Publications by authors named "Oliver Kepp"

252 Publications

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

Cell Death Dis 2021 Oct 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
October 2021

A genotype-phenotype screening system using conditionally immortalized immature dendritic cells.

STAR Protoc 2021 Sep 12;2(3):100732. Epub 2021 Aug 12.

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

Here, we describe a protocol for CRISPR/Cas9-mediated gene knockout in conditionally immortalized immature dendritic cells (DCs), which can be limitlessly expanded before differentiation. This facilitates the genetic screening of DC functions including assessment of phagocytosis, cytokine production, expression of co-stimulatory or co-inhibitory molecules, and antigen presentation, as well as evaluation of the capacity to elicit anticancer immune responses . Altogether, these approaches described in this protocol allow investigators to link the genotype of DCs to their phenotype. For complete details on the use and execution of this protocol, please refer to Le Naour et al. (2020).
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http://dx.doi.org/10.1016/j.xpro.2021.100732DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8365513PMC
September 2021

Vesicular trafficking in cellular responses to stress.

Methods Cell Biol 2021 ;165:xix-xxiii

Université de Paris, Paris, France; Department of Radiation Oncology, Weill Cornell Medical College, New York, NY, United States; Sandra and Edward Meyer Cancer Center, New York, NY, United States; Caryl and Isreal Englander Institute for Precision Medicine, New York, NY, United States; Department of Dermatology, Yale School of Medicine, New Haven, CT, United States. Electronic address:

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http://dx.doi.org/10.1016/S0091-679X(21)00081-9DOI Listing
January 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
January 2021

Multiplexed quantification of autophagic flux by imaging flow cytometry.

Methods Cell Biol 2021 27;165:59-71. Epub 2021 Mar 27.

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

Imaging flow cytometry allows for the quantitative assessment of fluorescent signals at the subcellular level. Here, we describe the use of a biosensor cell line, namely, U2OS osteosarcoma cells equipped with a fusion protein comprising monomeric red fluorescent protein (mRFP), green fluorescent protein (GFP) and microtubule-associated proteins 1A/1B light chain 3B (best known as LC3), for the assessment of autophagic flux by imaging flow cytometry. We detail all analysis tools required to distinguish autophagosomes (that emit both a red and a green fluorescence) and autolysosomes (that emit a red fluorescence, yet lose the green fluorescent signal) and to quantitate autophagic flux in a convenient fashion.
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http://dx.doi.org/10.1016/bs.mcb.2021.02.005DOI Listing
March 2021

Assessment of EGFP-Q74 degradation for the measurement of autophagic flux.

Methods Cell Biol 2021 27;165:31-38. Epub 2021 Apr 27.

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

Autophagy plays a major role in physiological and pathological processes. The quantitation of the abundance of autophagy-specific substrates constitutes an efficient strategy for assessing autophagic activity. Here, we provide a detailed protocol for quantifying the decay of a fusion protein composed by enhanced green fluorescent protein (EGFP) and glutamine repeats (Q74) using regular or high-throughput fluorescence microscopy. This method provides a direct measurement of autophagic flux in a Huntington's disease model.
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http://dx.doi.org/10.1016/bs.mcb.2020.10.018DOI Listing
April 2021

A novel tool for detecting lysosomal membrane permeabilization by high-throughput fluorescence microscopy.

Methods Cell Biol 2021 20;165:1-12. Epub 2020 Nov 20.

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

Lysosomes are placed at the center of cellular trafficking and degradative pathways. They also function as a signaling platform for nutrient sensing and metabolic reprogramming. Lysosomes play crucial roles in cellular adaptation in response to stress and are tightly connected to a variety of cell death modalities. Several stimuli can initiate the permeabilization of the lysosome membrane, thus causing cell death when the cellular adaptive system fail to repair or replace damaged lysosomes. The induction of lysosomal membrane permeabilization (LMP) triggers the rapid translocation of Galectin 3/LGALS3 from the cytosol to the lysosomal lumen, making it a valuable marker of LMP. However, Galectin 3 can also be recruited to damaged endo/phagosomal membranes. To make sure that Galectin 3 labels damaged lysosomes, it is therefore important to verify its colocalization with lysosomal markers such as lysosome-associated membrane protein 1 (LAMP1). Here, we describe a simple, fast and robust protocol that allows the detection of LMP of individual lysosomes in U2OS cells expressing mCherry-tagged Galectin 3 and mGFP-tagged LAMP1. This method permits the high-throughput detection and quantification of damaged lysosomes by fluorescence microscopy. It also offers the advantage of studying, in the same experiment, the alterations in size, shape and subcellular localization of intact and damaged lysosomes.
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http://dx.doi.org/10.1016/bs.mcb.2020.10.004DOI Listing
November 2020

Pharmacological inhibitors of anaplastic lymphoma kinase (ALK) induce immunogenic cell death through on-target effects.

Cell Death Dis 2021 07 16;12(8):713. Epub 2021 Jul 16.

Centre de Recherche des Cordeliers, INSERM UMRS1138, Université de Paris, Sorbonne Université, Team "Metabolism, Cancer & Immunity", 75006, Paris, France.

Immunogenic cell death (ICD) is clinically relevant because cytotoxicants that kill malignant cells via ICD elicit anticancer immune responses that prolong the effects of chemotherapies beyond treatment discontinuation. ICD is characterized by a series of stereotyped changes that increase the immunogenicity of dying cells: exposure of calreticulin on the cell surface, release of ATP and high mobility group box 1 protein, as well as a type I interferon response. Here, we examined the possibility that inhibition of an oncogenic kinase, anaplastic lymphoma kinase (ALK), might trigger ICD in anaplastic large cell lymphoma (ALCL) in which ALK is activated due to a chromosomal translocation. Multiple lines of evidence plead in favor of specific ICD-inducing effects of crizotinib and ceritinib in ALK-dependent ALCL: (i) they induce ICD stigmata at pharmacologically relevant, low concentrations; (ii) can be mimicked in their ICD-inducing effects by ALK knockdown; (iii) lose their effects in the context of resistance-conferring ALK mutants; (iv) ICD-inducing effects are mimicked by inhibition of the signal transduction pathways operating downstream of ALK. When ceritinib-treated murine ALK-expressing ALCL cells were inoculated into the left flank of immunocompetent syngeneic mice, they induced an immune response that slowed down the growth of live ALCL cells implanted in the right flank. Although ceritinib induced a transient shrinkage of tumors in lymphoma-bearing mice, irrespective of their immunocompetence, relapses occurred more frequently in the context of immunodeficiency, reducing the effects of ceritinib on survival by approximately 50%. Complete cure only occurred in immunocompetent mice and conferred protection to rechallenge with the same ALK-expressing lymphoma but not with another unrelated lymphoma. Moreover, immunotherapy with PD-1 blockade tended to increase cure rates. Altogether, these results support the contention that specific ALK inhibition stimulates the immune system by inducing ICD in ALK-positive ALCL.
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http://dx.doi.org/10.1038/s41419-021-03997-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8285454PMC
July 2021

Belantamab Mafodotin (GSK2857916) Drives Immunogenic Cell Death and Immune-mediated Antitumor Responses .

Mol Cancer Ther 2021 Oct 12;20(10):1941-1955. Epub 2021 Jul 12.

Oncology R&D, GlaxoSmithKline, Collegeville, Pennsylvania.

B-cell maturation antigen (BCMA) is an attractive therapeutic target highly expressed on differentiated plasma cells in multiple myeloma and other B-cell malignancies. GSK2857916 (belantamab mafodotin, BLENREP) is a BCMA-targeting antibody-drug conjugate approved for the treatment of relapsed/refractory multiple myeloma. We report that GSK2857916 induces immunogenic cell death in BCMA-expressing cancer cells and promotes dendritic cell activation and GSK2857916 treatment enhances intratumor immune cell infiltration and activation, delays tumor growth, and promotes durable complete regressions in immune-competent mice bearing EL4 lymphoma tumors expressing human BCMA (EL4-hBCMA). Responding mice are immune to rechallenge with EL4 parental and EL4-hBCMA cells, suggesting engagement of an adaptive immune response, immunologic memory, and tumor antigen spreading, which are abrogated upon depletion of endogenous CD8 T cells. Combinations with OX40/OX86, an immune agonist antibody, significantly enhance antitumor activity and increase durable complete responses, providing a strong rationale for clinical evaluation of GSK2857916 combinations with immunotherapies targeting adaptive immune responses, including T-cell-directed checkpoint modulators.
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http://dx.doi.org/10.1158/1535-7163.MCT-21-0035DOI Listing
October 2021

Prolonged SARS-CoV-2 RNA virus shedding and lymphopenia are hallmarks of COVID-19 in cancer patients with poor prognosis.

Cell Death Differ 2021 Jul 6. Epub 2021 Jul 6.

Gustave Roussy Cancer Campus, Villejuif, France.

Patients with cancer are at higher risk of severe coronavirus infectious disease 2019 (COVID-19), but the mechanisms underlying virus-host interactions during cancer therapies remain elusive. When comparing nasopharyngeal swabs from cancer and noncancer patients for RT-qPCR cycle thresholds measuring acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in 1063 patients (58% with cancer), we found that malignant disease favors the magnitude and duration of viral RNA shedding concomitant with prolonged serum elevations of type 1 IFN that anticorrelated with anti-RBD IgG antibodies. Cancer patients with a prolonged SARS-CoV-2 RNA detection exhibited the typical immunopathology of severe COVID-19 at the early phase of infection including circulation of immature neutrophils, depletion of nonconventional monocytes, and a general lymphopenia that, however, was accompanied by a rise in plasmablasts, activated follicular T-helper cells, and non-naive Granzyme BFasL, EomesTCF-1, PD-1CD8 Tc1 cells. Virus-induced lymphopenia worsened cancer-associated lymphocyte loss, and low lymphocyte counts correlated with chronic SARS-CoV-2 RNA shedding, COVID-19 severity, and a higher risk of cancer-related death in the first and second surge of the pandemic. Lymphocyte loss correlated with significant changes in metabolites from the polyamine and biliary salt pathways as well as increased blood DNA from Enterobacteriaceae and Micrococcaceae gut family members in long-term viral carriers. We surmise that cancer therapies may exacerbate the paradoxical association between lymphopenia and COVID-19-related immunopathology, and that the prevention of COVID-19-induced lymphocyte loss may reduce cancer-associated death.
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http://dx.doi.org/10.1038/s41418-021-00817-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8259103PMC
July 2021

Vesicular trafficking in cellular responses to stress.

Methods Cell Biol 2021 ;164:xvii-xxi

Université de Paris, Paris, France; Department of Radiation Oncology, Weill Cornell Medical College, New York, NY, United States; Sandra and Edward Meyer Cancer Center, New York, NY, United States; Department of Dermatology, Yale School of Medicine, New Haven, CT, United States; Caryl and Isreal Englander Institute for Precision Medicine, New York, NY, United States. Electronic address:

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http://dx.doi.org/10.1016/S0091-679X(21)00069-8DOI Listing
January 2021

Autophagic flux assessment by immunoblot.

Methods Cell Biol 2021 1;164:63-72. Epub 2021 Mar 1.

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; Metabolomics and Cell Biology Platforms, Gustave Roussy Comprehensive Cancer Institute, Université Paris Saclay, Villejuif, France; Suzhou Institute for Systems Medicine, Chinese Academy of Medical Sciences, Suzhou, China; Pôle de Biologie, Hôpital Européen Georges-Pompidou, AP-HP, Paris, France; Department of Women's and Children's Health, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden. Electronic address:

Autophagy is one of the main adaptive mechanisms to maintain cellular homeostasis in response to multiple stresses. During autophagy diverse cellular components such as damaged organelles or superfluous proteins are targeted for lysosomal degradation. Importantly, during the initiation of autophagy MAP1LC3B (better known as LC3) lipidates into the membrane of the forming phagophore, which facilitates the formation and lengthening of autophagosomes. In addition, the autophagy receptor SQSTM1 (better known as p62) selectively recruits various cargos to autophagosomes for lysosomal degradation. Both, the conversion of LC3 as well as the degradation of p62 can be assessed as means of monitoring autophagy. Here we detail a protocol for assessing these key events of the autophagic flux via immunoblot.
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http://dx.doi.org/10.1016/bs.mcb.2020.10.005DOI Listing
March 2021

Live cell imaging of LC3 dynamics.

Methods Cell Biol 2021 1;164:27-38. Epub 2021 Mar 1.

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; Metabolomics and Cell Biology Platforms, Gustave Roussy Comprehensive Cancer Institute, Université Paris Saclay, Villejuif, France; Pôle de Biologie, Hôpital Européen Georges-Pompidou, AP-HP, Paris, France; Suzhou Institute for Systems Medicine, Chinese Academy of Medical Sciences, Suzhou, China; Department of Women's and Children's Health, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden. Electronic address:

Macroautophagy (hereafter referred to as autophagy) serves the liberation of energy resources through the degradation of cellular components and is characterized by the formation of double-membraned vesicles, commonly referred to as autophagosomes. Microtubule-associated proteins 1A/1B light chain 3B (hereafter referred to as LC3) plays a crucial role during autophagosome formation, as cleavage of its immature form and subsequent conjugation to phosphatidylethanolamine facilitates autophagosomal membrane biogenesis. Indeed, the redistribution of green fluorescent protein (GFP)-conjugated LC3 from a diffuse cytosolic pattern into forming autophagosomes constitutes a morphological phenotype (commonly referred to as LC3 puncta) applicable to phenotypic analysis. The quantification of LC3 puncta in end-point assays has extensively been used in the past, allowing for the identification of autophagy modulators. Here, we describe a robust method employing automated confocal live cell imaging for the study of time-resolved LC3 dynamics. Furthermore, this method can be used to differentiate between phenotypes such as the homogeneous distribution of LC3 puncta in the cytoplasm, and the aggregation of LC3 clusters juxtaposed to the nucleus thus allowing for functional predictions.
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http://dx.doi.org/10.1016/bs.mcb.2020.10.003DOI Listing
March 2021

The intracellular metabolome of starving cells.

Methods Cell Biol 2021 1;164:137-156. Epub 2021 May 1.

Metabolomics and Cell Biology Platforms, Gustave Roussy Comprehensive Cancer Institute, Université Paris Saclay, Villejuif, France; 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; Suzhou Institute for Systems Medicine, Chinese Academy of Medical Sciences, Suzhou, China; Department of Women's and Children's Health, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden; Pôle de Biologie, Hôpital Européen Georges-Pompidou, AP-HP, Paris, France. Electronic address:

Fasting induces vast metabolic adaptations on the cellular level and leads to an organism-wide induction of autophagy. Autophagic degradation subserves resource recycling and facilitates the maintenance of energetic homeostasis. Mass spectrometry offers the possibility to assess changes in the metabolome that occur in conditions of nutrient deprivation and to profile such adaptations. Here we describe a detailed workflow for the targeted quantitation and untargeted profiling of metabolites that can be used to assess the intracellular metabolome of starving cells. Moreover, we outline a workflow for the use of non-radioactive isotope labeled metabolites. Altogether, we show that mass spectrometry is a powerful tool for monitoring metabolic changes in conditions of fasting.
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http://dx.doi.org/10.1016/bs.mcb.2021.04.001DOI Listing
May 2021

Monitoring TFEB translocation.

Methods Cell Biol 2021 25;164:1-9. Epub 2020 Nov 25.

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; Metabolomics and Cell Biology Platforms, Gustave Roussy Comprehensive Cancer Institute, Université Paris Saclay, Villejuif, France; Suzhou Institute for Systems Medicine, Chinese Academy of Medical Sciences, Suzhou, China; Pôle de Biologie, Hôpital Européen Georges-Pompidou, AP-HP, Paris, France; Department of Women's and Children's Health, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden.

The transcription factor EB (TFEB) plays a critical role in autophagy induction and lysosomal biogenesis by orchestrating the expression of autophagy- and lysosome-related genes. In response to a series of stresses such as nutrient starvation, TFEB translocates from the cytoplasm to the nucleus, where it exerts its regulatory function. The activity of TFEB is tightly regulated by multiple phosphorylation and acetylation sites. Methods that rely on the analysis of posttranslational modification as a proxy for TFEB activation are often misleading. Here, we elaborate on protocols for monitoring nuclear translocation of TFEB by fluorescence microscopy.
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http://dx.doi.org/10.1016/bs.mcb.2020.10.017DOI Listing
November 2020

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

ATP and cancer immunosurveillance.

EMBO J 2021 Jul 14;40(13):e108130. Epub 2021 Jun 14.

Department of Radiation Oncology, Weill Cornell Medical College, New York, NY, USA.

While intracellular adenosine triphosphate (ATP) occupies a key position in the bioenergetic metabolism of all the cellular compartments that form the tumor microenvironment (TME), extracellular ATP operates as a potent signal transducer. The net effects of purinergic signaling on the biology of the TME depend not only on the specific receptors and cell types involved, but also on the activation status of cis- and trans-regulatory circuitries. As an additional layer of complexity, extracellular ATP is rapidly catabolized by ectonucleotidases, culminating in the accumulation of metabolites that mediate distinct biological effects. Here, we discuss the molecular and cellular mechanisms through which ATP and its degradation products influence cancer immunosurveillance, with a focus on therapeutically targetable circuitries.
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http://dx.doi.org/10.15252/embj.2021108130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8246257PMC
July 2021

Autophagy induction by IGF1R inhibition with picropodophyllin and linsitinib.

Autophagy 2021 08 10;17(8):2046-2047. Epub 2021 Jun 10.

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

Induction of macroautophagy (hereafter termed autophagy) is a strategy to improve the outcome of antineoplastic therapies by facilitating the induction of immunogenic cancer cell death and the consequent immune recognition of malignant cells. We analyzed 65,000 distinct compounds by means of a phenotypic discovery platform for autophagy induction and identified the IGF1R (insulin like growth factor 1 receptor) inhibitor picropodophyllin (PPP) as a potent inducer of autophagic flux. We found that PPP acts on-target, as an inhibitor of the tyrosine kinase activity of IGF1R and enhances the release of adenosine triphosphate, ATP, from stressed and dying cancer cells in vitro, thereby improving the therapeutic efficacy of chemoimmunotherapy in cancer-bearing mice. This PPP effect was phenocopied by another IGF1R inhibitor, linsitinib. Moreover, in human triple-negative breast cancer, phosphorylation of IGF1R correlates with reduced autophagy, an unfavorable local immune profile and poor prognosis. In summary, IGF1R inhibition may constitute a novel strategy for the treatment of cancer in the context of chemoimmunotherapy.
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http://dx.doi.org/10.1080/15548627.2021.1936934DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8386749PMC
August 2021

Translocation of chromatin proteins to nucleoli-The influence of protein dynamics on post-fixation localization.

Cytometry A 2021 Jun 10. Epub 2021 Jun 10.

Department of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Kraków, Poland.

It is expected that the subnuclear localization of a protein in a fixed cell, detected by microscopy, reflects its position in the living cell. We demonstrate, however, that some dynamic nuclear proteins can change their localization upon fixation by either crosslinking or non-crosslinking methods. We examined the subnuclear localization of the chromatin architectural protein HMGB1, linker histone H1, and core histone H2B in cells fixed by formaldehyde, glutaraldehyde, glyoxal, ethanol, or zinc salts. We demonstrate that some dynamic, weakly binding nuclear proteins, like HMGB1 and H1, may not only be unexpectedly lost from their original binding sites during the fixation process, but they can also diffuse through the nucleus and eventually bind in nucleoli. Such translocation to nucleoli does not occur in the case of core histone H2B, which is more stably bound to DNA and other histones. We suggest that the diminished binding of some dynamic proteins to DNA during fixation, and their subsequent translocation to nucleoli, is induced by changes of DNA structure, arising from interaction with a fixative. Detachment of dynamic proteins from chromatin can also be induced in cells already fixed by non-crosslinking methods when DNA structure is distorted by intercalating molecules. The proteins translocated during fixation from chromatin to nucleoli bind there to RNA-containing structures.
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http://dx.doi.org/10.1002/cyto.a.24464DOI Listing
June 2021

Multifaceted modes of action of the anticancer probiotic Enterococcus hirae.

Cell Death Differ 2021 Jul 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

AKT as a therapeutic target for autophagy induction and cancer therapy.

Oncoscience 2021 19;8:34-36. Epub 2021 Mar 19.

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.

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http://dx.doi.org/10.18632/oncoscience.526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8045973PMC
March 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

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
June 2021

Quantification of eIF2α Phosphorylation Associated with Mitotic Catastrophe by Immunofluorescence Microscopy.

Methods Mol Biol 2021 ;2267:217-226

Cell biology and metabolomics platforms, Gustave Roussy Cancer Center, Villejuif, France.

Mitotic catastrophe is an oncosuppressive mechanism that drives cells toward senescence or death when an error occurs during mitosis. Eukaryotic cells have developed adaptive signaling pathways to cope with stress. The phosphorylation on serine 51 of the eukaryotic translation initiation factor (eIF2α) is a highly conserved event in stress responses, including the one that is activated upon treatment with mitotic catastrophe inducing agents, such as microtubular poisons or actin blockers. The protocol described herein details a method to quantify the phosphorylation of eIF2α by high-throughput immunofluorescence microscopy. This method is useful to capture the 'integrated stress response', which is characterized by eIF2α phosphorylation in the context of mitotic catastrophe.
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http://dx.doi.org/10.1007/978-1-0716-1217-0_15DOI Listing
June 2021

Calreticulin Exposure in Mitotic Catastrophe.

Methods Mol Biol 2021 ;2267:207-215

Cell biology and metabomics platforms, Gustaver Roussy Cancer Campus, Villejuif, France.

Mitotic catastrophe is a modality of cell death (or occasionally senescence) that occurs after cells enter, and fail to resolve, abnormal mitosis, for instance after DNA damage or perturbations of the cell cycle. Mitotic catastrophe can avoid the generation of neoplastic cells from premalignant precursors, yet may also occur in cancer cells as a result of radiotherapy or chemotherapy. Of note, vinca alkaloids and taxanes, which are both known for affecting the stability of microtubules, can trigger mitotic catastrophe. Such agents can also cause cancer cells to undergo immunogenic cell death (ICD), which allows therapeutic responses to last beyond treatment discontinuation due to the induction of an antitumor immune response. ICD is commonly characterized by the exposure of the endoplasmic reticulum protein calreticulin on the cell surface. Here we describe an immunofluorescence-based cytofluorometric technique to detect calreticulin exposure on tumor cells exposed to drugs that induce mitotic catastrophe.
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http://dx.doi.org/10.1007/978-1-0716-1217-0_14DOI Listing
June 2021

Cytokine-like protein 1-induced survival of monocytes suggests a combined strategy targeting MCL1 and MAPK in CMML.

Blood 2021 Jun;137(24):3390-3402

INSERM U1287, Gustave Roussy Cancer Campus, Villejuif, France.

Mouse models of chronic myeloid malignancies suggest that targeting mature cells of the malignant clone disrupts feedback loops that promote disease expansion. Here, we show that in chronic myelomonocytic leukemia (CMML), monocytes that accumulate in the peripheral blood show a decreased propensity to die by apoptosis. BH3 profiling demonstrates their addiction to myeloid cell leukemia-1 (MCL1), which can be targeted with the small molecule inhibitor S63845. RNA sequencing and DNA methylation pattern analysis both point to the implication of the mitogen-activated protein kinase (MAPK) pathway in the resistance of CMML monocytes to death and reveal an autocrine pathway in which the secreted cytokine-like protein 1 (CYTL1) promotes extracellular signal-regulated kinase (ERK) activation through C-C chemokine receptor type 2 (CCR2). Combined MAPK and MCL1 inhibition restores apoptosis of monocytes from patients with CMML and reduces the expansion of patient-derived xenografts in mice. These results show that the combined inhibition of MCL1 and MAPK is a promising approach to slow down CMML progression by inducing leukemic monocyte apoptosis.
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http://dx.doi.org/10.1182/blood.2020008729DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8233685PMC
June 2021

In Vivo Imaging of Orthotopic Lung Cancer Models in Mice.

Methods Mol Biol 2021 ;2279:199-212

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

The success of anticancer interventions relies on their ability to ignite an anticancer immune response and to reinstate cancer immunosurveillance. Thus, high dose crizotinib can induce immunogenic cell death (ICD) in cancer cells. If combined with cisplatin, crizotinib sensitizes non-small cell lung cancers (NSCLC) to subsequent (but not simultaneous) immunotherapy with PD-1 immune checkpoint blockade, facilitating the cure of more than 90% of established orthotopic cancers in mice. Here, we detail protocols for the establishment and monitoring of transplantable orthotopic NSCLCs in syngeneic immunocompetent animals. Indeed, TC1 cells establish lung cancer upon their intravenous injection into the tail vein, while Lewis lung carcinoma (LLC) cells can be implanted intrathoracically to generate lung cancers. If transduced with luciferase, both TC1 and LLC cells form tumors that can be conveniently monitored by chemiluminescence. This type of NSCLC model is highly useful for the development of novel curative anticancer therapies.
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http://dx.doi.org/10.1007/978-1-0716-1278-1_16DOI Listing
April 2021

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

Ferroptosis becomes immunogenic: implications for anticancer treatments.

Oncoimmunology 2020 12 29;10(1):1862949. Epub 2020 Dec 29.

Equipe Labellisée Par la Ligue Contre le Cancer, Université de Paris, Sorbonne Université, INSERM U1138, Centre de Recherche des Cordeliers, Paris, France.

Ferroptosis is an iron-dependent form of non-apoptotic cell death that has recently been attributed with antitumor immune effects. Thus, early ferroptotic cells underwent immunogenic cell death that was accompanied by the emission of damage-associated molecular patterns (DAMPs) and triggered dendritic cell maturation in vitro. Furthermore, ferroptotic cells were able to vaccinate against a rechallenge with fibrosarcoma in preclinical models.
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http://dx.doi.org/10.1080/2162402X.2020.1862949DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7781761PMC
December 2020

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
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