarcoma behavior.

Experimental Design: By using a mouse model of osteosarcoma induced by paratibial injection of cells, we assessed the impact of Smad7 overexpression or SD-208 on tumor growth, tumor microenvironment, bone remodeling, and metastasis development.

Results: First, we demonstrated that TGFβ levels are higher in serum samples from patients with osteosarcoma compared with healthy volunteers and that TGFβ/Smad3 signaling pathway is activated in clinical samples. Second, we showed that Smad7 slows the growth of the primary tumor and increases mice survival. We furthermore demonstrated that Smad7 expression does not affect in vitro osteosarcoma cell proliferation but affects the microarchitectural parameters of bone. In addition, Smad7-osteosarcoma bone tumors expressed lower levels of osteolytic factors such as RANKL, suggesting that Smad7 overexpression affects the "vicious cycle" established between tumor cells and bone cells by its ability to decrease osteoclast activity. Finally, we showed that Smad7 overexpression in osteosarcoma cells and the treatment of mice with SD208 inhibit the development of lung metastasis.

Conclusion: Taken together, these results demonstrate that the inhibition of the TGFβ/Smad signaling pathway may be a promising therapeutic strategy against tumor progression of osteosarcoma, specifically against the development of lung metastasis.
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http://dx.doi.org/10.1158/1078-0432.CCR-13-3191DOI Listing
October 2014

High frequency and clinical prognostic value of MYD88 L265P mutation in primary cutaneous diffuse large B-cell lymphoma, leg-type.

JAMA Dermatol 2014 Nov;150(11):1173-9

French Study Group for Cutaneous Lymphomas, France9Department of Dermatology, Hôpital Robert Debré, Centre Hospitalier Universitaire Reims, Reims, France.

Importance: The activating mutation of MYD88 L265P is a frequent feature of primary cutaneous diffuse large B-cell lymphoma, leg-type (PCLBCL-LT), reported in up to 69% of the cases. Whether patients with MYD88 mutation display specific clinical and evolutive features has not been evaluated.

Objective: To identify the clinical characteristics associated with MYD88 mutation, confirm its high prevalence, and evaluate its effect on prognosis in patients with PCLBCL-LT.

Design, Setting, And Participants: A retrospective multicenter study was conducted using the medical records of patients from dermatology departments belonging to the French Study Group for Cutaneous Lymphomas. Sixty-one patients with a diagnosis of PCLBCL-LT made between 1988 and 2010 who were available for molecular study were included. Of these, 58 patients displaying interpretable results constituted the study group. Median follow-up was 33 months, and 39 patients (67%) were monitored until death.

Main Outcomes And Measures: Clinical features (age, sex, number of skin lesions, tumor stage, and location as leg vs elsewhere), MYD88 mutation (allele-specific TaqMan polymerase chain reaction assay), treatment regimen, and outcome were recorded. Baseline characteristics and outcome were compared according to the status of MYD88.

Results: The median age of the patients was 79 years, and 59% were female. Skin lesions were located on the leg in 76% of the cases. Thirty-four of 58 patients (59%) harbored the MYD88 L265P mutation. Patients had similar clinical characteristics at presentation regardless of their MYD88 status, except that those harboring the MYD88 mutation were older (P = .006) and had more frequent involvement of the leg (P = .008). Patients harboring the MYD88 mutation had 3- and 5-year-specific survival rates of 65.7% and 60.2% vs 85.4% and 71.7% in patients with the wild-type allele. The MYD88 mutation was significantly associated with shorter disease-specific survival in univariate (P = .03) and multivariate (odds ratio, 3.01; 95% CI, 1.03-8.78; P = .04) analysis. There was no significant difference between the groups in their treatment regimens. Considering overall survival, in univariate (P = .002) and multivariate (odds ratio, 2.94; 95% CI, 1.18-7.30; P = .02) analysis, MYD88 L265P mutation was an independent adverse prognostic factor.

Conclusions And Relevance: This study confirms the high prevalence of MYD88 L265P mutation in PCLBCL-LT and shows its association with shorter survival. The clinical effect of MYD88 mutation activating the nuclear factor-κB pathway supports the use of targeted therapies at the time of relapse after conventional therapies.
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http://dx.doi.org/10.1001/jamadermatol.2014.821DOI Listing
November 2014

Improvement of survival in patients with primary cutaneous diffuse large B-cell lymphoma, leg type, in France.

JAMA Dermatol 2014 May;150(5):535-41

Importance: Primary cutaneous diffuse large B-cell lymphoma, leg type (PCDLBCL-LT), occurs in elderly patients and has been considered as a lymphoma with a poor prognosis, with estimated 5-year specific survival rates of approximately 50%. The hypothesis of an improvement in prognosis over time has not been studied.

Objectives: To evaluate this hypothesis in a large series of patients and investigate factors associated with prognosis as well as improvement in the prognosis.

Design, Setting, And Participants: A retrospective multicenter study was conducted including dermatology departments belonging to the French Study Group on Cutaneous Lymphoma. Participants were 115 patients with PCDLBCL-LT diagnosed between 1988 and 2003 (period 1) or between 2004 and 2010 (period 2).

Main Outcomes And Measures: Age, sex, period of diagnosis, number of skin lesions, tumor stage, tumor location (leg vs nonleg), lactate dehydrogenase level, type of therapy (with or without a combination of rituximab and polychemotherapy [PCT]), and outcome were recorded. Baseline characteristics and outcome were compared according to period of diagnosis and type of therapy. Prognosis factors were identified by univariate and multivariate survival analyses.

Results: The mean age of the patients was 76.9 years, and 47% of the patients were older than 80 years. The 3- and 5-year specific survival rates improved between period 1 and period 2, from 55% to 74% and from 46% to 66%, respectively (P = .01). Patients had similar baseline characteristics during both periods, but rituximab-PCT regimens were administered to 88.5% of the patients in period 2 vs 16.7% in period 1 (P < .001). The 3- and 5-year specific survival rates were 80% and 74%, respectively, in patients who received a rituximab-PCT regimen compared with 48% and 38% in those who received less-intensive therapies. No significant difference was observed between both groups in age and baseline prognostic factors. In multivariate analysis, treatment without rituximab-PCT was the only adverse prognostic factor (odds ratio, 4.6 [95% CI, 2.4-9.1]; P < .001), whereas the number of skin lesions (P = .06) and location on the leg (P = .07) had only borderline significance.

Conclusions And Relevance: A major improvement in the survival of patients with PCDLBCL-LT has occurred over time in France, mainly as a result of the use of intensive rituximab-PCT regimens in most patients, including very elderly ones. Until further prospective clinical trials are conducted, such regimens should be considered as the standard of care in these patients.
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http://dx.doi.org/10.1001/jamadermatol.2013.7452DOI Listing
May 2014
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